Association Study And Functional Analysis Of SNPs In Long Non-coding RNA LINC00520 With Breast Cancer Susceptibility

Breast Cancer?BC?remains a public health problem worldwide.In women,BC is the most common cancer and the leading cause of death.It was found that BC was the result of the interaction of individual lifestyle,environment,genetic and reproductive factors.Long non-coding RNA?lncRNA?plays an important biological role in the development of tumors.More and more evidence showed that susceptibility genes played an important role in tumor development.The structural complexity of lncRNAs also determines the functional complexity.Although it has been reported that lncRNA LINC00520 is associated with breast cancer,the association between LINC00520 SNPs and genetic susceptibility of BC and its mechanism of action remain unclear.ObjectivesDatabase,document retrieval,and bioinformatics technology were comprehensively united to screen lncRNA associated with BC and its Single Nucleotide Polymorphism?SNP?loci.The study used the method of molecular epidemiology,combined with the case control study to investigate the association between genetic variations of long chain noncoding RNA and susceptibility studies of breast cancer.Then a preliminary study was conducted on its biological function.Methods1.Based on bioinformatics,combined database and literature search,lncRNA and functional SNPs related to breast cancer were screened comprehensively,after which the functional prediction of SNPs was conducted.2.The sample size was calculated by using the molecular epidemiology method in combination with the case-control study and PASS 8.0.14?http://www.ncss.com/pass.html?.A total of 504 new cases of BC and 505 healthy controls were included matched by age?±2 years?based on frequency in this study.According to different loci,rs11622641,rs7157819,rs12880540 and rs2152275were classified by SNPscan,rs4144657,rs2152278 and rs7142488 were classified by PCR-RFLP,rs8008130 and rs8012083 were classified by CRS-RFLP.The gene expression frequency of screened SNPs in BC patients and normal population was detected.Hardy-weinberg software was used to analyze whether the genotype distribution of the control group conformed to the hardy-weinberg equilibrium.MDR2.0 software was used to analyze the interaction between polymorphic loci on lncRNA genes and reproductive factors.SPSS 21.0?SPSS Inc.,Chicago,USA?software was used for statistical analysis to analyze the difference in allele frequency distribution of SNPs and its correlation between BC susceptibility and hormone levels,so as to find functional SNPs and conduct further functional studies.3.Real-time fluorescent quantitative PCR?qRT-PCR?was used to detect the relative expression level of lncRNA in different functional SNP genotypes group,so as to analysis the functional link between SNPS and the lncRNA expression level.Meanwhile,luciferase assays test was used to verify whether SNP allele variation would affect the binding of lncRNA and the predicted miRNA with potential binding sites.Results1.Correlation analysis:The number of alleles at two loci,rs11622641 and rs2152278 can affect the risk of BC,rs11622641 genotype CT reduced the risk of BC by 33.4%compared with its wild genotype CC?OR:0.666,95%CI:0.453-0.979?,both the dominant model?OR:0.650,95%CI:0.445-0.949?and the overdominant model?OR:0.670,95%CI:0.455-0.984?reduced the risk of BC.Compared with wild-type TT,rs12880540 genotype GG increased the risk of BC by 55.1%?OR:1.551,95%CI:1.034-2.328?,rs12880540 increased the risk of BC in the recessive model?OR:1.566,95%CI:1.069-2.295?.rs2152278 genotype TT was associated with 70.0%increased risk of BC compared with wild-type GG?OR:1.700,95%CI:1.278-2.261?,both the dominant model?OR:1.619,95%CI:1.239-2.114?and the overdominant model?OR:1.556,95%CI:1.194-2.026?increased the risk of BC.However,rs7157819,rs2152275,rs8008130,rs4144657,rs8012083 and rs7142488 were not statistically correlated with the risk of BC either in co-dominant,dominant,recessive and superdominant models?P>0.05?.2.Stratified analysis:In the population of younger than 50?OR:0.549,95%CI:0.327-0.923?,age of menarche less than 14?OR:0.549,95%CI:0.342-0.880?,number of pregnancies more than 2?OR:0.596,95%CI:0.363-0.980?and number of abortions less than or equal to 2?OR:0.637,95%CI:0.434-0.935?,individuals with rs11622641genotype CT or TT had a lower risk of BC than those with genotype CC?P<0.05?.rs7157819 genotype CT or TT increased the risk of BC in the population with abortion times more than 2?OR:4.192,95%CI:1.135-9.486?.rs12880540 genotype TG or GG increased the risk of BC in postmenopausal patients whose age less than 50 years?OR:1.800,95%CI:1.055-3.070?.rs4144657 genotype CT or TT could reduce the risk of BC compared with genotype CC?OR:0.230,95%CI:0.061-0.871?.rs2152278 could increase the risk of BC with the age?50,age of menarche?14,age of menarche>14,unmenopause,number of pregnancies?2,number of pregnancies>2,number of miscarriages?2,number of miscarriages>2,history of breastfeeding,no history of breastfeeding,no family history of BC carrying genotype GT or TT.rs12880540genotype TG?OR:1.617,95%CI:1.037-2.522?,rs2152275 genotype AA?OR:2.735,95%CI:1.008-7.418?and rs8012083 genotype GG?OR:0.305,95%CI:0.135-0.693?were all statistically correlated with positive Her-2 receptor status.Rs8012083genotype GG?OR:3.575,95%CI:1.318-9.698?significantly increased the risk of Triple negative BC.3.Haplotype analysis:The haplotype of LINC00520 gene Crs_7157819 T_rs12880540Ars2152275 Trs11622641 could reduce the risk of BC.4.Interaction analysis:The number of pregnancies,miscarriages and rs2152278had gene-reproduction factor interaction during the occurrence of BC.The results showed that the number of pregnancies>2,miscarriages>2 and the population with the allele T of rs2152278 increased the risk of BC by 5.239 times.5.qRT-PCR:The relative expression of LINC00520 in rs12880540 genotype TG and GG was significantly higher than that in genotype TT?P<0.01?.The relative expression of LINC00520 in rs12880540 genotype GG was also significantly higher than that in genotype TG?P<0.01?.The results of trend test showed that relative expression of LINC00520 showed a linear change with the increase number of rs12880540 mutation(P_trend<0.001).6.Functional verification:After the G allele of rs12880540 mutated into T,the secondary structure of LINC00520 was changed.Meanwhile two binding sites of miR-92a-2-5p and miR-4648 were generated,and four binding sites of miR-3122,miR-3913-5p,miR-4259 and miR-4425 were lost.Luciferase reporter gene analysis showed that the rs12880540 allele was related to the interaction between LINC00520 and miR-3122at T,but the rs12880540 G/T variation had no effect on the binding ability of LINC00520 to miR-3122.Conclusions1.For the first time,we found that the site rs11622641 on LINC00520 could reduce the risk of BC,rs12880540 and could increase the risk of BC.rs12880540 TG,rs2152275 AA and rs8012083 GG were statistically correlated with Her-2 receptor positive status,rs8012083 GG significantly increased the risk of Triple negative BC.2.The haplotype of LINC00520 gene Crs7157819 Trs12880540 Ars2152275 Trs11622641 could reduce the risk of BC.The number of pregnancies,miscarriages and rs2152278 had gene-reproduction factor interaction during the occurrence of BC.3.The relative expression of LINC00520 increased linearly with the number of rs12880540 mutations.The rs12880540 allele was related to the interaction between LINC00520 and miR-3122 at T,but the rs12880540 G/T variation had no effect on the binding ability of LINC00520 to miR-3122.4.In this study,the association and functional verification of SNPs in LINC00520with BC susceptibility were explored for the first time,which provided potential biomarkers for screening and early diagnosis of BC,and also provided a basis for the prevention of BC.