Blockage Of The PI3k/Akt Signaling Pathway Reverses Sorafenib-derived Chemo-resistance In Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma was very common in the worldwide,and has high mortality rate.Liver cancer in North America almost reached 10 patients per 100000 patients,in Western Europe,Asia and Africa,reaching 100000 people have 50 to 150 patients.Liver is a tumor that is rich in blood vessels,and is prone to drug resistance.A stumbling block to the efficacy of chemotherapeutic agents in the production of multi drug resistance.Only in depth understanding of the molecular mechanisms of drug resistance in hepatocellular carcinoma,to identify drug targets,can effectively reverse the drug resistance,radical cure of liver cancer.Objective1.To establish sorafenib resistance cell model from human hepatocellular carcinoma(HCC)cell lines after long-term exposure to sorafenib and identify sorafenib resistance and study the characteristics of molecular and biology.2.To examine the changes of sorafenib resistance after treating resistant cells with PI3 k inhibitors LY294002 and study the association sorafenib with PI3k/Akt signaling pathway.3.To inhibit the PI3k/Akt signaling pathway and study the association of migration and invasion with PI3k/Akt signaling pathway.Methods1.HCC cell lines were exposed to sorafenib persistently.The morphological changes were detected under phase-contrast microscopy.CCK8 assay was used to examine drug sensitivity.Using gene chip to detect the differential expression of LncRNA and mRNA in hepatocellular carcinoma cells and parental cells.2.Real-time PCR was performed to test resisitance-associated expression(ABCB1ABCB1 ABCG2),and mesenchymal makers(Snail,Slug)in gene expression were alsoexamined.3.Testing the changes of protein expression on epithelial makers,mesenchymal makers,PI3k/Akt signaling pathway and ERK signaling pathway after long exposure to sorafenib in hepatocellular carcinoma through WB assay.4.Transwell assay was used to examine the changes of migration and invasion after long exposure to sorafenib in parental HCC cells and resistant sorafenib HCC cells.5.WB was performed to test the key protein expression level in PI3k/Akt signaling pathway and ERK signaling pathway after the PI3 k inhibitors LY294002 was added to resistant sorafenib HCC cells.6.The tail vein metastatic assay was used to study the metastatic changes of HCC cells after long exposure to sorafenib.Results1.Long-term exposure to sorafenib can induce resistance in HCC,and resistant cells show inhibition efficacy to sorafenib compared to parental cells.2.HCC cells underwent epithelial-to-mesenchymal transition(EMT)in morphology and molecular,and the tail vein metastatic assay showed resistant cells had enhanced metastatic ability.3.PI3k/Akt signaling pathway and ERK signaling pathway were activated after long-term exposure to sorafenib.4.LY294002 can reverse sorafenib resistance and weaken the ability of migration and invasion in sorafenib resistant HCC cell lines.Conclusion1.We successfully developed three sorafenib resistant HCC cell lines(HepG2R?Huh7R ? MHCC-97H-R).Sorafenib resistance can be associated with the activation of PI3k/Akt signaling pathway.2.HCC cell lines underwent epithelial-to-mesenchymal transition(EMT),which had enhanced migration and invasion can be associated with PI3k/Akt signaling pathway.3.The expression difference of LncRNA and mRNA in human hepatocellular carcinoma cells induced by long-term exposure to sorafenib.