基于ARHGAP9基因的人参皂苷Rg3抗肝癌作用机制研究

发布时间：2018-04-01 10:13

本文选题：ARHGAP9　切入点：Rg3　出处：《第二军医大学》2017年硕士论文

【摘要】：高侵袭转移是原发性肝癌(简称“肝癌”)重要的生物学特性,也是导致肝癌高死亡率的重要原因。然而,其发病机制并不明确,阻碍了肝癌的有效诊治。近几年研究表明,Rho GAP蛋白家族的许多成员,如p190Rho GAP、DLC1和ARHGAP在人肝癌组织中明显上调。本实验室前期研究发现,ARHGAP9基因在肝癌组织中明显低表达,且低表达患者相对于高表达患者预后差。ARHGAP9的高表达还抑制肝癌侵袭转移相关信号通路的激活。由此我们推测,ARHGAP9基因与肝癌的发生发展密切相关。人参皂苷Rg3是人参的重要组成部分,对多种恶性肿瘤均具有抑制作用。由于其显著的抗癌疗效,人参皂苷Rg3的单体已被开发成我国一类抗癌新药—参一胶囊。实验和临床研究均已证实,人参皂苷Rg3在治疗肝癌的复发转移中发挥重要作用。然而,人参皂苷Rg3抗肝癌的作用机制研究并不深入,缺乏临床治疗依据,限制了人参皂苷Rg3在临床上的广泛应用。鉴于我们前期发现,ARHGAP9与肝癌的发生发展相关,人参皂苷Rg3抗肝癌是否与ARHGAP9基因相关值得深入探讨。因此本课题的研究主要分为两个部分:(1)分析ARHGAP9基因在肝癌生长及侵袭转移中的作用;(2)确定人参皂苷Rg3是否通过ARHGAP9基因抑制肝癌细胞的侵袭迁移。(一)ARHGAP9在肝癌生长转移中的作用研究我们首先通过细胞转染和慢病毒感染构建了ARHGAP9基因干扰的肝癌细胞株和过表达的肝癌细胞株。然后采用CCK-8、流式细胞术、Transwell等一系列实验检测分析ARHGAP9基因干扰和过表达对肝癌细胞增殖、周期、凋亡、侵袭和迁移能力的影响。体内实验,通过腋下注射ARHGAP9过表达的Hep G2肝癌细胞株建立裸鼠皮下成瘤模型,分析ARHGAP9对裸鼠皮下瘤生长情况的影响。取得的实验结果如下:1、ARHGAP9在肝癌细胞系中的表达6种肝癌细胞株中,ARHGAP9基因表达最高的是MHCC-97L细胞,其余5种细胞株中ARHGAP9基因的表达相对较低且相互之间无显著性差异。因此选择MHCC-97L细胞进行基因干扰研究,选择Hep G2和MHCC-97H细胞进行基因过表达研究。2、干扰ARHGAP9基因对肝癌细胞增殖、周期和凋亡的影响干扰ARHGAP9基因显著促进了MHCC-97L细胞增殖,抑制细胞凋亡,且S期细胞比率增加。3、过表达ARHGAP9基因对肝癌细胞增殖、周期和凋亡的影响过表达ARHGAP9基因显著抑制了Hep G2和MHCC-97H细胞增殖,促进了细胞凋亡,并将细胞周期阻滞在G0/G1期。4、ARHGAP9对肝癌细胞侵袭和迁移的影响干扰ARHGAP9基因显著促进了MHCC-97L细胞的迁移和侵袭,过表达ARHGAP9基因显著抑制了Hep G2和MHCC-97H细胞的迁移和侵袭。5、ARHGAP9对裸鼠肝癌生长的影响ARHGAP9过表达组肿瘤生长速度明显低于对照组。从第24天开始,ARHGAP9过表达组肿瘤体积较对照组明显降低(p0.05)。结论:我们的研究结果显示,ARHGAP9基因高表达显著抑制肝癌发展过程。(二)基于ARHGAP9的人参皂苷Rg3对肝癌细胞侵袭迁移的影响我们采用CCK-8、Transwell、PCR、Western blot以及基因干扰等实验观察不同浓度20(R)-Rg3对肝癌细胞侵袭迁移能力以及对细胞内ARHGAP9表达的影响。取得的实验结果如下:1、人参皂苷Rg3对肝癌细胞活力的影响采用CCK-8实验检测不同浓度人参皂苷Rg3对肝癌细胞活力的影响。当Rg3处理Hep G2和MHCC-97L细胞24 h后,实验组(1.25、2.5和5μg/m L)的细胞活力与对照组相比无统计学差异。2、人参皂苷Rg3抑制肝癌细胞的侵袭迁移人参皂苷Rg3在浓度为1.25、2.5和5μg/m L时均显著抑制了Hep G2和MHCC-97L细胞的侵袭和迁移。3、人参皂苷Rg3提高肝癌细胞中ARHGAP9的表达与对照组相比,人参皂苷Rg3(2.5和5μg/m L)显著提高了Hep G2和MHCC-97L细胞中ARHGAP9蛋白的表达。4、人参皂苷Rg3通过ARHGAP9抑制肝癌细胞侵袭迁移沉默ARHGAP9基因后显著缓解了人参皂苷Rg3(2.5和5μg/m L)对Hep G2和MHCC-97L细胞的抗侵袭迁移作用。结论:我们的研究结果显示,人参皂苷Rg3可抑制肝癌细胞的侵袭迁移,且此过程与人参皂苷Rg3诱导肝癌细胞内ARHGAP9的表达有关。
[Abstract]:High invasion and metastasis of primary liver cancer ("cancer") important biological properties, is an important cause of cancer mortality. However, its pathogenesis is not clear, hinder the effective diagnosis and treatment of liver cancer. In recent years, research shows that many members of the Rho family of GAP proteins, such as p190Rho DLC1 and GAP. ARHGAP in human hepatocellular carcinoma was up-regulated. Our previous study showed that ARHGAP9 gene expression in hepatocellular carcinoma tissues was significantly lower, and patients with low expression relative to the activation of high high expression and poor prognosis of.ARHGAP9 also inhibited the invasion and metastasis of HCC related signaling pathway. We conclude that closely related to the occurrence and development of ARHGAP9 gene and hepatocellular carcinoma. Ginsenoside Rg3 is an important part of ginseng, has inhibitory effect on many malignant tumors. Because of its remarkable anticancer effect of ginsenoside Rg3 has been developed into our country A class of new anticancer drug - capsule. The experimental and clinical studies have confirmed that ginsenoside Rg3 plays an important role in the treatment of liver cancer recurrence and metastasis. However, the mechanism of ginsenoside Rg3 on hepatocellular carcinoma is not thorough, the lack of evidence for clinical treatment, limited the wide application of ginsenoside Rg3 in clinic. In view of our early discovery, occurrence and development of ARHGAP9 and hepatocellular carcinoma, ginsenoside Rg3 against hepatocellular carcinoma is associated with the ARHGAP9 gene which is worthy of further exploration. This research is mainly divided into two parts: (1) analysis of ARHGAP9 gene in hepatocellular carcinoma growth and invasion and metastasis; (2) to determine whether the invasion and migration of ginsenoside Rg3 inhibition of ARHGAP9 gene in hepatocarcinoma cells. (a) role of ARHGAP9 in metastasis of hepatocellular carcinoma we begin by cell transfection and lentivirus infection to construct ARHGAP9 gene interference small hepatocellular carcinoma Cell lines and expression in hepatocellular carcinoma cells. Then using CCK-8, flow cytometry, Transwell and a series of experimental detection and analysis of ARHGAP9 gene interference and overexpression on the proliferation of hepatocellular carcinoma cell cycle, apoptosis, and influence the invasion and migration ability. In vivo, through axillary injection of ARHGAP9 over expression of Hep G2 cell line establishment the nude mice tumor model, analysis of the influence of ARHGAP9 on the growth of subcutaneous tumors. The experimental results are as follows: 1, ARHGAP9 expression in hepatocellular carcinoma cells in the 6 hepatoma cell lines, ARHGAP9 gene expression was highest in MHCC-97L cells, expression of the 5 ARHGAP9 genes in cell lines is relatively low and the mutual there was no significant difference between MHCC-97L cells. So the choice of gene interference, gene overexpression of.2 Hep and G2 MHCC-97H cells, the interference of ARHGAP9 gene on proliferation of hepatocellular carcinoma cell cycle, and Effect of ARHGAP9 gene apoptosis significantly promoted MHCC-97L cell proliferation, inhibition of apoptosis, and the cell ratio in S phase increased.3, overexpression of ARHGAP9 gene on proliferation of hepatocellular carcinoma cell cycle and apoptosis, effects of overexpression of ARHGAP9 significantly inhibited the proliferation of Hep G2 cells and MHCC-97H cells, promote cell apoptosis and cell cycle arrest G0/G1.4, the interference effect of ARHGAP9 gene ARHGAP9 on the invasion and migration of hepatocellular carcinoma cells significantly promoted the migration and invasion of MHCC-97L cells, overexpression of ARHGAP9 significantly inhibited the Hep G2 and MHCC-97H cell migration and invasion of.5, the influence of ARHGAP9 on the growth of human hepatocellular carcinoma in nude mice ARHGAP9 overexpression group the tumor growth rate was significantly lower than the control group. From the beginning of the twenty-fourth day, ARHGAP9 overexpression group tumor volume was significantly lower than the control group (P0.05). Conclusion: Our results suggest that high expression of ARHGAP9 gene was significantly inhibited The development process of liver cancer. (two) the effect of ginsenoside Rg3 on hepatocellular carcinoma cell invasion and migration of ARHGAP9 based on Transwell, we use CCK-8, PCR, Western and blot gene interference experiment observation of different concentrations of 20 (R) the effects of -Rg3 on the invasion and migration ability of hepatoma cells on the expression of ARHGAP9 in cells. The experimental results are as follows 1, the effect of ginsenoside Rg3 on the hepatocellular carcinoma cell viability effect on liver cancer cell viability by CCK-8 assay with different concentrations of ginsenoside Rg3. When Rg3 G2 and MHCC-97L Hep cells after 24 h, the experimental group (1.25,2.5 and 5 g/m L) cell viability compared with the control group there was no significant difference in.2, ginsenoside Rg3 inhibition of cancer cell invasion and migration of ginsenoside Rg3 in the concentration of 1.25,2.5 and 5 g/m L significantly inhibited the invasion and migration of.3 Hep G2 and MHCC-97L cells, ginsenoside Rg3 increased ARHGA in liver cancer cells The expression of P9 compared with the control group, ginsenoside Rg3 (2.5 and 5 g/m L) significantly increased the expression of.4 G2 and Hep ARHGAP9 protein in MHCC-97L cells, ginsenoside Rg3 inhibited the migration and invasion of ARHGAP9 gene silencing significantly alleviated the ginsenoside Rg3 by ARHGAP9 cells (2.5 and 5 g/m L) anti invasion effect on Hep G2 and migration of MHCC-97L cells. Conclusion: Our results show that ginsenoside Rg3 can inhibit the invasion and migration of hepatocellular carcinoma cells, and this process with ginsenoside Rg3 induced expression in hepatocellular carcinoma cell line ARHGAP9.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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