Oxidative Stress–MicroRNA Interactions in Colorectal Cancer and Their Possible Relationships with Estrogen Receptor β

Abstract

The pathogenesis of colorectal cancer (CRC) is characterized by significant alterations in redox homeostasis, epigenetic regulation and hormone-dependent signaling pathways. Oxidative stress (OS) promotes DNA damage, genomic instability, chronic inflammation, and the activation of several signaling pathways. Furthermore, dysregulation of various microRNAs (miRs) increases proliferation, invasion, metastasis, epithelial–mesenchymal transition, and angiogenesis, and promotes resistance to apoptosis and therapy. Increasing data suggest that there is a two-way interaction between reactive oxygen species (ROS) and miRs, where OS can influence the expression of miRs and, at the same time, miRs can affect intracellular redox state. This interrelationship plays a role in CRC initiation, development, and response to treatment. Estrogen receptor beta (ERβ) represents one of the defensive elements in the development of CRC. Its expression gradually decreases in colorectal cancerogenesis; conversely, its activation suppresses inflammation, proliferation, migration, invasion, and angiogenesis, and enhances apoptosis and DNA repair. In addition, ERβ is capable of modulating the expression of different miRs related to CRC and is involved in the modulation of the intracellular redox state. Limited and often indirect data indicate the role played by miRs and OS in regulating ERβ expression and vice versa. However, disturbing the complex regulatory network involving OS, miRNAs and ERβ may favor CRC development, while restoring it may represent a promising therapeutic strategy.

1. Introduction

Colorectal cancer (CRC) is the third most common cancer among men and the second most common among women. It is also one of the leading causes of cancer-related deaths worldwide, with higher rates observed in high-income countries [1,2]. The current increase in CRC incidence can be attributed to lifestyle changes among younger people under the age of 50 who are not yet covered by screening programs [3]. CRC usually develops from adenomatous polyps through a multifactorial process involving several steps that are influenced by genetic, epigenetic, metabolic and environmental factors [4]. Age, smoking, a sedentary lifestyle, alcohol consumption, obesity, a diet high in saturated fats and low in fiber, gut microbiome, and the presence of chronic inflammatory bowel diseases (IBD) represent the main non-hereditary risk factors for CRC, while familial adenomatous polyposis (FAP) and a genetic predisposition to hereditary non-polyposis colorectal cancer (HNPCC) are the hereditary factors involved in CRC [5,6]. Compared with normal mucosa, many metabolic pathways in CRC are altered. These include the Krebs cycle, nucleotide biosynthesis and glycolysis, as well as those involved in amino acid, lipid and steroid metabolism [7]. The activation of mitogen-activated protein kinases (MAPKs) and the Wnt/β-catenin signaling pathway are involved in the pathogenesis and progression of CRC, to the extent that they are considered to be potential therapeutic targets [8,9]. The primary prevention of CRC aims to avoid risk factors and enhance protective factors. The secondary prevention involves screening programs, early diagnosis and innovative therapies for removing precancerous colorectal neoplastic polyps [10,11]. Given that CRC is more prevalent in males than in females and that the survival rate is higher in young female patients in the same age group (aged 18–44), gender and female hormones play an important role in the onset and survival of CRC [12]. Furthermore, postmenopausal women who do not undergo hormone replacement therapy (HRT) are more likely to develop CRC than those who do [13]. For these reasons, estrogens, and in particular 17β-estradiol (17β-E2), are involved in preventing CRC. This prevention occurs via the predominant estrogen receptor isoform in colon tissue, the estrogen receptor β (ERβ) subtype, which exhibits anti-proliferative, anti-inflammatory and pro-apoptotic properties [14,15]. Studies have shown that low levels of ERβ are present in CRC and that its downregulation is associated with a less favorable prognosis, an increased risk of tumor recurrence, and a more advanced stage of disease [16,17]. In contrast, the expression of the estrogen receptor α subtype (ERα), which is traditionally associated with a proliferative response, is low in normal colon mucosa. However, it is upregulated in CRC, where it activates the Wnt/β-catenin signaling pathway and promotes metastasis [18]. The role of the G protein-coupled estrogen receptor (GPER, also known as GPR30) in CRC is controversial, as it can either promote or suppress tumor growth depending on the context, often modulating rapid non-genomic proliferative pathways [19]. Similarly, ERα signaling is frequently associated with pro-tumorigenic responses. In contrast, the ERβ receptor exerts a protective role in CRC by directly counteracting these oncogenic pathways. Specifically, ERβ inhibits the Wnt/β-catenin pathway to prevent cell proliferation, promotes apoptosis via Bax upregulation, inhibits the inflammatory NF-κB pathway, and regulates redox homeostasis via the regulation of antioxidant enzymes [14,15]. Thus, it is evident that ERβ plays a protective role in CRC, given that its deletion enhances adenoma formation, while its activation has anti-tumorigenic properties [15].

MicroRNAs (miRs), which are single-stranded non-coding RNA molecules constituted by 18–25 nucleotides, can regulate gene expression at the post-transcriptional level by promoting the degradation of specific messenger RNA (mRNA) or inhibiting its translation into protein [20]. Altered miR expression profile and oxidative stress (OS), which is caused by an imbalance between reactive oxygen species (ROS) production and antioxidant levels, are recognized as major cancer promoters and potential therapeutic targets [21]. Dysregulated miR expression contributes to CRC initiation and progression by affecting key processes, including proliferation, apoptosis, invasion, angiogenesis, epithelial–mesenchymal transition (EMT), and treatment response. It may also have diagnostic, prognostic, and therapeutic relevance [22,23,24,25]. The upregulation of ROS levels promotes DNA damage, contributing to tumor-promoting mutations and the inhibition of tumor suppressors [26]. In CRC, mitochondrial impairment and the activation of NADPH oxidases are the main causes of increased ROS levels. These latter play an important role in mutagenesis and the progression of CRC by activating key oncogenic pathways, including the nuclear factor kappa B (NF-κB) pathway, which regulates genes involved in cell survival, proliferation and metastasis [27,28]. Literature data demonstrate the existence of cross-talk between miRs and OS [29,30], as well as a bidirectional interplay between them and ERβ [31,32,33,34]. In particular, miRs can regulate intracellular redox state by affecting signaling pathways that induce or reduce OS, while OS can also alter miR expression [35,36]. Genomic ERβ signaling can reduce OS by increasing antioxidant defenses, such as the expression of antioxidant enzymes like superoxide dismutase and catalase, and by reducing ROS levels [37,38,39]. Additionally, ERβ has been shown to promote apoptosis and prevent malignant transformation in response to OS [40]. However, high ROS levels can alter the conformation and expression of ERβ, which can in certain cases cause an increase in proliferative processes [31,41]. ERβ can upregulate specific miRs through distinct mechanisms. Direct regulation occurs when, upon 17β-E2 binding, this receptor translocates in to the nucleus and binds to the estrogen response elements (EREs), located in the proximity of the promoters of specific miR genes [42,43]. In contrast, indirect regulation involves the transcriptional repression of oncogenes (i.e., Myc, which activates the transcription of several miRs) or through epigenetic mechanisms [44]. This receptor can affect miR levels in human colon cancer cells, thereby contributing to the prevention and treatment of CRC [45]. On the contrary, miRs can also regulate the expression of genes encoding ERα (ESR1) and ERβ (ESR2). Specific miRs decrease expression of ERβ levels or promote the activation of ERβ signal pathways by downregulating specific factors, causing an increase in tumor aggressiveness or delaying cancer development, respectively [46,47]. Several miRNAs have been experimentally validated to target the 3′UTR of ESR2 transcripts via dual-luciferase reporter assays. In vitro functional experiments using miRNA mimics have proven that miR-29b-1-5p and let-7f-1-3p directly repress ESR2 expression, which results in the significant inhibition of downstream ERβ signaling, as observed in mouse hypothalamus under environmental stress [48]. The regulation of the ERβ signaling pathway is usually mediated by epigenetic modifiers or competitive endogenous RNA (ceRNA) network. One such indirect epigenetic regulation is represented by the miR-22-5p/TET2 axis. Unlike other indirect pathways where miRNAs target ESR2 directly, in this case, miR-22-5p regulates the ten-eleven translocation 2 (TET2) enzyme, which is an important epigenetic regulator of DNA demethylation, and thus indirectly modulates chromatin accessibility and represses ESR2 transcription. Importantly, this indirect regulation does not apply only to oncogenesis, but plays a key pathological role in other conditions that are dependent on estrogens; for example, in infertile women with minimal/mild endometriosis during the implantation window, it causes the deregulation of the progesterone response and infertility associated with implantation failure [49]. Figure 1 shows the intricate OS–miR–ERβ network that is balanced in normal cells, but which, when disrupted, could contribute to CRC initiation and progression.

Figure 1. An emerging complex network involving OS–miR–ERβ that controls cellular homeostasis and cancer development.

This review aims to highlight the key molecular mechanisms involved in the roles of OS and miRs and the protective role of ERβ-mediated estrogen in CRC. Despite the fact that extensive studies have been conducted on OS, miR dysregulation and ERβ signaling pathways as factors involved in CRC, their interconnections have never been examined in a comprehensive manner. Our review clarifies the potential connections between these factors that could lead to shared mechanisms regulating inflammation, proliferation, apoptosis, EMT, invasion, metastasis and therapeutic responses. It should be emphasized that distinguishing well-established molecular mechanisms from indirect evidence and mechanistic hypotheses has highlighted a lack of clear information regarding the presence and role of an integrated OS–miR–ERβ axis in CRC development. However, the information contained in this review could facilitate the identification of new potential biomarkers and therapeutic approaches targeting the interactions among these three regulatory components, with the aim of preventing and treating CRC.

2. Oxidative Stress and CRC

Under physiological conditions, ROS are produced during redox reactions that form part of the normal metabolism and during the inflammatory response [50]. Their levels are highly regulated by the body’s antioxidant systems with the aim to maintain redox homeostasis [51], and ROS are involved in numerous cellular processes by regulating several signaling pathways [52]. These include those mediated by the transcription factor NF-κB, mitogen-activated protein kinases (MAPKs), phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt), nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), antioxidant response elements (AREs), toll-like receptor 4 (TLR4) and signal transducer and activator of transcription 3 (STAT3) [53,54,55]. At intestinal level, the physiological redox state regulates signaling pathways, including Wnt/β-catenin and Notch, involved in proliferation, crypt-to-villus migration and stem cell differentiation [56]. However, if ROS production becomes excessive and is not offset by antioxidant systems, OS occurs. This causes cellular damage and serious metabolic disorders by reacting with biological molecules, such as nucleic acids, lipids, and proteins [57]. It also alters cell signaling pathways, which can lead to the development of cancer and resistance to treatment [27]. However, the OS is involved in processes ranging from tumor formation to tumor cell death via mechanisms involving mitochondrial and endoplasmic reticulum stress [58]. In addition, the accumulation of ROS and disorders of iron metabolism together with lipid peroxidation cause ferroptosis, a form of iron- and ROS-dependent cell death [59]. Thus, the role of OS in cancer is dual and complex. On the one hand, it causes the onset of cancer; on the other hand, if OS levels are high, it can also induce cell death [60]. Notably, the biological effect of ROS on CRC progression is highly dictated by a fine homeostatic regulation; although physiological levels of ROS act as necessary second messengers in signaling pathways, excessive pathological ROS cause genomic instability and promote aberrant oncogenic pathways. In CRC progression, chronic OS is mainly caused by mitochondrial malfunction and upregulation of NOXs, resulting in an abnormal increase in ROS production. This leads to extensive DNA damage and mutations and chromosomal instability, thus promoting malignancy [61]. On the other hand, ROS may change the expression, biogenesis, stability, and functions of miRs in different cancer types, including CRC, through several mechanisms [62]. Among these are the activation of redox-sensitive transcription factors such as NF-κB and HIF-1α, directly responsible for inflammatory and hypoxic responses. Moreover, OS negatively affects the stability and activity of miRs by controlling the expression of proteins participating in miR biogenesis and maturation, such as the RNA-binding protein DiGeorge critical region 8 (DGCR8) and Dicer, and by inducing epigenetic changes, like DNA methylation, histone acetylation, and phosphorylation [62,63].

Ferroptosis, an iron- and ROS-dependent form of cell death, may suppress or promote tumor development depending on the cellular context [64,65,66]. Lipid peroxidation through ROS pathway is an essential mediator in ferroptosis in CRCs. The iron-dependent oxidative metabolism of lipid leads to the oxidation of polyunsaturated fatty acids (PUFAs) in the cell and mitochondrial membrane phospholipids, causing damage to membrane and ferroptotic cell death. In a healthy state, the GSH–GPX4 pathway acts as an antioxidant against lipid peroxidation; however, in the absence of GSH and GPX4, the accumulation of lipid peroxides causes ferroptosis. In CRC, this process is important because of the possibility of using ferroptosis induction as a tool of tumor cell elimination, including treatment-resistant populations [67].

Chronic inflammation associated with IBD can significantly promote the development, progression and onset of CRC. In fact, IBDs are characterized by an overproduction of ROS, which is exacerbated by high levels of inflammatory mediators, such as cytokines and chemokines. These, in turn, induce ROS generation enhancing cancer progression [68,69]. Furthermore, CRC development can be triggered by certain carcinogenic compounds that cause OS [70,71].

Analysis of OS markers shows decreased levels of reduced glutathione (GSH), glutathione reductase, glutathione peroxidase (GPX) and catalase (CAT), and increased levels of oxidized glutathione (GSSG) and superoxide dismutase (SOD) in CRC patients [72,73,74]. In fact, serum OS parameters, such as total oxidant status, total antioxidant status, and the oxidative stress index, are related to CRC status and may be associated with CRC severity [75]. Levels of NADPH oxidase (NOX1), cyclooxygenase-2 (COX-2), and tumor necrosis factor-α (TNFα), all of which are related to OS, are also high in the serum of patients with CRC compared with healthy patients [76]. Furthermore, the presence of elevated levels of 8-oxo-2′-deoxy-7,8-dihydroguanosine (8-oxodG), one of the main products of DNA oxidation, in the plasma, urine, and serum also suggests elevated levels of ROS in CRC [77,78]. It has been demonstrated that OS stimulates intestinal carcinogenesis by increasing the levels of 8-oxodG in MUTYH-deficient mice, a model characterized by impaired base excision repair and increased susceptibility to CRC [79]. 4-hydroxynonenal (4-HNE), a very reactive aldehyde that is generated by lipid peroxidation resulting from OS, causes DNA cross-links and mutations, as well as altering protein function [80,81]. High levels of 4-HNE are found in CRC cells and the tumor environment, where it activates the non-canonical Hedgehog (HH) signaling pathway in cancer stem cells, increasing CRC progression [82]. Moreover, 4-HNE can also activate COX-2, a key molecule in the development and progression of CRC [83,84], and promote the activation of Nrf2 and the expression of heme oxygenase-1 (HO-1) [85]. Following the surgical removal of the tumor, a reduction in biomarkers of OS and an increase in antioxidant defenses were observed. These changes may slow the progression of CRC by improving redox homeostasis [86].

2.1. Nrf2 Signaling Pathways

Under OS, Nrf2 dissociates from Keap1, translocates into the nucleus, binds to DNA sequences called antioxidant response elements (AREs) and induces the transcription of important antioxidant genes that protect cells. For this reason, Nrf2 is often considered essential for cancer prevention [87]. However, although Nrf2 provides protection against carcinogenesis in the early stages of the disease [88,89], numerous studies have shown that high levels of Nrf2 are present in various tumors, including CRC, and are associated with tumor size and poor prognosis [90,91]. In particular, transient Nrf2 activation protects normal colonocytes from oxidative DNA damage and malignant transformation and plays a protective role in the early stages of tumorigenesis. However, persistent activation of Nrf2 plays a disruptive role in CRC cells in later stages. In fact, ROS and increased DNA damage can cause hyperactivation of Nrf2, resulting in increased cancer cell survival and reduced apoptosis [92].

The activation of the Nrf2/Keap1 and Nrf2/ARE pathways protects against the cytotoxicity that arises due to the anticancer drugs and radiation-induced damage, and Nrf2 could play an important role in chemoresistance to CRC [93,94,95]. In CRC, activation of the Nrf2/Keap1 signaling pathway by mitochondrial ROS affects glycolysis, oxidative phosphorylation, mitochondrial biogenesis, and mitophagy [96]. Adenylosuccinate lyase (ADSL), an essential enzyme for de novo purine biosynthesis, is considered an oncogene for CRC, given that its expression is elevated in this tumor and promotes OS and activation of the Nrf2/Keap1 pathway, resulting in increased proliferation and migration of CRC cell lines [97]. In CRC, the transcription factor forkhead box A2 (FOXA2), which is involved in tumor cell proliferation, is overexpressed and positively correlates with the activation of Nrf2/GPX4, which is a ferroptosis inhibitor. In fact, inhibiting FOXA2 causes the inactivation of the Nrf2/GPX4 signaling pathway, resulting in the induction of ferroptosis and the suppression of CRC [98]. The activation of Nrf2 by OS promotes the expression of HO-1, a stress-responsive enzyme, which is highly expressed in CRC tissue and is mainly located in cell nuclei [99,100]. It seems that the expression of nuclear HO-1 is associated with the development of CRC, and that HO-1 upregulation is involved in progression and metastasis by promoting CRC cell invasion and angiogenesis, while reducing apoptosis and ferroptosis [100,101,102,103,104]. However, HO-1 can act as both an anti- and pro-tumorigenic agent, as HO-1 activation has been shown to increase survival rates in patients with CRC and suppress colorectal carcinogenesis [103,105]. In fact, activation of the endoplasmic reticulum stress-related kinase (PERK)/Nrf2/HO-1 signaling pathway by OS leads to ferroptosis, alongside decreased GSH levels and increased lipid peroxidation [106].

2.2. Wnt/β-Catenin Signaling Pathway

The Wnt/β-catenin signaling pathway is upregulated by OS and stimulates colon cancer development by acting on its cancer-related downstream targets, as well as by interacting with the tumor microenvironment and cancer stem cells [107,108]. It is activated in CRC alongside a reduction in the function of adenomatous polyposis coli (APC), a tumor suppressor protein that prevents carcinogenesis by degrading β-catenin, thereby avoiding uncontrolled cell growth and proliferation [109,110,111]. However, under conditions of moderate OS, β-catenin and APC expression increases in primary CRC cells and decreases in metastatic cells [112]. Furthermore, the inhibition of Jun N-terminal kinase (JNK), which is also activated by mitochondrial ROS [113], restores β-catenin and APC to control values in primary CRC cells but not in metastatic cells, indicating that moderate OS differentially modulates the interplay between Wnt/β-catenin and JNK in these cells [112]. In CRC cells, treatment with exogenous H₂O₂ increases the expression of β-catenin and leucine-rich repeat G protein-coupled receptor 5 (LGR5), which is also involved in cancer development and progression [114,115]. However, although the LGR5 gene is a target of Wnt signaling, the overexpression of LGR5 is associated with JNK signaling activation by ROS [114]. Levels of solute carrier family 7 member 11 (SLC7A11), a transmembrane cystine/glutamate transporter protein which plays a key role in controlling ferroptosis and glutathione homeostasis, are elevated in tissue samples from CRC patients and are associated with tumor stage and lymph node metastasis due to reduced ferroptosis [116]. The primary metabolite of the commonly used pesticide p-dichlorodiphenyltrichloroethane (DDT) stimulates the growth of human colorectal adenocarcinoma cell lines by activating NADPH oxidase and decreasing GSH levels, as well as the activities of SOD and CAT. This subsequently increases ROS production and activates the Wnt/β-catenin and Hedgehog/Gli1 signaling pathways [117]. Increased proliferation of cells in CRC due to activation of the Wnt/β-catenin pathway under OS can be mediated by STAT3 activation [118]. Sirtuin 1 (Sirt1), an NAD⁺-dependent deacetylase, which deacetylases histone and non-histone proteins, is overexpressed in CRC cells in response to OS and plays a role in CRC development and progression by promoting migration, proliferation, and metastasis, while reducing apoptosis [119,120,121]. In particular, Sirt1 in CRC contributes to fatty acid oxidation by deacetylating β-catenin, and to increased proliferation by inhibiting apoptosis through deacetylating p53 tumor suppressor protein [122,123].

2.3. PI3K/Akt and NF-κB Signaling Pathways

The activation of the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signaling pathway plays a key role in increasing cell proliferation and invasion, and reducing apoptosis in CRC [124,125,126]. There is cross-talk between this signaling pathway and ROS which activate the PI3K cascade by inhibiting phosphatase and tensin homolog (PTEN), a negative regulator of the pathway and tumor suppressor. OS also activates AKT and stimulates receptor tyrosine kinases (RTKs). Conversely, PI3K/Akt contributes to increased ROS levels by activating NADPH oxidases or influencing mitochondrial bioenergetics [127]. COX-2, which is involved in inflammation via prostaglandin production, is induced by ROS and promotes CRC cell proliferation and invasion, while it inhibits apoptosis by downregulating the expression of PTEN and activating the PI3K/Akt pathway [128,129]. Tumorigenesis is closely associated with NF-κB, which influences a range of processes, including infiltration, metastasis, apoptosis, cell cycle regulation and differentiation [130]. NF-κB activation by OS is particularly involved in carcinogenesis due to inflammation, and an increase in this transcription factor is present in inflammation-related CRC [131,132,133]. Additionally, NF-κB can be activated by signaling pathways such as TLR4, MAPK, STAT and PI3K, which are affected by OS. This involves a complex network that can influence CRC development and progression [134]. In fact, NF-κB plays an anti-apoptotic role by interacting with genes and signaling pathways in CRC [28,135,136]. It has been observed that NF-κB activation occurs in the tissues of CRC patients alongside an increase in metalloproteinase (MMP) 9 [137]. Indeed, OS is also able to activate MMPs, and elevated levels of MMP2 and MMP9, which play a role in ECM degradation responsible for cell migration and metastatic and invasive growth, have been found in the serum of patients with CRC and are associated with tumor stage [138,139,140].

Figure 2 schematizes the principal redox-regulated pathways activated by OS in CRC.

Figure 2. Principal redox-sensitive signaling pathways activated by increased levels of ROS in CRC. ROS, which increase under conditions of oxidative stress, activate several redox-sensitive signaling pathways, including the Nrf2/Keap1, Wnt/β-catenin, PI3K/Akt and NF-κB pathways. These affect cell proliferation, survival, apoptosis, invasion, angiogenesis, stemness, metabolic reprogramming and therapy resistance. Although transient Nrf2 activation protects cells by maintaining redox homeostasis, sustained activation in cancer cells may promote tumor progression. Combined activation of these signaling networks ultimately promotes the initiation, progression and metastatic dissemination of CRC. This image was created with Created in BioRender software. Gaia Palmini. (2026) (https://www.biorender.com/). Abbreviations: ROS, reactive oxygen species; Nrf2, nuclear factor erythroid 2-related factor 2; Keap1, Kelch-like ECH-associated protein 1; ARE, antioxidant response element; CAT, catalase; SOD, superoxide dismutase; HO-1, heme oxygenase-1; GPX, glutathione peroxidase; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; APC, adenomatous polyposis coli; TCF/LEF, T-cell factor/lymphoid enhancer factor; SLC7A11, solute carrier family 7 member 11; PI3K/Akt, phosphoinositide-3-kinase/protein kinase B; PTEN, phosphatase and tensin homolog; NF-κB, nuclear factor kappa B; COX-2, cyclooxygenase 2; MMPs, metalloproteinases; IL-6, interleukin-6; TNFα, tumor necrosis factor-α.

3. miRs and CRC

miRs act by binding to complementary sequences in the 3′-untranslated region (3′-UTR) of target mRNAs, which can then be degraded or repressed [141]. Dysregulation of miRs alters the expression of their target mRNAs, thereby increasing or decreasing the levels of related proteins. The onset and progression of CRC are affected by epigenetic factors, with miRs playing a significant role by modulating the expression of genes involved in cell proliferation, apoptosis, angiogenesis, and metastasis. However, whether miRs act as oncogenes (oncomiRs) or tumor suppressors depends on their target gene [142,143]. The expression of various miRs is altered in CRC, so detecting these can help with diagnosis, prognosis, and therapeutic approaches [144]. For instance, the upregulation of miR-141-3p and the downregulation of miR-10b-5p in tissue samples from CRC patients have diagnostic value in identifying the disease. This suggests that colonoscopy, alongside determining the level of miR expression in colon mucosa, can provide more accurate information for early CRC diagnosis [145]. Overexpression of miR-549a, miR-552, and miR-592 in peripheral blood and tissue samples from CRC patients can be considered as a useful biomarker for diagnosing CRC and differentiating it from other tumors [146].

3.1. Principal miRs Upregulated in CRC and Their Role

3.1.1. miR-21

The oncomiR miR-21 is highly expressed in CRC and is one of the key miRs involved in CRC progression, contributing to cell proliferation, invasion, and extracellular matrix construction [147,148], and promoting epithelial–mesenchymal transition (EMT), which is involved in cancer progression and metastasis, by inhibiting the expression of mothers against decapentaplegic homolog 7 (SMAD7), programmed cell death 4 (PDCD4), and PTEN [147,149,150,151]. PTEN acts as a negative regulator of the PI3K/Akt signaling pathway by reducing Akt phosphorylation. Thus, high levels of miR-21 in CRC cells result in reduced PTEN levels, thereby increasing Akt phosphorylation and promoting the activation of the PI3K/Akt signaling pathway. The latter lead to increased cell invasion, migration and proliferation, and reduced apoptosis [152]. High circulating levels of miR-21, alongside increased levels of miR-29a and miR-92a, could help to diagnose CRC at an early stage and improve the effectiveness of diagnosis [153,154]. The combined upregulation of miR-21 and downregulation of miR-145, involving the Kirsten rat sarcoma viral oncogene homolog (K-Ras) protein, creates a modified gene network that plays a key role in progression, metastasis, invasion, and the growth of cancer stem cells (CSCs) [153,155,156].

3.1.2. miR-223

High levels of miR-223 in CRC tissues are linked to an increased risk of disease progression, as well as being associated with the tumor-node-metastasis (TNM) stage, lymph node involvement, distant metastasis, and poorer survival [157,158,159,160,161]. In particular, miR-223 induces cell proliferation, migration and invasion and reduces apoptosis by downregulating tumor suppressors such as PR/SET domain 1 (PRDM1), BCL2-like protein 11 (BCL2L11), forkhead transcription factor O3 (FOXO3), Ras homolog family member B (RhoB), F-box and WD repeat domain-containing 7 (FBXW7), RAS p21 protein activator 1 (RASA1), and F-box only protein 8 (FBX8) [162,163,164,165,166,167,168]. In addition, miR-223 promotes drug resistance by reducing FBXW7 and inducing EMT [169]. Together with upregulation of miR-106a-5p, miR-223 is involved in the downregulation of solute carrier family 4 member 4 (SLC4A4), which has tumor-suppressive properties and is expressed at low levels in CRC [170]. However, literature data show that miR-223 may also inhibit NLR family pyrin domain-containing 3 (NLRP3), which is involved in CRC due to inflammation, but this alone is insufficient to prevent CRC metastasis [171].

3.1.3. miR-31

Increased levels of miR-31 in CRC tissue samples are associated with advanced stages and poor tumor differentiation [172]. They contribute to the development and progression of CRC by promoting cell migration and invasion, and by suppressing the expression of T-cell lymphoma invasion and metastasis 1 (TIAM1) [173]. They also activate the RAS, MAPK kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) and hypoxia-inducible transcription factor 1α (HIF-1α) signaling pathways [174,175,176,177]. In CRC cell lines, miR-31 reduces sensitivity to 5-fluorouracil (5-FU) and increases migration and invasion [178]. However, it also induces radiosensitivity by suppressing serine/threonine-protein kinase 40 (STK40), suggesting that miR-31 could eventually be considered a biomarker of the radiation response [179].

3.1.4. miR-155

Compared with adjacent normal tissue, miR-155 is overexpressed in tumor tissues from CRC patients and plays an important role in promoting or inhibiting tumor progression [180,181]. In human CRC samples, the targets of miR-155 include several tumor suppressors, such as suppressor of cytokine signaling 1 (SOCS1), protein tyrosine phosphatase receptor type J (PTPRJ) and PTEN, resulting in the promotion of tumor growth, invasion, and migration [182,183,184]. In addition, miR-155 activates cancer-associated fibroblasts, thereby promoting metastasis and invasion in CRC by targeting SOCS-1 and subsequently promoting the activation of Janus kinase 2 (JAK2)/STAT3 signaling pathway and the transcriptional upregulation of NF-κB/p65 [185]. Elevated levels of miR-155 increase claudin-1 and β-catenin expression, downregulate E-cadherin, upregulate zinc finger E-box binding homeobox 1 (ZEB1), and activate Wnt/β-catenin signaling pathway in human CRC cell lines. This promotes an EMT-like transformation, increases cell migration, proliferation and invasion, and reduces cell-to-cell adhesion [180,186]. They also increase colon cancer resistance to cisplatin by decreasing FOXO3 levels [187]. However, literature data have shown that the overexpression of miR-155 reduces the proliferation of CRC cells, causes cell cycle arrest, induces apoptosis, and regulates cell migration by inhibiting the expression of casitas B-lineage lymphoma (CBL) and collagen triple helix repeat containing 1 (CTHRC1) [181,188].

3.1.5. miR-135b

miR-135b is also overexpressed in CRC tissue, and its levels in stool samples from CRC patients are higher than those detected in healthy patients. Therefore, miR-135b can be considered a possible non-invasive biomarker for CRC detection [189]. Upregulation of miR-135b causes proliferative, anti-apoptotic, invasive, and metastatic effects by reducing the expression of its targets, including APC, transforming growth factor β receptor 2 (TGFβR2), death-associated protein kinase 1 (DAPK1), factor inhibiting hypoxia-inducible factor 1-alpha (HIF-1α) (FIH-1), metastasis suppressor 1 (MTSS1), and tumor suppressor kinase 2 (LATS2) [190,191,192]. Furthermore, it reduces the expression of ubiquitin-specific peptidase 13 (USP13), resulting in the ubiquitination and degradation of PTEN and the activation of the PI3K/Akt pathway [193]. In CRC cells, upregulation of miR-135b increases resistance to cisplatin by targeting Krüppel-like factor 13 (KLF13) [194], while it reduces apoptosis induced by oxaliplatin (Oxa) by decreasing the expression of FOXO1 and B-cell lymphoma 2 (BCL2) family members, BCL2L11 and Noxa [195]. Indeed, the expression of miR-135b-5p was found to be significantly increased in Oxa-resistant CRC cells and in the serum of CRC patients. It promotes Oxa resistance by inducing autophagy through the suppression of mitochondrial E3 ubiquitin protein ligase 1 (MUL1), which normally degrades the key autophagy molecule, Unc-51-like kinase 1 (ULK1), via ubiquitination [196]. Furthermore, miR-135b-5p promotes the proliferation, invasion and cetuximab resistance in CRC by negatively regulating the transcription factor forkhead box N3 (FOXN3), thereby activating the EMT and the Wnt/β-catenin signaling pathway [197].

3.1.6. miR-27a-3p

Upregulation of miR-27a-3p levels in CRC tissues and cell lines causes apoptosis inhibition and increases proliferation, migration, and invasion by reducing the expression of B-cell translocation 1 (BTG1), calcium voltage-gated channel auxiliary subunit alpha-2/delta 3 (CACNA2D3), transcription factor 7-like 2 (TCF7L2) and TGFβR2 [198,199,200]. Moreover, miR-27a-3p promotes CRC progression through the inhibition of retinoid X receptor alpha and the activation of the Wnt/β-catenin signaling pathway [201].

3.1.7. miR-17-92 Cluster

Six distinct miRs, namely miR-17, miR-18a, miR-19a, miR-20a, and miR-92a, are derived from the transcription of the polycistronic miR-17-92 cluster, which is often amplified in various cancer types, including CRC [202]. In CRC, several members of the miR-17-92 cluster are mainly active in promoting tumor cell growth, survival, migration and invasion and inhibiting apoptosis by targeting tumor suppressor genes, such as PTEN, BCL2L11, Krüppel-like factor 4 (KLF4), forkhead box F2 (FOXF2), and FBXW7 [203,204,205,206,207]. One key mechanism underlying the dysregulation of the miR-17-92 cluster in CRC involves the activation of the Wnt/β-catenin signaling pathway [208]. Conversely, β-catenin directly enhances transcription of the miR-17-92 cluster, whose members then reinforce Wnt signaling by inhibiting their negative regulators, creating a feedback loop that sustains tumor growth and EMT [203,209]. The miR-17-92 cluster also interacts with other oncogenic pathways, including the epidermal growth factor receptor (EGFR)/PI3K/Akt and TGFβ/Smad pathways. This contributes to angiogenesis, extracellular matrix remodeling, and metastasis [210,211]. The miR-17-92 cluster promotes angiogenesis primarily by suppressing anti-angiogenic factors, such as thrombospondin-1 (TSP-1) and connective tissue growth factor (CTGF) [212]. It also induces chemoresistance, especially against 5-FU, via mechanisms linked to PTEN repression and altered mitochondrial homeostasis [213,214].

Table 1 summarizes the main upregulated miRs and their underlying molecular mechanisms and effects in CRC.

Table 1. Principal miRs upregulated in CRC.

miRs	Molecular Mechanisms	Major Effects	Refs.
miR-21	PTEN (−) PDCD4 (−) SMAD7 (−) Notch 1 (+) PI3K/Akt (+)	Proliferation (+) Invasion (+) EMT (+) Metastasis (+) Apoptosis (−)	[147,148,149,150,151,152]
miR-223	PRDM1 (−) BCL2L11 (−) FOXO3 (−) RhoB (−) FBXW7 (−) NLRP3 (−) SLC4A4 (−) RASA1 (+) FBX8 (+)	Metastasis (+) Proliferation (+) Migration (+) Invasion (+) EMT (+) Apoptosis (−)	[160,162,163,164,165,166,167,168,169,170,171]
miR-31	TIAM1 (−) RASA1 (−) SATB2 (−) FIH-1 (−) STK40 (−) RAS (+) MEK5/ERK5 (+) HIF-1α (+)	Proliferation (+) Migration (+) Invasion (+) Metastasis (+) Drug resistance	[173,174,175,176,177,178,179]
miR-155	SOCS1 (−) PTPRJ (−) PTEN (−) FOXO3 (−) E-cadherin (−) ZEB1 (+) JAK2/STAT3 (+) NF-κB/p65 (+) Wnt/β-catenin (+) CBL (−) CTHRC1 (−)	Invasion (+) Metastasis (+) Proliferation (+/−) Migration (+/−) EMT (+) Drug resistance Apoptosis (+)	[180,181,182,183,184,185,186,187,188]
miR-135b	APC (−) PTEN (−) TGFβR2 (−) DAPK1 (−) FIH-1 (−) USP13 (−) MTSS1 (−) LATS2 (−) KLF13 (−) FOXO1 (−) BCL2 family member (−) PI3K/Akt (+) SRC (+)	Invasion (+) Metastasis (+) Proliferation (+) Apoptosis (−) Glycolysis (+) Drug resistance	[190,191,192,193,194,195]
miR-135b-5p	MUL1 (−) FOXN3 (−) Wnt/β-catenin (+)	Proliferation (+) Invasion (+) Autophagy (+) EMT (+) Drug resistance	[196,197]
miR-27a-3p	BTG1 (−) CACNA2D3 (−) TCF7L2 (−) TGFβR2 (−) Retinoid X receptor alpha (−) Wnt/β-catenin (+)	Invasion (+) Migration (+) Proliferation (+) Apoptosis (−)	[198,199,200,201]
miR-17-92 cluster	PTEN (−) BCL2L11 (−) KLF4 (−) FOXF2 (−) FBXW7 (−) TSP-1 (−) CTGF (−) Wnt/β-catenin (+) EGFR/PI3K/Akt (+) TGFβ/Smad (+)	Migration (+) Invasion (+) Angiogenesis (+) Metastasis (+) Apoptosis (−) Drug resistance	[203,204,205,206,207,208,209,210,211,212,213,214]

Abbreviations: PTEN, phosphatase and tensin homolog; PDCD4, programmed cell death 4; SMAD7, mothers against decapentaplegic homolog 7; Notch 1, neurogenic locus notch homolog protein 1; EMT, epithelial–mesenchymal transition; PI3K/Akt, phosphatidylinositol-3-kinase/protein kinase B; PRDM1, PR/SET domain 1; BCL2L11, B-cell leukemia 2-like 11; FOXO3, forkhead transcription factor O3; RhoB, Ras homolog family member B; FBXW7, F-box and WD repeat domain-containing 7; NLRP3, NLR family pyrin domain-containing 3; SLC4A4, solute carrier family 4 member 4; RASA1, RAS p21 GTPase activating protein 1; FBX8, F-box only protein 8; TIAM1, T-cell lymphoma invasion and metastasis 1; SATB2, special AT-rich sequence-binding protein 2; FIH-1, factor inhibiting HIF-1α; STK40, serine/threonine-protein kinase 40; hypoxia-inducible transcription factor 1α; MEK5/ERK5, MAP kinase kinase 5/extracellular signal-regulated kinase 5; SOCS1, suppressor of cytokine signaling 1; PTPRJ, protein tyrosine phosphatase receptor type J; ZEB1, zinc finger E-box binding homeobox 1; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3; NF-κB, nuclear factor kappa B; CBL, casitas B-lineage lymphoma; CTHRC1, collagen triple helix repeat containing 1; APC, adenomatous polyposis coli; TGFβR2, transforming growth factor β receptor 2; DAPK1, death-associated protein kinase 1; USP13, ubiquitin-specific peptidase 13; MTSS1, metastasis, suppressor 1; LATS2, tumor suppressor kinase 2; KLF13, Krüppel-like factor 13; FOXO1, forkhead transcription factor O1; SRC, proto-oncogene tyrosine-protein kinase Src; MUL1, mitochondrial E3 ubiquitin protein ligase 1; FOXN3, forkhead box N3; BTG1, B-cell translocation 1; CACNA2D3, calcium voltage-gated channel auxiliary subunit alpha-2/delta 3; TCF7L2, transcription factor 7-like 2; KLF4, Krüppel-like factor 4; FOXF2, forkhead box F2; TSP-1, thrompbospondin-1; CTGF, connective tissue growth factor; EGFR, epidermal growth factor receptor. (−) Downregulated. (+) Upregulated.

3.2. Principal miRs Downregulated in CRC and Their Role

3.2.1. miR-143/145 Cluster

Downregulation of miR-143/145 cluster in CRC tissue samples and human colon cancer cell lines is associated with tumor initiation and contributes to CRC progression and differentiation by regulating epigenetic, proliferative, and survival pathways [215,216,217,218,219,220,221,222,223]. In addition, it increases the expression of the insulin-like growth factor 1 receptor (IGF1R), Krüppel-like factor 5 (KLF5), and cluster of differentiation 44 (CD44) proteins, promoting cell proliferation, angiogenesis, migration, EMT, and metastasis [224,225,226]. Despite their coordinated action, accumulating evidence shows that miR-143 and miR-145 also have distinct targets but complementary roles in CRC. In particular, low levels of miR-143 are associated with high levels of Kirsten rat sarcoma virus (KRAS) protein in CRC, subsequently activating the MAPK/ERK1/2 pathway [227]. Additionally, transcription of the Ras-responsive element-binding protein (RREB1), which is activated downstream of KRAS, inhibits expression of the miR-143/145 cluster, creating a negative feedback loop that increases KRAS signaling [228]. In fact, the carotenoid crocin suppresses proliferation and promotes apoptosis in CRC by upregulating the miR-143/145 cluster, which leads to the downregulation of KRAS, RREB1 and the inhibition of PI3K/Akt signaling [229]. Reduced levels of the miR-143/145 cluster increase CRC cell proliferation by increasing the expression of cell cycle regulators, such as cyclin-dependent kinase 6 (Cdk6), and ERK5 via the negative regulation of miR-145 or miR-143, respectively [230]. Low levels of miR-143 promote cell proliferation, metastasis, and angiogenesis by increasing DNA methyltransferase 3A (DNMT3A) and metastasis-associated protein 1 (MACC1), and activating the PI3K/AKT/HIF-1/vascular endothelial growth factor (VEGF) pathway [217,219,231]. In addition to its effect on proliferation in CRC cells, deregulated miR-145 enhances EMT, migration, and invasion by increasing E-cadherin, vimentin, Snail 1, zinc finger e-box binding homeobox (ZEB1), LIM and SH3 protein 1 (LASP1), and fascin 1 (FSCN1) [232,233]. However, it has been shown that sulfiredoxin (Srx), a specific oxidoreductase that also plays a role in oncogenic signaling, promotes the invasion of CRC cells by downregulating miR-143 and consequently increasing FSCN1 [234]. These data suggest that the FSCN1 mRNA is targeted by both miR-145 and miR-143, which, when downregulated, are also involved in drug resistance in CRC by increasing hexokinase 2 (HK2), IGF1R, and RAD18 expression [219,235,236]. A meta-analysis study of an elevated number of CRC patients has reported that low miR-143 expression is particularly related to disease progression. In contrast, low miR-145 expression correlates with a poorer prognosis and shorter overall survival, identifying miR-145 as a more accurate prognostic factor in CRC [237].

3.2.2. miR-34 Family

The miR-34 family (comprising miR-34a, miR-34b and miR-34c) is downregulated in CRC, and its reduced levels upregulate the expression of proteins associated with lymph node metastasis and poor patient survival, such as the inhibin subunit beta B (INHBB), the tyrosine kinase membrane receptor AXL, the fibroblast growth factor receptor 1 (FGFR1), and the platelet-derived growth factor receptor beta (PDGFRB) [238]. The simultaneous reduction of all members of the miR-34 family upregulates the DREAM complex in the CRC cell line HCT116, leading to increased migration, invasion, autophagy, chemoresistance, and EMT [239]. In particular, downregulation of miR-34a in CRC promotes proliferation, by upregulating the expression of formin-like 2 (FMNL2), E2F transcription factor 5 (E2F5) and high mobility group box 1 (HMGB1) proteins, and reduces apoptosis, by upregulating anti-apoptotic genes (BCL2) [240,241,242]. In addition, it increases the migration of CRC cells by upregulating the expression of Notch1 and Jagged1 proteins and the Sirt1/p53 signaling pathway [243,244]. Low levels of miR-34a induce an increase in the expression of interleukin (IL)-6 receptor (IL-6R), which, after binding with IL-6, activates STAT3, which in turn reduces levels of miR-34a, resulting in EMT, migration, and invasion of CRC cells [245]. In CRC cell lines, the levels of miR-34c are inversely related to those of the KIT proto-oncogene ligand (KITLG) and the transcription factor Myc. This results in increased proliferation, cellular transformation, migration and invasion, and decreased apoptosis [246,247].

3.2.3. miR-126

There is accumulating evidence that miR-126 is significantly downregulated in CRC tissue compared with adjacent normal mucosa, and that it plays a pivotal role in CRC development and progression [248,249]. The downregulation of miR-126 activates the RhoA, Akt and ERK1/2 signaling pathways, thereby increasing cell proliferation and invasion [249,250]. Hypermethylation of the epidermal growth factor-like domain 7 (EGFL7) gene/miR-126 locus leads to the silencing of miR-126 transcription in CRC, resulting in the upregulation of pro-angiogenic factors, such as VEGF, and causing tumor vascularization and invasive growth [251]. In models of colitis-associated CRC, reduced miR-126 expression enhances tumor development by increasing the expression of C-X-C motif chemokine ligand 12 (CXCL12), which promotes the recruitment of macrophages and the activation of inflammatory pathways, including IL-6 signaling. This suggests that miR-126 is implicated in inflammation-associated colorectal (IAC) tumorigenesis [252]. From a clinical perspective, miR-126 has proven to be a promising prognostic and predictive biomarker. Higher levels of miR-126 expression in tumors have been linked to better overall survival and a more favorable prognosis in patients with CRC [253].

3.2.4. miR-138

In human CRC tissues and cell lines, it was found that the expression of miR-138 is reduced, and this correlates inversely with the levels of the twist family bHLH transcription factor 2 (TWIST2), Sirt1, podocalyxin-like protein 1 (PODXL), programmed death ligand 1 (PD-L1), human telomESR2ase reverse transcriptase (hTERT), and Myc proteins. In addition, downregulation of miR-138 activates the nuclear factor I B (NFIB)/Snail 1 and specificity protein 1 (SP1)/leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) pathways. All of this promotes cell proliferation, viability, invasion, migration, metastasis, and EMT, and reduces apoptosis [254,255,256,257,258,259,260,261,262]. Low levels of miR-138 are also involved in drug resistance by increasing the expression of 3-phosphoinositide-dependent protein kinase 1 (PDK1), which is responsible for resistance to Oxa in CRC cell lines [263].

3.2.5. miR-133a and miR-133b

The reduction of miR-133a and miR-133b expression in CRC tissue has been correlated with advanced tumor stage and lymph node metastasis, suggesting that these miRs can be useful as prognostic biomarkers [264,265]. In particular, reduced levels of miR-133a have been linked to an increase in the expression of the ring finger and FYVE-like domain-containing E3 ubiquitin protein ligase (RFFL), which in turn inhibits the expression of the p53 tumor suppressor protein and its downstream target, the p21 protein, thereby inducing CRC cell growth. As p53 correlates with cell responses to chemotherapeutic agents, low levels of miR-133a may be implicated in the reduced sensitivity of CRC cells to drug treatment. Increased levels of miR-133a have been shown to sensitize HCT116 cells to doxorubicin and Oxa by triggering early apoptosis [266]. Low levels of miR-133a are related to increased expression of ubiquitin-like modifier-activating enzyme 2 (UBA2), eukaryotic initiation factor 4A1 (eIF4A1), LASP1, CXCR4, TATA-box binding protein-like 1 (TBPL1), homeobox A9 (HOXA9), ZEB1, and E-cadherin, as well as MAPK signaling activation. This results in enhanced proliferation, invasion, migration, metastasis and apoptosis inhibition in CRC tissue and cell lines [267,268,269,270,271,272]. The downregulation of miR-133b upregulates disruptor of telomeric silencing 1-like (DOT1L), a histone H3K79 methyltransferase, which is involved in maintaining stem cell-like properties and chemoresistance, leading to CSC differentiation and decreased sensitivity to 5-FU and Oxa [273]. Furthermore, loss of miR-133b by increased expression of SRY-box transcription factor 13 (SOX13) and ATP-binding cassette subfamily C member 5 (ABCC5) induces resistance to Oxa and 5-FU, respectively, in CRC cells [274,275].

3.2.6. miR-497

In CRC, miR-497 loss upregulates the expression of kinase suppressor of Ras1 (KSR1), which is associated with metastasis and chemoresistance, while it activates the expression of IGF1R, insulin receptor substrate 1 (IRS1), VEGF, MMP-9, as well as the ERK1/2 and PI3K/Akt signaling pathways, resulting in cell proliferation, invasion, metastasis, and resistance to apoptosis [276,277,278,279,280]. In addition, downregulation of miR-497 is related to upregulation of Fos-related antigen 1 (Fra-1) resulting in the induction of EMT in HT29 cells and in vivo experiments [281].

3.2.7. Let-7 Family

The let-7 family is significantly downregulated in CRC tissue, and the levels of its members are associated with major tumor aggressiveness and poor differentiation [282,283]. The downregulation of some members of the Let-7 family is related to perineural invasion and advanced tumor stage through activation of the IGF signaling pathway [282], while the downregulation of Let-7g promotes proliferation, migration and invasion in CRC by increasing extracellular calcium influx through store-operated calcium channels [283]. Furthermore, Let-7e expression in CRC cells increases the levels of the ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 (ST8SIA1) and IGF1R proteins, which are associated with tumor progression, chemoresistance and radioresistance [284,285,286].

Table 2 summarizes the main downregulated miRs and their underlying molecular mechanisms and effects in CRC.

Table 2. Principal miRs downregulated in CRC.

miRs	Molecular Mechanisms	Major Effects	Refs.
miR-143/145	IGF1R (+) FLF5 (+) CD44 (+)	Proliferation (+) Migration (+) Angiogenesis (+) EMT (+) Metastasis (+)	[215,216,217,218,219,220,221,222,223,224,225,226]
miR-143	KRAS (+) MAPK/ERK1/2 (+) ERK5 (+) DNMT3A (+) PI3K/Akt/HIF-1/VEGF (+) MACC1 (+) FSCN1 (+) HK2 (+)	Proliferation (+) Epigenetic aberration Metastasis (+) Chemoresistance	[217,219,227,230,231,234,235]
miR-145	Cdk6 (+) EMT markers (+) LASP1 +) FSCN1 (+) IGFR1 (+) RAD18 (+)	Proliferation (+) EMT (+) Chemoresistance	[230,232,233,236]
miR-34 family	INHBB (+) AXL (+) FGFR1 (+) PDGFRB (+) DREAM complex (+) FMNL2 (+) E2F5 (+) HMGB1 (+) BCL2 (+) Notch1(+) and Jagged1 (+) IL−6R/STAT3 (+) KITLG (+) Myc (+)	Metastasis (+) Poor patient survival Migration (+) Invasion (+) Autophagy (+) EMT (+) Chemoresistance Proliferation (+) Apoptosis (−)	[238,239,240,241,242,243,244,245,246,247]
miR-126	CXCR4 (+) RhoA/Akt/ERK1/2 signaling pathways (+) VEGF (+) CXCL12 (+)	TNM stage Metastasis (+) Proliferation (+) Invasion (+) Angiogenesis (+) IAC tumorigenesis	[249,250,251,252]
miR-138	TWIST2 (+) Sirt1 (+) PODXL (+) Myc (+) PDK1 (+) PD-L1 (+) NFIB/Snail1 (+) SP1/LGR5 (+) hTERT (+) MCU (+)	Invasion (+) Metastasis (+) Proliferation (+) Migration (+) EMT (+) Apoptosis (−) Chemoresistance ROS increase	[254,255,256,257,258,259,260,261,262]
miR133a	RFFL (+) p53 and p21 (−) UBA2 (+) eIF4A1 (+) LASP1 (+) MAPK signaling (+)	Proliferation (+) Invasion (+) Migration (+) Chemoresistance Apoptosis (−)	[264,266,267,268,269]
miR-133b	CXCR4 (+) TBPL1 (+) HOXA9 (+) ZEB1 and E-cadherin (+) HOXA9 (+) DOT1L (+) SOX13 (+) ABCC5 (+)	Invasion (+) Metastasis (+) Proliferation (+) Stemness Chemoresistance	[265,270,271,272,273,274,275]
miR-497	KSR1 (+) IRS1 (+) PI3K/Akt (+) Fra-1 (+) VEGF (+) ERK1/2 (+) ACSL5 (+)	Proliferation (+) Invasion (+) Migration (+) EMT (+) Chemoresistance	[276,277,278,279,280,281,287,288]
Let-7 family	IGF signaling (+) Extracellular calcium influx (+) ST8SIA1 (+) IGF1R (+)	Invasion (+) Migration (+) Proliferation (+) Chemoresistance (+)	[282,283,284,285,286]

Abbreviations: IGF1R, insulin-like growth factor 1 receptor; KLF5, Krüppel-like factor 5; CD44, cluster of differentiation 44; KRAS, Kirsten rat sarcoma virus; EMT, epithelial–mesenchymal transition; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; DNMT3A, DNA methyltransferase 3A; PI3K/Akt, phosphatidylinositol-3-kinase/protein kinase B; HIF-1, hypoxia-inducible factor 1; VEGF, vascular endothelial growth factor; MACC1, metastasis-associated protein 1; FSCN1, fascin 1; HK2, hexokinase 2; Cdk6, cyclin-dependent kinase 6; LASP1, LIM and SH3 protein 1; INHBB, inhibin subunit beta B; FGFR1, fibroblast growth factor receptor 1; PDGFRB, platelet-derived growth factor receptor beta; FMNL2, formin-like 2; E2F5, E2F transcription factor 5; HMGB1, high mobility group box 1; BCL2, B-cell lymphoma 2; IL-6R, interleukin-6 receptor; STAT3, signal transducer and activator of transcription 3; KITLG, KIT proto-oncogene ligand; CXCR4, chemokine (C-X-C motif) receptor 4; RhoA, Ras homolog family member A; CXCL12, C-X-C motif chemokine ligand 12; IAC, inflammation-associated colorectal; TWIST2, twist family bHLH transcription factor 2; Sirt1, sirtuin 1; PODXL, podocalyxin-like protein 1; PDK1, phosphoinositide-dependent protein kinase 1; PD-L1, programmed death ligand 1; NFIB, nuclear factor I B; SP1, specificity protein 1; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; hTERT, telomerase reverse transcriptase; MCU, mitochondrial calcium uniporter; ROS, reactive oxygen species; RFFL, ring finger and FYVE-like domain-containing E3 ubiquitin protein ligase; UBA2, ubiquitin-like modifier-activating enzyme 2; eIF4A1, eukaryotic initiation factor 4A1; TBPL1, TATA-box binding protein-like 1; ZEB1, zinc finger E-box binding homeobox 1; HOXA9, homeobox A9; DOT1L, disruptor of telomeric silencing 1-like; SOX13, SRY-box transcription factor 13; ABCC5, ATP-binding cassette subfamily C member 5; KSR1, kinase suppressor of Ras1; IRS1, insulin receptor substrate 1; Fra-1, Fos-related antigen 1; ACSL5, acyl-CoA synthetase long-chain family member 5; IGF, insulin-like growth factor; ST8SIA1, ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1. (−) Downregulated. (+) Upregulated.

4. Interplay Between miRs and OS in CRC

There is a two-way relationship between miRs and ROS: ROS can affect the processing and expression of miRs, while miRs can influence ROS-related signaling pathways. In fact, miRs can modulate intracellular redox homeostasis and carcinogenesis by affecting the production of ROS and regulating several signaling pathways, including the Nrf2/Keap1, SOD/catalase and mitochondrial regulatory pathways, as well as the activity of various essential enzymes. Conversely, ROS can alter the expression, biogenesis and function of miRs in various cancer types through various mechanisms. These include activating transcription factors such as NF-κB and HIF-1α, regulating the expression of proteins involved in miR synthesis and maturation, such as the RNA-binding protein DiGeorge critical region 8 (DGCR8) and Dicer, and inducing epigenetic modifications including DNA methylation, histone acetylation and phosphorylation [62,63].

In particular, OS alters the epigenetic profile by promoting the hypermethylation of promoter regions of miR genes, thereby leading to their transcriptional silencing [62]. In addition, the OS may influence the stability and regulatory activity of miRs by expressing genes involved in antioxidant defense, mitochondrial function, inflammation, cell proliferation and apoptosis. This contributes to the development and progression of CRC [288].

In colitis-associated carcinoma (CAC), high levels of miR-222-3p are associated with OS and increased cytokine production causing the downregulation of Brahma-related gene 1 (BRG1), a chromatin remodeling factor, and the inhibition of the Nrf2/HO-1 antioxidant pathway [289]. Conversely, inhibiting miR-222-3p reduces OS and inflammation in mice with ulcerative colitis (UC) by activating the BRG1/Nrf2/HO-1 signaling pathway. This results in the mitigation of UC injuries and the prevention of transition to CAC [290]. The simultaneous treatment of HCT116 cells with mimics of miR-186, miR-2216b, miR-337-3p, and miR-760 stimulates senescence by decreasing the expression of protein kinase CKII, resulting in an enhancement of ROS and upregulation of p53 and p21^Cip1/WAF1 protein expression [291]. In CRC cell lines, the overexpression of miR-210 increases ROS production, induces cell cycle arrest at the G2/M phase and promotes apoptosis by upregulating the expression of the pro-apoptotic protein Bim and activating caspase-2. Interestingly, inhibiting ROS production prevents cells from undergoing apoptosis in response to miR-210 overexpression, thus establishing a direct link between this miR and ROS in apoptotic pathways [292]. Furthermore, it has been demonstrated that hypoxia increases the expression of miR-210 in CRC cell lines, resulting in an increase in glycolysis and a decrease in mitochondrial function. Thus, miR-210 increases the sensitivity of cancer cells to glycolysis suppressor and represents a link between microenvironmental stress, oxidative phosphorylation, ROS and iron homeostasis, as it reduces the expression of two important factors of the mitochondrial electron transport chain and the tricarboxylic acid cycle: iron-sulfur cluster scaffold homolog (ISCU) and cytochrome c oxidase assembly protein (COX10) [293]. High levels of miR-27a-3p, which are observed in CRC cell lines and human CRC tissue, are associated with reduced expression of the calcium voltage-gated channel auxiliary subunit alpha2delta-3 (CACNA2D3). This leads to cell proliferation and reduced apoptosis, due to the downregulation of intracellular Ca²⁺ and ROS levels, as well as the inactivation of the mitochondrial apoptotic pathway [199]. Additionally, miR-20a is upregulated in CRC cell lines, and its knockdown enhances the sensitivity of these cells to cisplatin via a pathway involving ROS, ASK1 overexpression and phosphorylation, and JNK activation [294]. The induced overexpression of certain miRs, including miR-124, miR-1 and miR-133b, in CRC cell lines leads to OS which promotes apoptosis and inhibits cell growth [295,296]. In particular, the overexpression of miR-143 downregulates SOD1, peroxiredoxin 2 (PRDX2) and PRDX6, and, in addition to reducing cell proliferation, increases sensitivity to oxaliplatin-induced apoptosis [297]. Additionally, the upregulation of miR-22-3p induces CRC cell apoptosis by increasing ROS levels and decreasing Bcl-xL and mitochondrial membrane potential [298]. On the contrary, miR-1225-5p overexpression inhibits migration and invasion in HT-29 cells and reduces ROS production by activating the Keap1/Nrf2/HO-1 signaling pathway [299]. In CRC tissue and cell lines, the downregulation of miR-369-3p expression is associated with the overexpression of the transcription factor 4 (TGF4) gene. This results in increased cell proliferation and invasion, as well as increased OS, by upregulating E-cadherin and VEGF proteins and reducing SOD activity, while also raising lactate dehydrogenase (LDH) activity and malondialdehyde (MDA) levels [300]. Additionally, downregulation of miR-138-5p in CRC promotes cell growth by increasing the mitochondrial calcium uniporter (MCU) protein levels, thereby enhancing mitochondrial biogenesis and ROS production [301]. Low levels of miR-212 and miR-128 were detected in CRC tissue and cell lines [302,303]. However, when upregulated, miR-212 reduces the expression of the antioxidant enzyme manganese superoxide dismutase (MnSOD), thereby suppressing invasion, migration, EMT and metastasis [302]. In contrast, high levels of miR-128 promote tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by decreasing Sirt-1 expression, resulting in increased ROS and decreased death receptor 5 (DR5) expression [303].

miR-335-5p, miR-26b-5p, and miR-34a-5p are shear-sensitive and ROS-mediated miRs [304]. In HCT116 cells, shear stress induces specific intracellular ROS production, the upregulation of miR-355-5p and downregulation of miR-34a-5p. However, it has been shown that the expression of all three miRs is inversely related to hydroxyl radical levels, meaning that high levels of miR-335-5p, miR-26b-5p, and miR-34a-5p in the presence of low levels of hydroxyl radicals increase apoptotic processes and inhibit migration, invasion, EMT, proliferation and metastasis [304]. Treating the CRC cell line CT26 with linoleic acid (LA) suppresses cell proliferation by inducing mitochondrial OS, which is involved in the upregulation of miR-494. This, in turn, reduces the expression of Myc and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), which are involved in the regulation of glycolysis and oxidative phosphorylation, respectively [305].

Curcumin’s anticancer role in colon cancer cells is due to its ability to induce ROS production, resulting in reduced expression of miR-27a, miR-20a and miR-17-5p. This leads to the subsequent upregulation of ZBTB10 and ZBTB4, which are zinc finger and BTB domain-containing proteins that repress genes involved in cell growth and division [306]. In HCT116 cells treated with various concentrations of H₂O₂, OS regulates the interaction between miR-138 and the HIF1α gene. In particular, under low OS, the levels of the long non-coding RNA RP11-431M3.1 are high and it sequesters miR-138, which is unable to bind HIF-1α, resulting in cell migration and proliferation. Conversely, under high OS, RP11-431M3.1 levels are low and miR-138 can interact with the HIF1α gene, inducing cell apoptosis. In both conditions, an increase in Nrf2 occurs [307]. In cancer cells, H₂O₂ promotes an upregulation of miR-21 levels, which is blocked by antioxidants, thus suggesting that OS is principally involved in the upregulation of this miR. These observations have actually been described in various cancer models, and could also occur in CRC, given that miR-21 is upregulated in this tumor and enhances proliferation, invasiveness, EMT and chemoresistance [308]. However, it has been shown that the inhibition of aldose reductase reduces cell growth by downregulating miR-21 and increasing its target PTEN and FOXO3 through the inhibition of ROS production and consequent inactivation of the transcription factor activator protein 1 (AP1) in human CRC cell lines [309]. Although several miRs have been reported to respond to OS in different cancer types, direct experimental evidence demonstrating ROS-mediated regulation of their expression specifically in CRC is limited. Current knowledge is therefore largely based on indirect evidence or extrapolated from studies performed in non-CRC models [308]. It is important to note that evidence supporting one direction of this interaction does not necessarily prove the existence of a complete bidirectional feedback mechanism. Although altering ROS levels or miR expression may reveal a specific regulatory relationship, establishing a reciprocal feedback loop requires evidence that ROS regulate the miR expression, which in turn can modulate ROS levels through a defined molecular mechanism. Therefore, the above interactions should be interpreted according to the type and strength of the available experimental evidence.

Figure 3 summarizes the relationship between OS and the principal miRs in CRC.

Figure 3. Bidirectional relationship between oxidative stress and microRNA dysregulation in CRC. Oxidative stress (OS) affects the expression of various microRNAs (miRs); conversely, dysregulated miRs can modulate intracellular reactive oxygen species (ROS) levels. The top panel shows upregulated or downregulated miRs that promote OS by affecting ROS generation and/or antioxidant mechanisms. The bottom panel summarizes miRs that have been reported to be either upregulated or downregulated in response to OS. The solid red arrows connecting these panels to the OS are directional. However, these findings emphasize the existence of a regulatory loop between OS and miRs that contributes to the development and progression of CRC. Created in BioRender software. Gaia Palmini. (2026) (https://www.biorender.com/).

5. Protective Role of ER-β in CRC

The incidence and prognosis of CRC differ between sexes. 17β-E2 plays a protective role through ERβ, contributing to the maintenance of epithelial architecture, gastrointestinal physiology, and immune responses in normal colon tissue [310,311]. Its loss has been linked to increased gut permeability, colitis and CRC risk [312]. Both ERβ and ERα are expressed in normal colorectal tissue, but ERβ is more prevalent. In CRC an increased ERα/ERβ ratio is associated with tumor stage and grade, particularly in men [18,313]. ERβ-mediated protection against CAC involves the inhibition of TNFα and NF-κB signaling pathway and macrophage infiltration, as well as the modulation of gut microbiota composition [314,315]. Additionally, 17β-E2 can reduce DNA damage via ERβ by decreasing ERα and pro-inflammatory mediators, while increasing antioxidant enzymes and ERβ levels. Estrogen also stimulates ERβ expression in colon cancer cells, partly via p38 MAPK signaling [316,317,318,319]. Upon activation, ERβ interacts with chromatin at ERE and regulatory motifs, including those associated with cell adhesion, apoptosis and the Wnt signaling pathway, all of which play a central role in CRC [320]. Its antitumoral roles include the promotion of DNA repair, the suppression of oncogenes, the inhibition of cell proliferation, the induction of apoptosis, and the inhibition of metastasis [15,321]. The loss of the mismatch repair (MMR) system, which includes MutL Homolog 1 (MLH1), is often the cause of mutations in CRC, and 17β-E2 is able to enhance MLH1 in colon cancer cells overexpressing ERβ [322,323]. ERβ also reduces the expression of oncogenes, such as c-Myb, c-Myc and PROX1, and this could be due to the role of this receptor in changing the pool of miRs in colon cancer cells [45,324,325].

Activation of ERβ in CRC cells reduces proliferative and anti-apoptotic capacity by modulating genes involved in cell cycle control and apoptosis [317,321,326,327]. In particular, the overexpression of ERβ in CRC cells downregulates cyclin D1, promoting its degradation and activating autophagy by inhibiting the mammalian target of rapamycin (mTOR) or activating Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) [317,321]. It also downregulates the expression of cyclin E, polo-like kinase 1 (PLK1) and cMyc and upregulates the expression of the cyclin-dependent kinase inhibitor p21 (CDKN1A), blocking the cell cycle in the G1-S phase [317,326,327]. ERβ activation inhibits the proliferation, migration and survival of CRC cells by reducing nuclear β-catenin levels, thereby inhibiting the Wnt/β-catenin signaling pathway, and increasing the expression of anti-tumorigenic molecules, such as 15-hydroxyprostaglandin dehydrogenase (15-PGDH) [328]. It promotes the apoptosis of colon cancer cells by upregulating p53 expression, activating caspases 8 and 9, and increasing the expression of pro-apoptotic genes such as Bax, Noxa and BCL2L11 [329]. It also reduces c-Myb protein levels, which consequently leads to decreased Bcl-2 expression [330,331,332]. Furthermore, ERβ can induce apoptosis via the p38 MAPK pathway, promoting caspase-3 activation and poly(ADP-ribose) polymerase cleavage [332,333]. The inhibitory role of 17β-E2 on the migration of LoVo colon cancer cells is due to the reduced expression of migration-related factors such as urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), MMP-2, and MMP-9 without altering the expression of plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of MMPs (TIMPs). This effect is mediated by Erβ, p38 MAPK and the p53 signaling pathway [334,335]. In CRC cells, ERβ downregulates genes related to angiogenesis and invasion, such as platelet-derived growth factor subunit A (PDGFA) and angiopoietin-like 4 (ANGPTL4). It also suppresses the hypoxia-responsive pathway and the consequent activation of HIF, which play a role in tumor resistance to chemotherapy and radiotherapy [336].

A variety of natural and synthetic compounds that are structural analogues of estrogen, such as phytoestrogens and xenoestrogens, bind to ERβ and have different effects depending on their structure. Xenoestrogens, for example, are synthetic compounds that can disrupt estrogen signaling and adversely affect human physiology, increasing susceptibility to diseases such as CRC [337,338]. For example, the synthetic plasticizer bisphenol A interacts with ERβ and, as a 17β-E2 antagonist, blocks the activity of the receptor, thereby promoting CRC progression [339]. Conversely, phytoestrogens, which are found in fruit and vegetables, exert anticancer effects in the colon by binding to ERβ [15]. In particular, in colon cancer cells, the inhibition of cell growth and the upregulation of ERβ expression due to genistein are mediated by its binding to the receptor [340,341]. It has been shown that genistein treatment in ovariectomized rats induces the apoptosis and reduces the proliferation of colonocytes [342]. Quercitin and the kaempferide triglycoside, a glycosylated flavonol purified from a Dianthus caryophyllus cultivar, show anti-proliferative properties mediated by ERβ [340,343]. Calycosin inhibits proliferation and invasion, and activates apoptosis, by downregulating IGF-1R, increasing PTEN, and inhibiting the activation of PI3K/Akt signaling pathways in CRC cells, via ERβ [344,345,346]. Recently, it has been shown that natural δ-tocopherol and two of its semi-synthetic derivatives, bis-δ-tocopheryl sulfide and bis-δ-tocopheryl disulfide, reduce proliferation and upregulate the expression of the ERβ receptor in HCT8 cells overexpressing this receptor, in a manner similar to that of estrogen [317]. Figure 4 provides a summary of the principal mechanisms involved in the protective role of ERβ in CRC.

Figure 4. The principal molecular mechanisms involved in the ERβ-mediated anti-tumorigenic role in CRC. 17β-E2, phytoestrogens and ERβ-selective agonists bind to ERβ and activate the receptor, which then translocates to the nucleus and promotes the transcription of genes that contribute to the prevention and treatment of CRC. ERβ regulates the tumor environment by inhibiting pro-inflammatory signaling pathways, enhancing immune response and regulating microbiota composition. At the cellular level, ERβ suppresses tumor cell proliferation, promotes apoptosis, enhances genomic stability and limits tumor progression by inhibiting invasion and metastasis. Created in BioRender software. Gaia Palmini. (2026) (https://www.biorender.com/). Abbreviations: NF-κB, nuclear factor kappa B; CDKN1A, cyclin-dependent kinase inhibitor p21; PLK1, polo-like kinase 1; mTOR, mammalian target of rapamycin; 15-PGDH, 15-hydroxyprostaglandin dehydrogenase; MLH1, MutL Homolog 1; BCL2L11, BCL-2-like protein 11; ANGPTL4, angiopoietin-like 4; uPA, urokinase plasminogen activator; tPA, tissue plasminogen activator; HIF, hypoxia-inducible factor; MMPs, matrix metalloproteinases; PDGFA, platelet-derived growth factor subunit A.

6. Relationship Between ERβ and Oxidative Stress

Although there is a lack of direct evidence linking ERβ to intracellular redox regulation in CRC, several studies suggest that reducing ERβ creates an environment that promotes CRC development by inducing inflammation that is known to promote OS during colorectal carcinogenesis [15,347]. Conversely, in CRC, ERβ inhibits pro-inflammatory signaling pathways such as NF-κB and PI3K/AKT, which are important in the development of OS and tumor progression [347]. Furthermore, some experimental evidence from other organs indicates that ERβ may act as an antioxidant by altering mitochondrial processes and OS-related pathways [348,349]. Consequently, this receptor may contribute to maintaining redox balance in colonocytes. The role of ERβ in the regulation of intracellular redox state is related to cell type and experimental conditions. This is demonstrated by the way in which it mediates ROS production in TCam-2 seminoma cells [350] while leaving unaffected ROS levels in starved human colon adenocarcinoma HCT8 cells overexpressing ERβ protein [317]. In the human colorectal adenocarcinoma cell line DLD-1, 17β-E2-mediated ERβ activation induces neuroglobin (NGB) expression. NGB is an oxygen sensor principally present in neurons to protect them from OS and to impair apoptosis induced by estrogen. Thus, the upregulation of NGB protects colon cancer cells against damage due to OS, but it also reverses the pro-apoptotic role of 17β-E2, reducing the anti-tumorigenic role of estrogen [351]. The anti-proliferative effect of the ethanolic extract of Fagonia cretica, which is rich in polyphenols, is due to decreased expression of DNA methyltransferase 1 (DNMT1) and increased ROS production, as well as increased ERβ expression [352]. To the best of our knowledge, there is no direct evidence in the literature demonstrating that OS induces ERβ downregulation in CRC. However, OS is a recognized driver of epigenetic instability, promoting alterations in DNA methylation, histone modifications, and non-coding RNA expression in cancer and may therefore indirectly contribute to the suppression of ERβ expression that can be regulated by epigenetic mechanism [353,354], but this relationship has not been characterized in CRC so far. This could cause a feedback loop in which higher ROS leads to downregulation of ERβ, thus making cells more susceptible to OS and inflammation. Therefore, it is possible to assume that ERβ helps to maintain redox homeostasis in the colon, and that its loss may contribute to a redox imbalance in CRC. However, the potential links between OS and ERβ have not been specifically characterized in CRC and can thus far only be suggested by indirect studies. Much more research is needed to explore how OS affects ERβ function in CRC and vice versa. Such studies will help to identify new treatment targets and develop strategies to regulate the redox balance and ERβ expression in CRC.

7. Relationship Between ERβ and miRs

It has been shown that ER activity is closely interconnected with miRs via regulatory networks, promoting a reciprocal circuit that influences cell growth, cell death, inflammation, EMT and metastasis [355]. ERβ can modulate the expression of specific miRs linked to cell growth, death, EMT, and metastasis [356], while miRs can directly regulate ERβ expression or that of components related to ERβ-dependent signaling pathways [357]. Indeed, the upregulation of ERβ expression in colon cancer cells can significantly alter the CRC-related miRs, suggesting that the anticancer role attributed to ERβ may be partly due to miR-dependent mechanisms [45]. The expression of ERβ in CRC cells regulates the expression of 27 miRs, with miR-205 and miR-10a/b being upregulated and the rest being downregulated. Some miRs regulated by Myc, including miR-200a/b, miR-9, miR-106a and miR-135a-1, can be regulated directly by both ER-beta and MYC [45]. However, it has been shown that, in CRC, ERβ-mediated repression of the miR-17-92 cluster and miR-106 is due only to Myc downregulation, while Myc is not involved in the reduced levels of miR200a/b and miR-221. In particular, ERβ-mediated repression of the miR-17-92 cluster is due to the downregulation of Myc and promotes an increase in clusterin (CLU) and nuclear receptor coactivator 3 (NCOA3) expression, resulting in the apoptotic capacity enhancement of CRC cells, as well as prolonged survival and a decreased probability of death in colon cancer [45]. It is possible that mechanisms involved in the increase in apoptosis in CRC due to downregulation of Myc and miR 17-92 are due to the increase in DNA repair and pro-apoptotic p53, which are effectively increased by ERβ activation and Myc downregulation [358,359]. ERβ also causes low expression of miR-200a/b, which is linked to reduced migration in the human colorectal adenocarcinoma cell line SW480 [45]. Furthermore, ERβ-induced downregulation of miR-196a-1 results in increased annexin 1 expression, which in turn inhibits NF-κB and inflammation, thus contributing to an anti-tumorigenic effect [45,360]. The treatment of CRC cell lines with 17β-E2 promotes the downregulation of oncogenic miRs, such as miR-135b, miR-31 and miR-155, via ERβ, and this effect is reversed by the estrogen receptor antagonist ICI182,780 [361]. In the HT-29 colon cancer cell line, the fermented extract of naringenin promotes an increase in ERβ expression, inducing a reduction in the levels of the miR-200c and miR-141, even in the presence of bisphenol A, which is related to cancer progression. This results in an increase in PTEN, which downregulates proliferation and migration [362]. Calycosin, a natural isoflavone extracted from Radix Astragali, promotes apoptosis and inhibits the growth of SW480 cells by inducing ERβ expression, inhibiting the PI3K/Akt signaling pathway, and downregulating the expression of IGF-1R and miR-95. Reduction in miR-95 levels or an increase in apoptosis when ERβ is inhibited do not occur, suggesting a relationship between this microRNA and the estrogen receptor in the pro-apoptotic and anti-proliferative role of calycosin [344]. In addition, calycosin has been shown to exhibit anti-tumor effects by decreasing miR-17 and upregulating PTEN in another human colorectal carcinoma cell line (HCT116) [346].

Emerging literature evidence shows that miRs may also regulate ERβ expression. This has been demonstrated in other hormone-responsive tissues, such as breast cancer, where the downregulation of miR-92 in MCF-7 cells induces ERβ1 expression, and in endometriosis, where some miRs can regulate the expression of ERα and ERβ, contributing to progesterone resistance [34,46]. To our knowledge, there is little data in the literature regarding the regulation of ERβ expression by miRs. It has been demonstrated that miR-129, which is highly expressed in tissue and serum samples from CRC patients, downregulates the expression of ERβ, acting as an oncogene involved in CRC progression [363]. Conversely, the downregulation of this miR increases ERβ levels, indicating that this receptor is a target of miR-129 [363].

Overall, the existing data suggest that ERβ influences the expression and function of miRs in patients with CRC; however, few studies exist regarding the impact of ERβ downregulation via miRs. Hence, the downregulation of ERβ during the onset of colorectal cancer may be influenced more by other regulatory processes than by miRs. It will be useful in the future to investigate whether some deregulated miRs can reduce ERβ levels by targeting proteins that positively regulate signaling pathways involved in ERβ expression and function. Understanding the complex regulatory circuit between ERβ and miRs may allow one to identify ERβ agonists or factors that can modulate individual miRs, which could be useful in developing a promising therapeutic approach for CRC.

8. Conclusions and Future Perspectives

The present data suggest that the OS, miRs, and ERβ take part in CRC pathogenesis and development. They function independently by regulating biological processes associated with tumor development. In particular, OS plays a dual role in CRC. It is involved in the onset of tumor and, at the same time, it reduces CRC development and progression. miRs promote CRC development and progression by regulating the genes involved in key cellular processes, including proliferation, apoptosis, angiogenesis, and metastasis. Their dysregulation can contribute to tumor formation, and the expression profile of specific miRs shows promise as a biomarker for the early detection and monitoring of CRC. ERβ mainly has protective and anti-tumorigenic effects in CRC. It inhibits inflammation, proliferation, invasion and angiogenesis, while promoting DNA repair and apoptosis. Consequently, loss or reduced expression of ERβ may contribute to CRC development and progression, establishing ERβ as a potential therapeutic and preventive target. The growing body of literature proves that they act as components of a complicated regulatory network, where ROS regulate the expression of miRs, while the latter regulate redox-sensitive pathways, and ERβ regulates inflammatory and tumor-related signaling. Depending on the genes they target, miRs can either promote or inhibit CRC progression by regulating OS, cell proliferation, apoptosis and response to treatment. Conversely, OS regulates miRNA expression, thereby altering their downstream effects on cellular signaling and tumor progression. A potential bidirectional relationship between ERβ and OS may exist in CRC, whereby ERβ could contribute to redox homeostasis by limiting inflammation and oxidative stress, while its loss could promote a pro-oxidative and pro-inflammatory environment. However, there is limited direct evidence for OS-mediated regulation of ERβ in CRC, highlighting the need for further investigation. ERβ acts as a CRC suppressor by maintaining redox homeostasis, limiting inflammation by inhibiting the NF-κB and PI3K/AKT pathways, and regulating the expression of several miRs involved in apoptosis, proliferation, epithelial–mesenchymal transition, and DNA mismatch repair. It has been suggested that ERβ and miRs form a complex bidirectional regulatory circuit that contributes to the control of CRC progression. ERβ can modulate miR expression to promote anti-tumorigenic effects, while the alteration of specific miRs, such as the upregulation of miR-129, may negatively regulate ERβ expression in CRC. Specifically, miR-129 directly binds to the ESR2 3′UTR, leading to ERβ repression, and contributes to the regulation of tumorigenesis in CRC through the control of cell behavior using various molecular targets, such as ERβ and SOX4. The role of miR-129 in the pathogenesis of CRC has been shown to control the malignant properties of cells, such as proliferation, migration, invasion, and EMT [363,364]. As in previous cases, the evidence regarding the regulation of ERβ by miRs in CRC is limited. This suggests that further studies are needed to clarify this interaction and its potential therapeutic relevance. Another open problem is whether OS, miRs, and ERβ form a consistent regulatory pathway in CRC. While individual links between the components of the pathway have been described in the literature, there are currently no studies examining the entire pathway. Several proposed interactions, in particular, are supported by indirect observations or studies conducted in different experimental models. Future research should investigate these relationships within the same CRC models to clarify their causal and functional relevance. It should also determine whether modulating them together might offer therapeutic advantages over targeting each component individually. This integrated approach provides a framework for identifying important knowledge gaps and generating testable hypotheses for future mechanistic and translational studies. Future studies should aim to clarify the relationships in the OS–miR–ERβ axis and determine whether targeting just one of them could restore the pathway. Consequently, elucidating this novel regulatory pathway will shed light on new potential biomarkers and pave the way for the development of tailored CRC prevention and therapy approaches. Table 3 summarizes the principal miRs associated with OS and ERβ in CRC.

Table 3. Principal miRs associated with OS and ERβ in CRC.

miRs	Expression in CRC	Validated/Proposed Molecular Target	Direct Effect on Redox Homeostasis	Relationship with ERβ	Major Biological Outcome	Experimental Model Evaluated	Refs
miR-21	Upregulated	PTEN, PDCD4	No direct evidence	No direct evidence	Proliferation, invasion, EMT, and chemoresistance	Human tissue samples, human cell lines (HCT116, HT29, T84, HCT116)	[148,149,150,151,152,207]
miR-27a-3p	Upregulated	CACNA2D3	Downregulates intracellular Ca2+ and ROS levels	No direct evidence	Inhibits apoptosis, enhances cell proliferation	Human CRC tissues, human cell lines (HT-29, SW480, HCT116, and SW620)	[199,362]
miR-129	Upregulated	ESR2, SOX4	No direct evidence	Directly targets and downregulates ERβ expression	Cell proliferation, migration, apoptosis, and EMT	Patient tissue/serum, human cell lines	[363,364]
miR-143	Downregulated	KRAS, SOD1, PRDX2, DNMT3A	miR-143 overexpression reduces SOD1/PRDX2, decreasing antioxidant shield	No direct evidence	Cell proliferation, apoptosis	Human cell lines (228, CaCO2, clone A, HTC116, HT-29, MIP101, and SW480)	[217,297]
miR-17-92	Upregulated	CTNNB1, PTEN	No direct evidence	Strongly downregulated by ERβ through Myc inhibition	Cell proliferation, migration	Animal tumor models, human and mouse cell lines (CT26, CMT-93, SW480, and MCA38)	[45,203]
miR-222-3p	Upregulated	BRG1	Inhibits Nrf2/HO-1 pathway, increasing OS	Linked to chronic inflammation driving CRC risk	Promotes transition from colitis (UC) to CAC	Mouse models of ulcerative colitis, human cell lines (NCM460)	[289,290]
miR-138-5p	Downregulated	MCU, TWIST2	Loss promotes ROS production via increasing MCU expression	No direct evidence	Cell growth, migration, and invasion	Human cell lines (LoVo, DLD1, HCT116, SW620, HT29, and SW116), clinical tissues	[254,301]

Abbreviations: Kirsten rat sarcoma virus; EMT, epithelial–mesenchymal transition; MCU, mitochondrial calcium uniporter; OS, oxidative stress; UC, ulcerative colitis; PTEN, phosphatase and tensin homolog; BRG1, Brahma-related gene 1; PDCD4, programmed cell death 4; CACNA2D3, calcium voltage-gated channel auxiliary subunit alpha-2/delta 3; SOD1, superoxide dismutase 1; SOX4, SRY-box transcription factor 4; PRDX2, peroxiredoxin 2; DNMT3, DNA methyltransferase 3; TWIST2, twist family bHLH transcription factor 2; ESR2, gene encoding Erβ; CTNNB1, gene encoding β-catenin.