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中华肝脏外科手术学电子杂志 ›› 2024, Vol. 13 ›› Issue (02) : 220 -226. doi: 10.3877/cma.j.issn.2095-3232.2024.02.017

基础研究

组蛋白伴侣VPS72驱动H2AFZ的表达并协同促进肝癌进展
吕垒1, 冯啸1, 何凯明1, 高成立1, 杨洲1, 贾昌昌1, 傅斌生1,()   
  1. 1. 510630 广州,中山大学附属第三医院肝脏外科暨肝移植中心
  • 收稿日期:2024-01-03 出版日期:2024-04-10
  • 通信作者: 傅斌生
  • 基金资助:
    广东省基础与应用基础研究基金(2020A1515010302)

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

Lei Lyu1, Xiao Feng1, Kaiming He1, Chengli Gao1, Zhou Yang1, Changchang Jia1, Binsheng Fu1,()   

  1. 1. Department of Hepatobiliary Surgery & Liver Transplantation Center, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China
  • Received:2024-01-03 Published:2024-04-10
  • Corresponding author: Binsheng Fu
引用本文:

吕垒, 冯啸, 何凯明, 高成立, 杨洲, 贾昌昌, 傅斌生. 组蛋白伴侣VPS72驱动H2AFZ的表达并协同促进肝癌进展[J/OL]. 中华肝脏外科手术学电子杂志, 2024, 13(02): 220-226.

Lei Lyu, Xiao Feng, Kaiming He, Chengli Gao, Zhou Yang, Changchang Jia, Binsheng Fu. Histone chaperone VPS72 up-regulates the expression of H2AFZ and synergistically promotes the progression of hepatocellular carcinoma[J/OL]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2024, 13(02): 220-226.

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-shVPS72-1 and Lenti-shVPS72-2 groups were significantly suppressed compared with that of Lenti-shNC in the control group. H2AFZ overexpression rescue experiment showed that the inhibited proliferation and migration phenotypes of Hep3B and SNU449 were not completely alleviated, suggesting a potential synergistical role between VPS72 and H2AFZ.

Conclusions

Histone chaperone VPS72 can up-regulate the expression of histone variant H2AZ1 of HCC, and probably plays a synergistical role with H2AFZ in promoting the progression of HCC. VPS72 can be used as an epigenetic therapy target for HCC.

图1 敲低VPS72抑制肝癌中H2AFZ的表达注:a、b、c分别为qRT-PCR和Western blot检测肝癌SNU398、Hep3B、SNU449细胞的Lenti-shNC、Lenti-shVPS72-1、Lenti-shVPS72-2组VPS72、H2AFZ表达水平,*为P<0.001;d为SNU398细胞Lenti-shNC和Lenti-shVPS72-2组的免疫荧光,绿色代表慢病毒感染成功,红色代表H2A.Z,蓝色代表染色的细胞核,MERGE代表三色荧光的合成图
图2 敲低VPS72抑制肝癌的增殖和迁移注:a为CCK8实验检测VPS72的敲低对肝癌细胞Hep3B、SNU449增殖能力的影响,*为P<0.001;b为细胞划痕实验检测VPS72的敲低对肝癌细胞Hep3B、SNU449迁移能力的影响;c为克隆形成实验检测VPS72的敲低对肝癌细胞Hep3B、SNU449增殖能力的影响;d为Transwell迁移实验检测VPS72的敲低对肝癌细胞Hep3B、SNU449迁移能力的影响
图3 过表达H2AFZ无法完全回复VPS72敲低对肝癌增殖和迁移能力的影响注:a为qRT-PCR验证肝癌细胞Hep3B、SNU449的Lenti-shVPS72组的H2AFZ过表达效率,ns为P>0.05,*为P<0.05;b为细胞划痕实验检测H2AFZ过表达对肝癌细胞Hep3B、SNU449的Lenti-shVPS72组的迁移能力影响;c为克隆形成实验检测H2AFZ过表达对肝癌细胞Hep3B、SNU449的Lenti-shVPS72组的增殖能力影响;d为Transwell迁移实验检测H2AFZ过表达对肝癌细胞Hep3B、SNU449的Lenti-shVPS72组的迁移能力影响
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