| Breast cancer(BC) is one of the most common malignant tumor in women, the incidence is increasing and patients are tending to become younger. It is a serious threat to women’s physical and mental health. Epidemiological studies generally believe that genetic, environmental and reproductive factors contribute to the development of BC, some genome-wide association studies(GWAS) indicated that single nucleotide polymorphisms(SNPs) in homologous recombination repair(HRR) pathway genes is the molecular genetic basis of individual differences in the ability of DNA damage and repair, micro RNAs regulate gene expression and function by binding to the 3’ UTRs of target m RNAs. Genomic instability is induced by DNA damage repair ability decline or lack is an important cause of breast cancer. ObjectiveFive mi RNA-binding-site single nucleotide polymorphisms(SNPs) in homologous recombination repair(HRR) pathway genes(MRE11A rs2155209, NBS1 rs2735383, RAD51 rs963917 and rs963918 and RAD52 rs7963551) were analyzed to explore the association between five SNPs and breast cancer and the role of gene-reproductive factors interactions in the development of BC, in order to provide the theoretical basis for individualization prevenion. And in order to provide the theoretical basis for the regulatory mechanism of mi RNA on HRR pathway target genes through the verification of mi RNA let-7b function. MethodCombined with the existing domestic and foreign literature database, screening SNPs in the HRR pathway genes through bioinformatics methods and mi RNA target prediction software. The case-control study included 450 patients with BC from hospital and 450 population-based healthy controls matched to cases by age(±2)was carried out in Henan Han population. After the collection of information of the cases and controls, genomic DNA was extracted from peripheral blood samples, the genotypes of rs2155209、rs2735383 and rs963918 were detected by using PCR-RFLP. The genotypes of rs963917 and rs7963551 were detected by using AS-PCR. SPSS 21.0 software was used for statistical analysis. The difference in factors distributions between cases and controls, and between carriers and non-carriers of the SNPs were tested using t-test, χ2-test and unconditional binary logistic regression analysis. Hardy–Weinberg equilibrium for genotypes in controls was tested by Pearson’s goodness-of-fit χ2-test. Haplotypes were evaluated by using SHEsis software. The gene-environment interactions was analyzed with the MDR software.Over expression vector of let-7b was generated by using molecular cloning technique, then combined with let-7b inhibitor, and tranfected into MCF-7 and SKBR3 cells, and q RT-PCR technique was used to analysis let-7b and m RNA RAD52 expressions, western-blot was used to analyze RAD52 protein expression levels, and then revealed whether RAD52 really is the target gene of Let-7b. Results(1) The individuals with TC(OR: 1.87; 95% CI: 1.23-2.86) and TC+CC(OR: 1.86; 95% CI: 1.23-2.80) genotypes of rs2155209 in MRE11 A showed increased risk to BC, patients with AC(OR: 0.67, 95% CI: 0.48-0.87), CC(OR: 0.36; 95% CI: 0.24-0.58) and AC+CC(OR: 0.71; 95% CI: 0.59-0.82) genotype of rs7963551 in RAD52 had a significantly lower risk for BC.(2) The combined TC+CC genotype of rs2155209(OR: 1.49; 95%CI: 1.06-2.08) and TC+CC of rs963917(OR: 1.78; 95%CI:1.04-3.05) had significantly higher risk of BC among women with family history of BC, GC+GG genotype of rs2735383 had a significantly lower risk of breast cancer among pre-menopausal women(OR: 0.47; 95%CI: 0.33-0.66), AG+GG genotype of rs963918 was associated with the risk of BC in women with number of pregnant ≤2 times(OR: 1.64; 95%CI: 1.10-2.45), AG+GG genotype of rs7963551 had a significantly higher risk of breast cancer patients who have breast-fed experience(OR: 1.49; 95%CI: 1.13-1.96), AC+CC genotype of SNP rs7963551 was associated with PR positive of BC patients(OR: 1.82; 95%CI: 1.33-2.48).(3) Combined Effect of risk alleles showed that the five SNPs were associated with increased breast cancer risk in a dose-dependent manner(Ptrend = 0.003).(4) Haplotype analysis showed that haplotypes of Crs963917Ars963918 decreased the risk of BC(ORadjusted: 0.53, 95%CI: 0.4-0.68), while the Trs963917Ars963918 and Trs963917Grs963918 haplotypes could increase the risk of BC(ORadjusted: 1.28, 95%CI: 1.05-1.57 and ORadjusted: 1.31, 95%CI: 1.09-1.62).(5) Gene-reproductive factors interactions analysis showed that risk of BC among “high risk women” carried rs2155209 C allele, rs7963551 A allele, family history of BC and breast-feeding has increased 3.59-fold, compared to the ‘‘low risk women’’.(6) Over expression vector of let-7b was constructed successfully verified by single, double enzyme digestion and sequencing. q RT-PCR results showed that the significant difference for relative expression of let-7b between over expression groups and inhibitors groups in MCF-7 cells and SKBR3 cells(P<0.05).(7) Western-blot results showed that the expression of RAD52 protein down regulated in the over expression groups and up regulated in the inhibitors groups in MCF-7 cells, while there were no significant differences of RAD52 protein between over expression groups and inhibitors groups in SKBR3 cells.Conclusion(1) rs2155209 TC and TC+CC genotype of MRE11 A could increase the risk of BC, RAD52 rs7963551 C allele could reduce the BC risk, haplotypes of RAD51 gene were associated with BC risk, gene-reproductive factors interaction between rs2155209, rs7963551, breast-feeding and family history of BC might be play important role in the development of BC.(2) Over expression vector of let-7b was successfully constructed and increased the expression level of let-7b in human breast cells MCF-7 and SKBR3. RAD52 gene may be a target gene of let-7b, and let-7b may negative regulate the expression of RAD52 protein. |
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