Cancer-Related Single Nucleotide Polymorphisms In MiRNA Binding-Sites And The Risk Of Breast Cancer

Objective: Breast cancer(BC)is one of the most common malignant tumor in female.Recently,the BC incidence displays a trend of rapid rise of worldwide and the patients are younger than before.The majority of breast cancers are sporadic,while familial breast cancer(FBC)comprises 5–10% of all breast cancers.BRCA1/2 are the known high risk-breast cancer genes in familial breast cancer,but they can explain,at best,20–25% of the overall excess familial risk.Micro RNAs(mi RNAs)are an abundant class of small,19-25 nt long single-stranded non-coding RNA molecules,which can act as regulators at post-transcriptional level.Mi RNAs are involved in crucial biological processes including development,proliferation,differentiation,apoptosis and stress reaction.Single nucleotide polymorphisms(SNP)in mi RNA binding-sites contributes to genetic variations and it occurs due to the transition,transversion,insertion or missing of basic group,which can influence the complementarity between the miRNA and its target gene to alter the expression of target genes,so as to influence cancer risk,treatment efficacy and patient prognosis.We genotyped 5 mi RNA binding SNPS including RYR3(rs1044129),KIAA0423(rs1053667),C14orf101(rs4901706),GOLGA7(rs11337),KRT81(rs3660)in a case-control study to evaluated the impact of these SNPs on sporadic breast cancer(SBC)and FBC risk,furthermore,to explore the relationship between these SNPs and patients' clinical characteristics.Methods:1 Samples: Blood samples for experimental group were collected at breast cancer center of the Tumor Hospital of Hebei Province between January of 2013 and March of 2016 including 152 SBC patients and 30 FBC patients who underwent tumor resection in the same hospital.Blood samples werecollected from 130 healthy people without cancer history or breast disease in the Tumor Hospital of Hebei Province between October of 2013 and October of 2016.2 DNA extraction: Genomic DNA was carried out by using the TIANamo Genomic DNAki(TIANGEN Beijing China)from blood samples.Then the DNA was frozen immediately under-80? until used.3 DNA amplification and miR-SNP screening: According to the NCBI SNP database(http://www.ncbi.nlm.nih.gov/snp/)to design the sequences of the five paired primers including RYR3(rs1044129),KIAA0423(rs1053667),C14orf101(rs4901706),GOLGA7(rs11337),KRT81(rs3660).Then use a PCR Master Mix Kit according to manufactor's instructions to perform polymerase chain reaction(PCR).Use agarose gel electrophoresis to confirm the products.Sequencing was done by Generay Biotech(Shanghai)Company.4 Statistical analysis: We used student's t-test to analyze quantitative data.The ?2 test was used to analyze any individual SNP in SBC or FBC patients and healthy controls,and to explore the relationships between KRT81(rs3660)and patients' clinical characteristics.Use the SPSS 21.0 software(SPSS Inc.,Chicago,IL,USA)to analyze statistical dates.For all the statistical tests,P<0.05 was considered to indicate a statistically significant difference.Results:1 No statistical difference exist referring to age between SBC or FBC patients and heathy controls(P>0.05).2 The genotype distribution frequency for each SNP(rs1044129,rs1053667,rs4901706,rs11337)showed no statistical difference between SBC patients and matched controls.For the rs3660 located in the gene KRT81,the frequencies of genotype GG and CG+CC were 70.4% and 29.6% in SBC patients whereas 42.9% and 57.1% in the controls.The GG genotype carrier of rs3660 was associated with a 5.548-fold increased risk when compared with that of CG+CC genotype carrier(odds ratio,5.548;95% CI,3.326-9.256;P=0.000).3 The genotype distribution frequency for each SNP(rs1053667,rs4901706,rs11337)showed no statistical difference between FBC patients and matched controls.For the rs3660 located in the gene KRT81,the frequencies of genotype GG and CG+CC were 60.0% and 40.0% in FBC patients whereas 42.9% and 57.1% in the controls.The GG genotype carrier of rs3660 was associated with a 3.497-fold increased risk when compared with that of CG+CC genotype carrier(odds ratio,3.497;95% CI,1.541-7.937;P=0.002).As for the rs1044129 located in the gene RYR3,the frequencies of genotype AA and AG+GG were 63.3% and 26.7% in the FBC patients,41.5%and 58.5% in controls.The AA genotype carrier show a significantly increased risk for FBC compared with AG+GG carrier(odds ratio,2.431;95% CI,1.070-5.522;P=0.031).4 No association exist for the genotype distribution of rs3660 with clinical characteristics(Age,Tumor Size,Lymph node status,Tumor histology,AJCC stages,immunohistochemical(IHC)staining,Immunophenotype)in BC patients(P>0.05).Conclusions:1 For the SNPs rs3660 targeting in KRT81,the frequency distribution was significant difference between SBC or FBC patients and healthy controls.The SNP for rs3660 was associated with the cancer risk for BC development.2 In addition to the SNPs rs3660 targeting in KRT81,the frequency distribution of the SNPs rs1044129 targeting in RYR3 was significant difference between FBC or FBC patients and healthy controls.The SNP for rs1044129 was associated with the cancer risk for FBC development.3 The different genotypes of the SNPs rs3660 are not significantly associated with the different clinical characteristics of BC.