ZNF545在結(jié)直腸癌中的甲基化改變及其抑制結(jié)直腸癌生長的作用和機制
發(fā)布時間：2018-09-02 08:12
【摘要】：目的:本實驗主要檢測基因ZNF545在人結(jié)直腸癌細(xì)胞株和結(jié)直腸癌組織中的表達情況、甲基化狀態(tài),研究基因ZNF545對結(jié)直腸癌細(xì)胞的增殖、克隆形成、凋亡和周期等功能影響,并初步探討ZNF545是通過何種機制發(fā)揮抑癌功能。方法和結(jié)果:1、通過逆轉(zhuǎn)錄PCR(RT-PCR)檢測ZNF545在結(jié)直腸癌細(xì)胞中的表達情況,結(jié)果顯示其mRNA在癌細(xì)胞中表達降低或缺失。2、通過實時熒光定量PCR(qPCR)檢測ZNF545在32對結(jié)直腸癌組織和對應(yīng)癌旁組織中的表達水平,結(jié)果顯示其在結(jié)直腸癌組織中的表達普遍低于癌旁組織,且具有統(tǒng)計學(xué)意義。3、甲基化特異性PCR(MSP)檢測ZNF545在32例原位結(jié)直腸癌組織和6例正常結(jié)直腸組織中的啟動子甲基化狀態(tài),結(jié)果顯示癌組織中的甲基化率比正常組織中的甲基化率高(p=0.0116);對結(jié)直腸癌細(xì)胞去甲基化處理后,ZNF545 mRNA表達均有不同程度的恢復(fù)。4、將ZNF545質(zhì)粒轉(zhuǎn)染到表達缺失的SW480和HT-29細(xì)胞中,用MTS細(xì)胞增殖實驗、克隆形成實驗和流式細(xì)胞儀檢測過表達ZNF545對結(jié)直腸癌細(xì)胞的增殖、凋亡和周期等的影響,結(jié)果顯示ZNF545基因能抑制結(jié)直腸癌細(xì)胞增殖和克隆形成,促進癌細(xì)胞凋亡,阻滯細(xì)胞周期于G0/G1期;裸鼠體內(nèi)實驗證實ZNF545能抑制結(jié)直腸癌細(xì)胞在體內(nèi)的生長。5、采用蛋白免疫印跡技術(shù)(Western-blot)分析ZNF545對Wnt/β-catenin和PI3K/AKT通路的影響,了解其在結(jié)直腸癌中可能的作用機制,結(jié)果示ZNF545可能通過Wnt/β-catenin和PI3K/AKT信號通路影響細(xì)胞生物學(xué)功能。結(jié)論:在結(jié)直腸癌細(xì)胞和組織中,ZNF545可受甲基化調(diào)控作用而表達降低;能抑制癌細(xì)胞增殖和克隆形成能力,誘導(dǎo)細(xì)胞凋亡和G0/G1期阻滯,且能抑制結(jié)直腸癌細(xì)胞的體內(nèi)生長,提示其在結(jié)直腸癌中為抑癌基因。其可能是通過影響Wnt/β-catenin和PI3K/AKT信號通路來發(fā)揮抑癌功能。ZNF545可能成為未來結(jié)直腸癌研究中的生物標(biāo)記物和治療靶基因。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34
本文編號：2218778
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34
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相關(guān)期刊論文 前4條
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2 Giampaolo Ugolini;Federico Ghignone;Davide Zattoni;Giacomo Veronese;Isacco Montroni;;Personalized surgical management of colorectal cancer in elderly population[J];World Journal of Gastroenterology;2014年14期
3 Norio Inoue;Naoki Sato;Susumu Matsumoto;Hitoshi Kanno;Yuko Hashimoto;Kazuhiro Tasaki;Kinya Sato;Shun Sato;Katsutoshi Kaneko;;Comparison between surgical outcomes of colorectal cancer in younger and elderly patients[J];World Journal of Gastroenterology;2011年12期
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