造血细胞激酶在肝癌患者癌组织中的表达以及对肝癌细胞株增殖和凋亡的影响

Expression of hematopoietic cell kinase in hepatocellular carcinoma and its effect on proliferation and apoptosis of hepatoma cells

目的探讨造血细胞激酶(HCK)在肝癌患者癌组织中的表达以及对肝癌细胞株增殖和凋亡的影响。方法选取中国人民解放军北部战区总医院2015—2019年收治的80例肝癌患者和既往体健的80例肝外伤破裂患者,收集其手术切除后的液氮冻存组织标本。人肝癌细胞系HepG2细胞培养传代后分为3组,空载体组转染HCK阴性对照质粒,HCK过表达组转染HCK过表达的质粒,HCK+LY2109761组转染采用转化生长因子(TGF)信号通路特异性抑制剂LY2109761处理的HCK过表达质粒。应用实时荧光定量聚合酶链反应检测HCK mRNA的表达,蛋白质印迹法检测HCK蛋白和TGF-β_(1)信号通路相关指标的表达,酶联免疫吸附试验法检测TGF-β_(1)水平,细胞计数法检测细胞增殖情况,应用流式细胞仪检测细胞凋亡情况,Edu实验观察细胞Edu阳性情况,Transwell实验检测细胞侵袭能力。实验均重复3次。结果肝癌组织中HCK mRNA和蛋白表达水平均高于正常肝组织[(2.48±0.42)比(1.00±0.08)、(0.68±0.14)比(0.37±0.09)],HCK过表达组HCK mRNA表达水平高于空载体组[(2.26±0.31)比(0.96±0.05)](均P<0.05)。HCK过表达组TGF-β_(1)水平和TGF-β_(1)、磷酸化母亲信号蛋白同源物通路2蛋白水平均高于空载体组(均P<0.05)。培养72、96、120 h后,HCK过表达组细胞存活率均高于空载体组,而HCK+LY2109761组均低于HCK过表达组(均P<0.05)。HCK过表达组细胞凋亡率低于空载体组,Edu阳性率高于空载体组,细胞侵袭数多于空载体组(均P<0.05);HCK+LY2109761组细胞凋亡率高于HCK过表达组[(9.65±0.15)%比(1.96±0.08)%],Edu阳性率低于HCK过表达组,细胞侵袭数少于HCK过表达组[(44±6)%比(67±7)%、(120±10)个比(276±17)个](均P<0.05)。结论HCK在肝癌组织和细胞中均呈高表达,HCK可能通过激活TGF-β_(1)信号通路促进肝癌细胞的增殖、侵袭,抑制其凋亡。

Objective To investigate the expression of hematopoietic cell kinase(HCK)in hepatocellular carcinoma and its effect on proliferation and apoptosis of hepatoma cells.Methods The clinical data of 80 patients with hepatocellular carcinoma and 20 patients with liver trauma rupture admitted to General Hospital of Northern Theater Command of Chinese People′s Liberation Army from 2015 to 2019 were retrospectively analyzed.The liquid nitrogen frozen tissue samples after surgical resection were collected.Human hepatoma cell line HepG2 was divided into 3 groups after culture and passage.The empty body group was transfected with HCK negative control plasmid,the HCK overexpression group was transfected with HCK overexpression plasmid,and the HCK+LY2109761 group was transfected with HCK overexpression plasmid treated with LY2109761,a specific inhibitor of transforming growth factor(TGF)signaling pathway.The expression of HCK mRNA was detected by real-time fluorescence quantitative polymerase chain reaction,HCK protein and TGF-β_(1)signaling pathway indicators were detected by western blotting,and the expression level of TGF-β_(1)was detected by enzyme-linked immunosorbent assay.Cell proliferation was detected by cell counting method,apoptosis was detected by flow cytometry,Edu positive cells were observed by Edu test,and cell invasion was detected by Transwell test.The experiment was repeated 3 times.Results The expression levels of HCK mRNA and protein in hepatocellular carcinoma tissues were higher than those in normal liver tissues[(2.48±0.42)vs(1.00±0.08),(0.68±0.14)vs(0.37±0.09)],and the expression level of HCK mRNA in HCK overexpression group was higher than that in empty body group[(2.26±0.31)vs(0.96±0.05)](all P<0.05).Serum levels of TGF-β_(1),TGF-β_(1)protein and phosphorylated Smad2 protein were higher in HCK overexpression group than those in empty body group(all P<0.05).After 72,96 and 120 h of culture,the cell survival rates in HCK overexpression group were higher than those in empty body group,and the cell survival rates in HCK+LY2109761 group were lower than those in HCK overexpression group(all P<0.05).The apoptosis rate in HCK overexpression group was lower than that in empty body group,and the positive rate of Edu and the number of cell invasion were higher than those in empty body group(all P<0.05);the apoptosis rate in HCK+LY2109761 group was higher than that of HCK overexpression group[(9.65±0.15)%vs(1.96±0.08)%],and the positive rate of Edu and the number of cell invasion were lower than those in HCK overexpression group[(44±6)%vs(67±7)%,(120±10)vs(276±17)](all P<0.05).Conclusions HCK is highly expressed in hepatocellular carcinoma tissues and cells.HCK may activate TGF-β_(1)signal pathway to promote the proliferation and invasion of hepatoma cells and inhibits their apoptosis.