| Part Ⅰ.The expression and function of TCF21 gene in breast cancerObjective:The purpose of this study was to examine the expression of TCF21 gene in human breast cancer cell lines and tissues,investigate the relationship between TCF21 gene expression and clinicopathological characteristics and prognosis of breast cancer patients,and assess the biological significance of TCF21 gene overexpression in MDA-MB-231 breast cancer cells.Methods:(1)The expression of TCF21 gene were measured by real-time quantitative PCR(RT-qPCR)and Western blot in human breast cancer cells(MDA-MB-231 and MCF-7),normal human breast epithelial cells(MCF-10A)and thirty-nine paired cancerous and adjacent normal breast tissues.In addition,the relationship between TCF21 mRNA expression and the clinicopathological characteristics of breast cancer patients was analyzed by Chi-square test.(2)KM-plotter tool was used to assess the relationship between TCF21 mRNA expression and prognosis of breast cancer patients.(3)Reconstructed pcDNA3.1-TCF21 and pcDNA3.1-empty plasmids(control group)were respectively transfected into MDA-MB-231 cells by the lipofectamine 2000.After 24h of culture,TCF21 gene expression levels were examined by RT-qPCR and Western blot.(4)Cell Counting Kit-8(CCK-8)and Annexin V-FITC/PI were used to investigate the effects of TCF21 gene overexpression on the proliferation and apoptosis of MDA-MB-231 cells.Results:(1)TCF21 gene expression was at a very low level in breast cancer cells compared with normal breast epithelial cells.The expression levels of TCF21 mRNA in 39 paired normal and cancerous breast tissues were detected by RT-qPCR.Results showed that expression levels of TCF21 mRNA were low in 36 breast cancer tissues,not significantly different in 1 breast cancer tissue,and high in 2 breast cancer tissues-Western blot method was used to furtherly detect samples with the decreased expression of TCF21 mRNA.Results showed that the expression levels of TCF21 protein were also decreased in cancerous breast tissues,which is similar with mRNA detection results.In addition,analysis of clinicopathological characteristics showed that expression levels of TCF21 mRNA was not associated with age,TNM stage,tumor differentiation,and expression of ER,PR and HER2,but with lymph node metastasis and tumor size.Low expression of TCF21 mRNA was significantly associated with positive lymph node metastasis(P=0.048)and larger tumor size(P=0.014).(2)KM-plotter results showed that expression levels of TCF21 mRNA was not associated with overall survival,but with relapse-free survival in breast cancer patients.Compared with high TCF21 expression patients,patients with low TCF21 expression had poorer relapse-free survival(P--4.7e-07).(3)After pcDNA3.1-TCF21 transfection,TCF21 gene expression levels were significantly increased in MDA-MB-231 cells.(4)Compared with control group,TCF21 gene overexpression suppressed proliferation and induced apoptosis of MDA-MB-231 cells.Conclusion:As a tumor suppressor gene,TCF21gene expression is decreased in breast cancer cell lines and tissues.Low expression of TCF21 gene is associated with positive lymph node metastasis,larger tumor size and poor relapse-free survival.In vitro studies suggest that TCF21 can suppress proliferation and induce apoptosis of MDA-MB-231 cells.Part Ⅱ.Association of TCF21 gene polymorphisms with breast cancer risk in Chinese Han womenObjective:To assess the association between TCF21 tagSNPs and breast cancer risk in Chinese Han women,and investigate the functional significance of different polymorphism using genotype-phenotype correlation as well as in vitro cell experiments.Methods:(1)Haploview 4.2 software and HapMap database were used to obtain TCF21 tagSNPs in Chinese Han population.(2)Multiplex polymerase chain reaction-ligase detection reaction(PCR-LDR)technology was used to detect genotypes of 901 breast cancer patients(case group)and 1225 healthy individuals(control group).Chi-square test of goodness-of-fit was used to analyze Hardy-Weinberg equilibrium(HWE)in control group.(3)Logistic regression model was used to evaluate the association between TCF21 tagSNPs and breast cancer risk in Chinese Han women.(4)RT-qPCR was used to exa,mine the expression of TCF21 mRNA in breast cancer tissues and adjacent normal breast tissues.One-way ANOVA was used to assess the relationship between rs12190287 genotypes and TCF21 mRNA expression.(5)In view of gene location of rs12190287,the TargetScan and miRanda softwares were used to predict potential function of rs12190287 polymorphism.(6)RT-qPCR technology was used to detect the expression of specific miRNA in human breast cancer cell lines and tissues.(7)The genotype of rs12190287 in MDA-MB-231 cells was investigated by direct sequencing.(8)MiRecords software and TSGene database were used to acquire potential target genes of specific miRNA in human breast cancer.(9)Specific miRNA mimic and inhibitor were chemically synthesized and were used to analyze the effects of specific miRNA on the expression of TCF21 and potential target genes(SMAD4,PPP2R1B,GGNBP2,NUAK1)in MDA-MB-231 cells.(10)pmirGLO-TCF21-3’UTR-C and pmirGLO-TCF21-3’UTR-G vectors were constructed by DNA recombination and site-directed mutagenesis,and were used to investigate the effect of rs12190287 polymorphism on specific miRNA function.Results:(1)A total of 6 tagSNPs(rs2327429 T>C,rs2327430 T>C,rs2327433 A>G,rs12190287 C>G,rs7766238 G>A,rs4896011 T>A),including 2 tagSNPs(rs2327429 T>C,rs2327430 T>C)in the promoter region of TCF21 gene,1 tagSNP(rs2327433 A>G)in TCF21 intron,3 tagSNPs(rs12190287 C>G,rs7766238 G>A,rs4896011 T>A)in TCF21 3’UTR,were obtained by Haploview 4.2 software as well as HapMap database.(2)Genotyping results indicated that genotype frequency distribution of TCF21 tagSNPs in control group conformed to HWE:PHwE= 0.61 for rs2327429,PHWE= 0.79 for rs2327430,PHWE= 0.74 for rs2327433,PHWE= 0.94 for rs12190287,PHWE= 0.06 for rs7766238,PHWE= 0.08 for rs4896011.(3)The results of case-control study showed that TCF21 rs12190287 polymorphism was significantly associated with the risk of breast cancer in Chinese Han women(G vs.C,OR=0.80,95%CI=0.71-0.91,P=0.001;GG vs.CC,OR=0.64,95%CI=0.49-0.84,P=0.001;CG vs.CC,OR=0.81,95%CI=0.68-0.98,P=0.03;GG+CG vs.CC,OR-0.77,95%CI=0.64-0.92,P=0.003;GG vs.CG+CC,OR=0.72,95%CI=0.56-0.92,P=0.007).Further stratified analyses based on age indicated that TCF21 rs12190287 polymorphism was not only associated with the risk of breast cancer in Chinese Han women under age 50(G vs.C,OR=0.85,95%CI=0.73-0.99,P=0.04;GG+CG vs.CC,OR=0.80,95%CI=0.64-0.99,P=0.04),but also associated with the risk of breast cancer in Chinese Han women age 50 and older(G vs.C,OR=0.72,95%CI=0.58-0.89,P=0.002;GG vs.CC,OR=0.52,95%CI=0.34-0.80,P=0.003;GG+CG vs.CC,OR=0.72,95%CI=0.54-0.97,P=0.03;GG vs.CG+CC,OR=0.58,95%CI=0.39-0.86,P=0.01).Stratified analyses based on pathological type indicated that TCF21 rs12190287 polymorphism was only associated with the risk of infiltrative ductal carcinoma(G vs.C,OR=0.78,95%CI=0.68-0.89,P<0.001;GG vs.CC,OR=0.59,95%CI=0.45-0.79,P<0.001;CG vs.CC,OR=0.82,95%CI=0.67-0.99,P=0.04;GG + CG vs.CC,OR=0.76,95%CI=0.83-0.91,P=0.003;GG vs.CG + CC,OR=0.67,95%CI=0.51-0.86,P=0.002).Stratified analyses based on tumor stage indicated that TCF21 rs12190287 polymorphism was not only associated with the risk of stage Ⅰ+Ⅱ breast cancer(G vs.C,OR=0.80,95%CI=0.70-0.92,P=0.002;GG vs.CC,OR=0.59,95%CI=0.44-0.80,P=0.001;GG+CG vs.CC,OR=0.79,95%CI=0.65-0.96,P=0.02;GG vs.CG+CC,OR=0.64,95%CI=0.49-0.85,P=0.002),but also associated with the risk of stage Ⅲ+Ⅳ breast cancer(G vs.C,OR=0.79,95%CI=0.65-0.97,P=0.03;GG vs.CC,OR=0.66,95%CI=0.44-0.99,P=0.05;CG vs.CC,OR=0.57,95%CI=0.41-0.79,P--0.001;GG+CG vs.CC,OR-0.60;95%CI=0.44-0.80,P=0.001).(4)Genotype-phenotype correlation analyses indicated that rs12190287 genotypes were associated with TCF21 mRNA expression levels in normal breast tissues,and TCF21 mRNA levels were significantly higher in normal breast tissues with rs12190287 GG genotypes than in those with rs12190287 CC genotype.(5)Bioinformatics analysis showed that rs12190287 polymorphism was located within the seed region of TCF21 mRNA 3’UTR and hsa-miR-224 hybridization.The rs12190287 C allele contributed to hsa-miR-224-mediated regulation of TCF21 gene expression.(6)RT-qPCR results indicated that hsa-miR-224 expression was at a very high level in MDA-MB-231 cells compared with normal breast epithelial cells.In addition,hsa-miR-224 expression levels in 30 paired normal and cancerous breast tissues were detected by RT-qPCR.Results showed that hsa-miR-224 expression levels were high in 14 cancerous breast tissues,not significantly different in 7 cancerous breast tissues,and low in 9 cancerous breast tissues.(7)Genotyping results showed that genotype of rs12190287 in MDA-MB-231 cells was CC,suggesting that hsa-miR-224 may regulate TCF21 gene expression in MDA-MB-231 cells.(8)Further analysis of bioinformatics indicated that hsa-miR-224 may also regulate expression of SMAD4,PPP2R1B,GGNBP2 and NUAK1 genes in MDA-MB-231 cells.(9)Gain-of-function study indicated that hsa-miR-224 overexpression could suppress expression of TCF21,PPP2R1B,GGNBP2 and NUAK1 genes in MDA-MB-231 cells.Loss-of-function study showed that hsa-miR-224 inhibition could increase expression of TCF21 and GGNBP2 genes in MDA-MB-231 cells.(10)Double luciferase activity analysis showed that the presence of rs12190287 G allele could disrupt the binding of hsa-miR-224 and TCF21 mRNA 3’UTR,which resulted in the increased expression of TCF21 gene.Conclusion:The rs12190287 in TCF21 3’UTR is significantly associated with the risk of breast cancer in Chinese Han women.Compared with individuals carrying rs12190287 C allele,individuals carrying G allele have a reduced risk of breast cancer.Further in vitro studies indicate that the rs12190287 polymorphism can affect the TCF21 gene expression by interfering with the binding efficiency of hsa-miR-224 and TCF21 mRNA 3’UTR,which results in breast cancer risk.Hence,rs12190287 polymorphism may act as a biomarker of breast cancer risk in Chinese Han women.Identifying rs12190287 polymorphism will contribute to individualized prevention,clinical decision and individualized treatment. |
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