Regulation of Inflammasome by microRNAs in Triple-Negative Breast Cancer: New Opportunities for Therapy

During the past decade, researchers have investigated the molecular mechanisms of breast cancer initiation and progression, especially triple-negative breast cancer (TNBC), in order to identify specific biomarkers that could serve as feasible targets for innovative therapeutic strategies development. TNBC is characterized by a dynamic and aggressive nature, due to the absence of estrogen, progesterone and human epidermal growth factor 2 receptors. TNBC progression is associated with the dysregulation of nucleotide-binding oligomerization domain-like receptor and pyrin domain-containing protein 3 (NLRP3) inflammasome, followed by the release of pro-inflammatory cytokines and caspase-1 dependent cell death, termed pyroptosis. The heterogeneity of the breast tumor microenvironment triggers the interest of non-coding RNAs’ involvement in NLRP3 inflammasome assembly, TNBC progression and metastasis. Non-coding RNAs are paramount regulators of carcinogenesis and inflammasome pathways, which could help in the development of efficient treatments. This review aims to highlight the contribution of non-coding RNAs that support inflammasome activation and TNBC progression, pointing up their potential for clinical applications as biomarkers for diagnosis and therapy.


Introduction
The breast is a complex structure, which is organized in 15 to 25 lobes of different sizes that are connected with lactiferous ducts that terminate in the nipple, ductal formations and glandular tissue, all of these being surrounded by fibro-adipose tissue [1]. Specifically, the adult breast is a tissue highly variable in conformation, adiposity and volume, due to the different proportions between adipose, fibrous and glandular tissue, among which are also found blood and lymphatic vessels. The corresponding distribution of fat and collagenous components differs among women and is influenced by hormonal, physiologic and environmental factors [2].
Breast cancer (BC) is a dynamic, aggressive and heterogeneous disease that is the principle cause of death among women worldwide, but that also affects men [3]. This neoplasm has the potential to be determined by exposure to both genetic and non-genetic risk factors such as gender, age, menopause, nulliparity, obesity, alcohol abuse and exposure to hormones, radiation or therapy [1]. The early detection and diagnosis of BC is based on screening techniques (ultrasound, mammography, contrast-enhanced digital mammography, magnetic resonance imaging and positron emission tomography), microwave imaging techniques (microwave tomographic, radar-based microwave imaging and radiometry), biomarker-based techniques (radioimmunoassay, immunohistochemistry, enzyme-linked immunosorbent assay and fluoroimmunoassay) and breast tissue biopsies, which are used to differentiate between malign and benign tumors [4].
Following a diagnosis of BC, it is necessary to stage it according to the American Joint Committee on Cancer (AJCC) tumor, nodes, and metastasis (TNM) system, as TNM staging is used to define and stratify the size of the tumors (T), the status of regional lymph nodes (N), and distant metastasis (M) [5]. TNM staging is divided into four classes: (I) clinical staging, which includes information from clinical examination; (II) pathological staging, which includes the affected anatomical formations; (III) post-therapy staging, which includes clinical and pathologic information; and (IV) restaging, if necessary [6].
In order to establish the most efficient treatment for patients, the histological classification is completed via the molecular classification of BC that is based on the expression profiles of the estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki67 antigen. The main molecular subtypes of breast cancer are luminal A (ER positive, PR positive and HER2 negative), luminal B (ER positive, PR negative and HER2 negative), HER2-enriched (ER negative, PR negative and HER2 positive), triple-negative breast cancer (TNBC), basal-like (ER negative, PR negative and HER2 negative), claudin low (ER negative, claudin negative, vimentin positive, E-cadherin low) and normal breast-like (adipose tissue gene signature) [7]. Luminal and HER2-enriched subtypes are associated with good prognoses and are highly responsive to therapy, resulting in a greatly improved outcome, while TNBC is the most aggressive subtype of BC, which is characterized by a high cell proliferation rate and a tendency to relapse [8]. Experimental studies indicate the presence of two main pathways involved in low-grade and high-grade breast tumorigenesis; low-grade BCs are regularly ER positive, PR positive and HER2 negative, while high-grade BCs are ER negative, PR negative and HER2 positive [9].
The mechanism of BC is dependent on the genetic modification and molecular processes that determine initiation, transformation and progression from normal tissue to tumor tissue [10]. More than 90% of diagnosed BCs are associated with the mutation of specific genes, such as breast cancer genes 1 and 2 (BRCA1 and BRCA2), TP53, phosphatase and tensin homolog (PTEN), serine/threonine kinase 11 (STK11), ataxia-telangiectasia mutated (ATM), BRCA1 interacting protein 1 (BRIP1) or the partner and localizer of BRCA2 (PALB2), etc. [11].
In addition, BC aggressiveness is supported by chronic inflammation considering the up-regulation of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-18 or tumor necrosis factor-α (TNF-α), growth factors or free radicals [12]. The secretion and release of pro-inflammatory cytokines are associated with inflammasome complex activation. Inflammasome is a cytoplasmic multiprotein complex, composed of a sensor (NOD-like receptor protein (NLRP)), an adaptor (an apoptosis-associated speck-like protein containing a caspase-activation and recruitment domain (CARD) (ASC)) and effector molecules (pro-caspase). The inflammasome complex is involved in numerous physiological and pathological mechanisms, including different types of cancer, although the activation of this complex can have both a positive or negative impact on carcinogenesis [13]. First of all, inflammasome activity stimulates epithelial mesenchymal transition (EMT), metastasis and angiogenesis, while inhibiting apoptosis and enhancing tumor development. On the other hand, the inflammasome pathway is also associated with immune reactions and the programmed death of tumor cells through pyroptosis [14].
The dynamic interaction between BC and inflammasome is regulated by a complex molecular network, including non-coding RNAs (ncRNAs). NcRNAs can be classified into short non-coding RNAs (sncRNAs) or long non-coding RNAs (lncRNAs), based on their number of nucleotides [15]. Among the most studied sncRNAs are microRNAs (miRNAs), which are single-stranded ribonucleic acid molecules involved in diverse cellular processes (cell survival, proliferation and adhesion, motility, cell death, inflammation, carcinogenesis, etc.). According to their implication in tumorigenic mechanisms and metastasis, miRNA molecules can be classified into oncogenic miRNAs and suppressor miRNAs [16]. Increasing evidence has indicated that many miRNAs are involved in the regulation of the inflammasome complex (miR-7, miR-9, miR-20a, miR-21, miR-23a, miR-30e, miR-33, miR-132, miR-133, miR-146, miR-155, miR-223, miR-296, miR-377, miR-711, etc.) and that these molecules represent a link between inflammasome activity and BC, especially TNBC [17]. Recently, researchers' attention has been directed towards targeting these molecules as possible therapeutic strategies for the treatment of BC, due to their significance in post-transcriptional regulation [18].
Considering the importance of this topic and the ongoing expertise, this review aims to highlight the molecular mechanisms involved in BC correlated with the significance of the inflammasome pathway and miRNAs. Furthermore, another target is to accentuate the complex network and dual role of miRNAs (oncogenic and suppressor), in doing so emphasizing RNA-based techniques' potential for clinical applications for diagnosis and therapy.

Molecular Characteristics of TNBC
TNBC is a highly invasive and aggressive type of BC, which is characterized by the absence of ER, PR and HER2 expression. TNBC represents almost 20% of all diagnosed BC, being associated with resistance to chemotherapy, a predisposition to metastasis, poor prognoses and reduced survival rates [19]. Over the years, researchers have studied the molecular signatures of TNBC using advanced techniques and, according to gene expression profiles, Lehmann et al. [20], Burstein et al. [21] and Jezequel et al. [22] have all proposed different classification of TNBC in order to investigate possible therapeutic targets to improve the outcomes of TNBC (Table 1) [22]. Classifying and understanding the particularities of TNBC allow for the development of personalized medicine, because each subtype has different characteristics and responses to anti-tumor therapy [23].  The breast tumor microenvironment (TME) is associated with chronic inflammatory reactions, with inflammatory infiltrate being a prognostic marker of cancer. Pro-inflammatory molecules together with their specific receptors are involved in the development of TNBC due to their capability to promote cell differentiation and angiogenesis, to recruit immune cells and to influence the immune system [46]. Lymphocytes, macrophages and fibroblasts are the most common cell types of the TME and secrete different factors (such as cytokines, chemokines, enzymes, growth factors, etc.), which are associated with an aggressive malignancy and high risk of metastasis. Pro-inflammatory mediators (IL-6, CCL2, COX2, and TNF-α) are generally up-regulated in breast tumor stroma; IL-1, IL-6, IL-8, IL-11, IL-18 and IL-23 are among the most studied inflammatory factors involved in inflammation, TNBC, invasion and metastasis [47]. In close correlation with the development of inflammatory reactions and TNBC is the activation of the NLRP3 inflammasome complex, with IL-1β and IL-18 being the two major cytokines activated by NLRP3 inflammasome, which promote tumor cell proliferation and invasion [48].
Over the years, new signaling pathways within breast TME have been discovered, which have opened up new perspectives for the development of strategies that aim to block these signals that promote tumorigenesis, angiogenesis and metastasis. Specifically, conventional therapeutic strategies are the gold standard for neoadjuvant treatment represented by a combination between anthracyclines and taxanes, capecitabine and taxane and ixabepilone monotherapy (paclitaxel, 5-fluorouracil, doxorubicin, cisplatin, carboplatin, abraxane, bevacizumab, cyclophosphamide, ixabepilone, capecitabine, etc.), adjuvant treatment (anthracycline-based drugs), surgery (mastectomy and lumpectomy) and radiotherapy [49].
Despite the heterogeneity and aggressiveness of TNBC, the corresponding standard of care (SOC) requires neoadjuvant chemotherapy, followed by surgery and adjuvant chemotherapy ( Table 2). There are several treatment schemes for early diagnosed TNBC that are based on anthracycline and taxane administration, but which cause numerous adverse effects (nausea, fatigue, gastrointestinal toxicity, myelosuppression, alopecia, hypothyroidism, hyperthyroidism, pneumonitis, skin reactions, adrenal insufficiency, peripheral neuropathy, neutropenia, pyrexia, anemia, thrombocytopenia, electrolyte abnormalities, infection, etc.) and do not lower the relapse rate [50].  [49,52] Although neoadjuvant chemotherapy is the standard treatment approach for diagnosed TNBC, the therapeutic potential of other agents that interfere with various molecular mechanisms (such as angiogenesis, the immune response, the cell cycle, etc.) have been tested or are currently under testing and approval ( Table 3). The optimal treatment scheme for TNBC is a great challenge due to the disease's heterogeneity and increased risk of relapse, so the researchers have explored several promising anti-tumor agents (pembrolizumab, bevacizumab, olaparib, sacituzumab govitecan, etc.) [53]. Other therapeutic approaches are represented by platinum salts (carboplatin, cisplatin, etc.) immunotherapies that target the programmed cell death-1 (PD-1) receptor/PD-L1 pathway, immune-checkpoint inhibitors, poly-adenosine diphosphate ribose polymerase (PARP) inhibitors or AKT inhibitors [54]. Table 3. Novel strategies for TNBC treatment.

Class of Agents
Examples of Therapy Mechanism of Action Refs.

PD-1 and PD-L1 inhibitors
Pembrolizumab + Paclitaxel Doxorubicin + Cyclophosphamide Pembrolizumab + Paclitaxel + Carboplatin Durvalumab + Nab-paclitaxel Atezolizumab + Nab-paclitaxel Atezolizumab + Nab-paclitaxel + Carboplatin Reactivation of the anti-tumor immune response PD-1/PD-L1 complex formation inhibition [55] Platinum-based therapy Cell growth and migration inhibition Binding to the topoisomerase in DNA replication inhibition S-phase-specific cell death initiation DNA damage [66] The molecular characteristics of TNBC tumors (heterogeneity and the chemoresistance mechanism) raise difficulties in establishing an effective conventional therapeutic strategy; therefore advanced therapeutic strategies have been developed. There are two main types of advanced therapeutic treatments: passive transport (or enhanced permeability and retention (nanoparticles)) and active transport (miRNA and aptamers) [49].

Molecular Mechanisms of Inflammasome Activation during TNBC
Discovered in 2002 by Martinon et al., inflammasome represents the perfect structure bridging inflammation, tumorigenesis, angiogenesis and metastasis [45]. The inflammasome complex is a cytoplasmic multiproteic platform that comprises three components: the sensor molecule, the adaptor protein and the effector (Figure 1). The inflammasome pathway is activated by the recognition of specific stimuli, danger-associated molecular patterns (DAMPs), pathogen-associated molecular patterns (PAMPs), microbe-associated molecular patterns (MAMPs), and homeostasis-altering molecular processes (HAMPs) (such as ATP, toxins, K+ efflux, reactive oxygen species, etc.) by toll-like receptors (TLRs), the nucleotidebinding oligomerization domain (NOD) and leucine-rich repeat (LRR)-containing receptors (NLRs), the absent in melanoma-2 (AIM2)-like receptors (ALRs)), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) or C-type lectin receptors (CLRs). Inflammasome-related sensor molecules consist of pathogen recognition receptors (PRRs), such as TLRs, NLRs, ALRs, RLRs, CLRs and proteins that contain a tripartite motif, such as pyrin (also known as marenostrin or TRIM20) [45]. The mechanism of the NLRP3 inflammasome assembly. NLRP3 activation require two signals: priming and activation. The priming signal is induced by the binding of DAMPs or PAMPs and stimulates the up-regulation of inflammasome components through the NF-kB inflammatory signaling pathway. Together, these two signals determine the assembly of the NLRP3 inflammasome and the activation of CASP1, which supports the maturation and release of the pro-inflammatory cytokines (pro-IL-1β and pro-IL-18), and catalyzes the cleavage of GSDMD and pore formation into the membrane, leading to pyroptosis (created with BioRender.com).
Over the years, clinical trials have revealed that the increased expression of NLRP3 components is associated with unfavorable prognoses and lower survival rates. The upregulation of NLRP3 inflammasome in TNBC promotes cell migration and invasion, being associated with epithelial-mesenchymal transition (EMT) mechanisms and metastasis through CASP1-dependent mechanisms and the autocrine production of pro-inflammatory IL-1β [80].
Although the function of pyroptosis in the TME of TNBC has not yet been fully elucidated, studies have revealed that pyroptosis plays a dual role (both anti-and pro-tumorigenic). The activation of pyroptosis can thereby generate inflammatory reactions and immune responses that could enhance the sensitivity to immunotherapy [78]. Recent studies have further indicated that the activation of NLRP3 inflammasome stimulates the myeloid cells' infiltration in breast TME [80]. On the other hand, pro-inflammatory cytokines are released into the TME and promote tumor growth. Additionally, the up-regulation of gasdermin B (GSDMB) is associated with poor prognoses and less sensitivity to HER2targeted therapy [81].
Considering that inflammasome is implicated in TNBC development and progression, numerous studies have focused on discovering the potential anti-cancer strategies of inflammasome components, such as the identification of possible NLRP3, IL-1β or CASP1 Figure 1. The mechanism of the NLRP3 inflammasome assembly. NLRP3 activation require two signals: priming and activation. The priming signal is induced by the binding of DAMPs or PAMPs and stimulates the up-regulation of inflammasome components through the NF-kB inflammatory signaling pathway. Together, these two signals determine the assembly of the NLRP3 inflammasome and the activation of CASP1, which supports the maturation and release of the pro-inflammatory cytokines (pro-IL-1β and pro-IL-18), and catalyzes the cleavage of GSDMD and pore formation into the membrane, leading to pyroptosis (created with BioRender.com).
The activation of inflammasome is carried out by two main signals: priming and activation. The priming stimuli of the inflammasome are activate TLRs, the FAS-associated death domain protein and IL-1 receptor ligands, which activate the NF-kB signaling pathway, causing the up-regulation of inflammasome expression. In normal conditions, the expression of inflammasome components is relatively low and unable to initiate inflammasome activation. In this stage, two signaling molecules from the NF-kB pathway, MyD88 and TIR-domain-containing adapter-inducing interferon-β (TRIF), are involved in inflammasome regulation and pro-IL-1β secretion, but the expression of ASC, pro-caspase-1 and pro-IL-18 remains unmodified. Following the priming signal, the second signal, which is induced by the presence of DAMPs or PAMPs, is required in order to promote the ASC oligomerization, inflammasome assembly and caspase-1 (CASP1) activation [67].
These sensors can determine the oligomerization of the adapter protein ASC, also known as PYCARD, and the activation of the cysteine protease CASP1 [68]. After the detection of specific stimuli by the receptors, the ASC adapter proteins are assembled and form speck-like structures, which recruit pro-CASP1 that is cleaved into mature CASP1. Active CASP1 cleaves the pro-inflammatory cytokines (pro-IL-1β and pro-IL-18) and poreforming gasdermin D (GSDMD) in order to promote the CASP1-dependent cell death, termed pyroptosis, with the pyroptosis' mechanism being directed by the gasdermin family (GSDMA, GSDMB, GSDMC, GSDMD, GSDME and DFNB59) [69].
The classification of inflammasome is based on the structure of the receptor activating the signaling pathway, meaning that therefore this complex is divided into: canonical inflammasome-forming NLRs (NLRP1, NLRP2, NLRP3, NLRP6, NLRP7, NLRP9 and NOD-like receptor C4 (NLRC4) and NLRC5), non-canonical inflammasome-forming AIM2 (AIM2 and IFI16) and inflammasome-forming pyrin (MEFV) [70]. The canonical NLRP3 inflammasome is the most studied in the BC context, although several molecular aspects have yet to be investigated. Unlike canonical inflammasome, which involves CASP1 activity, non-canonical inflammasome requires the activation of human caspase-4/5 or murine caspase-11. Moreover, the study of non-canonical signaling is still ongoing and no details are yet known about the affected molecular mechanisms [71].
Breast tumor growth, progression and aggressiveness are promoted by NLRP3 inflammasome activation, pro-inflammatory cytokine secretion and the pyroptotic death of cancer cells. The overexpression of NLRP3, IL-1β, IL-18 and CASP1 is correlated with inflammatory signaling pathways, such as NF-kB, STAT1, STAT3, IL-1β or Wnt/β-catenin, developing an invasive TME, characterized by the release of reactive oxygen species (ROS), reactive nitrogen species (RNS), angiogenic factors, cytokines or chemokines [72].
Aggressive factors from the extracellular environment can cause alterations of cellular structures, leading to the release of pro-inflammatory mediators (cytokines, DAMPs or PAMPs, etc.). The presence of an inflammatory response in the TME is favorable for the initiation of programmed cell death (apoptosis, necroptosis, pyroptosis, etc.) [73].
Pyroptosis, or CASP1-dependent cell death, is characterized by cellular swelling, cell lysis, nuclear condensation and DNA fragmentation. Pyroptosis can have both protumorigenic and anti-tumorigenic roles. The pro-tumorigenic role of pyroptosis is correlated with the NLRP3 and ASC oligomerization mechanism, while the anti-tumorigenic effects of pyroptosis are associated with the activation of the immunogenic signals and immune responses [74].
Following the activation of the NLRP3 inflammasome and the maturation of CASP1, the proteolytic cleavage of GSDMD into N-terminal and C-terminal domains is stimulated. The N-terminal domains of GSDMD attach to the cell membrane and form pores of 10-20 nm in diameter, with the GSDMD-derived pores allowing the influx of calcium or sodium ions and efflux of potassium, thus stimulating the inflammatory process [75].
Over the last years, there has been a special interest in the research field of the contribution of inflammasome complexes to the onset and progression of various diseases, such as obesity, diabetes or cancer. Advanced molecular studies have exposed the dynamic role of inflammasomes and the overlapping with the immune system and inflammatory-associated mechanisms. The connection between inflammasome complexes and the immune system is represented by the secretion and maturation of pro-inflammatory IL-1β, which activates the NF-kB signaling pathway and angiogenic signals, leading to tumor development [76].
The complex relationship among TNBC, inflammasome signaling, the immunosuppressive TME and resistance to chemotherapy requires meticulous investigation. Specifically, breast TME is characterized by the interaction between infiltrated immune cells, inflammatory cells, cytokines, chemokines and growth factors. According to Table 1, two of the criteria for the TNBC classification are represented by the involvement of the immune system and inflammation in the tumorigenic mechanisms, which implies personalized treatment strategies based on inflammatory and immune particularities [77]. In breast malignancies, an immunosuppressive TME is closely correlated with inflammasome signaling. Activation of immune mechanisms (cytokine and chemokine signaling pathways and T-cell receptor or B-cell receptor signaling, etc.) stimulates NOD-like receptor signaling leading to inflammasome activation. In addition, the activation of immune pathways (such as TLRs) stimulates the detection of DAMPs and PAMPs and induces intracellular signals that contribute to the recruitment of ASC adaptor protein and the oligomerization of inflammasome [78,79].
Over the years, clinical trials have revealed that the increased expression of NLRP3 components is associated with unfavorable prognoses and lower survival rates. The upregulation of NLRP3 inflammasome in TNBC promotes cell migration and invasion, being associated with epithelial-mesenchymal transition (EMT) mechanisms and metastasis through CASP1-dependent mechanisms and the autocrine production of pro-inflammatory IL-1β [80].
Although the function of pyroptosis in the TME of TNBC has not yet been fully elucidated, studies have revealed that pyroptosis plays a dual role (both anti-and protumorigenic). The activation of pyroptosis can thereby generate inflammatory reactions and immune responses that could enhance the sensitivity to immunotherapy [78]. Recent studies have further indicated that the activation of NLRP3 inflammasome stimulates the myeloid cells' infiltration in breast TME [80]. On the other hand, pro-inflammatory cytokines are released into the TME and promote tumor growth. Additionally, the upregulation of gasdermin B (GSDMB) is associated with poor prognoses and less sensitivity to HER2-targeted therapy [81].
Considering that inflammasome is implicated in TNBC development and progression, numerous studies have focused on discovering the potential anti-cancer strategies of inflammasome components, such as the identification of possible NLRP3, IL-1β or CASP1 inhibitors (MCC950 or AcYVAD-cmk) or the identification of ncRNA molecules that intervene at the transcriptional level. For example, anakinra, canakinumab or rilonacept are inhibitors of the IL-1 signaling pathway, which contribute to the initiation of apoptosis and blockade of tumor cells' cycle progression [82]. As mentioned previously, inflammasome opens up new therapeutic perspectives for TNBC, which must be studied thoroughly.

Non-Coding RNAs in Oncology
The sequencing of the human genome indicated that approximately 98% of genes are non-functional or "junk DNA" while about 2% of them encode proteins. Moreover, technological advances in sequencing have revealed that the non-coding sequences are copied into RNA molecules that can assist fundamental biological functions, such as regulating gene expression, cell growth and organs' development, or play an important role in the evolution of a wide variety of human diseases, including cancer, autoimmune disease, metabolic diseases or inflammation. Regarding the implication of ncRNAs in cancer, mutation or aberrations in the promoters of ncRNAs and the dysregulation of the enzymes involved in their biogenesis (Drosha, Dicer) can lead to altered gene expression levels, the normal expression levels of which are crucial for the normal functioning of the human body. Furthermore, up-or down-regulation of ncRNA expression is associated with cancer progression and metastasis [83].
As mentioned previously, ncRNAs can be classified into sncRNAs or lncRNAs, based on their number of nucleotides [15]. Among the most studied sncRNAs are miRNAs, small interfering RNAs (siRNAs), small nucleolar RNAs (snoRNAs), transfer RNA (tRNA)derived small RNAs (tsRNAs), and PIWI-interacting RNAs (piRNAs). MiRNAs are singlestranded ribonucleic acid molecules that incorporate between 19 and 25 nucleotides and regulate the expression of the target messenger RNAs (mRNAs). In the eukaryotic cells, a primary transcript (pri-miRNA) with multiple secondary structures is formed and is cleaved by two enzymes (Drosha and Dicer) in order to form a single hairpin structure precursor of microRNA (pre-miRNA) and miRNA duplex. Subsequently, these molecules are incorporated into the RNA-induced silencing complex (RISC) and are associated with Argonaute RISC catalytic component 2 (AGO2), leading to the cleavage of the complementary mRNA or suppression of translation ( Figure 2) [15]. MiRNAs biogenesis and roles in TNBC progression. Genes are transcribed by RNA polymerase II/III into a pri-miRNA molecule, which is cleaved by DGRC8 and Drosha generating the pre-miRNA. The pre-miRNA molecule is exported into the cytoplasm via exportin-5, where Dicer and TRBP cleaves the pre-miRNA, resulting in the mature miRNA. The functional mature miRNA molecule is associated with AGO2 onto the RISC, leading to gene silencing via mRNA cleavage, translational repression and degradation. Additionally, the up-regulation of oncogenic miRNAs determines the suppression of target genes, while the down-regulation of tumor suppressor miRNAs stimulates the overexpression of target genes. MiRNAs assist essential biological processes such as cell growth, proliferation, migration and invasion, hypoxia, inflammation, apoptosis, pyroptosis and metastasis in TNBC (created with BioRender.com).
Among the most studied oncomiRs are miR-373, miR-135b, miR-210, miR-638, miR-301b, miR-663a, etc. (Figure 2). MiR-373 was first identified as a metastasis-promoting miRNA in breast cancer, being involved in tumor-cell proliferation, migration, invasion and metastasis via CD44 targeting [84]. The up-regulation of miR-373 determines the secretion and maturation of caspase-8, which is mainly responsible for the non-canonical activation of the inflammasome complex [85]. MiR-135b is an oncomiR that targets TGF-β in order to enhance cell proliferation and invasion [86], at the same time being involved in the inhibition of CASP1 expression and inflammatory reactions [87]. MiR-210 is also known to play a significant role in breast tumor biological functions, including tumor proliferation and metastasis via regulation of hypoxia and programmed cell death [88]. In the aberrant miR expression, miR-301 is up-regulated in TNBC and promotes tumor growth, cell proliferation, migration, invasion and tamoxifen resistance via the TLR4/NF-kB signaling pathway [89]. Moreover, recent studies have revealed that miR-663a is an inflammation-related miRNA that is frequently deregulated in TNBC, being involved in both apoptotic pathway and inflammasome activity through secretion and maturation of pro-inflammatory cytokines [90,91]. The findings of these studies discovered that miR-638 up-regulation might play a critical role in TNBC progression and inflammasome assembly via modulation of the ATP synthesis mechanism [92,93].
MiRNAs have been described as important regulators in tumor suppression, with miR-217 having been acknowledged as an inhibitor of tumor growth, inflammation and inflammasome, through its regulation of the inflammatory response and the induction of oxidative stress and programmed cell death [94]. Recent studies have indicated that miR-340 functions as a tumor suppressor and cell cycle regulator and that miR-340 could affect pyroptosis through NLRP3 inflammasome and the TLR4/NF-kB signaling pathway, subsequently leading to decreased inflammatory signaling [95,96]. MiR-31 is among the most frequently altered microRNAs in breast malignancies, and the altered expression of miR-31 is associated with the negative regulation of the NF-kB signaling pathway, inflammasome activation, the release of cytokines and cell death [97,98]. Additionally, miR-223 is an anti-inflammatory microRNA that acts as a tumor suppressor in TNBC. The published literature has demonstrated that miRNA-223 expression is associated with NLRP3 inflammasome inhibition and the inhibition of cell cycle progression [99,100].
The main anti-cancer strategy is to activate the programmed death of cancer cells. For example, pyroptosis is a caspase-and-gasdermins-mediated cell death, which can be regulated by drugs or genetic interventions. Recently, special interest has been given to the potential of pyroptosis to suppress TNBC through the recruitment of tumor-suppressed immune cells and the activation of anti-tumor immunity. Studies have indicated that CASP1-induced pyroptosis combined with immune checkpoint inhibitors (ICIs) can enhance anti-tumor immunity in vitro and in vivo [101]. In TNBC conditions, immune cells (CD8+ T cells, NK cells, etc.) stimulate the expression of gasdermins, then coordinate M1 macrophages and T lymphocytes infiltration, in order to induce pyroptosis to stimulate cell sensitivity and to activate tumor clearance [102]. In addition, the activation of pyroptotic pathway by anti-tumor drugs, such as paclitaxel and cisplatin, determines the inhibition of tumor proliferation and metastasis [103].
In the field of oncology, miRNA, tsRNA and piRNA are among the most studied sncRNAs, with numerous studies having focused on the investigation of the mechanisms of miRNAs regarding the molecular and cellular processes involved in cancer conditions. Their results have demonstrated that miRNAs are regulators that guide the expression of both coding and non-coding molecules, such as oncogenes (RAS, MYC, etc.) and tumor suppressors (TP53, BRCA1, etc.). More than that, miRNAs can function as oncogenes, inhibiting the tumor suppressor molecules' activity, promoting accelerated cancer cell growth and finally encouraging the development of tumor formations. Tumors are subordinate to oncogenic miRNAs (oncomiR) expression for their survival and growth, due to the phenomenon of oncomiR addiction, making them promising potential therapeutic targets in anti-cancer treatment, thereby clearing the way to miRNA-based therapy [104,105]. On the other hand, miRNAs can also act either as tumor suppressors or oncogenes, depending on the progression of associated mechanisms [106]. Additionally, miRNAs can be used as molecular biomarkers because the aberrant expression of several miRNAs has been detected in multiple types of cancer. The expression level of a specific miRNA can be used to indicate the state of the cancer, to differentiate malignant tumors from benign ones and to indicate the type of cancer. Additionally, the molecular signatures of miRNAs outline the prognosis, anticipating the potential for metastasis and the response to therapy [107].

MicroRNAs Interplay with TNBC and NLRP3
MiRNA molecules are important regulators that target and interfere with mRNA translation, thus influencing gene expression. The aberrant expression of miRNAs disturbs normal biological mechanisms, such as cell growth, proliferation and migration, programmed cell death, and immune or inflammatory responses. The progression of BC (especially TNBC) is promoted by the abnormal expression of several miRNAs (Table 4).
Wang et al. investigated the influence of miR-200b on NLRP3, ASC, GSDMD, CASP1, IL-1β and IL-18 expression. Their results indicated that up-regulation of miR-200b determined the pyroptosis activation via regulation of the JAZF1/NF-kB signaling pathway. The tumor promoter JAZF zinc finger 1 (JAZF1) is a repressor of TGF-β-activated kinase 1 (TAK1), which is involved in NF-kB signaling cascade. The research group also ob-served that high expression of JAZF1 constrained the effect of miR-200b on the pyroptosis mechanism [149].
MiR-155-5p is another miRNA molecule that was found to be up-regulated in TNBC. Xu et al. evaluated the capacity of miR-155-5p antagomir to enhance the cytotoxic effects of cetuximab on tumor cells in vitro. The results demonstrated that the combination between miR-155-5p antagomir and cetuximab regulates GSMDE and CASP1 in order to stimulate the pyroptotic cell death and to decrease tumor growth. The in vivo experiments also confirmed that down-regulation of miR-155-5p increased the cytotoxicity of cetuximab, leading to pyroptosis and apoptosis activation in TNBC cells [124].
Bioinformatic analysis of miRNAs' role in inflammasome-mediated breast tumorigenesis has revealed that certain miRNAs that are associated with the NLR family and consequently influence the prospect of TNBC. For example, NLRP3 is associated with miR-125, miR-146b, miR-200, miR-223-3p, miR-373, miR-520 or miR-548 [150,151], NLRP1 regulation is realized by miR-143-5p, miR-210 [152], and NLRP8 is modulated by miR-181 [153]. The modulation of inflammasome by these miRNAs has effects on TNBC through different signaling pathways, such as NF-κB, TGF-β or IL-6, which are also regulated by the same miRNAs (miR-146b, miR-520, miR-373, etc.) and in normal conditions inhibit TNBC cell growth and proliferation [154,155]. MiRNAs function as master modulators of inflammasome and pyroptosis-related TNBC by regulating the expression of multiple genes and mechanisms. Although some studies have revealed a piece of miRNA-associated networks, it is compulsory to expand the knowledge about inflammasomes and the molecular mechanisms of pyroptosis into the context of TNBC.
MiRNA molecules are involved in the regulation of NLRP3 components, leading to the overexpression of pro-inflammatory markers. MiR-223 is a non-coding molecule and NLRP3 regulator that can bind to the 3 UTR region of NLRP3 mRNA, inhibit the translation mechanism and alter the release and maturation of pro-inflammatory cytokines [156]. Among the numerous other microRNAs involved in NLRP3 regulation is miR-22, which acts as a tumor suppressor. The increased expression of miR-22 is correlated with NLRP3 assembly difficulties, decreased cell proliferation rate and metalloproteinase 2 (MMP2), MMP9, vimentin, and N-cadherin low-expression profiles [157]. The aggressive behavior of cancer is due to the miR-21 activity, which inhibits the NLRP3 assembly by regulating NLRP3 phosphorylation and lysine 63-ubiquitination and alters the normal functions of the innate immune system [158].
Among the microRNA molecules that regulate NLRP3 inflammasome is miR-9. As well, miR-9 has been reported to perform important functions in tumor initiation, inflammation and immune regulation [159]. MiR-9 can suppress the activation of the NLPR3 inflammasome and NF-kB signaling, thereby determining the inhibition of CASP1 expression and the secretion of pro-inflammatory factors (IL-1β and IL-18). The up-regulation of miR-9 is also associated with embryonic lethal abnormal vision-like protein 1 (ELAVL1) regulation, anti-inflammatory mechanisms and the inhibition of pyroptosis cell death [126].
MiRNAs have great potential to become effective therapeutic targets in TNBC treatment, considering the critical functions of microRNAs in TNBC progression and the modulation of NLRP3 components' expression. Patients diagnosed with TNBC are still lacking an effective therapeutic response to chemotherapy, radiotherapy, surgery and immunotherapy. Therefore, it is necessary to develop new powerful and modern therapies that target the molecular mechanisms involved in the development of triple-negative breast tumors.
MiRNA molecules are decisive for oncogenic processes that have been intensively studied in recent years. For example, numerous studies have focused on the investigation of targeted therapies to silence (for oncogenes) or overexpress (for tumor suppressors) specific miRNAs [111,160]. However, miRNA-based therapeutic applications represent a subject that has not yet been fully elucidated, although these types of treatments have been evaluated in several pre-clinical studies, with the yielded results being deemed satisfactory. MiRNA-derived therapies are based on nucleotide hybridization, so the advantage of using them is that targeted sequences can be easily approached and that they can easily target combinations of RNAs. Although miRNA-based therapy indeed offers many advantages, the method for their delivery still remains a challenging subject [161][162][163].
MiRNA-based therapeutic strategies used in TNBC are either nucleic-acid-based strategies or drug-based strategies. Nucleic-acid-based treatments are divided into two groups: miRNA replacement (miRNA mimics) and anti-miRNA therapy, with the anti-miRNA treatment also being subdivided into two categories depending on the mechanism of action (miRNA antagonists or antagomiRs and miRNA sponges) [129].
MiRNA replacement or miRNA mimic is used in order to restore the defective tumor suppressor miRNA expression and to inhibit breast tumorigenesis progression. The delivery system of miRNA mimics is well received by normal cells [163]. For example, Li and her team explored the mechanism and role of miR-1290 in a radio-treated TNBC context, employing the transfection method for both miR-1290 mimic and miR-1290 inhibitor. Specifically, miR-1290 is a critical mediator that induces tumor proliferation and metastasis, being a potential biomarker for the diagnosis and prognosis of BC. The results of the first experiments carried out indicated that after radiotherapy the levels of NLRP3, ACS and CASP1 did not significantly differ between the radiosensitive and radioresistant MDA-MB-231 cells. Subsequently, the researchers evaluated the profile of miR-1290 in radiosensitive and radioresistant cells and concluded that the expression of miR-1290 was higher in the resistant MDA-MB-231 cells than in those that were sensitive. In the final part of their experiments, the transfection of the miR-1290 mimic and inhibitor demonstrated that the miR-1290 mimic had a positive impact on cell viability and inhibited NLRP3 inflammasome assembly via ASC and CASP1 suppression. Regarding pyroptosis, the miR-1290 inhibitor determined the up-regulation of CASP1, IL-1β and IL-18 by targeting NLRP3. One conclusion of this complex study was that miR-1290 promoted the radioresistance of MDA-MB-231 cells [163]. In addition to this study, which highlights the potential of miR-1290 as a key regulator of pyroptosis, other studies have revealed that many miRNAs are involved in positive regulation of pyroptosis through NLRP3 inflammasome activation, such as miR-21 [164] or miR-155-5p [124].
Anti-miRNA therapy is based on three main approaches: genetic knockout, anti-sense oligonucleotides (meaning antagomiRs) and miRNA sponges. AntagomiRs are modified anti-sense oligonucleotides that consist of ribose residues 2 -O-methylation, cholesterol residues 3 -conjugated and phosphorothioate linkages. AntagomiRs affect miRNA-related pathways by binding and inhibiting the activity of specific oncomiRs involved in TNBC development and progression [165], and the effectiveness of antagomiR therapy varies depending on tissue type and the method of delivery to the targeted tissue. This approach can be used as complementary sequences attached to the miRNAs or in the form of locked nucleic acid oligonucleotides, which contain a bond between the 2 -oxygen and 4 -carbon at each nucleotide, in order to inhibit TNBC progression and metastasis [166]. In 2016, Conde and their research team developed a RNA-triple-helix structure comprised of a miR-205 mimic and antagomiR-221 in order to evaluate a new platform for local anti-cancer therapy using both oncomiR inhibition and tumor suppressor miR replacement approaches. After miR delivery, the obtained data suggested that miR-205 and miR-221 targeted laminin subunit gamma 1 (LAMC1) protein and influenced TNBC by stimulating cell adhesion, proliferation, migration and metastasis, in both in vitro and in vivo conditions; on the other hand, miR-205 up-regulation was a consequence of an increased p53 profile. The concluding remarks of this study revealed that miR-221 and miR-205 inhibition affected E-cadherin, Slug and Snail expression, reducing TNBC cell migration and invasion. This new strategy also determined VEGF down-regulation, suppressing angiogenesis in TNBC contexts [167]. MiR-205 supports breast tumors' growth and inhibits apoptosis due to its capacity to mediate the assembly mechanism of the NLRP3 complex. The activation of NLRP3 is possible due to the increase in NLRP3, ASC and CASP1 expression and the development of an inflammatory response under the control of miR-205 [168]. The upregulation of miR-205 has also been reported to decrease steroid receptor co-activator (SRC) expression, which is involved in inflammation through the regulation of pro-inflammatory IL-18 expression [169,170]. On the other hand, miR-221 is a key modulator of breast tumorigenesis and inflammatory responses [171]. The up-regulation of miR-221 has antiinflammatory effects, and suppresses oxidative stress and programmed cell death in TNBC, due to the decrease in Bcl-2 expression and the suppression of the NLRP3/ASC/CASP1 signaling pathway, as well as inflammatory reaction [114].
MiRNA sponges represent another anti-miRNA strategy, which contain multiple binding sites (between four and sixteen) complementary to the target miRNAs and which have the capacity to inhibit other miRNAs that contain between two and seven specific sequential nucleotides [172]. Advanced studies indicate that miRNA sponges can be transcribed by both RNA polymerase II and III promoters, because their transcripts are stable as a result of capped 5 and 3 polyadenylated tails [173]. Additionally, numerous endogenous miRNA sponges that influence the profiles of TNBC-specific biomarkers have been studied. For example, Zhang et al. designed a self-assembled DNA nanosponge embedded with doxorubicin, which mimics the role of miRNA sponge transcripts for the adsorption and clearance of miR-21. The obtained sponge-like delivery system determined an increased efficiency of anti-tumor therapy due to cell apoptosis-specific gene regulation [174]. More interestingly, the potential of such a miR-21 sponge-based strategy can also be used to prevent metastasis through the negative regulation of colony stimulating factor-1 (CSF-1), a biomarker for malignancy and metastasis [175].
On the other hand, drug-based strategies focus on the regulation of miRNAs through the activity of certain intercalating agents (stilbenes, curcumin, flavonoids, histone deacetylases inhibitors, etc.) that bind to miRNA [176]. The regulation of miRNAs by flavonoids has a negative impact on tumor cells' proliferation, metastasis and EMT, but also modulates important mechanisms, such as angiogenesis, inflammation, inflammasome, immune responses, etc. [177]. The research activities carried out in this field have demonstrated that quercetin can influence the expression of certain oncomiRs, such as miR-21, miR-155 or let-7, leading to BC regression by programmed cell-death stimulation and increasing the sensitivity of tumor cells to chemotherapy [178]. In addition, miR-146a, miR-146b, miR-605 and miR-381 expression is modulated by quercetin, determining tumor growth inhibition and apoptosome assembly [179].
Over the years, the role of miRNA in TNBC progression has been intensively studied, with the results of these studies having highlighted the existence of numerous miRNAs involved in the modulation of the inflammasome complex, such as miR-223-3p, miR-7-5p, miR-22-3p, miR-33, miR-9, miR-155, etc. Their mechanism of action is based on binding 3 UTR of NLRP3 mRNA conserved regions and interfering with translation [180]. For example, Xie et al. studied the influence of miR-33 on NLRP3 inflammasome activation. The obtained results confirmed that miR-33 attenuates mitochondrial oxygen consumption and increases the production of cellular ROS, given that it is responsible for the secretion of IL-1β and the increased expression of NLRP3 and caspase-1 in macrophages [181]. On the contrary, Zhang et al. studied the possibility of inactivating the inflammasome and implicitly the growth of breast tumor cells targeting miR-233. Firstly, the researchers demonstrated the NLRP3 knockdown results in a decrease in breast tumor cell proliferation and migration, due to apoptosis initiation. Secondly, they used miR-233 mimics in order to suppress NLRP3 expression and NLRP3-dependent mechanisms, such as expression levels of ASC, IL-1β, IL-18 and CASP1. At the end of their study, the researchers stated that miR-233 activity inhibits the NLRP3 activation pathway, leading to the suppression of tumor cell growth, migration and invasion [18]. Another study, conducted by Hu et al. highlighted the role of miR-155 in the aggressiveness of TNBC due to the NLRP3 inflammatory pathway. Additionally, miR-155 interference decreased the MDA-MB-231 cell proliferation and ability to form colonies, stimulated the apoptosis of tumor cells and inhibited secretion of pro-inflammatory mediators [182].

Conclusions
In summary, this review integrates miRNAs and their functions into the inflammasomedependent TNBC progression, as miRNAs are key regulators of gene expression and tumorigenesis, being involved in NLRP3 inflammasome assembly and pyroptosis cell death initiation. Due to the heterogeneity and aggressiveness of TNBC caused by genetic and environmental factors, the development and validation of effective targeted strategies has been challenging. Over the past few years, wider attention has been paid to the activation of NLRP3 in oncogenic pathways and to the targeted therapeutic approaches based on miRNAs combined with anti-tumor drugs, in order to increase the survival rate of patients. The results of the original experiments are encouraging, although the elucidation of the molecular manipulation of inflammasome via microRNAs in order to improve TNBC treatment is imperative.