| Breast cancer is a common female malignancy in many countries.It is a major public health problem for the high morbidity and mortality,which seriously threatens women's health.Breast cancer is a complex disease.Breast cancer is generally considered to be a result of environmental,reproductive and genetic factors.Recent studies have found that long non-coding RNA(lncRNA)can affect the development of tumors and increasing studies have reported the associations between the single nucleotide polymorphisms(SNPs)and tumor susceptibility.Recent reports indicate the important role of LncRNA MIR2052HG in pharmacogenomics that MIR2052HG affects drug resistance in breast cancer patients.However,there has been no report on the association between the genetic variant SNPs of MIR2052HG and breast cancer susceptibility.ObjectiveIn this study,breast cancer-related LncRNA MIR2052HG SNPs and their biological functions were screened by bioinformatics and molecular epidemiology.To clarify the molecular biological mechanism of MIR2052HG functional SNPs,the biological functions of the predicted breast cancer-related MIR2052HG SNPs were verified through a variety of cytological experiments.Methods(1)Bioinformatic screening of MIR2052HG SNPs and prediction of their functions:NCBI and Ensembl websites were applied to determine the location and sequence information of MIR2052HG,1000Genomes website and Haploview software were used to screen Chinese populations for MIR2052HG SNPs with MAF values greater than 0.05,RNAfold was employed to predict MIR2052HG SNPs secondary structure.Finally,1ncRNASNP2,DIANA and other websites were used to predict the targeted miRNAs of MIR2052HG SNPs.(2)Exploring MIR2052HG SNPs related to breast cancer susceptibility:A case-control study was conducted in this study,and 504 cases and 505 controls(frequency matching with cases at age±2)were erolled to extract the peripheral blood DNA of the research subjects.SNPscan method and GeneMapper software were used to genotype MIR2052HG SNPs(rs3802201,rs2553716,rs4259395,rs2588297,rs10957736,rs269183,rs269198,rs34841297 and rs12546233).(3)Detection of the expression of MIR2052HG:The relative expression of MIR2052HG in the plasma of the control population was detected by real-time fluorescence quantitative PCR among different genotypes of rs34841297,using relative quantification and SYBR staining.(4)Validation of MIR2052HG SNP binding to miRNA:Construction of MIR2052HG rs34841297 wild-type and mutant plasmids,which were co-transfected with miR-4456 and normal control mimics.The double luciferase assay confirmed that rs34841297 had multiple genes does the state affect the binding of MIR2052HG to miR-4456.(5)Biological function of miRNA:Construct breast cancer cells(MDA-MB-231 and MCF-7)of low-overexpression,over-expression and normal control lentiviral vectors of miR-4456 for stable transfection.The screened stable transfected cell lines were applied for quantitative PCR experiments to detect the expression of miR-4456.CCK8 experiments.Transwell experiments and scratch test were performed to detect the proliferation,invasion and migration ability of breast cancer cells with different miR-4456 expression levels.(6)Statistical analysis:Non-conditional logistic regression analysis was used to explore breast cancer-related MIR2052HG SNPs,adjusting age,menarche age,menopausal status,number of pregnancies,number of abortions,breastfeeding history,and family history.SHEsis online software was applied to conduct Haplotype analysis of MIR2052HG.Multi-factor dimensionality reduction(MDR)was used to analyze the interaction between gene and environment.Independent t test was applied to compare the relative expression of LncRNA MIR2052HG with different rs34841297 genotypes;t test compares the OD value through CCK8 experiment in two groups among the low expression of miR-4456 and the high expression of miR-4456 and normal control.Independent t test was used to analyze accurate counting of stained cells of two groups through Transwell experiment.Differences in invasion and transfer capabilities was calculated by t test,so does it in scratch test.Results(1)rs3 802201 CG+GG type has a lower risk of breast cancer than CC type(OR:0.756,95%CI:0.580-0.986).rs2553716(A>C)is a protective factor for breast cancer(OR:0.737,95%CI:0.565-0.931).Compared with rs4259395 AA genotype,the risk of AG+GG is reduced(OR:0.756,95%CI:0.573-0.999).rs2588297 gene mutation(G>T)can reduce breast cancer susceptibility(OR:0.606,95%CI:0.459-0.798).rs10957736(C>T)and rs12546233(A>C)gene polymorphisms reduce the breast cancer susceptibility.rs269183(T>C)is associated with Her-2 receptor status(OR:1.755,95%CI:1.030-2.991)and Her-2 overexpressing breast cancer(OR:0.677,95%CI:0.319-1.437).rs269198(C>A)is associated with Her-2 overexpressing breast cancer(OR:0.677,95%CI:0.319-1.437).The GG genotype of rs4259395 is related to the PR receptor status(OR:1.905,95%CI:1.012-3.585),and the TT genotype of rs2588297 is related to the luminal type(OR:3.716,95%CI:0.987-13.996).rs3802201(C>G)and rs2553716(A>C)are associated with PR receptors and luminal breast cancer,which are statistically significant.In addition,the rs12546233 AC+CC genotype is associated with triple negative(OR:0.497,95%CI:0.260-0.949).Meanwhile,the AA genotype is associated with Her-2 overexpression(OR:1.738,95%CI:1.033-2.921).(2)Haplotype Crs3802201Ars2553716Ars4259395Grs2588297Crs10957736Trs269183Crs269198 Wrs34841297Ars12546233 can increase the risk of breast cancer(OR:1.203,95%CI:1.001-1.445).Haplotype Grs3802201Crs2553716Grs4259395Trs2588297Trs10957736Trs269183Crs269198 Mrs34841297Ars12546233 and haplotype Grs3802201Crs2553716Grs4259395Trs2588297Trs10957736 Trs269183Crs269198Mrs34841297Crs12546233 can reduce the risk of breast cancer(OR:0.258,95%CI:0.073-0.908;OR:0.698,95%CI:0.549-0.887,respectively).There is a significant interaction between MIR2052HG rs2588297 and the number of pregnancies and abortions,and they will increase the risk of breast cancer(OR:2.934,95%CI:2.272-3.790).(3)The relative expression of MIR2052HG in individuals with homozygous deletion of rs34841297 A gene(1.68±1.37)and heterozygous deletion(0.95±0.94)were higher than that of individuals with AA genotype(0.26±0.12)(P<0.001,P=0.001,respectively).(4)Double fluorescence reporting experiment was performed in 293 T cells.The results suggest that the binding of MIR2052HG to miR-4456 is mediated by rs34841297.When a deletion mutation occurs in rs34841297,the binding does not exist.(5)CCK8 test was performed in.breast cancer cell lines(MCF-7 and MDA-MB-231).The results showed that miR-4456 was related to the proliferation of breast cancer.Compared with normal control,high-expression miR-4456 breast cancer cells had strong proliferation ability(P<0.05),and low-expression miR-4456 breast cancer cells had relatively weak proliferation ability(P<0.05).(6)Transwell invasion and scratch experiments showed that the number of invading MDA-MB-231 cells of miR-4456 low expression group was lower than that of normal control group(P<0.001),and the number of invading MDA-MB-231 cells of miR-4456 high expression group was higher than that of normal control group(P=0.002).(7)Transwell migration experiments and scratch test showed that the number of migrating cells of miR-4456 low expression group MDA-MB-231 was lower than that of normal control group(P=0.004,P=0.024,respectively)and the number of migrating cells of miR-4456 high expression group MDA-MB-231 was higher than normal control group(P=0.022,P=0.001,respectively).Conclusions(1)MIR2052HG rs3802201(C>G),rs2553716(A>C),rs2588297(G>T),rs10957736(C>T)and rs12546233(A>C)gene polymorphisms reduce the risk of breast cancer and the rs34841297 deletion mutation increases the risk of breast cancer.rs3802201,rs2553716 and rs4259395 are related to PR receptor,rs269183 is related to Her-2 receptor.rs3802201,rs2553716,and rs2588297 are associated with the luminal type.rs269183 and rs269198 are related to Her-2 overexpression.The rs12546233 AC+CC genotype is related to the triple negative type,meanwhile,the AA genotype is related to the Her-2 overexpression type.(2)Haplotype Crs3802201 Ars2553716Ars4259395Grs2588297Crs10957736Trs269183Crs269198 Wrs34841297Ars12546233 increases risk of breast cancer.Haplotype Grs3802201Crs2553716 Grs4259395Trs2588297Trs10957736Trs269183Crs269198Mrs34S41297Ars12546233 and haplotype Grs3802201Crs2553716Grs4259395Trs2588297Trs10957736Trs269183Crs269198Mrs34841297Crs12546233 can reduce the risk of breast cancer.There is a significant interaction between MIR2052HG rs25 88297 and the number of pregnancies and abortions.(3)The deletion of rs34841297 A allele increases MIR2052HG expression.(4)The binding between MIR2052HG and miR-4456 can be affected by rs34841297.MIR2052HG affects breast cancer proliferation,invasion and migration through miR-4456. |
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