Gut Microbiota–MicroRNA Interactions in Intestinal Homeostasis and Cancer Development

Pre-clinical models and clinical studies highlight the significant impact of the host–microbiota relationship on cancer development and treatment, supporting the emerging trend for a microbiota-based approach in clinical oncology. Importantly, the presence of polymorphic microbes is considered one of the hallmarks of cancer. The epigenetic regulation of gene expression by microRNAs affects crucial biological processes, including proliferation, differentiation, metabolism, and cell death. Recent evidence has documented the existence of bidirectional gut microbiota–microRNA interactions that play a critical role in intestinal homeostasis. Importantly, alterations in microRNA-modulated gene expression are known to be associated with inflammatory responses and dysbiosis in gastrointestinal disorders. In this review, we summarize the current findings about miRNA expression in the intestine and focus on specific gut microbiota–miRNA interactions linked to intestinal homeostasis, the immune system, and cancer development. We discuss the potential clinical utility of fecal miRNA profiling as a diagnostic and prognostic tool in colorectal cancer, and demonstrate how the emerging trend of gut microbiota modulation, together with the use of personalized microRNA therapeutics, might bring improvements in outcomes for patients with gastrointestinal cancer in the era of precision medicine.


Introduction
Host-microbiota interactions in tumorigenesis and cancer treatment are gaining ever more attention. Mounting evidence from pre-clinical and clinical studies has documented the critical role of the human gut microbiome in the development of different types of cancer, including gastrointestinal and breast tumors, lymphomas, lung cancer, and many others [1][2][3][4][5]. In addition, the association between gut microbiota composition and cancer treatment efficacy highlights the potential of a microbiota-related approach in clinical oncology [6].
The regulation of gene expression by microRNAs (miRNAs) has been widely studied, showing the significant impact of miRNA expression on cellular proliferation, differentiation, and metabolism, as well as on cell death. MiRNAs are suggested to be connected to inflammatory responses and dysbiosis and could serve as biomarkers for several human disorders, including cancer. Deregulated miRNAs potentially affect gene expression in Table 1. An overview of the most abundant miRNAs in the intestine.

Examples of Involvement of the miRNA Family in Processes and Functions
Ref.
miR-21-5p the small intestine, colon 143 Promotes survival and proliferation of cancer cells by directly inhibiting its targets, including PTEN, PDCD4, RECK, and SPRY2; in CRC, it promotes tumor invasion and metastasis via modulating the expression of multiple cancer-related genes, including TGFβR2, PDCD4, and PTEN. [49] miR-24-3p the small intestine, colon 276 Affects intestinal barrier integrity and function due to an effect on cell-cell junctions; its overexpression leads to a decrease in the tight junction-associated protein cingulin, followed by compromised barrier formation in intestinal epithelial cell lines from ulcerative colitis patients. [50] miR-26a-5p the small intestine, colon 484 Has a suppressive role in colitis-associated CRC; suppresses the intestinal inflammatory response in macrophages by decreasing NF-κB/STAT3 activation and interleukin 6 production. [51] miR-27a-3p the small intestine, colon 610 Associated with histological differentiation, clinical stage, distant metastasis, and survival of CRC patients; promotes proliferation, migration, and invasion of CRC cells; affects the Wnt/β-catenin pathway via retinoid X receptor targeting. [52] miR-29b-3p the small intestine, colon 423 Inhibits intestinal mucosal growth by repressing cyclin-dependent kinase 2 translation; represses translation of menin mRNA, and thus affects intestinal epithelial homeostasis by altering intestinal epithelial cell apoptosis. [53,54] miR-143-3p the small intestine, colon 201 Affects epithelial regeneration of the intestine after injury; is expressed and functions exclusively within the mesenchymal compartment of the intestine. [55] miR-145-5p the small intestine, colon 296 Affects expression of SOX9, an important transcription factor, that negatively regulates the expression of claudin 8, accompanying reduced intestinal permeability and mucosal barrier homeostasis in Crohn's disease; is expressed and functions exclusively within the mesenchymal compartment of the intestine. [55,56] miR-192-5p the small intestine, colon 34 Has an antitumor effect on CRC; represses glycolysis by regulating the expression of sushi repeat-containing protein X-linked 2 in colon cancer cells. [57,58] miR-199a-3p the small intestine, colon 149 Its loss aggravates CRC by the activation of EMT-related signaling and targeting DDK1; ameliorates the intestinal barrier in ulcerative colitis by downregulating the interleukin-17A/interleukin-23 axis; reduces the production of ROS and improves the expression of junction protein in the intestinal tissue of ulcerative colitis. [51,59] let-7a-5p the small intestine, colon 391 Regulates cell proliferation, cell cycle, apoptosis, metabolism, and stemness. [60] * according to the miRDB database [61]. Only targets with a prediction score >80 that represent the "most likely to be real" status are included. Abbreviations: DDK1, discoidin domain receptors; 1EMT, epithelial-to-mesenchymal transition; NF-κB, nuclear factor kappa B; PDCD4, programmed cell death 4; PTEN, phosphatase and tensin homolog; RECK, reversion-inducing-cysteine-rich protein with kazal motifs; ROS, reactive oxygen species; SOX9, SRY-Box transcription factor 9; SPRY2, Sprouty RTK signaling antagonist 2; STAT, signal transducer and activator of transcription; TGF-β, transforming growth factor β; TGFβR2, transforming growth factor beta receptor 2.

MicroRNA as a Marker of Intestinal Homeostasis and Microbial Fluctuations
The studies report that miRNAs, as molecular regulators, play an essential role in the maintenance of gut homeostasis and host-microbiota interactions [62,63]. Maintenance mechanisms of gut microbiota composition have been studied through miRNA expression in IECs [64]. Aguilar et al. described the crucial role of miRNAs in host-pathogen interactions, affecting the cytoskeleton, cell cycle, autophagy, cell death, and survival [65]. As shown, miRNA profiles varied in different IEC subtypes and correlated with microbial status. Elevated permeability of intestinal epithelial stem cells (IESC) was shown to be related to the bacteria-induced increase in miR-21-5p expression levels [66]. Peck et al. observed that gut microbiota composition was associated with miR-375-3p inhibition, leading to IESC proliferation [42]. The gut microbiota affects the host's health through the interaction between gut microbiota and miRNAs in the central nervous system, intestinal homeostasis, immune system, and cardiovascular disease [43]. The regulation of intestinal homeostasis arises by the interaction between miRNA and nucleotide-binding oligomerization domaincontaining protein (NOD2), remotely activating immune cells [41]. For the maintenance of intestinal homeostasis, the recognition of commensal and food molecules is essential. This process is provided by pattern recognition receptors (PRRs) [37].
Ye et al. studied the mechanism of increased miR-122a in enterocytes and intestinal tissues. The overexpressed miR-122a bound to the 3 -untranslated part of occludin mRNA, leading to its degradation and increased gut barrier permeability [75]. Inducible nitric oxide synthase (iNOS) plays a role in intestinal disorders by modulating miR-212 levels. The overexpression of miR-212 mediated zonula occludens protein (ZO-1) downregulation, triggering intestinal barrier disruption [69]. The integrity of tight junctions was impaired by miR-21 upregulation in the mucosa of ulcerative colitis and Caco-2 cells. In addition, the inulin permeability increased, together with decreased transepithelial electrical resistance. According to the results, miR-21-induced RhoB mRNA degradation was associated with increased gut barrier permeability [76].
The regulation of intestinal homeostasis is provided by gut microbiota-immune system interactions. Anzola et al. studied the overexpression of miR-146a-induced immune tolerance via the inhibition of bacterial cytokine production (MCP-1 and GROα/IL-8) in response to lipopolysaccharide (LPS) or IL-1β in IEC18 and Caco-2 cells, respectively [77]. Lactobacillus casei (LC01) enhanced barrier integrity via the downregulation of miR-144 and the upregulation of occludin (OCLN) and zonula occludens 1 (ZO1/TJP1) in IECs. This bacterium promoted mucosal barrier function and maintained intestinal homeostasis [78]. The suppression of miR381-3p led to IEC proliferation and improvements in intestinal barrier function [79]. Furthermore, miR-375 has also been linked to IESC proliferation and mucus layer production in the intestinal epithelium [80].
Chen et al. showed that miR-122 influenced the IEC inflammatory response in Crohn's disease by downregulating NOD2 expression [81]. In HT-29 cells, NOD2 suppression by miR-122 inhibited LPS-induced apoptosis. A muramyl dipeptide (MDP) is a component of the bacterial wall and activator of NOD2, inducing the activation of NF-kB [82]. Bakirtzi and colleagues studied the regulation of intercellular communication between neuropeptide and colonic epithelial cells. Substance P (neuropeptide/hormone) was secreted in colonic epithelial exosomes. In human colonic epithelial cells and murine colonic crypts, substance P and NK-1R signaling stimulated colonic epithelial cell proliferation and induced miR-21 Microorganisms 2023, 11, 107 6 of 23 sorting [83]. These results suggest that exosomal miR-21 might inhibit PTEN expression. In contrast, Zhang et al. have suggested that miR-21 regulates intestinal barrier permeability via the PTEN/PI3K/Akt signaling pathway. These authors found a higher expression of miR-21 in the TNF-α-induced intestinal barrier-defective model [70].

The Gut Microbiota-MicroRNA Interactions and Intestinal Immunity
The intestinal immune system is an important component of the intestinal environment. Innate intestinal immunity consists of NOD2 and Toll-like receptors (TLRs). In contrast, adaptive intestinal immunity consists of T-cell and B-cell subtypes. Both innate and adaptive immunity can be regulated by host miRNAs in the gut intestine through their impact on differentiation and maturation [84]. MiRNAs modulate gene expression, which is reflected in the activity of the intestinal immune system after interaction with gut microbiota [37]. MiR-155 has been shown to increase TGFβ and decrease the expression of IL-2 and IFNγ [85], while miR-29 correlates with attenuated IL-23/Th17 responses [86]. MiR-10a also maintains regulatory T (Treg) cells, preventing the plasticity to other T cell subsets [87].
In intestinal immune responses, downregulated miR-125a expression [88] and miR-155 inhibition [89] affect Th1/Th17 cell differentiation. MiR-155 regulates T cell differentiation via the Jarid2/Wnt/β-catenin pathway and decreases Th17 cells in the colonic mucosa [90]. Targeting the IL-6R and IL-23R by miR-34a prevents inflammation-induced stem cell proliferation and suppresses Th17 cell differentiation and expansion [91]. Sanctuary et al. hypothesized that miR-106a deletion impacted CD4+ T cell colitogenic potential through a reduction in inflammation brought about by a decrease in Th1 and Th17 cells [92]. Mikami et al. showed that the expression of miR-221 and miR-222, which was induced by proinflammatory cytokines, modulated the intestinal Th17 cell response. According to these findings, miR-221 and miR-222 targeted Maf and Il23r [93]. Future research should pay attention to miR-146a's regulation immunosuppressive and anti-inflammatory functions of immune cells, including macrophages, dendritic cells, and T cells [94]. A deficiency of miR-146a impacts the composition of gut bacteria and results in a major increase in IgA-producing B cells by inhibiting Smad2, Smad3, and Smad4 expression [94].
Diaz-Garrido et al. documented that IEC-originated microRNAs are exported through extracellular vesicles to the intestinal lumen [96]. Human miRNAs target bacterial nucleic acid sequences via complementary base pairing [10,97]. In vitro analysis showed that the E. coli growth was affected by miR-1226 through the knockout of Dicer1∆IEC [98]. Authors found interactions between specific bacterial genes and fecal miRNAs. Redweik et al. assumed that catecholamines signaled vesicles with miRNAs from the IECs of the intestinal lumen using the plasmid transfer [99]. MiRNAs bind to the 3 UTR of the target mRNAs and decrease the target stability and translation. In the case of RNAse H, types HI and HII digest the RNA in RNA-DNA hybrids [100].
Gut microbiota can impact the miRNome as well as host immune pathways [65]. MiR-NAs serve as physiological ligands for TLRs, affecting genes associated with inflammation. Bayraktar et al. summarized the regulation of immune cell function by focusing on the capability of miRNAs to bind to TLRs [101]. TLRs identify pathogen-associated molecular patterns (PAMPs) and detect the invading pathogens [102]. Taganov  miR-146 is involved in the regulation of TLR and cytokine signaling and added miRNAs to the list of potential negative regulators of inflammation [103]. The regulation of the crosstalk between miRNAs and metabolism was also documented in macrophage inflammatory responses [104].
Gut microbiota has been shown to affect the expression of circulating miRNAs [105] and thus determine intestinal epithelial proliferation and differentiation. Results from mouse models have demonstrated that miR-156 inhibited intestinal cell proliferation by the Wnt/β-catenin signaling pathway [106] and miR-31 promoted intestinal epithelial cell proliferation through the Wnt/Hippo signaling pathway to increase epithelial regeneration following injury [74]. Moreover, miR-31 can inhibit the expression of GP130, IL17RA, and IL7R receptors in response to TNF and IL6 by STAT3 and NF-κB [74]. The proliferation of IESCs can be regulated by miR-375-3p [42], showing that highly expressed miR-375-3p reduced IESC proliferation.
Chronic inflammation is known to contribute to malignant development. Porphyromonas gingivalis is associated with miR-46a and influences the innate immune response [107]. Porphyromonas gingivalis stimulated the increase of miR-146a expression, contributing to the elevated secretion of IL-1β, IL-6, and TNF-α [107]. According to the results, miR-146a prevented intestinal inflammation and CRC development by repressing IL-17 production and IL-17R signaling in IECs [108]. Lu et al. demonstrated that the inhibition of miR-21-5p mediated the IL-6/STAT3 pathway in a rat model of ulcerative colitis, leading to a decrease in inflammation and apoptosis in monocyte/macrophage-like cells RAW264.7 cells [109].
Fecal miRNAs regulate the gut microbiota by specifically targeting bacterial genes, but the precise mechanisms of miRNA processing in bacteria need further investigation. MiRNAs are involved in the modulation of the transcriptional response to microbiota. However, the authors did not explain how epigenetic modification was involved in the pathogenic process induced by ETBF.

MicroRNA as a Diagnostic and Prognostic Biomarker in Cancer
The stool miRNA profile can be used as a biomarker for gut pathology and the clinical diagnosis of intestinal disorders [110]. Viennois et al. studied fecal miRNA as a marker of the microbiota colitogenic potential by illustrating how the absence of microbiota impacts the fecal miRNA profile [111].
A high expression of miR-21 and miR-106a in the stool of CRC patients indicates the potential use of fecal miRNA signatures as a noninvasive screening test for colorectal malignancies [115]. Hibner et al. also suggested fecal miR-21 as a diagnostic and prognostic biomarker for CRC [116]. A large meta-analysis evaluated 17 eligible research articles containing 6475, 783, and 5569 fecal miRNA profiles in patients with colorectal carcinomas, adenomas, and healthy individuals, respectively. The results showed that fecal miR-21, miR-92a, and their combination might serve as promising noninvasive biomarkers for CRC [117]. Wu et al. found the overexpression of miR-21 and miR-92a in biopsies from colorectal tumors compared to adjacent normal tissues in a cohort of 88 CRC patients [118]. As previously reported, miR-21 and miR-92a upregulation promoted CRC cell migration, invasion, and proliferation [118,119].
Importantly, the gut microbiota plays a critical role in cancer progression via the regulation of noncoding RNAs in microbiota-mediated cancer metastasis [91]. MiR-15a and miR-16-1 were shown to be involved in B-cell-mediated immune suppression by colorectal tumors. Significantly, upregulated levels of miRNAs correlated with increased patient survival [120].
The serum miR-155 expression might be used as a biomarker for the diagnosis and prognosis of CRC, noting that its high levels correlated with lymph node metastasis, distant metastasis, tumor differentiation, and TNM stage in a cohort of 146 CRC patients and 60 control subjects [121]. The miR-21 expression levels in serum and stool are also suggested to be noninvasive diagnostic tools for CRC. According to the findings, miR-21 expression significantly distinguished tumor, node, and metastasis stages III-IV from stages I-II with 88.1% sensitivity and 81.6% specificity, respectively [119]. Li et al. evaluated the diagnostic effectivity of stool miR-135-5p for metastasis in CRC patients. Stool miR-135-5p expression was upregulated in CRC patients with 74.1% specificity and 96.5% sensitivity. In a comparison of stool and serum miR-135b-5p, it was discovered that stool miR-135-5p was more effective in distinguishing TNM stage III versus IV [122]. Furthermore, miR-663 was of diagnostic value in CRC patients with sensitivity and specificity of 83.1% and 73.8%, respectively [123]. The miR-663 expression was significantly associated with TNM stage, tumor differentiation, invasion, and lymph node metastasis [123].
A number of studies have identified the correlation between the differential expression profiles of up-and downregulated miRNAs in the stool of patients with gastrointestinal disorders [112,113,[124][125][126], suggesting the potential clinical utility of fecal miRNA profiling ( Figure 1).
taining 6475, 783, and 5569 fecal miRNA profiles in patients with colorectal carcinomas, adenomas, and healthy individuals, respectively. The results showed that fecal miR-21, miR-92a, and their combination might serve as promising noninvasive biomarkers for CRC [117]. Wu et al. found the overexpression of miR-21 and miR-92a in biopsies from colorectal tumors compared to adjacent normal tissues in a cohort of 88 CRC patients [118]. As previously reported, miR-21 and miR-92a upregulation promoted CRC cell migration, invasion, and proliferation [118,119].
Importantly, the gut microbiota plays a critical role in cancer progression via the regulation of noncoding RNAs in microbiota-mediated cancer metastasis [91]. MiR-15a and miR-16-1 were shown to be involved in B-cell-mediated immune suppression by colorectal tumors. Significantly, upregulated levels of miRNAs correlated with increased patient survival [120].
The serum miR-155 expression might be used as a biomarker for the diagnosis and prognosis of CRC, noting that its high levels correlated with lymph node metastasis, distant metastasis, tumor differentiation, and TNM stage in a cohort of 146 CRC patients and 60 control subjects [121]. The miR-21 expression levels in serum and stool are also suggested to be noninvasive diagnostic tools for CRC. According to the findings, miR-21 expression significantly distinguished tumor, node, and metastasis stages III-IV from stages I-II with 88.1% sensitivity and 81.6% specificity, respectively [119]. Li et al. evaluated the diagnostic effectivity of stool miR-135-5p for metastasis in CRC patients. Stool miR-135-5p expression was upregulated in CRC patients with 74.1% specificity and 96.5% sensitivity. In a comparison of stool and serum miR-135b-5p, it was discovered that stool miR-135-5p was more effective in distinguishing TNM stage III versus IV [122]. Furthermore, miR-663 was of diagnostic value in CRC patients with sensitivity and specificity of 83.1% and 73.8%, respectively [123]. The miR-663 expression was significantly associated with TNM stage, tumor differentiation, invasion, and lymph node metastasis [123].
A number of studies have identified the correlation between the differential expression profiles of up-and downregulated miRNAs in the stool of patients with gastrointestinal disorders [112,113,[124][125][126], suggesting the potential clinical utility of fecal miRNA profiling ( Figure 1). Tarallo et al. noted that dysbiosis of the gut microbiome in CRC patients might be characterized by altered miRNA profile in collected stool samples. The gut microbiome analysis of feces revealed an abundance of Alistipes putredinis in CRC patients, while Faecalibacterium prausnitzii was prevalent in stool samples from controls and patients with adenomas. At the phylum level, Firmicutes dominated the CRC group, while Verrucomicrobia was higher in patients with adenomas. As shown, miR-6738-5p expression decreased from controls through patients with adenomas to CRC patients. On the contrary, miR-200b-3p expression was higher in CRC patients compared to other groups [127]. Fecal miRNAs affect the growth and abundance of gut bacteria [128]. Meta-analyses uncovered that miR-20a and miR-92a upregulated in stool and blood samples of CRC patients might represent potential diagnostic markers for CRC [129,130]. Accordingly, miR-223 and miR-92a in blood and stool samples represent other potential CRC biomarkers with 96.8% sensitivity [131]. Based on Ji et al., synthetic miR-199a, miR-223-3p, miR-1226, miR-548ab, and miR-515-5p might regulate the proliferation of bacteria involved in the development of gut diseases, including Fusobacterium nucleatum, Escherichia coli, and segmental filamentous bacteria [132].
According to findings, bidirectional interactions exist between host miRNA and microbiota. Gut microbiota affects miRNA expression in intestinal cells; conversely, host miRNA can shape microbiota composition. However, the exact correlations and mechanisms behind the host-microbiota interactions are far from being sufficiently understood. Further research, through combined studies focusing on circulating blood/fecal miRNAs and microbiota determination, is highly warranted. Mounting evidence highlights that fecal miRNA detection might represent a potential trend in CRC diagnosis and individualized patient care.

Gut Microbiota-MicroRNA Crosstalk in Cancer Development
Besides the elucidation of genetic and epigenetic mechanisms in CRC development [133], the complex interplay between gut microbiota and human IECs is intensively studied [134]. In addition, fecal miRNAs derived mainly from IECs represent a potential diagnostic and prognostic tool in CRC (Figure 1). Yuan et al. provided some of the first evidence linking gut microbiota communities and miRNA expression in CRC [135]. The authors performed a comparison between the microbiome and miRNA expression levels in colorectal tumor samples and adjacent tissues, showing 76 differentially expressed miRNAs, including miR-182, miR-503, and miR-17~92 clusters. Importantly, specific bacteria taxa correlated with differentially expressed miRNAs. A positive correlation was detected between Blautia and miR-139 expression levels while Blautia negatively correlated with miR-20a, miR-96, miR-182, miR-21, miR-7974, and miR-183. Potential targets for deregulated miRNAs include proteins involved in peptidoglycan and terpenoid backbone biosynthesis as well as transporters [135].
According to these findings, F. nucleatum elevated the invasive pathways in CRC and affected the expression of inflammatory mediators and miRNAs in colonic neoplasms. Proenca et al. indicated that the upregulation of miR-34a in CRC proceeds via TLR2/TLR4 signaling and is dependent on F. nucleatum response [139]. The infection of cells with F. nucleatum led to a higher expression of miR-21 via activation of TLR 4 signaling to MYD88. MiR-21 reduced the levels of the GTPase RASA1. RASA1 binds to RAS oncoprotein, leading to its inactivation [140]. The combination of F. nucleatum and miR-21 prophesy increased morbidity and poor patient outcomes. The dysregulation of miR-4474, miR-4717, and miR-21 was observed in F. nucleatum-positive CRC tissues [141]. According to Feng et al., increased miR-4474 and miR-4717 correlated with early and advanced stages of CRC [142]. Increased miR-21 promotes carcinogenesis by F. nucleatum via the RAS-MAPK cascade [143].  Moreover, an increase in ETBF and BFAL1 correlates with clinicopathological param eters, including poor prognosis, reduced survival, and worse patient outcomes. The level of ETB and expression of BFAL1 might be used as potential predictors of CRC prognosis [138]. The figur was created with BioRender.com. Abbreviations: CRC, colorectal cancer; ETBF, enterotoxigenic Bac teroides fragilis; lncRNA1, long noncoding RNA1; mTOR, the mammalian target of rapamycin RHEB, Ras homolog enriched in brain According to these findings, F. nucleatum elevated the invasive pathways in CRC an affected the expression of inflammatory mediators and miRNAs in colonic neoplasms Proenca et al. indicated that the upregulation of miR-34a in CRC proceeds via TLR2/TLR signaling and is dependent on F. nucleatum response [139]. The infection of cells with F nucleatum led to a higher expression of miR-21 via activation of TLR 4 signaling to MYD88 MiR-21 reduced the levels of the GTPase RASA1. RASA1 binds to RAS oncoprotein, lead ing to its inactivation [140]. The combination of F. nucleatum and miR-21 prophesy in creased morbidity and poor patient outcomes. The dysregulation of miR-4474, miR-4717 and miR-21 was observed in F. nucleatum-positive CRC tissues [141]. According to Fen et al., increased miR-4474 and miR-4717 correlated with early and advanced stages of CRC [142]. Increased miR-21 promotes carcinogenesis by F. nucleatum via the RAS-MAPK cas cade [143].
The evaluation of the link between miRNA expression and microbiome compositio has revealed novel mechanisms related to miRNA-driven glycan production in pathogen and CRC tumorigenesis [135]. F. nucleatum modulates the tumor-immune microenviron Moreover, an increase in ETBF and BFAL1 correlates with clinicopathological parameters, including poor prognosis, reduced survival, and worse patient outcomes. The level of ETBF and expression of BFAL1 might be used as potential predictors of CRC prognosis [138]. The figure was created with BioRender.com (accessed on 16 November 2022). Abbreviations: CRC, colorectal cancer; ETBF, enterotoxigenic Bacteroides fragilis; lncRNA1, long noncoding RNA1; mTOR, the mammalian target of rapamycin; RHEB, Ras homolog enriched in brain.
The evaluation of the link between miRNA expression and microbiome composition has revealed novel mechanisms related to miRNA-driven glycan production in pathogens and CRC tumorigenesis [135]. F. nucleatum modulates the tumor-immune microenvironment by inhibiting T-cell responses [144]. MiR-21 elevates the releasing of IL-10 and prostaglandin E2. This molecular interaction can be helpful in CRC prevention and treatment. Gut microbiota-derived metabolites are involved in the development of various cancers. The study by Huang et al. showed that bacterial metabolites upregulated miR-192-5p, leading to the downregulation of BMPR2 and the inhibition of RhoA-ROCK-LIMK2. These correlations led to the inhibition of colon cancer cell growth [145]. Faecalibacterium prausnitzii is a known butyrate-producing bacteria [146]. Butyrate is produced by microbiota-driven fermentation of dietary fibers [147]. Mounting evidence indicates the relationship between butyrate and dysregulation of miRNA expression. According to the findings, F. prausnitzii-produced butyrate correlated with the suppression of CRC cell proliferation by upregulating miR-203 and subsequent inhibition of NEDD9 and Hakai expression. Hu et al. found that miR-92a overexpression in human CRC cells was repressed by butyrate treatment, leading to a rapid decrease in cMYC and pri-miR-17-92a levels [45].
Cao et al. determined the mechanism of ETBF-mediated miR-149-3p in colitis and CRC showing the ETBF-induced downregulation of miR-149-3p expression both in vitro and in vivo. This process depended on methylation, where METTL14 mediated the N6methyladenosine. The PHF5A gene transactivated the SOD2 by using the KAT2A signaling pathway. Since PHF5A is the miR-149-3p target gene, miRNA promotes the PHF5A expression by regulation of alternative splicing of KAT2A mRNA in CRC cells [148]. Parvimonas micra is linked to colorectal tumorigenesis by enhancing the oncogenic Wnt signaling pathway [149]. Some bacterial taxa, including Clostridium difficile, Campylobacter jejuni, Escherichia coli, Enterococcus faecalis, Helicobacter pylori, Fusobacterium nucleatum, Vibrio cholerae, and Porphyromonas gingivalis promoted the expression of miR-21 and reduced PTEN levels [150]. These bacteria were suggested to be associated with pancreatic cancer metastasis. However, cancer cells might follow an immune escape via the miR-21/PTEN axis and immune-suppressive cells. The intensive host-microbiota crosstalk in CRC development is placed in Figure 3. There is still a lack of studies proving a direct interaction between miRNA expression and gut microbiota composition in CRC cancer development and progression. However, recent findings highlight the existence of host miRNA-microbiota interactions and their clinical relevance should be further analyzed.

Targeting the Gut Microbiota-MicroRNA Interactions in Cancer Treatment
Exosomal miR-149-3p from ETBF-treated cells facilitates Th17 differentiation. Thus, the ETBF/miR-149-3p pathway may represent a potential target in CRC treatment [148]. Further, an association was found between the miR-21/PTEN axis and increased chemotherapy sensitivity of pancreatic cancer cells [150]. Infection with Escherichia coli, Helicobacter pylori, Porphyromonas gingivalis, Fusobacterium nucleatum, and Pseudomonas aeru- There is still a lack of studies proving a direct interaction between miRNA expression and gut microbiota composition in CRC cancer development and progression. However, recent findings highlight the existence of host miRNA-microbiota interactions and their clinical relevance should be further analyzed.

Targeting the Gut Microbiota-MicroRNA Interactions in Cancer Treatment
Exosomal miR-149-3p from ETBF-treated cells facilitates Th17 differentiation. Thus, the ETBF/miR-149-3p pathway may represent a potential target in CRC treatment [148]. Further, an association was found between the miR-21/PTEN axis and increased chemotherapy sensitivity of pancreatic cancer cells [150]. Infection with Escherichia coli, Helicobacter pylori, Porphyromonas gingivalis, Fusobacterium nucleatum, and Pseudomonas aeruginosa led to increased miR-21 and decreased PTEN levels [150]. Li et al. demonstrated that the production of a tRNA scaffold could be used to produce the chimeric pre-miR-1291 (tRNA/miR-1291) in Escherichia coli to miR-1291 functions in drug metabolism. According to the findings, tRNA-carried pre-miR-1291 suppressed the cell growth and increased the sensitivity of ABCC1-overexpressing PANC-1 cells to doxorubicin [151]. Accordingly, the recombinant tRNA fusion with pre-miR-34a (tRNA/mir-34a) led to the chimeric tRNA/mir-34a in Escherichia coli with a high degree of homogeneity and stability after the purification. The tRNA/mir-34a is processed to a mature miR-34a, and the tRNA scaffold metabolizes or degrades into tRNA fragments. The results showed that tRNA/miR-34a inhibited the proliferation of human carcinoma cells, including hepatocarcinoma. In mouse models, recombinant tRNA/miR-34a had no or minimal effect on blood chemistry and interleukin-6 level [152]. Tanooka et al. observed that bacterial genes were capable of exerting oncogenic activity via miRNAs. As shown, bacterial plasmid mucAB and Escherichia coli genomic homolog umuDC, carrying homologs for mouse anti-miR-145, were associated with the oncogene Nedd9 and its downstream Aurkb [153]. Another study showed that cord blood mesenchymal stem cell-derived exosomes containing anti-miRNA-221 inhibited proliferation and clonal formation of CRC cell lines Caco-2 and HCT-116. Subsequent in vivo analysis confirmed the targeting of exosomes into cancer cells, with a predominant location in the liver, spleen, and lung [154]. Xue et al. studied the effect of anti-miR-221 on CRC irradiation. Anti-miR-221 significantly downregulated miR-221, followed by increased expression of the PTEN protein, resulting in enhanced radiosensitivity of Caco2 cells [155]. The study concerning the potential therapeutic effect of anti-miR-223 showed the decreased cell proliferation, migration, and invasion of CRC cells after silencing miR-223 [156]. The influence of anti-miR-135b on metabolism in intestinal tumor organoids showed decreased glucose consumption and lactate production. After the transduction of anti-miR-135b lentivirus into organoids tumor-derived (OTD), decreased expression of miR-135b was detected in the OTD of CRC. Anti-miR-135b repressed the activities of luciferase and reduced SPOCK1, which influenced the proliferation and invasion of CRC [157]. Zhang et al. found an association between anti-miR-19a and resistance of CRC to oxaliplatin. The authors found that PTEN expression levels increased through the PI3K and AKT signaling pathways. Anti-miR-19a targeted the PTEN gene and suppressed the phosphorylation in CRC cell lines SW480/R and HT29/R [158].
Xiao et al. found a novel mechanism of colitis-induced oncogenesis regulation through the therapeutic effect of Clostridium butyricum, which impacted miR-200c expression and increased epithelial cell proliferation. The interaction between Clostridium butyricum and miR-200c affected proinflammatory TNF-α and IL-12 production and decreased transepithelial permeability by lengthening epithelial microvilli and increasing transepithelial electrical resistance (TEER), a marker of tight junction function [159]. Research into TLR4/miR-155 promoted novel strategies for colitis-associated cancer (CAC) prevention and control [160]. TLR4 is a receptor for Fusobacterium nucleatum and Salmonella, and is connected to oncogenic infection to colonic inflammatory and malignant processes. MiR-155 increased the TLR4 signaling through modulating the negative regulators SOCS1 and SHIP1. Conversely, TLR4 increases the miR-155 expression through transcriptional and post-transcriptional modulation. According to the findings, TLR4 activation and decreased cyclooxygenase 2 regulate apoptosis in CD4-TLR4-expressing intestinal tumors. T Moreover, the authors admitted that the inhibiting TLR4/MD2 signaling suppressed the metastatic capacity of colon cancer cells [160].
Bifidobacterium longum has been used in cancer gene therapy as a vehicle to transport anticancer genes [161]. The results showed that Bifidobacterium longum suppresses murine CRC via modulation of oncomiRs and tumor suppressor miRNAs. This interaction leads to the inhibition of cancer cell proliferation and invasion [162]. Bifidobacterium administration induces the expression of tumor suppressor miRNAs, including miR-145 and miR-15a. These miRNAs regulate IL-6 and IL-1β expression. Bifidobacterium decreased the NF-kB concentration and increased IL-1β mRNA and IL-1β concentration in CRC mice. Consequently, it decreased the IL-6 mRNA and IL-6 concentration [162].
Another publication on gut microbiota-miRNA interaction in cancer treatment studied the response of gastric epithelial cells to bacterial infections. It showed that Enterococcus faecalis was associated with miR-17-92 and miR-106-363 cluster expression. MiR-17-92 cluster was downregulated via a p53-dependent mechanism or during treatment with reactive oxygen species (ROS). This combination represents a potential strategy to combat gastric malignancy [163]. Since inflammation modulates miRNA expression, Mathé et al. induced systemic inflammation by treatment with Corynebacterium parvum in a mouse model. Following Corynebacterium parvum-induced inflammation in C57BL mice, the levels of miR-21, miR-29b, and miR-34a/b/c were enhanced while a decrease in miR-29c and miR-181a/c expression was observed. These miRNAs have protumorigenic features that affect the expression of cytokines (IL-6, IL-8, IL-10, and IL-12a), epidermal growth factor receptor (EGFR) signaling pathways, and the TP53 tumor suppressor, mediating p53induced apoptosis, cell cycle arrest, and increasing the population doubling of normal human fibroblasts [164].
As discussed, findings on the emerging role of gut microbiota-miRNA interactions in intestinal homeostasis, immunology, cancer development, and progression are presented in Table 2.

Conclusions and Future Perspectives
Complex molecular interactions at the host-microbiota interface play a significant role in maintaining intestinal homeostasis and immunity. MiRNAs are critical mediators of post-transcriptional regulation through binding to complementary regions of coding and noncoding transcripts. Mounting evidence identifies fecal miRNAs as potential biomarkers for intestinal disorders, including gastrointestinal malignancies. Besides consideration as a diagnostic tool, the analysis of miRNAs in stool can also facilitate the prediction of cancer prognosis. Further research focusing on the described associations between gut microbiota and specific miRNA levels, together with the identification of novel miRNAs and targets potentially linked to the composition of microbial communities, is highly warranted.
Although increasing evidence has demonstrated the impact of the gut microbiota-miRNA interplay on host pathophysiology, the exact mechanisms are yet to be identified. A deep understanding of signaling pathways by which the intestinal microbiota regulates miRNA and gene expression in individual IEC subtypes could shed the light on the molecular pathways involved in maintaining intestinal homeostasis. According to the evidence, microbiota-miRNA interactions are bidirectional. Fecal miRNA has been shown to regulate bacterial gene transcripts and thus shape the gut microbiome. Targeting miRNAs is also being considered in antitumor therapy. The preparation of synthetic miRNA mimics that imitate endogenous miRNAs or antagomiRs reducing oncogenic miRNA expression might represent novel therapeutic options, without increasing the treatment-related toxicity. However, miRNA-like off-target repression, caused by partial complementarity to mRNA other than the target, results in unwanted toxicity.
The impact of microRNAs and particular microbiota species on inflammation and cancer development remains to be elucidated. It is supposed that the interaction between microbiota and microRNAs is complex; currently, there is a lack of studies showing direct interactions between them. Therefore, it is still unclear whether miRNA expression patterns are only associated with microbiota composition or whether there is a causal relationship. Moreover, we need to consider the complexity of microbiota composition as well as the lack of long-term effects of microbiota modulations on miRNAs and vice versa.
Since small differences in miRNA expression can lead to alterations in the intestinal epithelium and the disruption of intestinal homeostasis, new strategies could help to improve outcomes for cancer patients. Moreover, the modification of the gut microbiota composition by probiotics, prebiotics, or fecal microbiota transplantation might represent a potential trend in modulating the cancer-related microbiota-miRNA interactions, aiming to reduce gastrointestinal cancer development. However, our knowledge of the impact of gut microbiota modulation on miRNA expression is still limited and largely unknown, and extensive research in this field is needed.