肝受体同源物-1通过转录激活多药耐药基因1调控肝癌细胞对奥沙利铂的敏感性

Orphan nuclear receptor LRH-1 promotes oxaliplatin resistance in hepatocellular carcinoma cells by regulating MDR1 gene

目的探索肝受体同源物-1(the liver receptor homolog-1,LRH-1)在调控肝癌细胞对奥沙利铂敏感性过程中的功能和分子机制,为肝癌治疗提供新思路。方法在肝癌细胞系中构建低表达及过表达LRH-1的细胞模型,通过检测半抑制浓度(half maximal inhibitory concentration,IC_(50))、细胞增殖和平板克隆形成实验等功能学实验探索LRH-1在肝癌细胞中对奥沙利铂敏感性的影响;通过定量PCR检测LRH-1对多药耐药基因1(multidrug resistance gene 1,MDR1)基因的转录调控作用;通过荧光素酶报告实验评估LRH-1对MDR1的转录激活能力。结果在HuH7细胞中过表达LRH-1后,细胞在奥沙利铂处理下,IC_(50)显著升高,达到18.012μmol/L,显著高于HuH-7对照组的2.042μmol/L,差异有统计学意义(P<0.05);同时其细胞增殖能力显著增强,MDR1 mRNA表达水平明显升高。在HepG2细胞中敲低LRH-1表达后,细胞在奥沙利铂处理下,IC_(50)显著降低,为1.012μmol/L,显著低于HepG2对照组的6.294μmol/L,差异有统计学意义(P<0.05);同时其细胞增殖能力和平板克隆形成能力显著降低,MDR1 mRNA表达水平明显降低。荧光素酶报告实验证实,LRH-1可以剂量依赖性激活MDR1启动子的转录活性,其特异性小分子抑制物ML-180能显著降低LRH-1对MDR1启动子的转录激活能力。结论LRH-1通过转录激活MDR1降低肝癌细胞对奥沙利铂的敏感性,因其特异性小分子抑制剂已经合成成功,LRH-1可作为肝癌耐药治疗的潜在靶点。

Objective To investigate the function and molecular mechanism of LRH-1 in regulating the sensitivity of hepatocellular carcinoma(HCC)cells to oxaliplatin,providing new ideas for the treatment of liver cancer.Methods Knockdown and overexpression of LRH-1 in HCC cell lines were constructed,and the effect of LRH-1 on oxaliplatin resistance of HCC cells was explored by detecting IC_(50),cell proliferation,and plate colony formation assay.The transcriptional regulation of the MDR-1 gene by LRH-1 was detected through quantitative PCR.The transcriptional activation ability of LRH-1 on the MDR1 gene was evaluated by luciferase reporter assay.Results In HuH7 cells overexpressing LRH-1,the IC_(50) significantly increased to 18.012μmol/L under oxaliplatin treatment,significantly higher than the 2.042μmol/L in the HuH-7 control group,showing statistically significant differences(P<0.05).After overexpression of LRH-1 in HuH-7 cells,the cell proliferation ability was significantly increased,with a noticeable increase in MDR1 mRNA level.In HepG2 cells with knockdown LRH-1 expression,the IC_(50) significantly dropped to 1.012μmol/L,significantly lower than the 6.294μmol/L in the HepG2 control group,with statistically significant differences(P<0.05).After knockdown of LRH-1 in HepG2 cells,the cell proliferation and plate colony formation ability were significantly inhibited,with a notable decrease in MDR1 mRNA expression level.Luciferase reporter assay confirmed that LRH-1 can activate the transcriptional activity of the MDR1 promoter in a dosedependent manner,and its specific inhibitor ML-180 can significantly reduce LRH-1′s transcription activation ability on the MDR1 promoter.Conclusions LRH-1 may promote oxaliplatin resistance in hepatocellular carcinoma cells by regulating the transcriptional activity of MDR1 gene.Since its specific small molecule inhibitor has been successfully synthesized,LRH-1 can potentially become a target for the treatment of drug resistance in hepatocellular carcinoma.