[关键词]
[摘要]
目的 探讨冬凌草甲素对人肝癌细胞Huh-7索拉非尼(Sor)耐药细胞株Huh-7/Sor的影响及其作用机制。方法 体外培养Huh-7/Sor细胞株,qRT-PCR检测miR-217和ABCC1 mRNA的表达水平,Western blotting检测ABCC1蛋白的表达水平;用冬凌草甲素处理Huh-7/Sor细胞后,CCK-8法检测细胞活力,Annexin V/PI染色检测细胞凋亡,Western blotting检测凋亡蛋白和ABCC1的表达水平;数据库预测miR-217与ABCC1的结合位点,转染miR-217 mimic或inhibitor后检测ABCC1的蛋白水平,荧光素酶报告基因检测ABCC1 3’UTR区域活性。结果 与Huh-7细胞相比,Huh-7/Sor细胞中miR-217的表达水平显著下调,ABCC1的表达水平显著上调(P<0.05);冬凌草甲素能抑制Huh-7/Sor细胞的增殖,促进细胞凋亡,上调miR-217的表达水平,抑制ABCC1的表达(P<0.05);miR-217能与ABCC1 3’UTR区域结合,抑制ABCC1的蛋白表达水平(P<0.05)。结论 冬凌草甲素能通过上调miR-217而抑制ABCC1的表达,进而抑制Huh-7/Sor细胞增殖,诱导凋亡。
[Key word]
[Abstract]
Objective To investigate the effect of oridonin on sorafenib resistant human hepatoma cell line Huh-7/Sor and its mechanism. Methods Huh-7/Sor cells were cultured in vitro. The mRNA expression levels of mir-217 and ABCC1 in Huh-7/Sor cells were detected by qRT-PCR and ABCC1 protein was detected by Western blotting. After treated with oridonin, the cell viability was detected by CCK-8 method, apoptosis was detected by Annexin V/PI staining, and the protein expression of apoptotic proteins and ABCC1 were detected by Western blotting. The binding sites of miR-217 and ABCC1 were predicted by database. The protein level of ABCC1 was detected by Western blotting after transfection with miR-217 mimic or inhibitor. The activity of ABCC1 3' UTR region was detected by luciferase reporter gene. Results Compared with Huh-7 cells, the expression level of miR-217 in Huh-7/Sor was down-regulated, and the expression level of ABCC1 was significantly up-regulated (P<0.05). Oridonin inhibited the proliferation of Huh-7/Sor cells and promoted apoptosis. The expression of miR-217 was up-regulated and the expression of ABCC1was down-regulated in Huh-7/Sor cells treated by Oridonin. miR-217 could bind with ABCC1 3' UTR region and inhibit the protein expression of ABCC1 (P<0.05). Conclusion Oridonin could inhibit the proliferation of Huh-7/Sor cells and induce apoptosis by up regulating the expression of ABCC1 and miR-217.
[中图分类号]
R285.5
[基金项目]