CXC趋化因子配体10对肝细胞癌SMMC-7721细胞增殖和迁移的影响及其机制

Effect of CXC chemokine ligand 10 on proliferation and migration of hepatocellular carcinoma SMMC-7721 cells and its mechanism

目的:探讨外源性CXC趋化因子配体10 (CXCL10)对肝细胞癌(HCC) SMMC-7721细胞增殖和迁移的影响,并阐明其作用机制。方法:按照CXCL10作用浓度,将人HCC SMMC-7721细胞分为0 mg·L^(-1)CXCL10组、10 mg·L^(-1)CXCL10组和30 mg·L^(-1)CXCL10组。上述部分细胞给予细胞外调节蛋白激酶(ERK)抑制剂PD98059 (80μmol·L^(-1))后,将SMMC-7721细胞分为0 mg·L^(-1)CXCL10+PD98059组、 10mg·L^(-1)CXCL10+PD98059组和30mg·L^(-1)CXCL10+PD98059组。CCK-8法检测各组SMMC-7721细胞增殖率,EdU法检测各组SMMC-7721细胞中EdU阳性表达率,Transwell小室实验检测各组SMMC-7721细胞迁移率,Western blotting法检测各组SMMC-7721细胞中ERK、磷酸化ERK (p-ERK)和细胞周期蛋白D1 (Cyclin D1)蛋白表达水平。结果:CCK-8法检测,培养24h后,与0mg·L^(-1)CXCL10组比较,10mg·L^(-1)CXCL10和30mg·L^(-1)CXCL10组SMMC-7721细胞增殖率升高(P<0.05或P<0.01)。EdU法检测,与0mg·L^(-1)CXCL10组比较,10mg·L^(-1)CXCL10和30mg·L^(-1)CXCL10组SMMC-7721细胞中EdU阳性表达率升高(P<0.01);Transwell小室实验检测,培养48 h后,与0 mg·L^(-1)CXCL10组比较,10 mg·L^(-1)CXCL10和30 mg·L^(-1)CXCL10组SMMC-7721细胞迁移率升高(P<0.01)。Western blotting法检测,细胞培养24 h后,采用CXCL10溶液处理24h,与0mg·L^(-1)CXCL10组比较,10mg·L^(-1)CXCL10组和30 mg·L^(-1)CXCL10组SMMC-7721细胞中ERK、p-ERK及Cyclin D1蛋白表达水平升高(P<0.01)。CCK-8法检测,加入ERK抑制剂PD98059后,与0 mg·L^(-1)CXCL10组比较,10 mg·L^(-1)CXCL10+PD98059组和30 mg·L^(-1)CXCL10+PD98059组SMMC-7721细胞增殖率降低(P<0.05);与10 mg·L^(-1)CXCL10组比较,10 mg·L^(-1)CXCL10+PD98059组SMMC-7721细胞增殖率降低(P<0.05);与30 mg·L^(-1)CXCL10组比较,30 mg·L^(-1)CXCL10+PD98059组SMMC-7721细胞增殖率降低(P<0.05)。结论:CXCL10能够促进HCCSMMC-7721细胞增殖和迁移,其作用机制与上调ERK/p-ERK/Cyclin D1通路蛋白表达有关。

Objective:To discuss the effect of exogenous CXC chemokine ligand 10(CXCL10)on the proliferation and migration of the hepatocellular carcinoma(HCC)SMMC-7721 cells,and to clarify its mechanism.Methods:The human HCC SMMC-7721 cells were divided into 0 mg·L^(-1) CXCL10 group,10 mg·L^(-1) CXCL10 group,and 30 mg·L^(-1) CXCL10 group according to the CXCL10 concentration.Some of the above cells were treated with extracellular regulated protein kinase(ERK)inhibitor PD98059(80μmol·L^(-1)),then the SMMC-7721 cells were divided into 0 mg·L^(-1) CXCL10+PD98059 group,10 mg·L^(-1) CXCL10+PD98059 group,and 30 mg·L^(-1) CXCL10+PD98059 group.The proliferation rates of the SMMC-7721 cells in various groups were detected by CCK-8 method;the EdU positive expression rates in SMMC-7721 cells in various groups were detected by EdU method;the migration rates of the SMMC-7721 cells in various groups were detected by Transwell chamber assay;the expression levels of ERK,phosphorylated ERK(p-ERK),and Cyclin D1 proteins in the SMMC-7721 cells in various groups were detected by Western blotting method.Results:The CCK-8 results showed that after cultured for 24 h,compared with 0 mg·L^(-1) CXCL10 group,the proliferation rates of the cells in 10 mg·L^(-1) CXCL10 group and 30 mg·L^(-1) CXCL10 group were increased(P<0.05 or P<0.01);the EdU detection results showed that compared with 0 mg·L^(-1) CXCL10 group,the positive expression rates of EdU in the cells in 10 mg·L^(-1) CXCL10 group and 30 mg·L^(-1) CXCL10 group were increased(P<0.01).The Transwell chamber assay results showed that after cultured for 48 h,compared with 0 mg·L^(-1) CXCL10 group,the migration rates of the cells in 10 mg·L^(-1) CXCL10 group and 30 mg·L^(-1) CXCL10 group were increased(P<0.01).The Western blotting results showed that after cultured for 24 h and treated with CXCL10 for 24 h,compared with 0 mg·L^(-1) CXCL10 group,the expression levels of ERK,p-ERK,and Cyclin D1 proteins in the SMMC-7721 cells in 10 mg·L^(-1) CXCL10 group and 30 mg·L^(-1) CXCL10 group were increased(P<0.05).The CCK-8 method results showed that after treated with ERK inhibitor PD98059,compared with 0 mg·L^(-1) CXCL10 group,the proliferation rates of the SMMC-7721 cells in 10 mg·L^(-1) CXCL10+PD98059 group and 30 mg·L^(-1) CXCL10+PD98059 group were decreased(P<0.05);compared with 10 mg·L^(-1) CXCL10 group,the proliferation rate of the SMMC-7721 cells in 10 mg·L^(-1) CXCL10+PD98059 group was decreased(P<0.05);compared with 30 mg·L^(-1) CXCL10 group,the proliferation rate of the SMMC-7721 cells in 30 mg·L^(-1) CXCL10+PD98059 group was decreased(P<0.05).Conclusion:CXCL10 can promote the proliferation and migration of the HCC SMMC-7721 cells,and its mechanism is mainly related to the up-regulation of the expressions of ERK/p-ERK/Cyclin D1 pathway proteins.