Molecular Mechanism Of Sorafenib Resistance In Hepatocellular Carcinoma

Objective:This study aims to explore the molecular mechanism of sorafenib resistance,and look for molecular markers to predict the efficacy of sorafenib in order to improve the sensitivity and overcome the resistance for better treatment of hepatocellular carcinoma patients.Methods:1.Establish sorafenib-resistance HepG2 cell model and screen resistant related genes by Affymetrix expression profile chip.2.Detect the expression of resistant related genes in sorafenib-resistance HepG2 cells and high celigo select the key resistant related genes affecting cell proliferation.3.Constructed the RNAi lentivirus plasmid of RPL28 gene,Real time qPCR and Western Blot were used to detect the efficiency of knock down for RPL28 gene.At the same time,Celigo,Caspase3/7 and flow cytometry were used to observe the biological behavior changes of sorafenib-resistance HepG2 cells which was infected by RNAi lentivirus,including the variation of cell proliferation,apoptosis and cell cycle in order to explore the role of RPL28 on resistance to sorafenib in hepatocellular carcinoma and the correlative cytological mechanism.4.Establish a rat model of Morris Hepatoma to study sorafenib resistance in vivo.Real time qPCR and Immunohistochemistry were used to test the expression change of RPL28 gene with the sensitive and resistant samples of sorafenib.Meanwhile,functional proteins changes were detected by Immunohistochemistry.All these were to further prove RPL28 gene to be key resistant related gene of sorafenib..Result:1.Sorafenib-resistance HepG2 cell model has been established successfully by the method of increasing concentration of sorafenib,IC₅₀0 was 9.988?M and resistance index?RI?was 5.97.Then,35 resistant related gene were selected and speculated to regulate FXR/RXR?Liver X receptor/Retinoid X receptor?activation?p53 signaling pathways,and played the role of accumulation of blood cells,coagulation and lipid metabolism and so on.2.We detected the expression of 35 resistant related genes in sorafenib-resistance HepG2 cells by the technology of Real time qPCR,and the result showed 32 genes among them had the constitutive higher expression level.Secondly,we knocked down20 genes randomly to observe their influence on cell proliferation.By this way,RPL28and MAP4K3 gene were selected preliminarily as the key resistant related genes.Thirdly,in order to further confirmed specific targets of these two genes affecting cell proliferation,the plasmids of three RNAi targets were packaged into lentivirus and done high celigo selection.Finally,the efficiency of down regulated gene expression by each single target was tested using Real time qPCR.We chose RPL28-1 target for follow-up study of cellular function because this target had most significant inhibition of cell proliferation?2.76?and efficiency of knock-down genes?86.1%?,compared with the controls.3.With RPL28-1 target as the template,we constructed the RNAi lentivirus plasmid and identified by PCR and sequencing.This RNAi lentivirus plasmid was packaged and infected sorafenib-resistance HepG2 cells.The efficiency of infection was up to80%.Then,we tested the efficiency of knock-down RPL28-1 gene by Real time qPCR and reduction of exogenous protein expression by Western Blot,the results both suggested knocking out RPL28-1 had the effect on the reduction of RPL-28 protein expression,which meant RPL28-1 was an effective RNAi target.Using the technology of celigo,we found the proliferation of sorafenib-resistance HepG2 cells was inhibited after knocking out RPL28-1.Meanwhile,the apoptosis of sorafenib-resistance HepG2cells increased with the method of Caspase3/7 and flow cytometry.Cell cycle analysis showed sorafenib-resistance HepG2 cells increased in S phase,no significant change in G2/M phase,and reduced in G1 phase.4.ACI rat model of Hepatocellular Carcinoma in situ was established to research sorafenib resistance in vivo by Morris hepatoma 3924A cell line.The results of Real time qPCR and Immunohistochemistry indicated the expression of RPL28 gene increased in sorafenib resistant sample,compared with the samples sensitive to sorafenib.In addition,Immunohistochemical results also showed that the expression of Ki-67 and CDK4 were higher in the samples sensitive to sorafenib than that of resistance to sorafenib.All these further verified RPL28 gene as the key gene related to sorafenib resistance.Conclusion:MAP4K3 and PRL28 are the key genes of resistance to sorafenib.And RPL28 gene lead to resistance to sorafenib in hepatocellular carcinoma by the way of proliferation,apoptosis and the regulation of cell cycle.