巯基氧化酶1对人SMMC-7721肝癌细胞生物学功能的影响研究

发布时间：2018-01-31 19:31

本文关键词： 肝癌 SMMC-7721细胞巯基氧化酶1 RNA干扰　出处：《广西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景与目的:肝癌是我国常见肿瘤之一,肝癌发病机制错综复杂,遗传、饮食、病毒和寄生虫感染等均可与肝癌发生发展有关。随着分子生物学发展,有多种肝癌相关基因经过相同或不同研究者多次研究,被认为可能参与到肝癌的发病过程中。Araujo等发现QSOX1在高分化的神经母细胞瘤肿瘤组织中高表达,且在复发率高的病人中也出现高表达。另外,他们还发现QSOX1在神经母细胞瘤的分化和侵袭中起重要作用。QSOX1也被认为可能参与到乳腺癌、前列腺癌等的发生发展过程。目前关于QSOX1与肝癌的发生发展间关系的研究报道甚少,本研究进一步验证QSOX1在肝癌细胞中的表达及其对肝癌细胞生物学功能的影响,揭示QSOX1对肝癌细胞生物学功能所发挥的作用,为肝癌早诊和治疗提供理论基础和实验依据。方法:针对QSOX1的shRNA慢病毒载体的构建由上海吉凯基因技术有限公司完成。将针对QSOX1的shRNA慢病毒载体,以及空病毒载体(带有阴性对照病毒)各转染至人SMMC-7721肝癌细胞,即分别为转染组和阴性对照组。空白对照组为不做任何转染的目的细胞。经筛选出稳定表达株后扩大培养,采用实时荧光定量核酸扩增(qPCR)检测系统,及免疫印迹法(Western-blot)分别检测细胞中QSOX1基因mRNA和蛋白质在肝癌细胞中的表达情况;运用cck-8法通过检测细胞活力以验证细胞增殖情况;采用细胞克隆形成实验测定细胞成瘤能力;利用流式细胞技术检测细胞周期;运用细胞transwell实验和划痕实验分别测定细胞转移和迁移能力。通过spss17.0统计学软件进行统计分析。结果:慢病毒转染组和对照病毒转染组均成功转染;慢病毒转染组qsox1基因mrna和蛋白质表达均分别明显低于空病毒转染组和空白对照组;经cck-8细胞增殖实验发现,慢病毒转染组细胞增殖相对于其他对照组明显减缓(p0.05);慢病毒转染组细胞克隆数量分别明显低于其他对照组(p0.05);流式细胞术分析表明,慢病毒转染组与空细胞组比较,慢病毒转染组g1期细胞所占比例明显减少(p0.001),而s期明显增多,在g2/m期无明显变化;慢病毒转染组与空病毒转染组细胞相比,慢病毒转染组g1期细胞也明显减少,而s期细胞无显著变化,g2/m期细胞明显增多。通过细胞划痕实验观察细胞在0小时、8小时、24小时的迁移情况,结果显示,慢病毒转染组迁移能力在24小时明显降低(p0.05)。细胞transwell转移实验显示,空细胞组和空病毒组细胞转移数无明显差异,而慢病毒转染组细胞转移数明显减少(p0.05)。结论:通过慢病毒转染,干扰qsox1基因在smmc-7721细胞中的表达,能降低smmc-7721肝癌细胞增殖能力,克隆形成实验亦表明肿瘤细胞成瘤能力明显减弱。基因敲减后,根据各组细胞周期,结合细胞增殖速度的结果,说明细胞周期可能被阻滞在s期或g2期。这些结果表明,QSOX1基因在肝癌进展中起重要调控作用,可能作为一个靶向指标,指导肝癌的诊断和治疗。
[Abstract]:Background & objective: liver cancer is one of the common tumors in China. The pathogenesis of liver cancer is complicated. Heredity, diet, virus and parasite infection are all related to the occurrence and development of liver cancer. With the development of molecular biology. Many genes related to liver cancer have been studied many times by the same or different researchers. It is believed that QSOX1 may be involved in the pathogenesis of HCC. Araujo et al have found that QSOX1 is highly expressed in well-differentiated neuroblastoma. They also found that QSOX1 plays an important role in the differentiation and invasion of neuroblastoma. QSOX1 is also thought to be involved in breast cancer. There are few reports on the relationship between QSOX1 and the occurrence and development of liver cancer. This study further verified the expression of QSOX1 in HCC cells and its effect on the biological function of HCC cells, and revealed the role of QSOX1 in the biological function of HCC cells. Methods: to provide theoretical and experimental basis for early diagnosis and treatment of liver cancer. The construction of shRNA lentivirus vector for QSOX1 was completed by Shanghai Qikai Gene Technology Co., Ltd. ShRNA lentivirus vector for QSOX1 will be constructed. The empty virus vector (with negative control virus) was transfected into human SMMC-7721 hepatoma cells. That is the transfection group and the negative control group respectively. The blank control group is the target cell without any transfection. The stable expression strain was screened out and the culture was expanded. The real-time fluorescence quantitative nucleic acid amplification system was used. The expression of QSOX1 gene mRNA and protein in hepatoma cells was detected by Western blotting. Cck-8 method was used to test cell viability to verify cell proliferation. The tumor-forming ability of the cells was measured by cell clone forming assay. Cell cycle was detected by flow cytometry. Cell metastasis and migration were measured by cell transwell test and scratch test respectively. Statistical analysis was carried out by spss17.0 software. Results:. The lentivirus transfection group and the control virus transfection group were successfully transfected. The expression of qsox1 gene mrna and protein in lentivirus transfection group was significantly lower than that in empty virus transfection group and blank control group. Cck-8 cell proliferation assay showed that the proliferation of lentivirus transfected group was significantly slower than that of other control groups. The number of cell clones in lentivirus transfection group was significantly lower than that in other control groups (P 0.05). The results of flow cytometry showed that the percentage of g1 phase cells in lentivirus transfected group was significantly lower than that in blank cell group, while the percentage of g1 phase cells in lentivirus transfection group was significantly lower than that in blank cell group. There was no significant change at g 2 / m; In lentivirus transfection group, compared with empty virus transfection group, g1 phase cells in lentivirus transfection group were significantly reduced, but no significant changes were found in s phase cells. The cell migration was observed at 0 h, 8 h and 24 h by cell scratch assay. The migration ability of lentivirus transfection group decreased significantly at 24 hours. Cell transwell transfer assay showed that there was no significant difference between empty cell group and empty virus group. Conclusion: lentivirus transfection interferes with the expression of qsox1 gene in smmc-7721 cells. After gene knockout, according to the cell cycle of each group, combined with the results of cell proliferation rate. These results suggest that QSOX1 gene plays an important role in the progression of HCC and may be used as a target marker to guide the diagnosis and treatment of HCC.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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