水通道蛋白9通过下调核内β-catenin抑制肝癌细胞增殖
发布时间：2019-05-16 03:33
【摘要】：目的:探讨AQP9基因过表达后对人肝癌细胞SMMC-7721增殖的影响及其可能的机制。方法:将空载体慢病毒(LV-Luciferase)和靶向AQP9基因过表达慢病毒(LV-AQP9)分别转染人肝癌细胞SMMC-7721,并用嘌呤霉素筛选细胞,从而得到空载体稳定细胞株(SMMC-7721/LV-Luciferase)和AQP9过表达稳定细胞株(SMMC-7721/LV-AQP9),最后通过Western blotting检测AQP9在肝癌细胞内表达。实验分组:阴性对照组(NC组)为空载体稳定细胞;AQP9过表达组(AQP9组)为AQP9过表达稳定细胞。并且运用流式细胞术检测过表达AQP9基因对人肝癌细胞周期的影响;实时荧光定量PCR检测SMMC-7721/LV-Luciferase细胞和SMMC-7721/LV-AQP9细胞的周期相关调控因子mRNA水平表达情况;Western blotting技术检测细胞周期相关调控因子,PCNA,pser675β-catenin,β-catenin蛋白水平表达。同时采用免疫荧光染色观察PCNA和β-catenin在两组肝癌细胞中表达情况。两组数据比较采用了独立样本t检验。结果:Western blotting检测结果显示AQP9过表达组较阴性对照组AQP9的蛋白水平有显著升高,差异有统计学意义(P值0.01)。通过流式细胞技术检测细胞周期,结果显示过表达AQP9基因后影响SMMC-7721细胞周期分布,具体地,SMMC-7721细胞G0-G1期细胞比例由52.24%±0.83%升高到65.68%±0.63%(P值0.05);S期细胞比例由34.3%±0.65%降低至16.56%±0.85%(P值0.05);G2-M期由13.46%±0.2%上升到17.4%±1.12%,差异有统计学意义(P值0.05)。对细胞周期调控因子,PCNA,β-catenin分别进行Western blotting检测,其结果显示:与阴性对照组相比,过表达AQP9组人肝癌细胞细胞周期调控因子Cyclin D1,CDK2,CDK4表达下调,P27表达上调,差异均有统计学意义(P均值0.05);同时细胞内PCNA,核内β-catenin,pser675-β-catenin表达均降低,差异同样有统计学意义(P值均0.05)。同样,细胞免疫荧光染色及光密度分析结果显示,与阴性对照组相比,AQP9过表达组PCNA蛋白表达水平下调,差异有统计学意义(P0.05)。AQP9过表达后影响β-catenin在细胞内的分布。结论:肝癌细胞中过表达AQP9基因后可抑制人肝癌细胞SMMC-7721细胞增殖,根据实验结果分析,其机制可能是AQP9基因下调细胞核内β-catenin的表达水平,进而抑制Cyclin D1蛋白表达水平,使得肝癌细胞阻滞在G1/S期。
[Abstract]:Objective: to investigate the effect of AQP9 gene overexpression on the proliferation of human hepatocellular carcinoma cell line SMMC-7721 and its possible mechanism. Methods: empty vector lentivirus (LV-Luciferase) and target AQP9 gene overexpression lentivirus (LV-AQP9) were transfected into human hepatocellular carcinoma cell line SMMC-7721, and screened by puromycin. The empty vector stable cell line (SMMC-7721/LV-Luciferase) and AQP9 overexpression stable cell line (SMMC-7721/LV-AQP9) were obtained. Finally, the expression of AQP9 in HCC cells was detected by Western blotting. Experimental groups: negative control group (NC group) was empty vector stable cells, AQP9 overexpression group (AQP9 group) was AQP9 overexpression stable cells. Flow cytometry was used to detect the effect of overexpression of AQP9 gene on human hepatocellular carcinoma cell cycle, and real-time fluorescence quantitative PCR was used to detect the expression of mRNA, a cycle-related regulator in SMMC-7721/LV-Luciferase cells and SMMC-7721/LV-AQP9 cells. The expression of cell cycle related regulatory factor and PCNA,pser675 尾-catenin, 尾-catenin protein was detected by Western blotting. At the same time, the expression of PCNA and 尾-catenin in two groups of HCC cells was observed by immunofluorescence staining. The independent sample t test was used to compare the two groups of data. Results the results of: Western blotting showed that the protein level of AQP9 in AQP9 overexpression group was significantly higher than that in negative control group (P < 0.01). The cell cycle was detected by flow cytometry. The results showed that the overexpression of AQP9 gene affected the distribution of SMMC-7721 cell cycle. The proportion of G0-G1 phase cells in SMMC-7721 cells increased from 52.24% 卤0.83% to 65.68% 卤0.63% (P < 0.05). The proportion of cells in S phase decreased from 34.3% 卤0.65% to 16.56% 卤0.85% (P < 0.05), and the proportion of cells in G2 鈮，
本文编号：2477984
[Abstract]:Objective: to investigate the effect of AQP9 gene overexpression on the proliferation of human hepatocellular carcinoma cell line SMMC-7721 and its possible mechanism. Methods: empty vector lentivirus (LV-Luciferase) and target AQP9 gene overexpression lentivirus (LV-AQP9) were transfected into human hepatocellular carcinoma cell line SMMC-7721, and screened by puromycin. The empty vector stable cell line (SMMC-7721/LV-Luciferase) and AQP9 overexpression stable cell line (SMMC-7721/LV-AQP9) were obtained. Finally, the expression of AQP9 in HCC cells was detected by Western blotting. Experimental groups: negative control group (NC group) was empty vector stable cells, AQP9 overexpression group (AQP9 group) was AQP9 overexpression stable cells. Flow cytometry was used to detect the effect of overexpression of AQP9 gene on human hepatocellular carcinoma cell cycle, and real-time fluorescence quantitative PCR was used to detect the expression of mRNA, a cycle-related regulator in SMMC-7721/LV-Luciferase cells and SMMC-7721/LV-AQP9 cells. The expression of cell cycle related regulatory factor and PCNA,pser675 尾-catenin, 尾-catenin protein was detected by Western blotting. At the same time, the expression of PCNA and 尾-catenin in two groups of HCC cells was observed by immunofluorescence staining. The independent sample t test was used to compare the two groups of data. Results the results of: Western blotting showed that the protein level of AQP9 in AQP9 overexpression group was significantly higher than that in negative control group (P < 0.01). The cell cycle was detected by flow cytometry. The results showed that the overexpression of AQP9 gene affected the distribution of SMMC-7721 cell cycle. The proportion of G0-G1 phase cells in SMMC-7721 cells increased from 52.24% 卤0.83% to 65.68% 卤0.63% (P < 0.05). The proportion of cells in S phase decreased from 34.3% 卤0.65% to 16.56% 卤0.85% (P < 0.05), and the proportion of cells in G2 鈮，
本文编号：2477984
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