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  • 孙非凡,李东,李华,王涛,刘煜,张涛.程序性死亡配体1在索拉非尼耐药肝癌细胞中的表达和功能[J].第二军医大学学报,2018,39(2):152-158    [点击复制]
  • SUN Fei-fan,LI Dong,LI Hua,WANG Tao,LIU Yu,ZHANG Tao.Expression and role of programmed cell death ligand-1 in sorafenib-resistant hepatocellular carcinoma cells[J].Acad J Sec Mil Med Univ,2018,39(2):152-158   [点击复制]
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程序性死亡配体1在索拉非尼耐药肝癌细胞中的表达和功能
孙非凡1,李东2,李华2,王涛2,刘煜2,张涛2*
0
(1. 陆军军医大学(第三军医大学)研究生院, 重庆 400038;
2. 成都军区总医院肿瘤诊治中心, 成都 610083
*通信作者)
摘要:
目的 探讨程序性死亡配体1(PD-L1)在索拉非尼耐药人肝癌细胞中的表达及其功能。方法 采用浓度递增法构建索拉非尼耐药人肝癌细胞株(Hep3B-SR和HepG2-SR),用CCK-8法检测半数抑制浓度(IC50)并计算耐药指数,蛋白质印迹法和qPCR检测P-糖蛋白(P-gp)、多药耐药相关蛋白1(MRP1)和PD-L1的表达。转染siRNA沉默耐药细胞PD-L1的表达并检测沉默效率。沉默PD-L1表达后,CCK-8法检测IC50、计算耐药指数,并检测耐药基因的表达,用细胞增殖实验检测细胞增殖情况,划痕实验检测细胞迁移情况,平板克隆形成实验检测细胞克隆形成情况,流式细胞术检测细胞凋亡情况。结果 Hep3B-SR和HepG2-SR的耐药指数分别为5.4和5.2。耐药细胞中P-gp、MRP1和PD-L1表达较亲本细胞上调(P均<0.01)。沉默PD-L1表达后,Hep3B-SR和HepG2-SR的耐药指数分别下降为1.8和1.5,P-gp和MRP1的表达下调(P均<0.01);沉默PD-L1表达可抑制耐药细胞的增殖、迁移、克隆形成并促进其凋亡(P均<0.01),合用索拉非尼时作用更为显著。结论 PD-L1在索拉非尼耐药人肝癌细胞中高表达;抑制PD-L1表达可部分逆转索拉非尼耐药人肝癌细胞的耐药性,提高索拉非尼的抗癌效果;抑制PD-L1表达可有效抑制索拉非尼耐药人肝癌细胞的生长,合用索拉非尼其抗癌效果更强。
关键词:  肝细胞癌  程序性死亡配体1  索拉非尼  耐药性  基因沉默
DOI:10.16781/j.0258-879x.2018.02.0152
投稿时间:2017-11-16修订日期:2017-12-25
基金项目:国家自然科学基金(81101634),四川省科技厅科技支撑计划项目(2015FZ0073),四川省科技厅重点研发项目(2017SZ0066),四川省卫计委科研课题(16PJ026).
Expression and role of programmed cell death ligand-1 in sorafenib-resistant hepatocellular carcinoma cells
SUN Fei-fan1,LI Dong2,LI Hua2,WANG Tao2,LIU Yu2,ZHANG Tao2*
(1. Graduate School, Army Medical University(Third Military Medical University), Chongqing 400038, China;
2. Cancer Center, Chengdu Military General Hospital, Chengdu 610083, Sichuan, China
*Corresponding author)
Abstract:
Objective To explore the expression and role of programmed cell death ligand-1 (PD-L1) in sorafenib-resistant human hepatocellular carcinoma cells. Methods Sorafenib-resistant human hepatocellular carcinoma cell lines (Hep3B-SR, HepG2-SR) were established by the procedure of stepwise increase in sorafenib concentrations. CCK-8 assay was used to detect half inhibition concentration (IC50). The expressions of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and PD-L1 were examined by Western blotting and qPCR. The expression of PD-L1 was silenced by transfecting siRNA into the drug-resistant cells, and the silencing efficiency was tested. After silencing the expression of PD-L1 in the drug-resistant cells, CCK-8 assay was used to detect IC50, the drug-resistance index was calculated, and the expressions of P-gp and MRP1 were examined. Then cell proliferation assay, wound healing assay, colony formation assay and flow cytometry were applied to examine cell proliferation, migration, clone formation and apoptosis, respectively. Results The drug-resistance indexes of Hep3B-SR and HepG2-SR were 5.4 and 5.2, respectively. The expressions of P-gp, MRP1 and PD-L1 in the drug-resistant cells were significantly up-regulated in comparison with the parental cells (all P<0.01). After inhibiting the expression of PD-L1, the drug-resistance indexes of Hep3B-SR and HepG2-SR decreased to 1.8 and 1.5, respectively, and the expressions of P-gp and MRP1 were down-regulated (all P<0.01). Silencing the PD-L1 expression could significantly inhibit the proliferation, migration and colony formation of drug-resistant cells, and could significantly promote the apoptosis (all P<0.01); and these effects were strengthened by combining sorafenib. Conclusion PD-L1 is highly expressed in sorafenib-resistant hepatocellular carcinoma cells. Inhibition of PD-L1 expression can partially reverse the drug-resistance of the cells and significantly enhance the anticancer effect of sorafenib. Inhibiting the expression of PD-L1 can effectively repress the growth of sorafenib-resistant hepatocellular carcinoma cells, which is more if combining with sorafenib.
Key words:  liver cell carcinoma  programmed cell death ligand-1  sorafenib  drug resistance  gene silencing