The association between genetic variation rs2292832 and the processing efficiency of pre-mir-149 affects the risk of breast cancer

microRNAs (miRNAs) play key roles in regulating cancer development, including breast cancer. Variation in miRNA genes can associate with the risk of cancer by alterations in the miRNA’s processing and maturation. Therefore, human blood samples and breast cancer cell line (MCF7) were analyzed to study any possible association between the genetic variant (rs2292832) in the miR-149 precursor and breast cancer susceptibility. To study the role of rs2292832 polymorphism in breast cancer, the miR-149 gene variant was genotyped using PCR-RFLP. For evaluating the effect of SNP on function and expression levels of mature miR-149, we inserted pre-miR-149 and flanking region with CC or TT genotype into a pEGFPN1 expression vector, and qPCR was accomplished. Cell survival, proliferation, and migration properties investigated by MTT and wound healing assay. Statistical analysis was carried out for data analysis. T allele in variant rs2292832 is associated with an increased risk of breast cancer. Such association was also obtained in co-dominant (OR = 2.5) and dominant (OR = 2.016) models. The variant allele led to reduced production of mature miR-149 and resulted in increased cell proliferation and migration of MCF7 cells. These findings suggest that miR-149 suppresses tumor cell proliferation, and the pre-mir-149 polymorphism affects the processing of miR-149, causing an alteration in the abundance of the miRNA mature form, which can regulate tumor progression and metastasis.


Introduction
Many studies have reported substantial effects of miRNAs in cancer progression. These factors can act as oncogene or tumor suppressor by degrading target genes transcripts or translation inhibition [1]. Besides, miRNAs have drawn much attention as potential diagnostic markers and therapeutic targets in human cancers [2]. The literature has reported that many miRNAs are involved in breast cancer [3]. The down-regulation or up-regulation of miRNAs levels in different cancers, including breast cancer, has been strongly verified. Changes in miRNAs expression can occur due to point mutation or Single nucleotide polymorphisms as head and neck, lung, stomach, breast, prostate, and renal cell carcinoma. The miR-149 targets different genes and pathways and plays a dual role in proliferation and apoptosis [11]. In some cancers such as prostate, leukemia, glioblastoma, and melanoma, mir-149 is upregulated and acts as an oncomiR, but in the hepatocellular, colorectal, lung, gastric, and breast cancers is downregulated and known as an important tumor suppressor and in some cases, its expression level has been further reduced during metastasis [11][12][13][14][15][16]. The human miR-149 has different variants, and rs2292832 is one of the most important variants, which is associated with the risk of several cancers, including lung [17], gastric and colorectal [18], head and neck, and squamous cell carcinoma [19]. Huang GL et al. Showed that the rs2292832 polymorphism contributes to the progression of esophageal cancer and significantly increases metastasis and cell proliferation [20]. However, Li L et al. observed that rs2292832 T / C polymorphism may decrease digestive cancer susceptibility [21]. In breast cancer, few studies have been performed on rs2292832 polymorphism only in the Chinese population. Since changes in miR-149 expression and its effects on tumorigenesis in breast cancer have been proved, such studies could shed light on the role of miR-149 and make it a useful therapeutic target. Therefore, this study aimed to investigate the association of rs2292832 polymorphism with breast cancer risk in a group of Iranian population, evaluate the effects of this variant on the expression of mature miRNA, and examine the viability and migration of cancer cells treated with this variant.

Study population
The subjects enrolled in this study were recruited from an ongoing breast cancer study. In this case-control study, blood samples were collected after providing written consent, and their clinicopathological data were provided using a questionnaire. All participants (healthy controls or the patients) were genetically unrelated. The individuals were randomly selected from 130 patients referring to the cancer ward of a hospital, and their breast cancer was pathologically confirmed by a specialist. Exclusion criteria included a positive familial history of breast cancer, secondary and recurrent malignancies, and prior radiotherapy or chemotherapy. The 155 healthy individuals who were matched by age (± 5 years) and ethnicity were enrolled as controls. Those healthy people who had a familial history of cancer or suspected symptoms were excluded from the study.

Genotyping analysis
The genomic DNA extraction was performed by the phenolchloroform method. The extracted DNA was qualitatively and quantitatively examined by a NanoDrop spectrophotometer. The genotypes of the rs2292832 variant in pre-miR-149 were detected by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. PCR primers include: F: 5´-GTCTTCACTCCCGTGCTTGT-3´, R: 5´-CCGAAACACCCGTAAGATATGG-3´. The PCR reaction conditions were as follows: Pre-denaturation at 95 °C for 5 min, 35 cycles comprising of denaturation at 95 °C for 30 s, 60 °C annealing temperature for 30 s, and synthesis of the fragments (extension) at 72 °C for 30 s, and terminal extension at 72 °C for 5 min. As a final confirmation for 248 bp amplicon, PCR products were electrophoresed on agarose gel 1% and visualized by green viewer staining. The 10 µl of all PCR products were digested at 37 °C overnight by 1 U of PvuII enzyme (Fermentase). The digested product was separated by 10% polyacrylamide gel electrophoresis, and the gel was stained with silver nitrate. The fragments were 186 bp and 62 bp for TT genotype, 248 bp, 186 bp, 62 bp for CT genotype, and 248 bp for CC genotype. Finally, 10% of samples randomly were also examined by sequencing.

Expression vector construction and transfection
To evaluate the role of rs2292832 polymorphism in the breast cancer cell line, the full-length of pre-miR-149 and ~ 300 bp flanking region harboring rs2292832T and rs2292832C alleles were amplified and cloned into pEGFP-N1 vectors. The pEGFPN1-miR-149-rs2292832T (V-mir149-T) and pEGFPN1-miR-149-rs2292832C (V-mir149-C) constructs were then confirmed by sequencing and FRLP method. Exponentially growing MCF-7 cells cultured in six-well plates were transfected in 70-80% confluency with V-mir149-T or V-mir149-C expression plasmid using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The original pEGFPN1 vector was used as the negative control.

qPCR analysis
MCF7 cells were seeded in 6-well plates and transfected with the V-mir149-T or V-mir149-C constructs. Treated cells were harvested 24 h later and used for total RNA extraction. Total RNA was isolated by Trizol reagent (Invitrogen). The RNA concentration was estimated by the Nanodrop spectrophotometer (Thermo Fisher). The reverse transcription reaction was performed using a specific stem-loop primer. For qPCR amplification, cDNAs were amplified by SYBR Green Real-time PCR Master Mix (QuantiTect SYBR Green PCR Kit), 0.3 µmol RNA and, reverse stem-loop primer. Amplification was conducted with an initial step for 5 min at 94˚C, followed by 40 cycles of amplification (94 ˚C for 15 s, annealing temperature for 10 s, and 72˚C for 10 s) using Mic qPCR cycler (BioMolecular Systems). All amplification reactions were performed in triplicate, and U6 snRNA was used for normalization.

MTT assay
MCF7 cells were plated into 96-well plates with 200 µL medium to assay the cell proliferation. After the V-mir149-C or V-mir149-T constructs were transfected the proliferation was evaluated by MTT (3 − 2, 5-diphenyl tetrazolium bromide) assay (Sigma, USA). Absorbance at 490 nm was measured using a 96-well plate reader (Beckman, Lodi, CA). The experiments were repeated in triplicate.

Wound healing assay
wound healing assay was implemented on MCF7 cell lines to evaluate cell migration. Cells were cultured in 24-well plates at 95% density. After 24 h, cell transfection was performed with V-mir149-C or V-mir149-T constructs and pEGFPN1 vector. Transfection was approved, and cells were scratched in the middle of the well using a pipette tip. The cells were then washed with PBS three times to remove cell debris. The images were taken at 0, 24, and 48 h, and the migration rate of the control and treated samples were evaluated with ImageJ software.

Statistical analysis
Statistical data analysis was conducted by the SPSS20.0 software package (SPSS Company, Chicago, Illinois, USA). The rs2292832 genotype distribution was analyzed to confirm Hardy-Weinberg equilibrium using the Chi-square test. Logistic regression analysis was employed to study the association between different SNP genotypes and risk of Breast cancer by odds ratio (OR) and 95% confidence interval (CI). The 2 −ΔΔCT method was used to analyze the real-time PCR data. Data were then presented as mean [± standard error of the mean (SEM)] and the Student's t-test was used to compare different groups. The Akaike information criterion (AIC) and Bayesian information criterion (BIC) were used for choosing the best predictor subsets in regression. All the experiments were carried out in triplicates and means ± standard errors were reported. Statistical tests were confirmed by the GraphPad Prism version 7.01 software, and graphs were drawn by this software. The significance level was considered < 0.05 for all statistical tests.

Results
The age of breast cancer patients and healthy controls was 48.2 ± 1.1 and 47.21 ± 8.5 years old. Most patients had ductal carcinoma with a tumor grade less than 3. The proportion of HER2 + patients was higher than HER2-. Clinical and demographic features of patients with breast cancer and the controls are shown in Table 1. Table 2 illustrates the frequency distribution of miR-149 rs2292832 polymorphism. No deviation from Hardy-Weinberg equilibrium was observed in the control and breast cancer groups (Fig. 1 A). The univariate analysis shows that the frequency of the TT genotype and the T allele in breast cancer patients is significantly higher compared to healthy controls (Fig. 1B), suggesting a potential relationship between the TT genotype of pre-mir-149 and breast cancer susceptibility (OR = 2.5). The over-dominant model did not show an association between TT genotype and breast cancer susceptibility. The dominant model analysis revealed a significant cancer risk of TT genotype for breast cancer susceptibility (p < 0.05) ( Table 2). Concerning the relationship between clinical factors and the polymorphisms, no association was found with age, BMI, menopause, age at menarche, metastasis, HER2 marker, and tumor grade. constructs, and empty vector. Then the expression of mature miRNA was evaluated for both T and C alleles by qPCR. The expression of mature mir-149 in V-mir149-C transfected cells was significantly 4 times higher than cells that had been transfected with V-mir149-T (P < 0.001) (Fig. 2 A) suggesting that this polymorphism could change the mature miRNA expression To evaluate the function of miR-149 and rs2292832 variant in cell proliferation, an MTT assay was performed for V-mir149-T, V-mir149-C constructs, and control. A significant difference was observed in their effects on cell growth, as V-mir149-C transfected cells significantly inhibited the growth rate of MCF7 cells 24 h after transfection (P < 0.05) (Fig. 2B). To investigate the effect of the miR-149 variant on the migration of breast cancer cells, V-mir149-T, V-mir149-C constructs and, empty vector were transfected into the MCF7 cell line. The wound-healing assay was performed; the results showed that the migration ability of V-mir149-C transfected cells was lower than V-mir149-T and empty vector transfected cells (P < 0.05) (Fig. 3).

Discussion
The vital importance of alteration in miRNA genes at pri, pre, and mature levels has been proved in the different biological processes by bioinformatics analyses and experimental studies. It has been reported that a variant in miR-125a alters pri-miRNA processing to pre-miRNA and inhibits mature miRNA expression. Besides, this SNP is located This genetic variant is located near the base of the stem hairpin, and optimal free energy was decreased from 56.49 Kcal/mol for C to 54.29 Kcal/mol for T alleles, proposing a less stable secondary structure for the T variant allele compared to the C allele. This genetic variant in pre-miRNA might change the conformation of the secondary structure and therefore alter the expression of mature miRNA. The MCF7 cells were transfected with V-mir149-T, V-mir149-C  risk factor for colorectal and hepatocellular carcinoma [25,26]. Also, polymorphisms in the regulatory sequences can affect miRNAs expression [27]. We studied a variation in the pre-mir-149 sequence to investigate its possible effect on the miR-149 maturation and function in breast cancer. Decreased expression of miR-149 has been shown in several cancers such as colon, gastric, lung, and head and neck squamous carcinoma [28,29]. The miR-149 expression is in the mature sequence and can interfere with the translation inhibition process [22]. Another study showed that two SNPs in the pri-mir-125a sequence affect the pre and mature levels, leading to recurrent pregnancy loss [23]. SNPs in the pre-miRNA sequence also can affect miRNA expression and function. A variant in the pre-mir-146a sequence can interfere with miRNA maturation and also affect target inhibition [24]. Polymorphism in pre-mir-196a2 has been reported as a  significantly reduced in advanced and metastatic breast cancer and can inhibit tumorigenesis and metastasis. Although the ectopic expression of miR-149 in MDA-MB-231 has not affected cell proliferation and has mainly worked as a migration/invasion inhibitor [30], Our study revealed a significant effect of the ectopic mir-149 expression on cell proliferation in the V-mir149-C transfected MCF7 cell line in addition to its inhibitory effects on cell migration. Important transcription factors such as SP1, FOXM1, and ZBTB2 have been identified as targets of miR-149 that justify its tumorsuppressing function [30,31]. Rs2292832 C > T polymorphism in miR-149 was first reported by Hu et al. in the study of non-small cell lung cancer [32]. Hu et al. also studied this variant in breast cancer, and no association was observed between breast cancer risk and this variant [33]. According to a meta-analysis in 2016 which had studied the association between different cancers and the rs2292832 polymorphism in miR-149, this polymorphism is significantly associated with breast cancer risk [34]. The results of this study also confirm the significant association of the rs2292832 variant with the risk of breast cancer, so that risk is 2 times higher in the patients with TT genotype. Decreased stability in the T-containing stem-loop structure can be a possible explanation for this C-T substitution effects on the expression of this tumor-suppressive miRNA and consequently breast cancer progression.
Author contributions F. Fakhrezare performed most of the experiments as part of his master's degree in biotechnology. S.O Ebrahimi performed the transfection and all cell culture related assay, genotyping analysis, and participated in intellectual discussions of the data. S.Reiisi coordinated the study, designed the experiments, and revised the manuscript.

Conflict of interest
We certify that there is no conflict of interest with any financial organization.
Ethical approval the authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. The samples with research ethics code 910 − 215 has been approved by research ethics committee at Shahrekord University of medical sciences, Iran. As well as, informed consent was obtained from all individual participants included in the study.