多聚腺苷酸结合蛋白互作蛋白1在肝癌发生中的作用机制研究

发布时间：2018-03-14 13:18

本文选题：PAIP1　切入点：肝细胞癌　出处：《南昌大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:检测肝癌组织及肝癌细胞株中PAIP1的表达水平,研究其在肝癌组织中的表达水平与肝癌患者的临床病理特征参数之间的关系。观察肝癌细胞株SMMC-7721中PAIP1基因的表达下调对肝癌细胞体内外生物学行为的影响。初步探讨PAIP1在肝细胞癌发生过程中的作用机制。方法:第一部分PAIP1在肝癌细胞及肝癌组织中的表达及临床意义采用免疫组织化学方法检测肝癌组织芯片PAIP1蛋白表达水平,比较肝癌组织和癌旁组织PAIP1的表达水平差异,分析肝癌组织中PAIP1的表达水平与肝癌患者临床病理参数之间的关系;运用实时荧光定量PCR(RT-PCR)技术观察肝癌细胞株中的PAIP1 mRNA的表达水平。第二部分PAIP1的表达下调对肝癌细胞体内外生物学行为的影响采用RNA干扰技术下调肝癌细胞株SMMC-7721中PAIP1的表达水平,研究PAIP1表达下调前后SMMC-7721细胞的生物学行为变化。Cellomics细胞计数检测SMMC-7721细胞的增殖能力;流式细胞仪分析SMMC-7721细胞的细胞周期和凋亡情况;克隆形成实验观察SMMC-7721细胞体外克隆形成能力;裸鼠成瘤模型探明SMMC-7721细胞裸鼠皮下移植瘤的生长情况。第三部分PAIP1基因表达沉默后差异表达基因的筛选及验证采用全基因组表达谱芯片筛选PAIP1表达敲减后的下游应答基因,利用生物信息学方法,对应答基因进行GO,Pathway分析,挑选出与细胞周期调控相关的关键基因进行Western blotting验证。结果:第一部分PAIP1在肝癌细胞及肝癌组织中的表达及临床意义在肝癌组织芯片中,PAIP1在肝癌组织中的表达水平显著高于癌旁组织;PAIP1的表达水平与肝癌患者的性别、年龄、肿瘤大小及TNM分期等无明显相关性,而与肝细胞癌的病理分级呈正相关,并与病人生存率呈负相关。此外,PAIP1在肝癌细胞株中也呈高表达状态。第二部分PAIP1的表达下调对肝癌细胞体内外生物学行为的影响下调PAIP1的表达后,肝癌细胞增殖能力减弱;细胞凋亡增多;处于S期、G1期的细胞无显著变化,处于G2期的细胞增多;细胞体外克隆形成能力减弱;裸鼠体内成瘤能力减弱。第三部分PAIP1基因表达沉默后差异表达基因的筛选及验证以FC2.0或FC0.5,P0.05为筛选标准,表达谱基因芯片结果显示PAIP1敲减后SMMC-7721细胞内基因发生改变的有333个,其中上调的有134个,下调的有199个。通过KEGG与BIOCARTA通路富集分析找到富集的差异基因所在的10条信号通路。Western blotting结果显示PAIP1表达下调后与细胞周期调控相关的基因CCND1、CCND2、CDK6、MDM2的表达水平发生明显变化。结论:(1)在肝癌组织及肝癌细胞株中PAIP1呈高表达状态,其可能在肝细胞癌的发生发展过程中发挥作用。(2)PAIP1的表达下调可显著抑制肝癌细胞的增殖,促进细胞的凋亡,抑制细胞的体外克隆形成,改变细胞周期的分布,抑制细胞的体内成瘤。(3)PAIP1可能通过影响调控细胞周期的关键基因的表达从而在肝细胞癌发生过程中发挥促进作用。
[Abstract]:Objective: to detect the expression of PAIP1 in hepatocellular carcinoma tissues and hepatoma cell lines. To study the relationship between the expression of PAIP1 gene in HCC and the clinicopathological parameters of HCC patients, and to observe the effect of down-regulation of PAIP1 gene expression on the biological behavior of HCC cells in vivo and in vitro. Methods: the expression and clinical significance of PAIP1 in hepatocellular carcinoma cells and tissues were studied by immunohistochemical method. The expression of PAIP1 protein in hepatocellular carcinoma tissue was detected by immunohistochemical method. To compare the expression level of PAIP1 between HCC and paracancerous tissues, and to analyze the relationship between the expression of PAIP1 and the clinicopathological parameters of HCC. The expression level of PAIP1 mRNA in hepatoma cell line was observed by real-time fluorescence quantitative PCR- RT-PCR.The second part, the effect of down-regulation of PAIP1 expression on the biological behavior of hepatoma cell line in vivo and in vitro. The expression level of PAIP1 in SMMC-7721 cell line was down-regulated by RNA interference technique. To study the changes of biological behavior of SMMC-7721 cells before and after down-regulation of PAIP1 expression. The cell count of Cellomics was used to detect the proliferative ability of SMMC-7721 cells; flow cytometry was used to analyze the cell cycle and apoptosis of SMMC-7721 cells; clone formation assay was used to observe the cloning ability of SMMC-7721 cells in vitro. The growth of subcutaneous xenografts of SMMC-7721 cells in nude mice was investigated in nude mice. The third part was the screening and verification of differentially expressed genes after silencing of PAIP1 gene expression. Whole genome expression microarray was used to screen downstream response genes of PAIP1 expression after knockout. By using bioinformatics method, GOP Pathway was used to analyze the response gene. The key genes related to cell cycle regulation were selected for Western blotting verification. Results: the first part was the expression of PAIP1 in HCC cells and HCC tissues and its clinical significance in HCC tissue microarray. The expression level of PAIP1 in paracancerous tissues was significantly higher than that in patients with hepatocellular carcinoma. Age, tumor size and TNM stage were not significantly correlated, but were positively correlated with the pathological grade of HCC. In addition, the expression of PAIP1 was also highly expressed in hepatoma cells. The second part of the down-regulation of PAIP1 on the biological behavior of hepatoma cells in vivo and in vitro down-regulated the expression of PAIP1, the proliferation capacity of hepatoma cells decreased. The cell apoptosis increased, the cells in S phase and G 1 phase did not change significantly, and the cells in G 2 phase increased, and the ability of cell clone formation was weakened in vitro. The ability of tumorigenesis in nude mice was weakened. The screening and verification of differentially expressed genes after silence of PAIP1 gene expression in nude mice were based on FC2.0 or FC0.5. The results of gene microarray showed that there were 333 genes in SMMC-7721 cells after PAIP1 knockout. Of these, 134 were up-regulated. By enrichment analysis of KEGG and BIOCARTA pathway, we found 10 signal pathways in which the enriched differentially expressed genes were located. Western blotting results showed that the expression level of CCND1- CCND2CDK6MDM2 gene related to cell cycle regulation was obvious after down-regulation of PAIP1 expression. Conclusion the expression of PAIP1 in liver cancer tissue and hepatoma cell line is high. The down-regulation of PAIP1 may play an important role in the development of hepatocellular carcinoma (HCC). The down-regulation of PAIP1 can significantly inhibit the proliferation of hepatoma cells, promote cell apoptosis, inhibit cell clone formation in vitro, and change the distribution of cell cycle. Inhibition of tumorigenesis in vivo may play a promoting role in the carcinogenesis of hepatocellular carcinoma by affecting the expression of key genes that regulate cell cycle.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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