调控p21和MTH1基因表达对肝癌HepG2细胞生长情况的影响

发布时间：2018-05-26 23:17

本文选题：RNA激活 + RNA干扰　；参考：《青岛大学》2017年硕士论文

【摘要】：目的原发性肝癌(hepato cellular carcinoma,HCC)是世界上最为常见的恶性肿瘤之一,其发病率和致死率都位居世界前列。肝癌具有发病隐匿,潜伏期长,发展速度快,恶性程度高等特点,手术治疗是目前治疗肝癌最好的方法。但由于大多数患者直至病程中晚期才得以确诊,因而错过了手术治疗的最佳时期。因此,寻找新的治疗工具和药物靶点对肝癌的治疗至关重要。本研究旨在探讨小激活RNA(small activating RNA,sa RNA)上调p21基因表达和小干扰RNA(small interfering RNA,si RNA)沉默MTH1基因表达后,对人肝癌Hep G2细胞增殖、迁移、侵袭和凋亡的影响。方法设计并合成靶向p21和MTH1基因的sa RNA和si RNA序列,并将其转染到体外培养的人肝癌Hep G2细胞中。实验分为5组:空白对照组(CT)、阴性对照组(NC)、sa RNA-p21转染组(p21)、si RNA-MTH1转染组(MTH1)和联合转染组(p21+MTH1)。q PCR检测各组p21和MTH1基因m RNA表达情况;Western blot检测各组p21,MTH1,以及凋亡相关蛋白表达水平;CCK8法检测各组Hep G2细胞增殖情况;划痕实验检测细胞迁移能力;Transwell侵袭实验检测细胞侵袭能力;流式细胞术检测细胞凋亡情况。结果与空白对照组和阴性对照组相比,sa RNA-p21转染组和联合转染组p21基因m RNA和蛋白表达水平明显上调(P0.01)。与空白对照组和阴性对照组相比,si RNA-MTH1转染组和联合转染组MTH1基因m RNA和蛋白表达水平明显下调(P0.01)。与空白对照组和阴性对照相比sa RNA-p21转染组和si RNA-MTH1转染组细胞存活率和划痕愈合率明显降低(P0.05),联合转染组的细胞存活率和划痕愈合率与对照组和单转组相比明显降低(P0.05)。流式细胞术实验结果显示与空白对照组和阴性对照相比sa RNA-p21转染组和si RNA-MTH1转染组细胞凋亡率明显升高(P0.01),联合转染组细胞凋亡率较对照组和单转组明显升高(P0.01)。Western blot结果显示与空白对照组和阴性对照相比sa RNA-p21转染组和si RNA-MTH1转染组caspase-3蛋白表达量明显上调(P0.05),联合转染组比单转组效果明显(P0.05);联合转染组Bak蛋白表达量与对照组相比明显上调(P0.01),单转组与对照组相比无差异(P0.05)。与空白对照组和阴性对照相比sa RNA-p21转染组和si RNA-MTH1转染组穿过基底膜的细胞数明显减少(P0.01),联合转染组穿过基底膜的细胞数较对照组和单转组明显升高(P0.01)结论sa RNA-p21可以有效的激活p21基因的表达,si RNA-MTH1可以有效的沉默MTH1基因的表达。通过RNA激活(RNAa)技术上调p21基因表达和RNA干扰(RNAi)技术下调MTH1基因表达可以抑制人肝癌Hep G2细胞的增殖、迁移和侵袭,并能诱导其凋亡。sa RNA-p21和si RNA-MTH1联合组的效果要比单转组好。
[Abstract]:Objective Primary liver cancer (HCC) is one of the most common malignant tumors in the world, and its morbidity and mortality are among the highest in the world. Liver cancer is characterized by occult onset, long incubation period, rapid development and high malignancy. Surgical treatment is the best method for the treatment of liver cancer at present. However, most patients were not diagnosed until the middle and late stage of the disease, thus missing the optimal period of surgical treatment. Therefore, the search for new therapeutic tools and drug targets is essential for the treatment of liver cancer. The aim of this study was to investigate the effects of up-regulation of p21 gene expression and silencing of MTH1 gene expression by small activated RNA(small activating RNA-siRNAs on proliferation, migration, invasion and apoptosis of human hepatoma Hep G2 cells. Methods the sa RNA and si RNA sequences targeting p21 and MTH1 genes were designed and synthesized and transfected into human hepatoma Hep G2 cells in vitro. The experiment was divided into five groups: the blank control group (CTL), the negative control group (RNA-p21) and the co-transfection group (p21 MTH1).q PCR) were used to detect the expression of p21 and MTH1 gene m RNA in each group. Western blot was used to detect the expression of p21 RNA-p21 and apoptosis-related protein (Apoptosis-related protein) in each group. The proliferation of Hep G2 cells was detected by CCK8 assay. The cell migration ability was detected by scratch assay and the cell invasion ability was detected by Transwell invasion assay, and apoptosis was detected by flow cytometry. Results compared with the blank control group and the negative control group, the expression of p21 gene m RNA and protein in the p21 RNA-p21 transfection group and co-transfection group were significantly up-regulated. Compared with the blank control group and the negative control group, the expression level of m RNA and protein of MTH1 gene in the RNA-MTH1 transfection group and co-transfection group were significantly down-regulated. Compared with the control group and the negative control group, the cell survival rate and scratch healing rate of the sa RNA-p21 transfection group and si RNA-MTH1 transfection group were significantly lower than that of the control group and the si RNA-MTH1 transfection group. The cell survival rate and scratch healing rate of the co-transfection group were significantly lower than those of the control group and the single transfer group. The results of flow cytometry showed that the apoptosis rate of sa RNA-p21 transfection group and si RNA-MTH1 transfection group was significantly higher than that of control group and negative control group. The apoptosis rate of co-transfection group was significantly higher than that of control group and single transfer group. The expression of caspase-3 protein in sa RNA-p21 transfection group and si RNA-MTH1 transfection group was significantly up-regulated than that in blank control group and negative control group, the effect of co-transfection group was significantly higher than that in single transfer group, and the expression of Bak protein in co-transfection group was significantly higher than that in control group. Compared with the control group, there was no difference between the single transfer group and the control group. Compared with the blank control group and the negative control group, the number of cells passing through the basement membrane in the sa RNA-p21 transfection group and the si RNA-MTH1 transfection group was significantly reduced, and the number of cells passing through the basement membrane in the co-transfection group was significantly higher than that in the control group and the single transfer group. The expression of MTH1 gene could be effectively silenced by activating p21 gene expression by sisi RNA-MTH1. Upregulation of p21 gene expression and down-regulation of MTH1 gene expression by RNA activator RNAa technique could inhibit the proliferation, migration and invasion of Hep G2 cells, and induce apoptosis of Hep G2 cells in combination with RNA-p21 and si RNA-MTH1.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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