Association Study And Functional Analysis Of SNPs In Long Non-Coding RNA MALAT1 And BCAR4 With Breast Cancer Susceptibility

Breast cancer（BC）is a common cancer in many countries including China.The high incidence and mortality rates of BC lead it to be a great public problem which would endanger the health of women.BC is a complex disease which is contributed by various environmental,reproductive,as well as genetic factors.Recent studies implied that lncRNA may play important roles in affecting the progressing of carcinogenesis and emerging reports between SNPs and cancer susceptibility have been reported.It was detected that MALAT1 is upregulated in multiple types of cancers including BC,and has been associated with the proliferation and metastasis of BC cells,making it a significant risk factor for BC.The high expression of lncRNA BCAR4 is discovered in non-small cell lung cancer and osteosarcoma,as well as breast cancer,and is associated with poor survival time of breast cancer patients.To the best of our knowledge,no studies have been reported about the effect of MALAT1 and BCAR4 variants on BC susceptibility.We proposed a hypothesis that the polymorphisms of lncRNA MALAT1 and BCAR4 might contribute to the risk of BC.Thus we conducted a case-control study to assess the association between tagging SNPs of MALAT1 and BCAR4 and BC susceptibility。ObjectiveThe objective of our study is to investigate the relationship of six tagging SNP（rs3200401,rs619586,rs7929113,rs4561483,rs11649623,rs13334967）in MALAT1and BCAR4 with breast cancer susceptibility.And the further aim is to examine the function of the significant loci found in our study in order to explore the effect of six SNPs of MALAT1 and BCAR4 on breast cancer.Methods（1）A case-control study containing 478 confirmed breast cancer cases from hospital and 489 health controls from health investigation project was conducted.（2）The tagging SNPs of MALAT1 and BCAR4 of CHB population were obtained by using haploview software according to the information from the websites including NCBI,Ensembl and GVS。（3）Polymerase chain reaction-restriction fragment-length polymorphism（PCR-RFLP）were used to genotype MALAT1 rs3200401、MALAT1 rs7929113 and BCAR4 rs11649623;created restriction site polymerase chain reaction-restriction fragment-length polymorphism,CRS-PCR-RFLP)were used to genotype MALAT1rs619586、BCAR4 rs4561483 and rs13334967.（4）The real time PCR was used to investigate the relative expression of lncRNA among different genotype for the significant loc.（5）SPSS21.0 was used to analyse data.The compare of basic characteristics between cases and controls were conducted by t-test andχ2 test;Hardy-Weinberg equilibrium（HWE）was performed to compare the genotype frequencies with the expected ones among cancer-free controls;Unconditional logistic regression model was applied to evaluate the association between three SNPs and breast cancer susceptibility;MDR method was applied to detect potential gene-reproduction factors interactions and online SHEsis was used to conduct haplotype analysis.（6）The 2^-ΔCT method was applied to calculate the relative expression of MALAT1and BCAR4..Results（1）MALAT1 rs3200401 is associated with lower breast cancer risk.The risk of breast cancer of carriers with CT genotype decreased 25%（CT VS CC,OR:0.75,95%CI:0.559-1.007）compared with carriers with CC genotpe;the risk of breast cancer of carriers with CC+TT genotype decreased 25.9%（CC+TT VS CT,OR:0.741,95%CI:0.552-0.993）compared with carriers with CT genotpe.MALAT1rs619586 is also associated with lower breast cancer risk.When compared with carriers with AA genotpe,the risk of breast cancer of carriers with AG genotype decreased 31.6%（AG VS AA,OR:0.684,95%CI:0.478-0.979）and carriers with AG+GG genotype decreased 32.5%（AG+GG VS AA,OR:0.675,95%CI:0.479-0.951）;the risk of breast cancer of carriers with AG genotype decreased 30.8%（AG vs AA+GG,OR:0.692,95%CI:0484-0.989）compared with carriers with AA+GG genotype.（2）BCAR4 rs13334967 is associated with lower breast cancer risk.When compared with carriers with AA genotype,the risk of breast cancer of carriers with AT genotype reduced 26.8%（AT VS AA,OR:0.732,95%CI:0.429-0.931）,carriers with TT genotype reduced 26.9%（TT VS AA,OR:0.732,95%CI:0.511-0.990）and carriers with AT+TT genotype reduced 20.2%（AT+TT vs AA,OR:0.798,95%CI:0.571-0.970）.（3）The genotype AG（OR:0.425,95%CI:0.239-0.755）and AG+GG（OR:0.425,95%CI:0.239-0.755）of MALAT1 rs619586 is associated with positive progesterone receptor.（4）Haplotype C_rs3200401A_rs619586G_rs7027113s7027113 of MALAT1 might increase breast cancer risk by 35.8%（OR:1.358,95%CI:1.106–1.668）;however,C_rs3200401G_rs619586G_rs7027113of MALAT1 might decrease breast cancer risk by 34.5%（OR:0.655,95%CI:0.475–0.904）.Haplotype Grs4561483Grs11649623Ars13334967 and Ars4561483Ars11649623Ars13334967of BCAR4 might increase breast cancer risk by 46.7%（OR:1.467,95%CI:1.217-1.768）and 37.0%（OR:1.370,95%CI:1.102-1.844）,respectively.However,Ars4561483Grs11649623Ars1333496 and Grs4561483Grs11649623Trs13334967 of BCAR4 might decrease breast cancer risk might increased breast cancer risk by 19.7%（OR:0.803,95%CI:0.653-0.989）and 41.5%(OR:0.585,95%CI:0.429-0.798,respectively.（5）Carriers with rs619586 AG（0.827±0.390）,GG（0.511±0.149）genotype have an lower MALAT1 expression when compared with AA（1.511±0.437）.Carriers with rs1333496 AA（2.171±0.130）genotype have an elevated BCAR4expression when compared carriers with AT（1.258±0.075）and TT（0.970±0.152）genotypes.Conclusions（1）Carriers with MALAT1 rs619586 AG and GG,MALAT1rs3200401 CT and BCAR4 rs13334967 AT and TT genotpye might decrease breast cancer risk.（2）Carriers with rs619586的AG,GG genotype have a lower MALAT1 expression compared with AA genotype.Carriers with rs13334967 AT,TT genotype have a lower BCAR4 expression compared with AA genotype.（3）MALAT1 rs619586 is correlated with positive progesterone receptor.（4）Haplotype Crs3200401Ars619586Grs7027113 of MALAT1,Grs4561483Grs11649623Ars13334967and A_rs4561483A_rs11649623A_rs13334967s13334967 of BCAR4 might raise the risk of breast cancer;Haplotype Crs3200401Grs619586Grs7027113 of MALAT1,Ars4561483Grs11649623Ars13334967 and G_rs4561483G_rs11649623T_rs13334967s13334967 of BCAR4 might decrease the risk of breast cancer.