赖氨酸乙酰转移酶6A对肝癌细胞增殖、侵袭和迁移的影响

Effect of lysine acetyltransferase 6A on the proliferation, invasion and migration of hepatoma cells

目的 观察赖氨酸乙酰转移酶6A(lysine acetyltransferase 6A,KAT6A)在肝细胞癌(hepatocellular carcinoma, HCC)中的表达并探讨其对肝癌细胞增殖、侵袭和迁移的影响。方法 收集2018年7月至2020年1月在武汉大学中南医院肝胆胰外科行肝切除术的HCC病人组织标本40份,通过实时荧光定量聚合酶链反应(PCR)技术和蛋白质印迹法(Western blot)技术检测KAT6A在40例HCC病人癌组织及癌旁组织、9株肝癌细胞系(SK-hep1、HepG2、Hep3B、Huh-7、HCCLM3、HCCLM6、MHCC-97H、MHCC-97L和PLC/PRF/5)和人正常肝细胞系(THLE-2)中的mRNA和蛋白表达差异,研究其表达高低与HCC病人临床病理参数之间的关联性。实验组为转染Si-KAT6A小干扰、oe-KAT6A质粒的肝癌细胞,对照组为转染了Si-NC小干扰oe-Vector质粒的肝癌细胞,48 h之后,采用CCK-8增殖实验、EdU增殖实验、划痕实验、Transwell侵袭实验和流式细胞术研究KAT6A敲低/过表达对MHCC-97H/Hep3B细胞增殖、迁移、侵袭能力和细胞周期的影响;采用单基因富集分析(GSEA)预测KAT6A涉及的下游信号通路并通过免疫印迹实验对其进行验证。结果 实时荧光定量PCR和Western blot结果提示,KAT6A在肝癌组织中的mRNA和蛋白表达水平较癌旁组织明显上调,且KAT6A在9株肝癌细胞系中的mRNA表达量明显高于人正常肝细胞系;KAT6A的表达水平与肝癌病人的肿瘤直径及是否合并门静脉癌栓显著相关;细胞功能实验结果表明,对KAT6A进行沉默后实验组MHCC-97H细胞相比于对照组其增殖、侵袭和迁移能力均明显下降,S期细胞比例显著减少,差异有统计学意义(P<0.05);对KAT6A进行过表达后实验组Hep3B细胞相比于对照组其增殖、侵袭和迁移能力均明显提高,S期细胞比例显著增多,差异有统计学意义(P<0.05);GSEA和Western blot结果表明,KAT6A与Wnt/β-catenin信号通路密切相关,沉默KAT6A后,Wnt/β-catenin通路也受到显著抑制。结论 KAT6A在HCC组织和细胞中相比于癌旁组织和正常肝细胞,表达明显升高,且与病人的肿瘤直径、是否合并门静脉癌栓相关。通过调节Wnt/β-catenin通路,沉默KAT6A之后显著削减了肝癌细胞的增殖、侵袭和迁移能力。

Objective To explore the expression of lysine acetyltransferase 6 A(KAT6 A) in hepatocellular carcinoma(HCC), examine the relationship between its expression and clinicopathologic parameters and observe its effects on the proliferation, invasion and migration ability of hepatoma cell lines.Methods Clinical samples were collected from 40 HCC patients who underwent hepatectomy at Department of Hepatoblliary & Pancreatic Surgery, Zhongnan Hospital of Wuhan University from July 2018 to January 2020.Real-time quantitative polymerase chain reaction(qRT-PCR) and immunoblotting assay were performed for detecting the expression of KAT6 A in 40 pairs of HCC tumor and peritumor tissues, 9 hepatoma cell lines(SK-hep1, HepG2, Hep3 B, Huh-7, HCCLM3, HCCLM6, MHCC-97 H, MHCC-97 L & PLC/PRF/5) and normal hepatocyte line(THLE-2). Based upon the expression tendency in HCC cell lines, MHCC-97 H/Hep3 B was selected as the knockdown/overexpression model for cellular phenotypic assays. Small interfering RNAs(siRNAs) targeting KAT6 A or plasmid overexpressing KAT6 A were transfected. At 48 h, cell counting kit-8, EdU incorporation, Transwell and scratch assays and flow cytometry were performed respectively for detecting cellular proliferative capacity, invasiveness, migration ability and cell cycle progression. Single gene set enrichment analysis(GSEA) was implemented for predicting the signaling pathway of KAT6 A.Results RT-PCR and Western blot indicated that KAT6 A mRNA and protein expression were higher than in HCC tumor tissues those in peritumor tissues. Similarly, 9 hepatoma cell lines had significantly higher KAT6 A expression than normal hepatocyte line THLE-2 at mRNA levels. Clinical data analysis showed that KAT6 A mRNA expression was correlated with tumor volume and portal vein tumor thrombus. Cellular phenotypic assays indicated that proliferation, invasion and migration ability and G1/S transition became markedly repressed after KAT6 A silencing in MHCC-97 H cells while ectopic expression of KAT6 A exerted the opposite effects in Hep3 B cells;The results of GSEA suggested that genes enriched in high KAT6 A expression group were closely associated with the Wnt/β-catenin pathway. It was further confirmed by Western blot. Statistically, all differences were significant.Conclusion KAT6 A is remarkably up-regulated in HCC tissues and cells. An over-expression of KAT6 A is associated with tumor volume and portal vein tumor thrombus in HCC patients. Moreover, interfering with the expression of KAT6 A can repress the proliferation, invasion and migration of hepatoma cells through regulating Wnt/β-catenin signaling pathway.