Molecular Mechanism Of Sorafenib- Resistant Liver Cancer Cells Based On P70S6K1 Target

Objective:To explore whether p70S6K1 is involved in the process of resistance to sorafenib in hepatocellular carcinoma,and to study the molecular mechanism of p70S6K1 on hepatocarcinoma cells after sorafenib-induced drug resistance,thereby p70S6K1 is identified as a potential therapeutic target for drug-resistant liver cancer,enriching the treatment of liver cancer.Methods:1.92 cases of surgical specimens and case data of liver cancer patients were collected,and their clinical biochemical test indexes were analyzed.20 in90 cases were fixed,taken,embedded,and prepared by 10% formalin.After conventional HE staining,typical wax blocks were selected.Immunohistochemical staining was used to detect the expression of endogenous p70S6K1,TOPO II,TP53,and Ki-67 protein in drug-resistant hepatocytes.Furthermore,the expression of endogenous p70S6K1 mRNAs in drug-resistant hepatocytes was detected by RT-PCR in liver cancer and adjacent tissues.2.Sorafenib-resistant HepG2 cell lines were constructed.The expression ofp70S6K1 mRNAs in sorafenib-resistant HepG2 cells was detected by real-time PCR.Furthermore,CCK-8 cell proliferation method and immunofluorescence staining method were used to investigate cell growth and proliferation,and then the effect of drug resistance on cell survival was evaluated.3.The shRNA technology was used to knock down p70S6K1,and the proliferation of p70S6K1 against drug-resistant hepatoma cells was further verified by transfection and cloning experiments.Then,the expression characteristics of PGP and target proteins p70S6K1,mTOR and MDM2 in p70S6K1 knockdown group and control group were detected by immunofluorescence,so as to find out the mechanism of action of p70S6K1 in sorafenib-resistant liver cancer cells.Results:1.Compared with non-cancerous tissue samples,the expression of p70S6K1 and specific resistance protein TOPO II in sorafenib-resistant liver cancer specimens was significantly up-regulated and proliferating protein Ki-67 protein was increased,while TP53 levels were decreased.The intracellular expression of p70S6K1 localized/quantitative protein was consistent with the up-regulation of p70S6K1 mRNA,which prompting the potential clinical application value of p70S6K1.2.The results of CCK-8 and immunofluorescence staining showed that PCNA cell proliferation protein was increased in drug-resistant liver cancer cells.In addition,compared with the control,the PGP resistance protein in sorafenib-resistant hepatoma cells increased significantly and was time-dependent.The results of real-time PCR showed that the content of p70S6K1 mRNA was up-regulated in sorafenib-resistant liver cancer cells.3.After transfection,the experimental data showed that theshRNA-specific knockdown of p70S6K1 significantly reduced the number of resistant hepatocarcinoma cells and blocked cell growth.Meanwhile,the expression of endogenous PCNA protein was significantly decreased after drug-resistant hepatoma cells knocked down p70S6K1.In addition,immunofluorescence staining analysis showed that the expression of PGP,mTOR,MDM2 and other proteins in drug-resistant hepatoma cells was significantly down-regulated after knocking down p70S6K1.It is suggested that p70S6K1 may be a molecular target for the treatment of potential drug-resistant liver cancer.Conclusion:1.p70S6K1 is highly expressed in sorafenib-resistant liver cancer tissues and cells;2.Knocking down p70S6K1 can increase the sensitivity of liver cancer cells to sorafenib;3.It is revealed that p70S6K1 negatively regulates TP53 by activating the mTOR/MDM2 pathway,thereby inducing the molecular mechanism of sorafenib resistance.