CAAP1抑制肝癌HepG2细胞凋亡而促进其增殖、迁移和侵袭

CAAP1 inhibits apoptosis and promotes proliferation, migration and invasion of hepatocellular carcinoma HepG2 cells

目的:探讨CAAP1对肝癌HepG2细胞凋亡、增殖、迁移和侵袭的影响及其作用机制。方法:构建CAAP1过表达载体pcDNA3/CAAP1和敲降载体p Silencer 2.1-U6 neo/shR-CAAP1,转染肝癌HepG2细胞后,qPCR和WB实验分别检测CAAP1mRNA和蛋白的表达水平。实验分为过表达对照组(pcDNA3)、过表达组(pcDNA3/CAAP1)、敲降对照组(pSilencer 2.1-U6 neo,pSilencer)和敲降组(pSilencer 2.1-U6 neo/shR-CAAP1,shR-CAAP1),流式细胞术检测各组细胞凋亡水平,WB检测cleaved caspase 3蛋白表达水平,CCK-8实验检测各组细胞的增殖情况,克隆形成实验检测各组细胞的集落形成能力,划痕和Transwell小室实验检测各组细胞迁移和侵袭能力。检索TCGA数据库,分析CAAP1对肝癌患者OS的影响。结果:成功构建CAAP1的过表达载体pcDNA3/CAAP1和敲降载体shR-CAAP1,转染后pcDNA3/CAAP1组和shR-CAAP1组细胞中CAAP1mRNA及蛋白表达水平均明显增高或降低(均P<0.05)。与pcDNA3组比较,pcDNA3/CAAP1组细胞凋亡率下降32%、cleaved caspase 3蛋白表达水平显著降低(均P<0.05);与p Silencer组比较,shR-CAAP1组细胞凋亡率上升73%,cleaved caspase 3蛋白表达水平显著升高(均P<0.05)。pcDNA3/CAAP1组细胞增殖显著增强(P<0.05),shR-CAAP1组细胞增殖显著降低(P<0.05)。pcDNA3/CAAP1组细胞迁移数增加48%、细胞迁移距离增加59%、细胞侵袭数增加52%(均P<0.05),shR-CAAP1组细胞迁移数减少53%、细胞迁移距离减少29%、细胞侵袭数减少45%(均P<0.05)。TCGA数据库数据分析显示,肝癌组织中CAAP1的高表达与肝癌患者OS呈负相关(P<0.05)。结论:CAAP1能够抑制肝癌HepG2细胞凋亡从而促进其增殖、迁移和侵袭,其可能与肝癌的发生发展密切相关。

Objective: To explore the effect of CAAP1 on apoptosis, proliferation, migration and invasion of hepatocellular carcinoma(HCC) HepG2 cells and its mechanism. Methods: The pcDNA3/CAAP1(CAAP1 over-expression) and pSilencer 2.1-U6 neo/shR-CAAP1(CAAP1 knockdown) plasmids were constructed and transfected into HepG2 cells. The mRNA and protein levels of CAAP1 were detected by qPCR and WB, respectively. The cells were divided into four groups, namely overexpression control group(pcDNA3),CAAP1 over-expression group(pcDNA3/CAAP1), silence control group(pSilencer 2.1-U6 neo, pSilencer) and CAAP1 silence group(pSilencer 2.1-U6 neo/shR-CAAP1, shR-CAAP1). Flow cytometry was used to analyze the apoptosis, and WB was used to detect the protein expression of cleaved caspase 3 in each group. CCK-8 assay was used to detect the proliferation of HepG2 cells, Colony formation assay was used to detect the clonogenesis, and Transwell assay and wound healing assay were used to detect the invasion and migration abilities of HepG2 cells in each group. The effect of CAAP1 on overall survival(OS) of HCC patients was analyzed after searching TCGA database. Results: PcDNA3/CAAP1 with CAAP1 over-expression and shR-CAAP1 with CAAP1 knockdown were successfully constructed. It was confirmed that pcDNA3/CAAP1 could increase the mRNA and protein expressions of CAAP1,while shR-CAAP1 could decrease the m RNA and protein expressions of CAAP1(all P<0.05). The cell apoptotic rate in pcDNA3/CAAP1 group decreased by 32% as compared to pcDNA3 group, and the cleaved caspase 3 protein expression was significantly decreased(all P<0.05);while the cell apoptotic rate in shR-CAAP1 group increased by 73% as compared to pSilencer group, and the cleaved caspase 3 protein expression was significantly increased(all P<0.05). The cell proliferation in pcDNA3/CAAP1 group significantly increased(P<0.05), while the cell proliferation in shR-CAAP1 group significantly decreased(P<0.05). The cell migration number increased by 48%, the cell migration distance increased by 59%(P<0.05) and the cell invasion number increased by52% in pcDNA3/CAAP1 group(all P<0.05). The cell migration number decreased by 53%, cell migration distance decreased by 29%and cell invasion number decreased by 45% in shR-CAAP1 group(all P<0.05). TCGA database analysis showed that the high expression of CAAP1 was negatively correlated with the OS of HCC patients(P<0.05). Conclusion: CAAP1 can promote the proliferation, migration and invasion of HepG2 cells by inhibiting its apoptosis, and it may be closely related to the occurrence and development of HCC.