Contribution of miRNAs in the Pathogenesis of Breast Cancer

Breast cancer is the most frequently diagnosed cancer among females. Gene expression profiling methods have shown the deregulation of several genes in breast cancer samples and have confirmed the heterogeneous nature of breast cancer at the genomic level. microRNAs (miRNAs) are among the recently appreciated contributors in breast carcinogenic processes. These small-sized transcripts have been shown to partake in breast carcinogenesis through modulation of apoptosis, autophagy, and epithelial–mesenchymal transition. Moreover, they can confer resistance to chemotherapy. Based on the contribution of miRNAs in almost all fundamental aspects of breast carcinogenesis, therapeutic intervention with their expression might affect the course of this disorder. Moreover, the presence of miRNAs in the peripheral blood of patients potentiates these transcripts as tools for non-invasive diagnosis of breast cancer.


INTRODUCTION
Breast cancer is the most frequently diagnosed cancer among females. With approximately 2.3 million new cases, breast cancer accounts for 11.7% of all diagnosed cancers. In terms of cancerrelated mortalities, female breast cancer is responsible for 6.9% of mortalities and ranks fifth. Notably, the mortality rate from female breast cancer is significantly higher in developing countries compared with that in developed countries (1). This cancer has been found to be associated with a number of lifestyle and reproductive risk factors, namely, early menarche age, late menopause age, first birth high age, lower period of breastfeeding, hormone replacement therapy after menopause, taking oral contraceptive pills, alcohol intake, and obesity (2). Approximately 5-10% of breast neoplasms are associated with inherited mutations in a number of genes, particularly the BRCA1 and BRCA2 genes (3). In addition, gene expression profiling methods have shown the deregulation of several genes in breast cancer samples and have confirmed the heterogeneous nature of breast cancer at the genomic level (4). More recently, several investigations have reported the dysregulation of microRNAs (miRNAs) in breast cancer samples or plasma samples from these patients in correlation with the functional aspects of tumorigenesis (5)(6)(7). miRNAs are produced through a multistep process mediated by two RNase III proteins, namely, Drosha and Dicer (8,9). These small-sized non-coding transcripts have been found to regulate the expression of a significant proportion of human genes and play fundamental roles in the development of human disorders (10). miRNAs mainly regulate gene expression at posttranscriptional level. Meanwhile, miRNA metabolism and functions are regulated through sophisticated mechanisms (10). Moreover, the expression of miRNA genes is regulated at the transcriptional level through mechanisms similar to the regulatory mechanisms of protein-coding genes. This type of regulation defines the tissue-or developmental stage-specific expression of miRNAs. Most notably, miRNAs can suppress the expression of mRNAs that code factors participating in miRNA biogenesis; thus, they contribute in autoregulatory feedback paths (10). The expression of miRNAs has been reported to be altered in breast cancer samples. As an illustration, recent studies have detected the aberrant expression of miR-221 and miR-222 in breast malignancy (11,12). In the current review, we describe the impact of miRNAs in breast carcinogenesis and explain their participation in the regulation of apoptosis, autophagy, epithelial-mesenchymal transition (EMT), and resistance to chemotherapy. These processes have important roles in the pathogenesis of cancer. EMT is regarded as a key participant in the invasion and metastasis of cancers. Thus, identifying the main regulators of this process has important implications in cancer treatment. Autophagy has dual roles in cancer progression. Its activation can provide energy and nutrient supplies during the metastatic process, which promotes cell survival in stressful situations (13). In contrast, autophagy can act as a cancer suppressor in the early phase of cancer progression and hinder metastasis through decreasing the expression of important transcription factors for EMT (13). Resistance to apoptotic signals is a key feature in cancer development (14). Moreover, defects in the apoptotic mechanisms enhance malignant transformation and induce the resistance of transformed cells to chemotherapy (14). Finally, resistance to chemotherapy is an important feature gained by tumor cells during tumor evolution, precluding cancer management.

REGULATION OF APOPTOSIS BY MIRNAS IN BREAST CANCER
Apoptosis is a coordinated process that happens in physiological and pathological contexts. Cancer is one of the contexts where lack of appropriate cell apoptosis results in the survival of malignant cells. Several pathways are involved in the regulation of apoptosis. Defects can happen at any portion of these pathways, resulting in the malignant transformation of cells, facilitation of tumor metastasis, and induction of resistance to anticancer agents (15). miR-7-5p is an example of miRNAs that regulate the apoptosis of breast cancer cells. This miRNA has been shown to target proteasome activator subunit 3 (REGg), an important modulator of breast cancer and activator of protein proteolysis. The upregulation of miR-7-5p has led to the suppression of proliferation and induction of cell apoptosis in breast cancer through influencing the expression of REGg (16). This member of the REG family has an oncogenic function which depends on the proteolysis of p21 and p53 (17,18). miR-15a and miR-16 are two other miRNAs that regulate the apoptosis of breast cancer cells. Luciferase reporter assay has confirmed the interaction between these miRNAs and 3′ UTR of BMI1 transcript. Both miRNAs could suppress the expression of BMI1 at the transcript and protein levels, resulting in the downregulation of anti-apoptotic protein BCL2 and the upregulation of pro-apoptotic proteins. The forced overexpression of these miRNAs has enhanced the levels of mitochondrial reactive oxygen species (ROS), leading to impairment of mitochondrial membrane potential, release of cytochrome c into the cytosol, and activation of Caspase-3 and Caspase-6/9. These events altogether induce the intrinsic pathway of apoptosis (19). miR-17-5p is another miRNA that has been found to induce apoptosis in breast cancer cells. Notably, the upregulation of miR-17-5p has enhanced the sensitivity of breast cancer cells to paclitaxel-associated cell apoptosis through the modulation of STAT3. Consistent with this finding, the upregulation of STAT3 has reduced the paclitaxel-associated apoptosis of MCF-7 cells. miR-17-5p has been found to enhance apoptosis through upregulating the p53 expression, which was suppressed by STAT3. Therefore, miR-17-5p suppresses STAT3 and upregulates p53 to increase breast cancer cell apoptosis (20).
Another study has demonstrated the impact of miR-23a on the suppression of apoptosis in breast cancer cells. Notably, this impact has been exerted in an independent manner from its inhibitory role on the X-linked inhibitor of apoptosis protein, the most potent anti-apoptotic member of the inhibitor-of-apoptosis proteins (21). Notably, the role of miR-23a on the enhancement of invasiveness of breast cancer cells has been verified in xenograft models (22). Several other upregulated miRNAs in breast cancer, such as miR-27a, miR-32, miR-205-3p, miR-221/ 222, and miR-1271, as well as downregulated miRNAs in breast cancer, such as miR-17-5p, miR-134, miR-139-5p, miR-200b, miR-214, miR-218, miR-543, miR-1301-3p, and miR-4458, have been found to regulate apoptosis in breast cancer cells. Table 1 shows the regulation of apoptosis by miRNAs in breast cancer. Figure 1 demonstrates that the aberrant expression of various miRNAs could contribute in adversely modulating the mitochondrial pathway of apoptosis which is involved in triggering human breast cancer.

REGULATION OF AUTOPHAGY BY MIRNAS IN BREAST CANCER
An autophagy mechanism is initiated by the establishment of autophagosomes that seizure degraded apparatuses and then fuse with lysosomes to induce recycling processes. Autophagy has dual impacts in tumor inhibition and promotion in several types of malignancies. Moreover, autophagy influences cancer stem cell properties through participating in the maintenance of stemness, regulation of tumor recurrence, and induction of  resistance to anticancer drugs (56). Autophagy is another subject of regulation by miRNAs in breast cancer cells. miR-20a is among the upregulated miRNAs in breast cancer, particularly in triple-negative breast cancer cells. The expression of miR-20a has been negatively correlated with the activity of the autophagy/ lysosome pathway. miR-20a suppresses the basal and nutrient starvation-associated autophagic flux and activity of lysosomalassociated proteolysis. Moreover, this miRNA enhances the intracellular ROS levels and DNA damage response through modulating numerous important regulators of autophagy; among them are BECN1, ATG16L1, and SQSTM1. The expression of miR-20a has been negatively correlated with the expressions of these target genes in breast cancer tissues.
Notably, triple-negative cancers have exhibited a particular downregulation of BECN1, ATG16L1, and SQSTM1 genes. The upregulation of miR-20a has also been associated with a higher occurrence of copy number variations and genetic mutations in breast cancer samples. The effects of miR-20a on the enhancement of tumor evolution and growth have also been confirmed in a xenograft model of breast cancer (57). Another study has shown the regulatory effects of miR-20a and miR-20b on the expression of RB1CC1/FIP200. Both miRNAs could decrease the expression of RB1CC1/FIP200 transcripts and proteins. The upregulation of these miRNAs has reduced basal and rapamycin-associated autophagy. Therefore, miR-20a and miR-20b can regulate autophagy through influencing the expression of RB1CC1/FIP200 (58). A high-throughput miRNA sequencing experiment has reported miR-25 as the most important target of isoliquiritigenin (ISL) in inducing autophagy flux. Moreover, mechanistical studies have shown that miR-25 silencing results in cell autophagy through enhancing the expression of ULK1, an early regulator of autophagy initiation. miR-25 upregulation blocks ISLassociated autophagy. ISL has been found to sensitize cancer cells to chemotherapeutic agents as demonstrated by the enhancement in LC3-II levels, decrease in ABCG2 levels, downregulation of miR-25, and activation of ULK1 (59).
The inhibitory roles of miR-26b, miR-129-5p, and miR-200c on autophagy are exerted through the modulation of DRAM1 (60), HMGB1 (61), and UBQLN1 (62) expressions, respectively. Notably, miR-129-5p and miR-200c could attenuate irradiationinduced autophagy and decrease the radioresistance of breast cancer cells through this route (61) (62). Table 2 shows the regulation of autophagy by miRNAs in breast cancer. Figure 2 presents the role of several miRNAs in breast cancer cells via regulating the autophagy pathway.

REGULATION OF EMT BY MIRNAS IN BREAST CANCER
EMT is a complicated developmental program that permits carcinoma cells to change the epithelial characteristics to mesenchymal features. This alteration permits them to obtain mobility and migration ability. EMT is involved in numerous stages of the metastatic program, from dedifferentiation to aggressiveness (74). TGF-b1-induced EMT has been shown to participate in the metastasis of breast cancer cells. This process is regulated by a number of miRNAs-for instance, miR-23a as an upregulated miRNA in breast cancer cells, particularly in metastatic samples, has been shown to be induced by TGF-b1. The TGF-b1-associated regulation of miR-23a is mediated by direct binding of Smads with the RNA Smad-binding element in miR-23a. The suppression of miR-23a expression has inhibited TGF-b1-associated EMT and attenuated the migration, invasiveness, and metastatic ability of breast cancer cells. miR-23a can directly inhibit the expression of CDH1, a key modulator of EMT. The miR-23a-mediated suppression of CDH1 has been found to activate Wnt/b-catenin signaling. Taken together, miR-23a enhances TGF-b1-associated breast cancer metastasis through influencing the expression of CDH1 and inducing Wnt/b-catenin cascade (75). miR−27a is another upregulated miRNA in breast cancer samples and cell lines. The upregulation of miR−27a has increased the migratory potential of breast cancer cells through induction of EMT. FBXW7 has been identified as a downstream target of miR−27a. The overexpression of FBXW7 in breast cancer cells could inhibit EMT and the migratory aptitude of these cells. Therefore, miR−27a can regulate the metastatic potential of breast cancer through the suppression of FBXW7 (76). miR-29a has also been found to be upregulated in breast cancer samples in correlation with distant metastasis and poor clinical outcome of patients. miR-29a silencing has suppressed the proliferation and migration of breast cancer cells. Ten eleven translocation 1 (TET1) has been identified as a target of miR-29a. The upregulation of TET1 has attenuated the proliferation and migration of breast cancer cells.
The miR-29a-mediated downregulation of TET1 enhances EMT (77). Several upregulated miRNAs in breast cancer, such as FIGURE 1 | A schematic diagram of the role of miRNAs in triggering the mitochondrial cascade of apoptosis in human breast cancer. Apoptosis pathway could be activated via both extrinsic and intrinsic cascades. The intrinsic pathway is generally occurring through the release of cytochrome c from the mitochondria and modulates mitochondrial outer membrane permeabilization via Bcl-2 family proteins. The activation of extrinsic cascade could be triggered via ligand binding to death receptor, including DR3, DR4, DR5, Fas, and TNFaR. Following that, caspase proteins have a significant part in cleaving target proteins as well as nuclear lamins to elevate DNA degradation, leading to apoptotic cells undergoing phagocytosis. Furthermore, P53, via triggering the upregulation of various proteins containing Bid, Bax, CD95, Puma, and TRAIL-R2, could get effectively involved in activating intrinsic and extrinsic apoptosis cascades. Therefore, any alterations or abnormalities occurring during apoptotic pathways could considerably contribute to the progression of human diseases, including cancer. Previous studies have authenticated that several miRNAs could have a crucial role in regulating the apoptosis pathway in breast cancer. All the information regarding the role of these miRNAs involved in the modulation of breast tumors can be seen in Table 1. miR-93, miR-125b, miR-199a-3p, and miR-221, as well as downregulated miRNAs, such as miR-34a, miR-92b, miR-124, miR-138-5p, miR-153, miR-516a-3p, and miR-524-5p, affect the EMT process. Table 3 shows the regulation of EMT by miRNAs in breast cancer. Figure 3 depicts the role of various miRNAs in the modulation of EMT via targeting receptors that convey signals from EMT inducers or multiple EMT components.

REGULATION OF CHEMORESISTANCE BREAST CANCER CELLS BY MIRNAS
Chemoresistance is a phenotype which is associated with several signaling pathways as well as cellular processes such as apoptosis, autophagy, and EMT. miRNAs have also been found to affect the resistance of breast cancer cells to important chemotherapeutic FIGURE 2 | A schematic representation of the role of several miRNAs in regulating the autophagy cascade in human breast cancer. The autophagy pathway is comprised of multiple sequential steps containing sequestration, transport to lysosomes, and degradation. The expression of Atgs could be triggered via AKT, MAKP-ERK, P53, and AMPK pathways. Autophagy is a fundamental substantial biological cascade by removing damaged organelles, but dysregulation of autophagy could contribute to several diseases, including cancers. Accumulating evidence has illustrated that various miRNAs could have a remarkable part in modulating the apoptosis cascade in breast tumors. All the information regarding the role of these miRNAs contributing to the regulation of breast cancer can be seen in Table 2. drugs-for instance, miR-7 has been shown to be downregulated in MCF-7 and adriamycin-resistant cells (MCF-7/ADR cells), particularly in MCF-7/ADR cells. The upregulation of miR-7 has enhanced sensitivity of MCF-7/ADR cells to ADR. The downregulation has led to the upregulation of EGFR and PI3K, while the upregulation of miR-7 has been associated with opposite effects. Moreover, the suppression of miR-7 has been associated with the enhancement of proliferation and inhibition of apoptosis. Therefore, miR-7 has been found to affect the resistance of breast cancer cells to ADR, and its upregulation can enhance the effects of ADR through the suppression of EGFR/ PI3K signaling (97). miR-30c is another miRNA that is involved in intrinsic adriamycin resistance in p53-mutated breast cancer (98). Moreover, another study in breast cancer has shown a correlation between high miR-7 levels and better pathological complete response to paclitaxel/carboplatin. Functionally, miR-7 has been shown to sensitize MCF-7 and MDA-MB-231 cells to the cytotoxic effects of paclitaxel and carboplatin through targeting MRP1 and BCL2. Taken together, miR-7 has been suggested as a predictive marker for the assessment of chemotherapy efficacy and therapeutic target for the enhancement of response of breast cancer patients to chemotherapy (99). The expression assays in an Src inhibitor saracatinib-resistant breast cancer cell line (SK-BR-3/SI) has shown the downregulation of miR-19b-3p in saracatinib-resistant cells compared with saracatinib-sensitive ones. The under-expression of miR-19b-3p not only has been associated with higher IC50 value of saracatinib but also has increased the migratory potential of breast cancer cells. Functionally, miR-19b-3p targets PIK3CA. Thus, the resistance to Src inhibitors might be due to the enhancement of the activity of PI3K/Akt pathway following miR-19b-3p downregulation (100). In addition, miR-34a could affect the sensitivity of breast cancer cells to sunitinib by regulating the Wnt/b-catenin signaling pathway (101). miR-24-3p is another miRNA which can regulate the sensitivity of breast cancer cells to tamoxifen. The upregulation of miR-24-3p has been shown to increase tamoxifen-induced cytotoxicity in breast cancer cells, while its silencing has decreased these effects. Bim has been identified as a target of miR-24-3p in breast cancer. Further experiments have shown the upregulation of miR-24-3p and the downregulation of BIM expression in tamoxifen-resistant MCF7 cells compared with original cells. Moreover, the suppression of miR-24-3p has enhanced the sensitivity of MCF7/TAM cells to tamoxifen through the enhancement of cell apoptosis (102). Besides this, miR-148a and miR-152, by downregulating ALCAM, could reduce tamoxifen resistance in ER+ breast cancer cells (33). miR-375 is another miRNA that could inhibit cancer stem cell phenotype and tamoxifen resistance in human ER+ breast cancer cells through degrading HOXB3 (103). Meanwhile, tamoxifen has been shown to regulate the expressions of miR-29b-1 and miR-29a (104). Table 4 shows the role of miRNAs in the regulation of response of breast cancer to therapeutic agents.

DISCUSSION
Non-coding RNAs can influence the expression of several groups of mRNAs through different mechanisms, such as modulation of chromatin structure as well as regulation of transcription and translation. miRNAs are mostly exerting their regulatory roles at the post-transcriptional level through binding to different regions of mRNAs to suppress their translation via mRNA degradation or translation inhibition. miRNAs have been found to regulate important aspects of breast carcinogenesis through the regulation of apoptosis, autophagy, and EMT. miRNAs affect the apoptosis of breast cancer cells through several mechanisms; among them are modulation of p53related pathways, expression of caspases, and regulation of response to ROS. Through modulating the expression of EMTrelated genes as well as those influencing cell motility and invasiveness, miRNAs regulate breast cancer metastasis. Notably, miRNAs can also influence the response of breast cancer cells to a wide array of therapeutic agents ranging from conventional chemotherapeutic drugs to tyrosine kinase inhibitors and hormone therapy agents. Based on in vitro experiments, miRNAs can regulate the cytotoxic effects of adriamycin, cisplatin, doxorubicin, docetaxel, paclitaxel, gemcitabine, trastuzumab, saracatinib, sunitinib, tamoxifen, and a number of other anti-cancer drugs. In addition to miRNAs whose direct effects on the modulation of response to therapeutic agents have been verified, other miRNAs that regulate cell apoptosis or autophagy can potentially influence therapeutic responses. The modulation of cellular DNA damage response and the activity of cancer stem cells are other routes of participation of miRNAs in the regulation of response of breast cancer cells to chemotherapy. A possible role of miRNAs in the determination of breast cancer stem cells has been suggested through the demonstration of differential expression of miRNAs in CD44+/CD24-/low breast cancer stem cells versus non- ) which have a significant role in inducing the EMT cascade. Thereby, the activation of EMT can be triggered through the overexpression of selected zinc finger, including ZEB1/2, snail, slug, or basic helix-loop-helix containing TWIST1 transcription factors (95,96). Recent studies have detected he regulatory role of multiple miRNAs in EMT and breast cancer cells. All the information regarding the influence of these miRNAs in EMT and the control that they exert in major signaling cascades in breast cancer can be seen in Table 3.   tumorigenic cancer cells (145). This kind of function of miRNAs has a practical significance in the determination of the behavior of breast cancer as well as its response to therapeutic modalities. In addition, a number of anti-cancer agents exert their effects through the modulation of the expression of miRNAs that affect apoptosis or autophagy-for instance, curcumol, via regulating the miR-181b-2-3p/ABCC3 axis, could enhance the sensitivity of triple-negative breast cancer cells to doxorubicin (133). Some miRNAs can affect several aspects of breast carcinogenesis-for instance, miR-34a can affect apoptosis, EMT, and drug resistance. miR-23a has an essential role in the regulation of apoptosis and EMT. Moreover, miR-15a and miR-16a regulate apoptosis and drug resistance. NF-kB, mTOR, and Wnt/bcatenin pathways are among the shared pathways between several miRNAs acting on these processes. Since miRNAs can target multiple transcripts, they can often modulate numerous pathways. Notably, miRNAs exert their inhibitory roles via a complex process which is dependent on cellular constituents, indicating tissue or cell type-specific features (146). The small molecular size of miRNAs and their capacity in the regulation of the expression of genes participating in the evolution of cancer have endowed miRNAs the potential to influence the treatment of breast cancer (147). As miRNAs can affect both the development of breast cancer and the response of cancerous cells to therapeutic options, intervention with their expression is regarded as an appropriate treatment modality for almost every stage of cancer development and progression. Forced overexpression or suppression of miRNA expression is a possible therapeutic modality for breast cancer. Examples of miRNAantagonism methods are 2′-O-methyl-modified oligonucleotides, locked nucleic acid anti-miRNAs, and cholesterol-conjugated antagomirs. These methods are being used as miRNA-inhibitory tools (148). In fact, the over-expression of miRNAs that induce cell apoptosis, such as miR-7-5p (16), miR-15a, miR-16 (19), and miR-17-5p (20), or inhibit cell cycle progression can suppress the progression of breast cancer. On the other hand, the suppression of expression of oncogenic miRNAs by oligo antisense mechanisms is a treatment modality. In vitro studies have provided a firm evidence for the specificity and efficacy of miRNA-based modalities in the modulation of the expression of target genes, yet future studies should focus on the improvement of delivery systems, enhancement of stability of the prescribed molecules, decreasing off-target effects, and assessment of longterm safety of these molecules (149). Only after solving these issues can miRNA-based therapeutics enter clinical practice.
The identification of the miRNA-associated network and interplay between miRNAs and other types of regulatory transcripts will open new opportunities for diagnostics and therapeutic modalities in breast cancer. System biology methods can be used to predict the role of miRNAs in the determination of response to anti-cancer therapies and prognostic approaches in clinical settings. Targeting miRNAs with essential roles in a drugresistant network has been suggested as a putative approach in overcoming chemoresistance in breast cancer (146). Finally, the combinations of conventional anticancer drugs with antioncogenic miRNA reagents are expected to enhance treatment responses. In fact, the recognition of miRNA profiles in different stages of breast cancer development and development of miRNAbased targeted therapies are two wings of miRNA studies which can introduce novel promising results in clinical settings.
In brief, the contribution of miRNAs in the regulation of cell death, cell motility and invasion, activity of cancer stem cells, regulation of EMT, and modulation of response to therapeutics potentiate miRNAs as proper targets for the treatment of breast cancer. However, the clinical application of miRNA-based therapies depends on the effective documentation of miRNA profiles in different subtypes of breast cancer and the construction of the interaction network between miRNAs and genes that regulate breast carcinogenesis and chemoresistance phenotype.