To Explore The Process Of Nuclear Receptor LRH-1 In Oxaliplatin Resistence Of HCC By Induction MDR1 Expression

Background:Oxaliplatin(OXA)was approved for chemotherapy of liver cancer in 2013,which is the first approved chemotherapy drug for liver cancer in China and even in the world.Oxaliplatin is a platinum-based cytotoxic drug,which has been widely used in Phase Ⅱ and Ⅲ hepatocellular carcinoma research trials.However,liver cancer patients will also develop different degrees of drug resistance when they receive oxaliplatin or oxaliplatin based combination drugs.Therefore,it is an urgent scientific problem to comprehensively study the molecular mechanism of oxaliplatin resistance of HCC cells,to guide the clinical development of more rational drug use regimens.Objective:Our previous study found that when oxaliplatin was used to treat hepatoma cells,the expression of LRH-1 and MDR1 was increased,while the LRH-1 specific antagonist ML-180 could reverse the MDR1 expression.Therefore,this study mainly focused on the molecular level to evaluate whether LRH-1 as a transcription factor has a transcriptional activation effect on MDR1 through dual luciferase reporting experiment,and to find the expression of genes related to oxaliplatin resistance through microarray biotechnological analysis.Thus,the regulatory mechanism of LRH-1 on MDR1 was analyzed,and the molecular mechanism of oxaliplatin resistance of LRH-1 was partially elucidated.Methods and contents1.After oxaliplatin treatment in HepG2LRH-1-and HepG2 cells,the influence of oxaliplatin on the cloning ability of hepatoma cells with different LRH-1 expression levels was evaluated by plate clone formation experiment,and the addition of ML-180 was used to evaluate whether the sensitivity of oxaliplatin to hepatoma cells could be increased.2.The migration ability of different HCC cell lines treated with oxaliplatin and ML-180 was detected by scratch test。3.CCK8 proliferation assay was used to evaluate the proliferation ability of different cell lines under different treatment conditions of oxaliplatin and ML-180,and IC50 value was calculated.4.The mRNA expression of MDR1 in HepG2 and HepG2LRH-1+ cells was detected by qPCR.5.The luciferase reporting assay was designed in HEK-293 and HepG2 cells to evaluate the transcriptional activation ability of LRH-1 on MDR1.6.Total RNA was extracted from HepG2LRH-1-and HepG2 cell lines for microarray bioinformation analysis,so as to find the expression of differential genes and genes related to drug resistance.Results1.The results of plate clone formation assay showed that ML-180 significantly reduced the clone formation rate of HepG2LRH-1-and HepG2 hepatoma cells compared with the oxaliplatin treated group.In addition,HepG2LRH-1-cells had a lower rate of clone formation than HepG2 cells under the same treatment conditions.2.The scratch test results showed that the scratch distance was detected in HepG2LRH-1-and HepG2 cells cultured without serum for 0h,4h,12h and 24h respectively,and it was found that the migration rate of the ML-180 treatment group was slower than that of the oxaliplatin treatment group.3.The results of scratch experiment showed that HepG2 cells migrated faster than HepG2LRH-1-cells.4.CCK8 results showed that,compared with HepG2 cells,oxaliplatin significantly reduced the proliferation ability of HepG2LRH-1-cells,and ML-180 increased the sensitivity of HepG2LRH-1-cells to oxaliplatin.5.CCK8 assay results showed that,compared with normal HepG2 cells,the inhibition rate of HepG2LRH-1-cells increased significantly with the increase of oxaliplatin and ML-180 concentrations.6.qPCR results showed that,compared with HepG2 cells,the mRNA expression of MDR1 in HepG2 cells overexpressed by LRH-1 was significantly increased.7.The results of double enzyme digestion and sequencing showed that the MDRl-Luc luciferase reporter plasmid was successfully constructed.8.The luciferase report assay showed that LRH-1 could dose-dependent activate the transcriptional activity of the MDR1 promoter,and its specific small molecule inhibitor ML-180 could significantly reduce the transcriptional activation ability of the MDR1 promoter.9.The luciferase report experiment results showed that luciferase activity was decreased in HepG2 cells with low LRH-1 expression.Luciferase activity was increased in HepG2 cells with high LRH-1 expression.In addition,ML-180 could decrease luciferase activity in HepG2 cells with high LRH-1 expression.10.The gene expression microarray Analysis revealed that there were 15 down-regulated genes related to drug resistance of liver cancer,namely ABCB1,ABCB4,ABCG1,ABCC13,RP11-374A4.1,RP11-6N13.1,RP11-231G15.1,RP11-143A12.3,CTD-2280E9.1,CTD-2193P3,RP11-392B6.1,HOXC-AS1,LINC01004,LINC01011 and LINC01271.Conclusion:LRH-1 may be involved in the mechanism of drug resistance in HCC cells by regulating the transcriptional activity of MDR1.Since its small molecule specific inhibitor ML-180 has been synthesized,LRH-1 may be a potential therapeutic target for the treatment of oxaliplatin-resistant HCC.