CAAP1对人肝癌细胞SMMC-7721增殖、迁移和侵袭的影响  

Effects of CAAP1 on Proliferation,Migration and Invasion of Hepatoma Cell Line SMMC-7721

作　　者：苏静慧 卢鸿健 王一同 孔艺璇 刘雨潭 王梅梅 熊亚南 章广玲 Hong-jian;WANG Yi-tong;KONG Yi-xuan;LIU Yu-tan;WANG Mei-mei;XIONG Ya-nan;ZHANG Guang-ling(Hebei Key Laboratory for Chronic Diseases,School of Basic Medical Sciences,North China University of Science and Technology,Tangshan 063210,China;School of Clinical Medicine,North China University of Science and Technology,Tangshan 063210,China)

机构地区：[1]华北理工大学基础医学院河北省慢性疾病基础医学重点实验室,唐山063210 [2]华北理工大学临床医学院,唐山063210

出　　处：《四川大学学报（医学版）》2021年第3期445-451,共7页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学青年基金资助项目(No.81201281);河北省自然科学基金项目(No.C2012401037);2020年政府资助临床医学优秀人才培养项目(冀财预复[2020]397号);河北省省属高等学校基本科研业务费研究项目(No.JQN2020005);河北省唐山市科学技术研究与发展计划(No.19130204C)资助。

摘　　要：目的探讨Caspase活性和凋亡抑制因子1(CAAP1)对人肝癌细胞SMMC-7721增殖、迁移和侵袭的影响。方法构建CAAP1的过表达载体pcDNA3/CAAP1和敲降载体pSilencer 2.1-U6 neo/shR-CAAP1(shR-CAAP1),并进行鉴定。SMMC-7721分为4组:pcDNA3/CAAP1组、pcDNA3对照组、shR-CAAP1组和pSilencer对照组。SMMC-7721培养后,将CAAP1的过表达载体pcDNA3/CAAP1(pcDNA3/CAAP1组)和敲降载体shR-CAAP1(shR-CAAP1组)以及它们的对照(pcDNA3对照组、pSilencer对照组)分别转染到SMMC-7721中,48 h后进行后续实验。实时定量PCR(qRT-PCR)检测各组细胞中CAAP1 mRNA的表达水平,Western blot检测CAAP1、cleaved Caspase-3蛋白的表达水平;CCK-8实验检测各组细胞的增殖,细胞集落形成实验比较各组细胞集落形成能力;划痕实验比较各组细胞运动能力,Transwell小室检测各组细胞的迁移和侵袭;流式细胞术检测各组细胞的凋亡。纳入TCGA数据库CAAP1低表达的患者75例、CAAP1高表达的患者295例随访48个月的数据,Kaplan-Meier生存曲线比较不同CAAP1表达水平对肝癌患者总生存(OS)的影响。结果双酶切鉴定表明CAAP1过表达载体pcDNA3/CAAP1和敲降载体shR-CAAP1构建成功;qRT-PCR和Western blot结果提示pcDNA3/CAAP1能够使SMMC-7721细胞中CAAP1 mRNA和蛋白表达水平增加(与pcDNA3对照组相比,P<0.05),而shRCAAP1使CAAP1 mRNA和蛋白表达水平降低(与pSilencer对照组相比,P<0.05)。与pcDNA3对照组比较,pcDNA3/CAAP1组SMMC-7721细胞的增殖能力、克隆形成能力、运动能力、迁移和侵袭能力增加,而细胞的凋亡受到抑制(均P<0.05);与pSilencer对照组相比较,shR-CAAP1组SMMC-7721细胞的增殖能力、克隆形成能力、运动能力、迁移和侵袭能力降低,而细胞凋亡增加(P<0.05)。TCGA数据库分析表明CAAP1低表达时肝癌患者的OS优于CAAP1高表达者。结论CAAP1能够促进肝癌细胞SMMC-7721增殖、迁移和侵袭,并抑制其凋亡。Objective To investigate the effect of caspase activity and apoptosis inhibitor 1(CAAP1)on the proliferation,migration and invasion of hepatoma cell SMMC-7721.Methods pcDNA3/CAAP1,the overexpression vector of CAAP1,and pSilencer 2.1-U6 neo/shR-CAAP1,the knockdown vector,were constructed and examined.The experiment included 4 groups of SMMC-7721 cells,pcDNA3/CAAP1 group,pcDNA3 control group,shR-CAAP1 group and pSilencer control group.After the SMMC-7721 cells were cultured,the overexpression vector pcDNA3/CAAP1(the pcDNA3/CAAP1 group),knockdown vector shR-CAAP1(the shR-CAAP1 group)and their controls(pcDNA3 control group and pSilencer control group)were transfected into SMMC-7721 cells respectively,and the follow-up experiments were carried out 48 h later.The mRNA expression of CAAP1 in each group was examined with qRT-PCR.The protein expression level of CAAP1 and cleaved Caspase-3 were checked with Western blot.The proliferation of cells was examined with CCK-8.The colony formation ability and the motility of cells in each group were assessed with colony formation assay and wound-healing assay,respectively.The migration and invasion of cells were examined with Transwell cell chamber and the apoptosis of cells was examined with flow cytometry.The data of 75 patients with low expression of CAAP1 and 295 patients with high expression of CAAP1 were downloaded from TCGA database and the data of 48 months follow-up were analyzed.Kaplan-Meier survival curve was used to compare the correlation between different levels of CAAP1 expression and overall survival(OS)of hepatocellular carcinoma(HCC)patients.Results Double enzyme digestion analysis showed that the overexpression vector pcDNA3/CAAP1 and knockdown vector shR-CAAP1 were constructed successfully.qRT-PCR and Western blot results showed that pcDNA3/CAAP1 increased the mRNA and protein expression level of CAAP1 in SMMC-7721 cells(in comparison with the pcDNA3 control group,P<0.05),while shR-CAAP1 decreased the mRNA and protein expression of CAAP1(in comparison with the

关 键 词：CAAP1 肝癌 增殖 迁移 侵袭 

分 类 号：R735.7[医药卫生—肿瘤]