Role of MiRNAs and it’s single nucleotide polymorphism in breast cancer

: MiRNAs are 20-22 nucleotide long single-stranded non-coding RNA sequences, which can regulate post transcriptional activity of mRNA by binding with it at 3’UTR region (untranslated region). Thus deregulation of miRNA expression is responsible for dysregulating mRNA function which contributes in developing various diseases as well as cancerous phenotypes. Alteration of single nucleotide in miRNA sequence is one of the reasons behind deregulation of miRNA expression. The most frequent carcinoma in current day is breast cancer which causes a high mortality among women around the world as well as India. Despite of the advancement of diagnostic tools, strategies and treatment, the cases of breast cancer is increasing every year. There are plenty of biomarkers like ER, PR, Her2, Ki-67, etc available which are frequently used in diagnosis and treatment of breast cancer. After the discovery of MiRNA in 1993 in Caenorhabiditis elegans , it is attracting all the limelight in diagnosis and treatment of different carcinomas as well as breast cancer. In this review we will discuss on involvement of different types of MiRNAs and miR SNPs in breast cancer occurrence and susceptibility in a detailed manner. A metanalysis study performed to correlate role of polymorphisms in miR-146a rs2910164, miR-196a rs11614913 and miR-499 rs3746444 with breast cancer risk showed rs11614913 CC genotype in miR-196a2 was linked with elevated level of breast cancer occurrence. This study is useful for early detection of breast cancer (229). Another metanalysis on these functional miRNAs’ SNPs reported increased breast cancer risk with miR-499 rs3746444 in Asians and in the overall populations, while miR-196a rs11614913 was found in decreased association with breast cancer risk among Caucasians. They did not find any relationship between miR-146a rs2910164 with breast cancer susceptibility On the other hand variant genotypes rs3746444 in miR-499 were reported to be associated with significantly increased risks of breast cancer in Chinese women According to researchers The G allele of miR-SNP rs895819 (A > G) of pre-miR-27a is associated with a significantly reduced risk

helicase into two single-stranded miRNAs named Guide strand and passenger strand. Later RISC complex releases passenger strand and directs guide strand which is miRNA, towards target mRNA. This mature single-stranded guide strand binds with the target mRNA, exerting it's inhibitory activity either by blocking the mRNA translation or mRNA degradation.

MiRNAs and Global Diseases:
Many studies have proved importance of Micro RNAs in different developmental processes metabolism, cell proliferation, apoptosis, developmental timing, and neuronal cell fate (14)(15)(16)(17)(18) along with other regulatory roles including neuronal gene expression (19), brain morphogenesis (20), muscle differentiation (21), and stem cell division Few studies claim that abundant expressions of miR-1, miR-16, miR-27b, miR-30d, miR-126, miR-133, miR-143, and the let-7 have been found in adult cardiac tissues (29). Generally in the heart three MiRNAs, miR-1, miR-133 and miR-208 are highly expressed (30,31) and have been found to regulate mostly heart development and myocyte differentiation, whereas deregulated expression of miR-1 and miR-133 have been found to be involved in human heart failure (32-35). In case of cardiac hypertrophy miR-23a, miR-23b, miR-24, miR -195, miR-199a, and miR-214 were found upregulated and their overexpression in cardiomyocytes in vitro may be responsible behind it (36). Changes of MiRNA expression levels in early or later brain development may be representative of biochemical signals for determining cellular fate, different cell division programming or/and apoptosis in brain (50). Therefore it can be speculated that the pathogenesis of neurodevelopmental disorders such as intellectual disability, autism, Attention Deficit Hyperactivity Disorder (ADHD) and epilepsy are linked with miRNA expression changes (51).

Inflammatory Diseases
Fragile X syndrome (FXS), a genetic disorder which was first found to be linked with miRNA pathway occurs due to interaction between FMRP (Fragile X Mental Retardation Protein, which is a product of FMR-1 gene) and Dicer-derived miRNAs. This interaction can regulate target mRNA translation by binding non coding miRNA structures within UTRs of target mRNAs in inducing the genetic Fragile X syndrome (52-64). Another neurodevelopmental syndrome is Rett syndrome which occurs due to de novo mutations in MECP2 gene which causes aberrations in the DNA methyl-CpG-binding protein, MeCP2. Postnatally cultured rat neurons have shown direct repression of MeCP2 expression by miR-132, which indicates the importance of miRNA role in regulating MeCP2 by which postnatal neuronal development and synaptic maturation are maintained (52,65,66). Potential evidence of MiRNA role in Down syndrome individuals also have been found in many studies. Few bioinformatics analyses have revealed that five miRNAs; miR-99a,let-7c, miR-125b-2, miR-155 and miR-802 are encoded in chromosome 21, triplication of all or part of which is responsible for developing Down Syndrome. These all above mentioned miRNAs have been found to be overexpressed in fetal brain and heart tissues of DS individuals, which may be responsible for developing cognitive and cardiac defects in Down syndromic individuals (52,67). According to Sethupathy P et al. miR-155 downregulates angiotensin II type 1 receptor (AGTR1) gene, associated with hypertension. The AGTR1 protein generally is also found in lower levels among DS patients than individuals without DS (68). These all findings indicates towards the importance of miRNA role in Down Syndromic individuals. Dysregulation of specific miRNA expressions has been elucidated to be linked also with Alzheimer Disease. Loss of miR-29 cluster associated with increased (beta-amyloid precursor protein-converting enzyme) BACE1/betasecretase expression in sporadic AD patients, direct interaction of miR-298 and miR-328 with 30-UTR of BACE1 transcript involved in b-amyloid production, regulation of the expression of Amyloid Precursor Protein (APP) by miR-20a family (e.g. miR-20a, miR-17-5p and miR-106b) or elevated expressions of miR-9, miR-128a and miR-125b in AD hippocampus, there are plenty of evidences of involvement of micro RNAs in AD development have been found (69-71). In case of Schizophrenia, researchers have found association between miRNA and BDNF (Brain-derived Neurotrophic Factor), which plays important role in regulating development and synaptic maintenance of a variety of neurons in the CNS including GABA and glutamatergic neurons. Few bioinformatics studies suggest that newly described miRNAs, has-mir-1 and has-mir-206 can regulate BDNF protein synthesis by interfering in BDNF mRNA translation during brain development which indicates towards importance of miRNA roles in Schizophrenic patients (72)(73)(74).  (80,81). In a microarray analysis for studying miRNA expressions in Systemic  Lupus Erythematosus seven miRNAs (miR-196a, miR-17-5p, miR-409-3p, miR-141, miR-383, miR-112, and miR-184) were found downregulated and nine miRNAs (miR-189, miR-61,   miR-78, miR-21, miR-142-3p, miR-342, miR-299-3p, miR-198, and miR-298) were found upregulated compared to healthy controls (82). Still more research is necessary for confirming association of miRNA function with autoimmune diseases.
Liver Diseases: Understanding the miRNA regulation in liver physiology and different liver diseases like Viral Hepatitis, polycystic liver diseases have gained a lot of attention. As previously discussed, viral protein coding genes have ability of encoding their own miRNAs which can regulate their own protein coding genes. Hepatitis B Virus can be a good example in this case. The pre-miRNA transcribed from HBV gene can control its own gene expression (83,84). Even miRNA from host cell also can regulate viral genes. Regulating the replication of HCV (Hepatitis C Virus) by miR-122 has already been shown. claim. Studies also have shown eight miRNAs (miR-1,miR-30, miR-128, miR-196, miR-296, miR-351, miR-431 and miR-448) among 30 cellular miRNAs, influenced by IFN-α/β or IFN-γ, were upregulated who have an almost perfect complementarity with HCV RNA genomes. This suggests the capability of these miRNAs in HCV replication and infection inhibition (85). According to Lee et al, 2008, miR-15a levels were decreased in livers of patients suffering from Autosomal Recessive Polycystic Kidney Disease (ARPKD), indicates that miRNA expression changes may be responsible in phenotypic changes found in cystic liver disease (86).

Other Diseases:
In regulation of muscle development along with hair follicle morphogenesis, autoimmune and chronic inflammatory diseases which affect skin, involvement of miRNAs have been noticed in many studies (87)(88)(89)(90)(91)(92). Having signature role of Muscle-specific miR-1, miR-133 and miR-20 in myogenesis, significant up-regulation of miRNAs-100, 103 and 107 in certain myopathies, diagnosing Facio scapula humeral muscular dystrophy (FSHD) distinguished from Duchenne muscular dystrophy (DMD) based on the level of miRNAs-381 and miRNAs-382 expressions prove the importance of miRNAs in muscular development (93)(94)(95). Similarly, many studies claim involvement of a cluster of 33 miRNAs in regulation of expression of target genes required for the entry of fibroblasts into the cell cycle and proliferation. In context dermal fibroblasts are important cells in wound healing process.
Another review also has outlined potential significance of involvement of miRNAs in wound angiogenesis and abnormal healing sequence in chronic wounds (96)(97)(98)(99). These show miRNAs play important role in developing skin disease. miRNAs in cancer: It has already been well established by many studies that overexpression or down-regulation of miRNAs which have oncogenic as well as tumor suppressor activity, play crucial role in human cancers (100)(101)(102). In reproductive cancers such as breast, ovary, and endometrioid adenocarcinoma along with colon and haematological cancer (ALL,AML,CLL,CML), esophagus, gastrointestinal, lung, bladder, and thyroid tumors, miRNA play crucial role. (103) ( Table 2,3)   Recent studies have shown importance of miRNAs also in angiogenesis as let-7b, miR-16, miR-21, miR-23a, miR-29, miR-100, miR-221, and miR-222 expressions were found to be associated with endothelial cells (104)(105)(106). Role of miRNAs in vascular cells has been elucidated by miRNA regulation during pathophysiological process. Researchers have conducted studies where downregulation and overexpression of miR-130 in quiescent HUVEC and in response to foetal bovine serum were shown respectively (107,108). Some research studies show induction of miR-210 expression in hypoxia, which also was reported to be overexpressed in the enhanced formation of capillary-like structures inhibited in decreased tube formation and migration (108). miR-221 and miR-222 who have anti-angiogenic effects are highly expressed in HUVEC as well as endothelial cells and reduction of endothelial Nitric Oxide Synthase's (eNOS) expression is occurred due to same reason (109,110). eNOS is known for modulating endothelial cell growth, migration, vascular remodelling , and angiogenesis, which are linked with various tumour tissues including bladder, colon, pancreatic cancers, etc. (111,112,113,114) Breast Cancer subtypes: On basis of gene expression profile and the phenotype the breast cancer be divided into six subtypes luminal A, luminal B, tumour enriched with human epidermal growth factor receptor 2 (Her2), basal-like, normal-like and claudin-low subtype (115-118).
Luminal A subtype: This is the most common subtype among all breast cancer subtypes, which accounts for 50%-60% of the total breast cancer cases. It is characterized by ER miRNA expression pattern specificity in certain cancer subtype: Expression of miRNAs are specific to specific cancer subtypes. miR-21, miR-210 and miR-221, who have been correlated with worse patient disease-free and overall survival, play a crucial role in triple negative breast cancer and also have been noticed to be significantly overexpressed, whereas miR-10b, miR-145, miR-205 and miR-122a have been found significantly downregulated in these cancer types (120). Comprehensive miRNA profiling in normal basal and luminal breast epithelium cells isolated from plastic surgery showed 116 out of 664 miRNAs were expressed in normal breast epithelium, whereas among them 8 miRNAs let7c, miR-125b, miR-126, miR-127-3p, miR-143, miR-145, miR-146-5p and miR-199a-3p were significantly overexpressed in normal basal cells and miR-200c and miR-429 were mostly luminal. In some studies, higher expression of miR-126,miR-127, miR-143, miR-145 and miR199 and downregulation of miR-200c and miR-429 were noticed in malignant myoepithelioma compared with luminal and basal-like subtypes (123). According to an artificial network analysis conducted for miRNA profiling of 29 early stage breast cancer patients for determining ER,PR and Her2 status by Lowrey et al., miR-342, miR-299, miR-217, miR-190, miR-135b, miR-218 were ER specific, miR-520 g, miR-377, miR-527-518a, miR-520f-520c were PR specific and miR-520d, miR-181c, miR-302c, miR-376b and miR-30e were Her2 specific. Furthermore, highest expression of miR-342 in ER and Her2positive luminal B tumours and lowest expression of miR-520g in ER and PR-positive tumours were revealed by further analysis (124). Oncogenic miRNA: Oncogenic miRNAs have been observed to be frequently upregulated and repress the expression of tumour suppressor gene/s, generally which remain involved in apoptosis, cell proliferation, cell migration and invasion and metastasis (138). Here we will discuss about some miRNAs or miRNA families which have already been studied and established as oncomiRs in breast cancer and their involvement in this cancer type. miRNA-10 family: This miR family consists of two miRNAs: miR-10a and miR-10b, and as they can regulate HOX transcripts, they have crucial role in developmental process (139).

Classification of MiRNAs involved in breast cancer
Retaining within the HOX cluster of developmental regulators these miRNAs also have been reported to be involved in various cancers such as glioblastoma (140), colon cancer (141), acute myeloid leukemia (142), melanoma (143), pancreatic cancer (144) and hepatocellular carcinoma (145) including breast cancer. In case of breast carcinoma miR-10 family has been found to be involved both in metastasis and development. According to reports miR-10b potentially targets E-cadherin and miR-10b expression level is positively correlated with tumour size, pathological grading, clinical staging, lymph node metastasis, Her2-positivity and tumour proliferation, whereas negative correlation was found with ER+, PR+ and E-cadherin levels (146,147,148). Researchers are claiming that miR-10b may modulate breast cancer metastasis by suppressing HOXD10, which results in elevation of pro-metastatic gene, RHOC, a Rho-GTPase family member, known for regulating actin dynamics, hence cell shape and motility. E-cadherin, which has pivotal role during inhibition of Tiam1 (T lymphoma invasion and metastasis)-mediated Rac activation and controlling cell-cell adhesion and EMT is suppressed by miR-10b, hence the ability of breast cancer cells to invade and migrate is suppressed. These indicate miR-10b can be used as biomarker for advanced progression and breast cancer metastasis (148,149). including miR-21 in breast tumours compared with normal breast tissues (150,151). Two important molecules, PDCD4 (programmed cell death 4) and maspin (mammary serine protease inhibitor), involved in invasion and metastasis in breast cancer have been identified as miR-21 targets (152). It has been observed that miR-21 expression is negatively correlated with PTEN (phosphatase and tensin homologue) expression and upregulated by transforming growth factor-β (TGF-β) in breast cancer. Despite of poor prognosis, samples from 344 patients with primary breast cancer showed elevated level of miR-21 expression (associated with aggressive disease status including high tumour grade, negative hormone receptor status and ductal carcinoma), which was found to be associated with poor disease-free survival in early stage breast cancer patients (153,154,155).

miRNA-17-92 cluster:
Locating in such region of DNA, which is amplified in human B-cell lymphomas, this miR cluster comprises of six mature miRNAs: miR-18b, miR-19b, miR-20a, miR-92, miR-93 and miR-106 (156,157). Along with having role in lymphoma, these miRNAs are markedly overexpressed in lung cancers , especially in small-cell lung cancer (158). In case of invasive MDA-MB-231 breast cancer cells, miR-17-5p was found to be highly expressed Studies have shown the inverse correlation between miR-155 and FOXO3a in breast cancer cell lines, suggesting that miR-155 is an essential therapeutic target in breast cancer (166).
Therefore miR-155 has a essential role in breast cancer metastasis as it is implicated in the acquisition of EMT and increases potential for invasion and metastasis. miRNA-373/520c: miR373 and 520c are two premetastatic miRNAs which were found to be involved in promoting migration and invasion in vivo and in vitro in many studies (170,171).
These miRNAs are also found to show migratory phenotype by inhibiting the CD44 expression, which is known as a metastatic repressor. MCF-7 cells migration, expressing miR-373 and miR-520c was reduced by ectopic overexpression of CD44 which indicates downregulation of CD44 is essential to the migration of these cells. Also upregulation of miR-373 has been noticed during reduced expression of CD44 in metastatic breast cancer (167,168,169). activation also has been reported. This study also suggested that RASD1 is a potential target for miR-375 and RASD1 has an important role in cell proliferation by negatively regulating ERα expression, hence miR-375 has an oncogenic role in breast cancer (172). In contrary tumour suppressive role of miR-375 also has been noticed. Investigation, where MCF-7 breast cells were exposed to continuous tamoxifen to understand the miRNA's role in tamoxifen resistance, showing invasiveness and mesenchymal characteristics of cancer cells, showed miR-375 as one of the top downregulated miRNAs in resistant. Also increased sensitivity of tamoxifen-resistant cells, and partly reversed EMT phenotype (tumour cell invasion and metastasis) by reexpression of miR-375 were reported in that study. This study also revealed metadherin (MTDH) as miR-375 target and showed inverse correlation between miR-375 and MTDH expression in primary breast cancer cell lines (173).  (177)(178)(179)(180). Downregulation of miR-200c is also have been found to be associated with drug resistance in human breast cancer (181). Also this miRNA family is involved in regulating PLCG1, BMI1, TGF-β2, FAP-1, ZEB and Suz12 and hence acts as tumour suppressor (179,(182)(183)(184)(185)(186)(187)(188)(189)(190). As earlier discussed, miR-200 family is involved in regulating transcription factor ZEB, an activator of EMT, overexpression of this miRNA in mouse breast cancer cells is associated with macroscopic metastasis (191). Later Korpal et al. strengthened this notion in clinical aspects. More research is needed to define this miRNAs' role in breast cancer metastasis (192). miRNA 205: Like miR-200 family, significant downregulation has been noticed also in miR-205 in cells, who have undergone EMT in response to TGF-β and in the triple-negative primary breast cancers (193,194). In many studies this miRNA expression has been reported to be restricted in basal epithelium of mammary ducts and lobules while compared with reduced or lost expression in matching tumour specimens and breast tumours while compared with the matched normal breast tissue (195). Ectopic expression of miR-205 while directly targeting Her3 and VEGF-A can inhibit invasion, proliferation and anchorage independent growth (196).
In triple negative breast cancer, p53-induced miR-205 has tumour suppressive role and it's reexpression is strongly responsible for reduction of cell proliferation, cell cycle progression and clonogenic potential in vitro and inhibition of tumour growth in vivo. This miRNA also targets E2F1 and LAMC1, who have ability to regulate cell cycle progression and cell adhesion, proliferation and migration, respectively (197).  (198). Studies suggest that this miRNA may also be used as novel cancer biomarker for early cancer detection due to its early manifestation altered expression.
miR-145 has shown pro-apoptotic as well as tumour suppression functions (195). According to Spizzo et al. miR-145 has tumour suppression function along with TP53 activation dependent pro apoptotic potential in breast cancer cell lines. Also this miRNA can directly interact with estrogen receptor-α (ER-α) expression and promote apoptosis in both ER-α positive and wild type TP53-expressing breast cancer cells (199). A well-known oncogene c-Myc, having potential of regulating cell growth and proliferation, which can inhibit tumour cell growth both in vivo and in vitro can be silenced by miR-145 (200). Inhibition of invasion property of breast cancer cells, while having no significant involvement in cell growth, shows that miR-145 also can suppress breast cancer metastasis. Silencing of metastasis gene mucin 1 (MUC1) is partially responsible for this breast tumour suppression (201). Furthermore, miR-145 also has observed to suppress cell-cell adhesion protein JAM-A and fascine in breast tumour cell lines, which is responsible for drastically decreasing cell motility (202).

Let-7 family:
In let-7 family there are total 10 members: let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, miR-98 and miR-202, which are basically involved in muscle formation, cell adhesion and gene expression and development regulation (203,204). Let-7 is generally downregulated in many cancers such as lung cancer (205), gastric cancer (206) decreased HUR levels, which is responsible for cell growth, and reduced cell migration and proliferation, suggests that HUR is a potential target of miR-125a for suppressing breast tumour (210,211,212). miR-125a and miR-125b both were downregulated also in breast cancer cases where HER2 (human epidermal growth factor receptor 2) which is common in most of the cancers including breast cancer patients, is over expressed (212). Researchers have proposed c-Raf as target of miR-125b for exerting it's antiproliferative effect (213). Studies suggest HER2 which is a significant tamoxifen resistant factor has a splice oncogenic isoform HER2Δ16 associated with metastatic breast cancer and resistance to endocrine therapy (224). Another study revealed contribution of miR-342 to tamoxifen resistance in several models, including cell lines that overexpress HER2Δ16. Furthermore, miR-342 is found to be downregulated in tamoxifen resistant breast cancer cell lines along with tamoxifen refractory breast cancers (225).

Role of miRNA polymorphisms in breast cancer occurrence or susceptibility:
One of the most famous and discussible genetic variations in current day is Single nucleotide polymorphism (SNP) which found almost in every 100-300 bases and plays crucial role in developing several cancers including breast cancer (231,232). SNPs found in miRNA coding genes or in the miRNA binding target genes either form new miRNA or alter target mRNA function due to overexpression of the target gene, which describes miRNAs as potential biomarkers for cancer/breast cancer prognosis and clinical management. (233)(234)(235)(236). Here role of miRSNPs in breast cancer occurrence and susceptibility will be discussed on the basis of three criteria.
1. SNPs in target genes affecting micro RNA-mRNA interaction.

SNPs in miR target genes affecting micro RNA-mRNA interaction: SNPs found in the 3'
Untranslated region (UTR) of miRNA target gene can play an important role in altering the mRNA-miRNA interaction, which may increase the risk of cancer including breast cancer, if the target gene is oncogene or tumour suppressor gene. (237)(238)(239). There are almost 27 SNPs which have been mentioned in the following table, which play crucial role in breast cancer.
( (T>A), another novel SNP found in 3' UTR region of Spleen tyrosine kinase (SYK), has been observed to play important role in increased breast cancer risk (244)(245)(246). Spleen tyrosine kinase (SYK), is a member of the nonreceptor tyrosine kinase family and normally expressed in the breast epithelial tissue (246). Studies have shown expression of this gene family in breast tissue in addition to cell lines of breast cancer (247). According to many studies increased level of SET8 expression can inhibit cell apoptosis by p53 transcriptional activation suppression; which promotes tumour migration along with invasion by enhancing the EMT (248,249). SET8 is known for catalysing the histone H4 K20 monomethylating and also non-histone proteins like p53. (250,251). Among 129 variants of SET8, rs16917496 (T>C), which is a novel SNP, has been observed to be located inside 3'-UTR region, capable of binding with miR-520, which can regulate breast cancer risk. (252,253). Palladin (PALLD) (an actin-associated protein risk. (261,262). In the 3'UTR region of BRCA1 gene miR-SNP rs8176318, miR-SNP rs12516 and miR-SNP rs3092995 have been identified to play crucial role in breast occurrence (260).
reported rs8176318 miR-SNP for predicting breast (including TNBC) and ovarian cancer risk in a population of Irish women. According to another study homozygosity for the A alleles in miR-SNPs rs12516 and rs8176318 was significant associated with familial breast and ovarian cancer in a Thai cohort (263). On the other hand, elevated breast cancer risk was observed among in African American breast cancer patients having rs3092995, with the G allele predominant as compared to control subjects (264). miR-SNP rs1042538 in the core binding region of miR-124 in IQGAP1 (encoding IQ motif-containing GTPase-activating protein 1) can be predictive for breast cancer risk and prognosis. In Chinese population, TT genotype of this SNP is associated with lower breast cancer risk when compared to the AA genotype.
Whether AA genotype has been found to be linked with higher breast cancer risk (265).
Brenden and colleagues found strong association of miR-SNP rs743554 in the ITGB4 3'UTR region with specific risk of developing ER-negative breast cancers and worse overall survival when compared to those with the wildtype alleles. They also showed impairment of miR-34a binding to the ITGB4 30UTR seed region by this SNP (266). A case control study in German population, where 1223 breast cancer families and 1495 controls were compared to study the effect of rs2747648 SNP in the 3'UTR miRNA binding site of ESR1, demonstrated that the T allele variant of this miR-SNP was associated with increased breast cancer risk in premenopausal women, whereas another group of researchers reported C allele genotypes of this SNP in same categoric women with lower breast cancer risk. (267). AG genotype of SNP rs334348 in the 3'UTR of TGFBR1 has been reported to be prone to breast cancer where, G allele is targeted by miR-628-5p with higher efficacy than its A allele counterpart in vitro (268).
On the other hand, in the same study, SNP rs1982073 in TGFB1 was observed to exert its effect on gene expression with miR-187 altered interaction, and CC carriers of this SNP was linked to increased risk of breast cancer (268). CT and TT variants of miR-SNP rs115160714 in the 3'UTR region of Topoisomerase IIb binding protein 1 (TOPBP1) are found to be associated with higher breast cancer risk, when compared to CC genotype in Caucasian populations, TOPBP1 is hypothesized to bind with three miRNAs (miR-3138, miR-4302, and miR-1207-5p) and reported to possess rs115160714 (269,270). bind and affect 3'UTR region of miR-491-5p targeted MMP9 which can prevent miRNAmediated inhibition (271,272). In an Iranian population this c allele has been observed to be linked with increased breast cancer (273). described more elaborately the effect of SNPs in miRNA biogenesis genes (274). SNPs rs644236 (C > T) and rs7737174 (A > G) in DROSHA (Nuclear RNAse III), which is a key factor in the execution of the initiation step of miRNA processing was found to be linked with elevated risk of developing breast cancer in Korean post-menopausal women whereas rs2291109 (A > T) SNP in the same gene was found to be associated with increased breast cancer susceptibility (275,276). DGCR8 is an essential component of the pri-miRNA processing complex with Drosha. rs9606250 (A > T) and rs417309 (A > G), these two polymorphisms have been found in elevated breast cancer risk in Korean and Chinese breast cancer patients respectively (276,277). Epigenetic state of Exportin 5, known as XPO5 is responsible for exporting pre-miRNAs from the nucleus. missense SNP rs11544382 (A > G) was observed to be significantly linked with breast cancer susceptibility when compared to homozygous controls, in Caucasian women. rs34324334 along with rs11544382 were found to be highly prone to develop breast cancer due to altered nucleocytoplasmic transport activity in post menopausal women (278). SNP rs1057035 (C > T), located in the 3'UTR of DICER1, a RNAIII protein in cytoplasm, having role in cleaving pre-miRNA to miRNA, has been observed to play significant role in disease-free survival, overall survival and elevation of breast cancer progression and breast cancer associated mortality (277,279). AGO2 a critical factor from in RISC (RNA interfering Silencing complex) complex, brings miRNA to the target site. Two SNPs rs11786030 (A > G) and rs2292779 (C > G),located in AGO2 were resulted in decreased free survival and poor overall survival. On the other hand SNP rs3864659 (A > C) in AGO2 was observed to have a protective role against breast cancer risk in a study conducted within Korean population. (275,277). to identify the correlation between rs2043556 in miR-605 and breast cancer. They performed the study using restriction fragment length polymorphism (RFLP) followed by Sanger sequencing and found rs2043556 to be associated with increased risk of breast cancer susceptibility. (226). A metanalysis study performed to correlate role of polymorphisms in miR-146a rs2910164, miR-196a rs11614913 and miR-499 rs3746444 with breast cancer risk showed rs11614913 CC genotype in miR-196a2 was linked with elevated level of breast cancer occurrence. This study is useful for early detection of breast cancer (229). Another metanalysis on these functional miRNAs' SNPs reported increased breast cancer risk with miR-499 rs3746444 in Asians and in the overall populations, while miR-196a rs11614913 was found in decreased association with breast cancer risk among Caucasians. They did not find any relationship between miR-146a rs2910164 with breast cancer susceptibility (230). On the other hand variant genotypes rs3746444 in miR-499 were reported to be associated with significantly increased risks of breast cancer in Chinese women (228). According to researchers The G allele of miR-SNP rs895819 (A > G) of pre-miR-27a is associated with a significantly reduced risk