组蛋白伴侣VPS72驱动H2AFZ的表达并协同促进肝癌进展  

Histone chaperone VPS72 up-regulates the expression of H2AFZ and synergistically promotes the progression of hepatocellular carcinoma

作　　者：吕垒 冯啸[1] 何凯明 高成立 杨洲 贾昌昌[1] 傅斌生[1] Lei Lyu;Xiao Feng;Kaiming He;Chengli Gao;Zhou Yang;Changchang Jia;Binsheng Fu(Department of Hepatobiliary Surgery&Liver Transplantation Center,the Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510630,China)

机构地区：[1]中山大学附属第三医院肝脏外科暨肝移植中心,广州510630

出　　处：《中华肝脏外科手术学电子杂志》2024年第2期220-226,共7页Chinese Journal of Hepatic Surgery(Electronic Edition)

基　　金：广东省基础与应用基础研究基金(2020A1515010302)。

摘　　要：目的:探讨组蛋白伴侣VPS72和H2AFZ在肝细胞癌(肝癌)的表达调控和促进肝癌发展的机制。方法:利用Lenti-shNC和Lenti-shVPS72-1、Lenti-shVPS72-2慢病毒感染相对高表达VPS72的肝癌细胞系SNU398、Hep3B、SNU449,构建VPS72稳定敲低肝癌细胞系,并使用qRT-PCR和Western blot在转录水平和蛋白水平验证敲低效率。采用CCK-8实验、平板克隆实验、划痕实验、Transwell迁移实验检测各种处理下肝癌细胞的增殖迁移能力,并通过回复实验验证VPS72与H2AFZ的调控关系。使用qRT-PCR、Western blot在转录水平和蛋白水平检测不同分组肝癌细胞VPS72、H2AFZ的表达情况。多组间实验数据比较采用方差分析。结果:Lenti-shNC和Lenti-shVPS72-1、Lenti-shVPS72-2慢病毒感染相对高表达VPS72的肝癌细胞系SNU398、Hep3B、SNU449,成功构建了VPS72稳定敲低肝癌细胞系。Lenti-shVPS72-1、Lenti-shVPS72-2相较于对照组Lenti-shNC均成功干扰了VPS72的表达,并且敲低VPS72明显抑制了H2AFZ的表达,这一结果在3种细胞系中均得到验证。在SNU398细胞中,相较于对照组Lenti-shNC,Lenti-shVPS72-2组的H2A.Z红色荧光减弱,并证明H2A.Z的亚细胞定位在细胞核。细胞增殖实验和克隆形成实验证明,敲低VPS72明显抑制Hep3B、SNU449细胞的增殖能力。细胞划痕实验和Transwell迁移实验证明,与对照组Lenti-shNC相比,Hep3B、SNU449细胞的Lenti-shVPS72-1、Lenti-shVPS72-2组的迁移能力被明显抑制。H2AFZ过表达回复实验显示Hep3B、SNU449被抑制的增殖、迁移表型并未完全缓解,提示VPS72与H2AFZ之间存在潜在协同作用。结论:组蛋白伴侣VPS72可驱动肝癌组蛋白变体H2AZ1的表达,并可能与H2AFZ协同促进肝癌进展,VPS72可作为肝癌表观遗传治疗靶点。Objective To investigate the mechanism of histone chaperones VPS72 and H2AFZ in regulating the expression levels and promoting the progression of in hepatocellular carcinoma(HCC).Methods The liver cancer cell lines SNU398,Hep3B and SNU449 with relatively high expression of VPS72 were infected with Lenti-shNC,Lenti-shVPS72-1 and Lenti-shVPS72-2 lentiviruses.The liver cancer cell lines with stable knockdown of VPS72 were constructed.The knockdown efficiency was validated by qRT-PCR and Western blot at the mRNA and protein levels.The proliferation and migration capabilities of liver cancer cells treated with different interventions were evaluated by CCK-8 assay,colony formation assay,scratch assay and Transwell chamber assay.The regulatory relationship between VPS72 and H2AFZ was verified by rescue experiment.The expression levels of VPS72 and H2AFZ mRNA and proteins of liver cancer cells in different groups were detected by qRT-PCR and Western blot.Experimental data among multiple groups were assessed by analysis of variance(ANOVA).Results Liver cancer cell lines SNU398,Hep3B and SNU449 with relatively high expression of VPS72 were infected with Lenti-shNC,Lenti-shVPS72-1 and Lenti-shVPS72-2 lentiviruses,and liver cancer cell lines with stable knockdown of VPS72 were successfully constructed.Lenti-shVPS72-1 and Lenti-shVPS72-2 successfully interfered with the expression of VPS72 compared with Lenti-shNC in the control group.VPS72 knockdown significantly inhibited the expression of H2AFZ,which was validated in all three cell lines.In SNU398 cells,compared with Lenti-shNC in the control group,the red fluorescence of H2A.Z was weakened in the Lenti-shVPS72-2 group,confirming that the subcellular site of H2A.Z was located in the nucleus.Cell proliferation assay and colony formation experiment found that VPS72 knockdown significantly inhibited the proliferation capabilities of Hep3B and SNU449 cells.Cell scratch assay and Transwell chamber assay showed that the migration abilities of Hep3B and SNU449 cells in the Lenti-

关 键 词：癌 肝细胞 组蛋白伴侣 组蛋白变体 VPS72基因 H2AFZ基因 

分 类 号：R735.7[医药卫生—肿瘤]