HepG2肝癌细胞中p24β1水平对高尔基体结构的影响  

Effect of p24β1 levels on Golgi apparatus in HepG2 hepatocellular carcinoma cells

作　　者：王迪 刘萱 WANG Di;LIU Xuan(Institute of Physical Science and Information Technology,Anhui University,Hefei 230601,China;Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]安徽大学物质科学与信息技术研究院,合肥230601 [2]军事科学院军事医学研究院,北京100850

出　　处：《军事医学》2023年第5期334-340,共7页Military Medical Sciences

基　　金：国家重点研发项目子课题(2018YFC200701)。

摘　　要：目的通过改变人肝癌细胞HepG2中p24β1表达水平,研究该细胞中p24β1表达水平是否对高尔基体产生影响。方法利用RNA干扰实验敲低HepG2细胞中p24β1表达(HepG2-KD),通过Western印迹检测蛋白干扰效率,实时荧光定量PCR(qPCR)验证转录水平;通过CRISPR/Cas9基因编辑系统构建同源染色体双敲除p24β12基因的HepG2细胞系(HepG2-KO),在基因组测序和Western印迹检测敲除表型后,通过CCK-8实验和克隆形成实验比较HepG2-KO与HepG2的细胞增殖活性;利用细胞免疫荧光实验分析高尔基体标志物α-N-乙酰半乳糖胺残基在细胞中的分布是否受p24β1水平影响。结果RNA干扰后能使HepG2细胞中p24β1基因的表达下调;成功构建了同源染色体双敲除p24β1的HepG2-KO细胞系,其细胞增殖活性较HepG2无明显差异;在HepG2-KD和HepG2-KO细胞中,高尔基体呈弥散分布,并伴随片层状顺式高尔基体形态改变。结论在肝癌细胞HepG2中,p24β1水平对于稳定高尔基体的结构至关重要,提示其对高尔基体功能具有重要调控作用。Objective To study the effect of p24β1 expressions on Golgi apparatus in HepG2 cells by changing the expression level of p24β1.Methods The expression of p24β1 was knocked down by RNA interference in HepG2 cells(HepG2-KD)before the knockdown efficiency of genes was detected by real time quantitative PCR(qPCR)and Western blotting.Furthermore,p24β1 genes in two homologous chromosomes were completely knocked out using CRISPR/Cas9 technique(HepG2-KO),which was confirmed by genomic sequencing and Western blotting.The proliferation of HepG2-KO and HepG2 wild type cells was also compared by means of CCK-8 assay and colony-forming assay.Moreover,the distribution and structure of Golgi apparatus in HepG2-KO and HepG2 wild type cells were analyzed and compared via immunofluorescence assay using Golgi markerα-N-acetylgalactosaminyl residues.Results p24β1-targeted RNA interference decreased p24β1 expressions.HepG2-KO cell clones with double knockout of p24β1 genes in two homologous chromosomes were also constructed,which showed no significant impact on cell proliferation compared to HepG2 wild type cells by CCK-8 assay and colony-forming assay.Most importantly,the distribution and structure of Golgi apparatus were significantly changed by p24β1 knockout.Loosened structure of Golgi apparatus,especially the dispersed lamellar morphology of cis Golgi bodies,was observed in the HepG2-KO cell line.Conclusion In HepG2 cells,p24β1 plays an important role in stabilizing the structure of Golgi apparatus,suggesting that p24β1 may facilitate the biological function of Golgi apparatus.

关 键 词：p24β1 HEPG2细胞 肝细胞癌 CRISPR/Cas9 RNA干扰 基因编辑 高尔基体 

分 类 号：R735.7[医药卫生—肿瘤]