TCF21基因多态性与中国汉族女性乳腺癌发病风险的关联研究及功能分析

发布时间：2018-05-16 17:01

本文选题：TCF21 + 抑癌基因　；参考：《东南大学》2017年博士论文

【摘要】：第一部分TCF21基因在乳腺癌中的表达及其功能目的:检测TCF21基因在人乳腺癌细胞和组织中的表达,并分析其表达与乳腺癌临床病理特征及预后的关系,同时观察TCF21基因过表达对乳腺癌MDA-MB-231细胞生物学行为的影响。方法:(1)通过实时定量 PCR(real-time quantitative PCR,RT-qPCR)和蛋白免疫印迹(Westernblot)方法检测TCF21基因在人乳腺癌细胞MDA-MB-231和MCF-7,人正常乳腺上皮细胞MCF-10A和39例术后乳腺癌组织及其癌旁正常组织中的表达,并分析癌组织中TCF21 mRNA表达与临床病理特征的关系。(2)利用生物信息软件KM-plotter分析乳腺癌组织中TCF21 mRNA表达与预后的关系。(3)采用脂质体介导法将重组质粒pcDNA3.1-TCF21和空质粒pcDNA3.1分别转染至乳腺癌MDA-MB-231细胞,并运用RT-qPCR和Western blot方法检测质粒转染后细胞中TCF21基因的表达情况。(4)CCK-8法和Annexin V-FITC/PI双染色法分别被用于检测TCF21基因过表达对乳腺癌MDA-MB-231细胞增殖和凋亡的影响。结果:(1)乳腺癌MDA-MB-231和MCF-7细胞中TCF21 mRNA和蛋白的表达水平均显著低于正常乳腺上皮细胞MCF-10A。随后采用同样的方法检测39例人乳腺癌组织及其癌旁正常组织中TCF21 mRNA的表达,结果显示TCF21 mRNA在36例乳腺癌组织中低表达,在1例乳腺癌组织中表达无差异,在2例乳腺癌组织中高表达。对TCF21 mRNA低表达的乳腺癌组织进行Western blot检验,发现TCF21蛋白在乳腺癌组织中的表达水平也显著低于癌旁正常组织,与mRNA检测结果一致。通过进一步分析乳腺癌组织中TCF21 mRNA表达水平与临床病理特征的关系,我们发现TCF21 mRNA表达水平与患者年龄、TNM分期、肿瘤分化程度、ER、PR及HER2的表达无关,但与肿瘤大小、淋巴结转移有关。TCF21 mRNA低表达更容易发生在大尺度肿瘤和淋巴结转移阳性的肿瘤中。(2)KM-plotter分析结果显示乳腺癌组织中TCF21 mRNA表达水平与患者总生存率无关,但与患者无复发生存率有关。与TCF21 mRNA高表达患者相比,TCF21 mRNA低表达患者的无复发生存率显著降低(P-=4.7e-07)。(3)重组质粒转染乳腺癌MDA-MB-231细胞后,细胞内TCF21 mRNA和蛋白的表达量明显增加。(4)与对照组相比,TCF21基因过表达不仅能抑制乳腺癌MDA-MB-231细胞的增殖,还能促进细胞凋亡。结论:TCF21基因作为抑癌基因在人乳腺癌细胞及大多数乳腺癌组织中低表达,并且TCF21基因低表达与乳腺癌淋巴结转移、肿瘤体积增大及不良预后有关。进一步体外实验证实TCF21基因过表达能够抑制乳腺癌MDA-MB-231细胞的增殖并促进其凋亡。第二部分TCF21基因多态性与中国汉族女性乳腺癌发病风险的关系及其机制目的:研究TCF21基因标签单核苷酸多态性(tag single nucleotide polymorphisms,tagSNPs)与中国汉族女性乳腺癌发病风险的关系,并利用基因型-表型关联分析及体外细胞实验研究差异多态性位点参与乳腺癌发生的分子机制。方法:(1)利用Haploview 4.2软件从HapMap数据库中获取中国汉族人群TCF21基因tagSNPs。(2)采用多重聚合酶链反应-连接酶检测反应技术对901例乳腺癌患者(病例组)和1225例健康个体(对照组)TCF21基因tagSNPs进行分型,并运用拟合优度卡方检验分析对照组中各多态性位点基因型分布是否符合哈迪-温伯格平衡(Hardy-Weinberg equilibrium,HWE)。(3)利用 logistic回归模型分析TCF21基因tagSNPs与中国汉族女性乳腺癌发病风险的关系。(4)通过RT-qPCR方法检测乳腺癌组织和癌旁正常组织中TCF21 mRNA的表达,并使用单因素方差分析检测差异多态性位点(rsl2190287 CG)基因型与TCF21 mRNA表达量的关系。(5)鉴于rs12I90287位点位于TCF21基因3'非翻译区(3'untranslated region,3'UTR),生物信息学软件 TargetScan 和 miRanda 被用于预测与该多态性位点结合的特异miRNA。(6)采用RT-qPCR方法检测乳腺癌细胞及组织中特异miRNA的表达。(7)通过直接测序法检测乳腺癌MDA-MB-231细胞中rs12190287位点基因型。(8)使用miRecords软件和TSGene数据库获取乳腺癌中特异miRNA的潜在靶基因。(9)化学合成特异miRNA mimic和inhibitor,并通过功能获得性研究和功能缺失性研究分析乳腺癌MDA-MB-231细胞中特异miRNA对TCF21基因及其潜在靶基因(SMA4D4,PPP2R1B,GGNBP2,NUAK1)表达的影响。(10)采用DNA重组和定点突变技术构建 pmirGLO-TCF21-3'UTR-C 和 pmirGLO-TCF21-3'UTR-G 双荧光素酶报告基因载体,分析rs12190287位点不同等位基因对特异miRNA发挥调控作用的影响。结果:(1)利用Haploview 4.2软件和HapMap数据库从TCF21基因中获得 6 个 tagSNPs(rs2327429 TC,rs2327430 TC,rs2327433 AG,rs12190287 CG,rs7766238 GA,rs4896011 TA),其中 2 个 tagSNPs(rs2327429 TC,rs2327430TC)位于基因启动子区,1个tagSNP(rs2327433AG)位于基因内含子区,3 个 tagSNPs(rs12190287 CG,rs7766238 GA,rs4896011 TA)位于基因3'UTR。(2)基因分型结果表明,对照组中tagSNPs位点基因型频率分布均符合HWE:rs2327429位点的PHWE值为0.61,rs2327430位点的PHWE值为0.79,rs2327433 位点的P PHWE值为 0.74,rs12190287 位点的 PHWE值为 0.94,rs7766238 位点的 PHWE值为 0.06,rs4896011 位点的P wE值为 0.08。(3)病例对照研究结果显示,TCF21 rs12190287位点多态性与中国汉族女性乳腺癌发病风险有关(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)。进一步基于年龄的分层分析结果表明,rs12190287位点多态性不仅与50岁以下女性乳腺癌发病风险有关(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),还与50岁及以上女性乳腺癌发病风险有关(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)。基于病理类型的分层分析结果表明,TCF21rs12190287位点多态性只与乳腺浸润性导管癌的发病风险有关(G vs.C,OR=0.78,95%CI=0.68-0.89,P0.001;GG vs.CC,OR=0.59,95%CI= 0.45-0.79,P0.001;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)。基于肿瘤期的分层分析结果表明,rs12190287位点多态性不仅与Ⅰ+Ⅱ期乳腺癌发病风险有关(Gvs.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),还与 Ⅲ+Ⅳ 期乳腺癌发病风险有关(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)基因型-表型关联分析显示,癌旁正常组织中rs12190287位点基因型与TCF21 mRNA表达量相关。与rs12190287 CC基因型癌旁正常组织相比,GG基因型癌旁正常组织中TCF21 mRNA表达量显著增加。(5)生物信息学分析结果表明,rs12190287位点位于hsa-miR-224与TCF21 mRNA 3'UTR结合的种子区域,其中rs12190287 C等位基因有助于hsa-miR-224对TCF21基因表达的调控。(6)RT-qPCR检测结果表明hsa-miR-224在乳腺癌MDA-MB-231细胞中的表达水平显著高于正常乳腺上皮细胞MCF-10A。通过检测30例乳腺癌组织及其癌旁正常组织中hsa-miR-224的表达,发现hsa-miR-224在14例乳腺癌组织中高表达,在7例乳腺癌组织中表达无差异,在9例乳腺癌组织中低表达。(7)测序分型结果表明乳腺癌MDA-MB-231细胞中rs12190287位点基因型为CC纯合子,提示hsa-miR-224可能参与调控乳腺癌MDA-MB-231细胞中TCT21基因的表达。(8)进一步生物信息学分析结果表明,hsa-miR-224也可能参与调控乳腺癌MDA-MB-231细胞中SMAD4,PPP2R1 GGNBP2和NUAK1基因的表达。(9)功能获得性研究结果表明,hsa-miR-224过表达能够显著抑制乳腺癌MDA-MB-231细胞中TCF21、PPP2R1B、GGNBP2和NUAK1基因mRNA的表达。功能缺失性研究结果表明,hsa-miR-224抑制能够显著上调乳腺癌MDA-MB-231细胞中TCF21和GGNBP2基因mRNA的表达。(10)双荧光素酶活性分析结果表明,rs12190287 G等位基因能够干扰hsa-miR-224与TCF21 mRNA 3'UTR的结合,从而增加TCF21基因的表达。结论:TCF21基因3'UTR中的rs12190287位点多态性与中国汉族女性乳腺癌发病风险有关。与携带有rs12190287C等位基因的个体相比,携带有G等位基因的个体患乳腺癌的风险显著降低。进一步功能研究表明,rs12190287 G等位基因能够干扰hsa-miR-224对TCF21基因表达的调控。因此该多态性位点可能作为中国汉族女性乳腺癌发病风险的生物标记物,鉴定该多态性位点将有助于实施明确的个体化预防、干预及治疗。
[Abstract]:Part 1: expression of TCF21 gene in breast cancer and its function objective: to detect the expression of TCF21 gene in human breast cancer cells and tissues, and to analyze the relationship between the expression and the clinicopathological features and prognosis of breast cancer, and to observe the effect of TCF21 gene overexpression on the biological behavior of breast cancer MDA-MB-231 cells. Methods: (1) through reality Quantitative PCR (real-time quantitative PCR, RT-qPCR) and protein immunoblotting (Westernblot) were used to detect the expression of TCF21 gene in human breast cancer cells MDA-MB-231 and MCF-7, normal mammary gland epithelial cells, MCF-10A and 39 cases of breast cancer tissues and the normal tissues adjacent to the cancer, and analyzed the TCF21 mRNA expression and Clinicopathology in the cancer tissues. (2) the relationship between the expression of TCF21 mRNA in breast cancer tissue and the relationship between the expression of mRNA and the prognosis. (3) transfection of recombinant plasmid pcDNA3.1-TCF21 and empty plasmid pcDNA3.1 to MDA-MB-231 cells of breast cancer by liposome mediated method, and RT-qPCR and Western blot method to detect TCF in the cells after transfection of the plasmid. The expression of 21 gene. (4) CCK-8 and Annexin V-FITC/PI double staining were used to detect the effect of TCF21 overexpression on the proliferation and apoptosis of MDA-MB-231 cells of breast cancer. Results: (1) the expression level of TCF21 mRNA and protein in MDA-MB-231 and MCF-7 cells of breast cancer were significantly lower than that of normal mammary epithelial cells after MCF-10A.. The same method was used to detect the expression of TCF21 mRNA in 39 cases of human breast cancer and normal tissues adjacent to cancer. The results showed that the expression of TCF21 mRNA was low in 36 cases of breast cancer tissue, and there was no difference in 1 breast cancer tissues, and high expression in 2 breast cancer tissues. The Western blot test of the breast cancer tissues with low TCF21 mRNA was detected by Western blot test and found to be found. The expression level of TCF21 protein in breast cancer tissues is also significantly lower than that of normal tissues adjacent to the carcinoma, which is consistent with the results of mRNA detection. By further analysis of the relationship between the expression level of TCF21 mRNA and the clinicopathological features in breast cancer tissue, we found that the expression level of TCF21 mRNA and the age of the patients, the TNM stage, the degree of tumor differentiation, the expression of ER, PR and HER2 The low expression of.TCF21 mRNA, related to tumor size, lymph node metastasis, was more likely to occur in large scale tumors and lymph node metastases. (2) KM-plotter analysis showed that the expression of TCF21 mRNA in breast cancer tissues was not related to the total survival rate of the patients, but was related to the non recurrent survival rate of the patients. The high expression of TCF21 mRNA was associated with the high expression of TCF21 mRNA. Compared with the low expression of TCF21 mRNA, the recurrence survival rate of the patients with low expression was significantly decreased (P-=4.7e-07). (3) the expression of TCF21 mRNA and protein in the cell transfected by recombinant plasmid was significantly increased. (4) compared with the control group, the overexpression of TCF21 gene could not only inhibit the proliferation of breast cancer MDA-MB-231 cells, but also promote cell withering. Conclusion: TCF21 gene is low expression in human breast cancer cells and most breast cancer tissues as tumor suppressor gene, and the low expression of TCF21 gene is associated with lymph node metastasis, tumor volume and bad prognosis. Further in vitro experiments have proved that TCF21 gene overexpression can inhibit the proliferation of MDA-MB-231 cells in breast cancer and promote the proliferation of breast cancer cells. The relationship between the second TCF21 gene polymorphism and the risk of breast cancer in Chinese Han women and its mechanism: the study of the relationship between the TCF21 gene label single nucleotide polymorphism (tag single nucleotide polymorphisms, tagSNPs) and the risk of breast cancer in Chinese Han women, and using genotype phenotypic correlation analysis and in vitro The molecular mechanism of polymorphic loci in the occurrence of breast cancer was investigated by cell experiments. Methods: (1) using Haploview 4.2 software to obtain TCF21 gene tagSNPs. (2) of Chinese Han population from HapMap database (2) using multiple polymerase chain reaction ligase detection reaction technology in 901 cases of breast cancer patients (case group) and 1225 healthy individuals. The TCF21 gene tagSNPs was typed and the genotype distribution of the polymorphic loci in the control group was analyzed by the TCF21 gene tagSNPs (Hardy-Weinberg equilibrium, HWE). (3) the relationship between the TCF21 gene tagSNPs and the risk of breast cancer in Chinese Han women was analyzed by logistic regression model (4). The expression of TCF21 mRNA in breast cancer tissues and adjacent normal tissues was detected by RT-qPCR method, and the relationship between the genotype of rsl2190287 CG and the expression of TCF21 mRNA was detected by single factor analysis of variance. (5) in view of the location of rs12I90287 loci in the non translation region of 3'of TCF21 gene (3'untranslated region,), bioinformatics Software TargetScan and miRanda were used to predict specific miRNA. (6) combining with the polymorphic loci (6) to detect the specific miRNA expression in breast cancer cells and tissues by RT-qPCR method. (7) the genotype of rs12190287 loci in breast cancer MDA-MB-231 cells was detected by direct sequencing. (8) use miRecords software and TSGene database to obtain milk. Potential target genes of specific miRNA in adenocarcinoma. (9) chemical synthesis of specific miRNA mimic and inhibitor, and the effects of specific miRNA on the expression of TCF21 gene and its potential target gene (SMA4D4, PPP2R1B, GGNBP2, NUAK1) in MDA-MB-231 cells of breast cancer by functional study and functional deletion. (10) DNA recombination and fixed-point mutation are used. The pmirGLO-TCF21-3'UTR-C and pmirGLO-TCF21-3'UTR-G double luciferase reporter gene vectors were constructed to analyze the effects of different alleles on the specific miRNA at the rs12190287 locus. Results: (1) 6 tagSNPs (rs2327429 TC, rs2327430 TC) were obtained from the TCF21 gene using the Haploview 4.2 software and the HapMap database. 7433 AG, rs12190287 CG, rs7766238 GA, rs4896011 TA), of which 2 tagSNPs (rs2327429 TC, rs2327430TC) are located in the gene promoter region, and 1 tagSNP are located in the intron of the gene, and the result of the gene typing (2) in the gene indicates that the locus gene is in the control group. The PHWE value of the type frequency distribution was 0.61, the PHWE value of the rs2327430 site was 0.79, the P PHWE value of the rs2327433 site was 0.74, the PHWE value of the rs12190287 site was 0.94, the PHWE value of the rs7766238 loci was 0.06. G vs.C, OR=0.80,95%CI=0.71-0.91, P=0.001; GG vs.CC, 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. The results showed that rs12190287 locus polymorphism was not only associated with the risk of breast cancer under 50 years of age (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 at the age of 50 and above (G vs.C. 4-0.80, 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). The pathological type based stratified analysis shows that TCF21rs12190287 locus polymorphism is only related to the risk of invasive ductal carcinoma of the breast. .79, P0.001; 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; the stratified analysis based on the tumor stage shows that the polymorphism of the locus is not only associated with the risk of stage I and II stage of breast cancer. G 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 related to the risk of breast cancer. Vs.CC, OR=0.60,95%CI=0.44-0.80, P=0.001). (4) genotype phenotype correlation analysis showed that the genotype of rs12190287 loci in the normal tissues adjacent to the cancer was associated with the TCF21 mRNA expression. The TCF21 mRNA expression in the normal tissues adjacent to the rs12190287 CC genotypes increased significantly. (5) the bioinformatics analysis result table The rs12190287 site is located in the seed region of the combination of hsa-miR-224 and TCF21 mRNA 3'UTR, in which rs12190287 C alleles contribute to the regulation of TCF21 gene expression. (6) RT-qPCR detection results show that hsa-miR-224 expression in breast cancer MDA-MB-231 cells is significantly higher than normal mammary epithelial cells. The expression of hsa-miR-224 in 30 cases of breast cancer tissue and its adjacent normal tissues showed that hsa-miR-224 was highly expressed in 14 breast cancer tissues. There was no difference in 7 breast cancer tissues and low expression in 9 breast cancer tissues. (7) sequencing results showed that the rs12190287 loci in MDA-MB-231 cells of breast cancer were CC homozygote, suggesting H Sa-miR-224 may participate in the regulation of the expression of TCT21 gene in breast cancer MDA-MB-231 cells. (8) further bioinformatics analysis shows that hsa-miR-224 may also be involved in the regulation of the expression of SMAD4, PPP2R1 GGNBP2 and NUAK1 in breast cancer MDA-MB-231 cells. (9) the results of functional acquisition showed that the overexpression of hsa-miR-224 could be significantly inhibited. The expression of TCF21, PPP2R1B, GGNBP2 and NUAK1 gene in breast cancer MDA-MB-231 cells. The results of functional deletion study showed that hsa-miR-224 inhibition could significantly increase the expression of TCF21 and GGNBP2 gene mRNA in breast cancer MDA-MB-231 cells. (10) the analysis of double luciferase activity indicates that the allele can interfere with the expression of the allele of the double luciferase activity. 24 combined with TCF21 mRNA 3'UTR to increase the expression of the TCF21 gene. Conclusion: the rs12190287 polymorphism in the TCF21 gene 3'UTR is associated with the risk of breast cancer in Chinese Han women. The risk of breast cancer carrying a G allele is significantly lower than that of individuals carrying rs12190287C alleles. The study shows that the rs12190287 G allele can interfere with the regulation of hsa-miR-224 on the expression of TCF21 gene. Therefore, the polymorphic loci may be used as a biomarker for the risk of breast cancer in Chinese women of Han nationality. Identification of the polymorphism site will be helpful for the implementation of specific individualized prevention, intervention and treatment.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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