HDAC6对HepG2细胞增殖与转移潜能影响机制研究

Effect and mechanism of HDAC6 on proliferation and metastasis ability of HepG2 Cell

目的肝癌相关基因突变的不断积累会导致肿瘤的发生,肝癌相关基因突变也涉及到细胞增殖、凋亡等的改变。组蛋白脱乙酰基酶6(histone deacetylase 6,HDAC6)参与肿瘤的发生与发展,本研究探讨HDAC6过表达质粒转染肝癌细胞株HepG2对肝癌细胞增殖、侵袭、迁移及凋亡的影响。方法将人肝癌细胞株HepG2(购自中国科学院上海生命科学研究院)分为空白组、阴性对照组和过表达组(P3-HDAC6组),空白组予以常规培养,阴性对照组和P3-HDAC6组则分别转染空质粒和过表达质粒P3。运用蛋白质印迹法检测HDAC6、过氧化物酶(PrxⅡ)蛋白及上皮-间质转化(epithdlid-meserchymal transition,EMT)相关蛋白E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)在肝癌细胞株中的表达量变化,运用细胞增殖实验(cell counting kit8,CCK8)和Transwell实验检测HepG2细胞的增殖、迁移及侵袭能力;流式细胞仪检测各组HepG2凋亡的变化情况。结果蛋白质印迹法检测结果提示,HDAC6在HepG2细胞株中表达量下调,HDAC6表达量在P3-HDAC6组与阴性对照组分别为2.761±0.304和1.037±0.249,F=46.410,P<0.001;其下游作用蛋白PrxⅡ表达分别为1.026±0.183和1.842±0.229,F=18.532,P=0.003;E-cadherin在HepG2细胞株中表达上调,其表达量在P3-HDAC6组与阴性对照组分别为1.858±0.293和0.924±0.211,F=16.745,P=0.004;N-cadherin、Vimentin在HepG2细胞株中表达下调,其表达量在P3-HDAC6组为0.854±0.193和1.565±0.286,在阴性对照组分别为1.315±0.204和2.011±0.356,两组比较差异均有统计学意义(F=8.120,P=0.020;F=8.114,P=0.020)。CCK-8实验表明P3-HDAC6组2.137±0.228的HepG2细胞增殖能力较空白组(2.508±0.169)和阴性对照组(2.451±0.225)降低,差异有统计学意义,F=9.479,P=0.014;Transwell实验则提示,HDAC6能够减弱肝癌细胞的迁移、侵袭能力,P3-HDAC6组(80.67±6.028)的HepG2细胞迁移能力较空白组(234.3±5.963)及阴性对照组(198.0±7.732)降低,差异有统计学意义,F=44.188,P<0.001;P3-HDAC6组(79.33±7.202)的HepG2细胞侵袭能力较空白组(158.3±5.764)及阴性对照组(137.7±4.453)降低,差异有统计学意义,F=46.807,P<0.001。细胞凋亡实验显示,P3-HDAC6组〔(40.77±1.123)%〕与空白组〔(17.67±0.602)%〕及阴性对照组〔(19.13±0.378)%〕细胞凋亡率增加;差异有统计学意义,F=834.361,P<0.001。结论 HDAC6过表达通过调节PrxⅡ,降低使其氧化还原酶活性,从而抑制肝癌细胞株HepG2的增殖能力,降低迁移和侵袭水平,促进凋亡。

OBJECTIVE The accumulation of cancer-related gene mutation can cause liver cancer occurrence.Cancer-related gene mutation is involved in the change of cell proliferation, apoptosis,and so on.Histone deacetylase 6 (HDAC6) is involved in tumorigenesis and development.The aim of this study was to observe the effect of recombinant plasmid expressing histone deacetylase 6( HDAC6) on the expression of Peroxidase Ⅱ(Prx Ⅱ), to investigate the effect of the Prx Ⅱ pathway on the proliferation, invasion, migration and apoptosis of hepatocellular carcinoma( HCC) cells.METHODS The human hepatoma cell line HepG2 was divided into three groups.The blank group(Blank) was routinely cultured.The negative control group(NC) and the overexpression group(P3-HDAC6) were transfected with empty plasmid and overexpression plasmid P3-HDAC6, respectively.The expression changes of HDAC6,Prx Ⅱ,and epithelial-mesenchymal transition(EMT)-related proteins including E-cadherin,N-cadherin,and vimentin were detected by western blot assay in hepatocellular carcinoma cell lines.The proliferation was evaluated by cell proliferation assay(CCK8) and the migration and invasion abilities of HepG2 cells were assessed by Transwell assay.The apoptosis rates of HepG2 cells were detected by flow cytometry.RESULTS Western blot analysis results suggested that the expression of HDAC6 in HepG2 cells was down-regulated.The expression levels of HD AC 6 in P3-HDAC6 group and NC group were 2.761±0.304 and 1.037±0.249, respectively (F=46.410,P<0.001).The expression levels of downstream functional protein Prx Ⅱ were 1.026±0.183 and 1.842±0.229,respectively (F=1.532,P=0.003).E-cadherin was up-regulated in HepG2 cells,and the expression levels of E-cadherin in P3-HDAC6 group and NC group were 1.858±0.293 and 0.924± 0.211, respectively (F=16.745,P=0.004).N-cadherin and Vimentin were down-regulated in HepG2 cells.The expression levels of N-cadherin and Vimentin in P3-HDAC6 group (0.854±0.193 and 1.565±0.286) and in NC group (1.315±0.204 and 2.011±0.356) were statistically significant (F=8.120,P=0.020;F=8.114,P=0.020).CCK8 assay indicated the proliferation ability in P3-HDAC6 group (2.137±0.228) was decreased compared with that in Blank group (2.508±0.169) or NC group (2.451±0.225),and the difference was statistically significant (F=9.479, P=0.014).Transwell assay suggested that HDAC6 suppressed HCC cell invasion and migration abilities.The migration ability in P3-HDAC6 group (80.67±6.028) was decreased compared with that in blank group (234.3±5.963) or NC group (198.0±7.732), and the difference was statistically significant (F=44.188, P<0.001).The invasion ability in P3-HDAC6 group (79.33±7.202) was decreased compared with that in blank group (158.3±5.764) or NC group (137.7±4.453),and the difference was statistically significant (F=46.807, P<0.001).Cell apoptosis assay showed that the apoptosis rate in P3-HDAC6 group (40.77±1.123)% was increased compared with that in blank group (17.67±0.602)% or NC group (19.13±0.378)%,and the difference was statistically significant (F=834.361,P<0.001).CONCLUSION HDAC6 inhibits the proliferation.repressed the migration and invasion abilities, a nd in duced apoptosis of HCC cell line HepG2 by down-regulating Prx U expression.