ZNF545在结直肠癌中的甲基化改变及其抑制结直肠癌生长的作用和机制

发布时间：2018-09-02 08:12

【摘要】：目的:本实验主要检测基因ZNF545在人结直肠癌细胞株和结直肠癌组织中的表达情况、甲基化状态,研究基因ZNF545对结直肠癌细胞的增殖、克隆形成、凋亡和周期等功能影响,并初步探讨ZNF545是通过何种机制发挥抑癌功能。方法和结果:1、通过逆转录PCR(RT-PCR)检测ZNF545在结直肠癌细胞中的表达情况,结果显示其mRNA在癌细胞中表达降低或缺失。2、通过实时荧光定量PCR(qPCR)检测ZNF545在32对结直肠癌组织和对应癌旁组织中的表达水平,结果显示其在结直肠癌组织中的表达普遍低于癌旁组织,且具有统计学意义。3、甲基化特异性PCR(MSP)检测ZNF545在32例原位结直肠癌组织和6例正常结直肠组织中的启动子甲基化状态,结果显示癌组织中的甲基化率比正常组织中的甲基化率高(p=0.0116);对结直肠癌细胞去甲基化处理后,ZNF545 mRNA表达均有不同程度的恢复。4、将ZNF545质粒转染到表达缺失的SW480和HT-29细胞中,用MTS细胞增殖实验、克隆形成实验和流式细胞仪检测过表达ZNF545对结直肠癌细胞的增殖、凋亡和周期等的影响,结果显示ZNF545基因能抑制结直肠癌细胞增殖和克隆形成,促进癌细胞凋亡,阻滞细胞周期于G0/G1期;裸鼠体内实验证实ZNF545能抑制结直肠癌细胞在体内的生长。5、采用蛋白免疫印迹技术(Western-blot)分析ZNF545对Wnt/β-catenin和PI3K/AKT通路的影响,了解其在结直肠癌中可能的作用机制,结果示ZNF545可能通过Wnt/β-catenin和PI3K/AKT信号通路影响细胞生物学功能。结论:在结直肠癌细胞和组织中,ZNF545可受甲基化调控作用而表达降低;能抑制癌细胞增殖和克隆形成能力,诱导细胞凋亡和G0/G1期阻滞,且能抑制结直肠癌细胞的体内生长,提示其在结直肠癌中为抑癌基因。其可能是通过影响Wnt/β-catenin和PI3K/AKT信号通路来发挥抑癌功能。ZNF545可能成为未来结直肠癌研究中的生物标记物和治疗靶基因。
[Abstract]:Objective: to investigate the expression and methylation status of gene ZNF545 in human colorectal cancer cell lines and tissues, and to investigate the effects of gene ZNF545 on the proliferation, clone formation, apoptosis and cell cycle of colorectal cancer cells. The mechanism by which ZNF545 can suppress cancer is discussed. Methods and results: the expression of ZNF545 in colorectal cancer cells was detected by reverse transcription PCR (RT-PCR). The results showed that the expression of mRNA was decreased or missing in cancer cells. The expression of ZNF545 in 32 pairs of colorectal cancer tissues and corresponding adjacent tissues was detected by real-time fluorescence quantitative PCR (qPCR). The results showed that the expression of ZNF545 in colorectal cancer tissues was generally lower than that in adjacent tissues and had statistical significance. Methylation specific PCR (MSP) was used to detect the promoter methylation status of ZNF545 in 32 cases of colorectal carcinoma in situ and 6 cases of normal colorectal tissues. The results showed that the methylation rate in cancer tissues was higher than that in normal tissues (p0. 0116), the expression of ZNF545 mRNA in colorectal cancer cells recovered to varying degrees after demethylation, and the ZNF545 plasmid was transfected into SW480 and HT-29 cells. MTS cell proliferation assay, clone formation assay and flow cytometry were used to detect the effects of overexpression of ZNF545 on the proliferation, apoptosis and cell cycle of colorectal cancer cells. The results showed that ZNF545 gene could inhibit the proliferation and clone formation of colorectal cancer cells. ZNF545 could inhibit the growth of colorectal cancer cells in vivo. The effect of ZNF545 on Wnt/ 尾 -catenin and PI3K/AKT pathway was analyzed by Western blot (Western-blot). The results showed that ZNF545 may affect cell biological function through Wnt/ 尾 -catenin and PI3K/AKT signaling pathway. Conclusion: ZNF545 can be reduced by methylation in colorectal cancer cells and tissues, can inhibit the proliferation and clone formation of cancer cells, induce apoptosis and G0/G1 arrest, and inhibit the growth of colorectal cancer cells in vivo. The results suggest that it is a tumor suppressor gene in colorectal cancer. ZNF545 may be a biomarker and therapeutic target gene for colorectal cancer in the future by affecting Wnt/ 尾 -catenin and PI3K/AKT signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

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