MTDH-siRNA下调MTDH基因表达对肝癌HepG2细胞增殖、凋亡、迁移的影响

发布时间：2018-09-16 20:21

【摘要】：研究背景:原发性肝癌(简称肝癌)是临床上常见的恶性肿瘤之一,其发病率逐年上升,据报道,全球每年新增50多万肝癌患者,其中约有一半在我国,病死率居我国恶性肿瘤的第二位。目前肝癌治疗方法有限,肝脏部分切除及肝移植是潜在的最佳选择,但仅仅只有10%-20%的患者有条件进行治疗;对不能手术切除的中晚期肝癌患者,微创治疗虽能一定程度控制局部肿瘤病灶生长,缓解局部症状但无法根治肿瘤,大多数肝癌患者最终死于肿瘤的侵袭和转移,因此,迫切需要寻求一种安全、更有效、低毒的治疗方法。近期研究发现MTDH/AEG-1具有癌基因的功能,能促进肿瘤细胞增殖、侵袭转移,增加肿瘤耐药性,在许多恶性肿瘤中参与肿瘤进展的各主要过程。相比良性肝肿瘤及正常肝组织细胞,MTDH/AEG-1在肝癌组织和培养的肝癌细胞中表达明显升高。本课题拟在建立采用Hep G2肝癌细胞系,通过小干扰RNA技术(small interfering RNA,si RNA)下调MTDH的表达,观察其对肝癌细胞增殖、迁移、凋亡等生物学行为的影响,通过以上研究来验证以MTDH/AEG-1作为靶点是否能有效预防和抑制肝癌生长、转移。目的:探讨研究干扰下调MTDH基因表达对肝癌Hep G2细胞增殖、迁移和凋亡的影响。方法:采用si RNA技术,下调肝癌Hep G2细胞MTDH基因的表达,免疫细胞化学及Western bolt实验检测干扰效果。MTT实验检测MTDH基因下调对Hep G2细胞增殖的影响,TUNEL细胞凋亡实验检测细胞凋亡情况,流式细胞术(FCM)检测细胞周期,划痕实验观察细胞迁移能力,RT-PCR检测Bcl-2、Bax m RNA表达验证促进细胞凋亡情况。结果:通过MTDH-si RNA干扰下调MTDH基因表达后,肝癌Hep G2细胞增殖能力明显下降,作用48小时和96小时的增殖抑制率分别达到43.8%和64.6%;细胞明显阻滞于静止期(G0)或DNA合成前期(G1),细胞凋亡速比例增加,凋亡率增加23.46%,细胞迁移能力显著降低,Bax基因表达明显上调,而Bcl-2表达下调,促进细胞凋亡。结论:MTDH-si RNA干扰下调MTDH基因表达可抑制Hep G2细胞增殖,阻滞其于G0/G1期,并诱导细胞凋亡,可明显抑制肝癌Hep G2细胞的迁移能力。MTDH基因有望成为肝癌治疗的新靶点。
[Abstract]:Background: primary liver cancer (HCC) is one of the most common malignant tumors in clinic, and its incidence is increasing year by year. It is reported that there are more than half a million liver cancer patients in the world every year, about half of which are in our country. The fatality rate is the second most common malignant tumor in China. At present, the treatment of liver cancer is limited. Partial hepatectomy and liver transplantation are the best potential options, but only 10% to 20% of the patients have the conditions to be treated. Although minimally invasive therapy can control the growth of local tumor focus to some extent, alleviate local symptoms but can not cure the tumor, most patients with liver cancer eventually die from invasion and metastasis of tumor. Therefore, it is urgent to seek a kind of safety and more effective. A low toxicity treatment. Recent studies have found that MTDH/AEG-1 has the function of oncogene, which can promote tumor cell proliferation, invasion and metastasis, increase tumor resistance, and participate in the main process of tumor progression in many malignant tumors. The expression of MTDH- AEG-1 in HCC and cultured hepatoma cells was significantly higher than that in benign and normal hepatoma cells. In this study, Hep G2 hepatoma cell line was established. The expression of MTDH was down-regulated by small interfering RNA (small interfering RNA,si RNA), and the effects of MTDH on proliferation, migration and apoptosis of HCC cells were observed. To verify whether MTDH/AEG-1 as a target can effectively prevent and inhibit the growth and metastasis of liver cancer. Aim: to investigate the effects of interference down-regulation of MTDH gene expression on proliferation, migration and apoptosis of Hep G2 cells. Methods: si RNA technique was used to down-regulate the expression of MTDH gene in Hep G2 cells. The effects of down-regulation of MTDH gene on the proliferation of Hep G2 cells were detected by immunocytochemistry and Western bolt assay. The apoptosis of Hep G2 cells was detected by Tunel cell apoptosis assay, and the cell cycle was detected by flow cytometry (FCM). The cell migration ability was observed by scratch test and the expression of Bcl-2,Bax m RNA was detected by RT-PCR. Results: after down-regulation of MTDH gene expression by MTDH-si RNA interference, the proliferation ability of hepatoma Hep G2 cells decreased significantly. The inhibition rates of proliferation were 43.8% and 64.6% at 48 and 96 hours, respectively. The cells were significantly blocked in the quiescent phase (G0) or DNA synthesis phase (G1), the rate of apoptosis increased, the apoptosis rate increased 23.46%, and the ability of cell migration decreased significantly the expression of Bax gene increased significantly. However, the expression of Bcl-2 was down-regulated and promoted apoptosis. Conclusion the down-regulation of MTDH gene expression by the interference of RNA from Hep to MTDH-si can inhibit the proliferation of Hep G2 cells, block their proliferation in G0/G1 phase, induce apoptosis, and inhibit the migration ability of Hep G2 cells. MTDH gene may be a new target for the treatment of HCC.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7

【相似文献】