Caveolin1 Reverse The Drug Resistance Of Colorectal Cancer Through OCTN2(SLC22A5)

Objective:1.To explore whether OCTN2 is involved in the process of drug resistance in colorectal cancer HCT15 cell line.2.Identify the relationship between OCTN2 and Cavl in drug-resistant cell lines and parent cell lines.3.Explore the role of OCTN2 in reversing drug resistance after Cavl is knocked down.4.Explore the mechanism of tumor drug resistance reversal after Cav1 is knocked down.Method:1.ADM induces recovery in the colon cancer HCT15/ADM resistant cell line that has been constructed in our laboratory in the early stage,so that it reaches the standard of high drug resistance,and CCK8 detects its drug resistance.2.Western Blot was used to detect the expression of Cavl and OCTN2 protein between HCT15 parent strain and MDR strain,and the result clear wether Cav1 and OCTN2 are involved in the mechanism of drug resistance in HCT15 cell line.3.There were three pairs of interference primers and one pair of negative control primer were designed.And then the interference plasmid of ShRNA-cav1 was constructed and the plamids were transfected into 293T cells,the supernatant was used to infect HCT15/ADM cells.The silencing efficiency of HCT15/ADM-shRNA3 was highest which were screened by RT-PCR and western blot.4.CCK8 was used to detect the proliferation inhibition rates before and after Cav1 knock-down,and calculates the multiple of reversal of drug resistance,at the same time western blot detect the expression of OCTN2 protein.5.CCK8 was used to detect and compare the optical density of HCT15/ADM cells under deferent concentrate of ADM incubation before and after meldonium treatment in Cav1 knock-down cells and control cells,and western blot detected the expression of Cavl and OCTN2 protein respectively.Result:1.The half maximal inhibitory concentration(IC50)under deferent concentrations gradient of ADM from 0 to 128ug/ml in HCT15/ADM MDR strains were significantly higher than the HCT15 parent strains,and the resistance index(RI)was 22.75>15.The expreesion of Cav1 in HCT15/ADM resistant strains were higher than HCT15 parent strains,and OCTN2(SLC22A5)in HCT15/ADM resistant strains ware lower than HCT15 parent strains.2.After knocking down the Cav1,the half maximal inhibitory concentration(IC50)was significantly lower than before with the reversal fold of 3.72,the expression of OCTN2(SLC22A5)protein was increased.The expression of Cav1 protein was increased respectively in HCT15 parent strains,HCT15/ADM MDR strains,HCT15/ADM-Vector cells,HCT15/ADM-shCav1 cells,in contrast the expression of OCTN2(SLC22A5)was reduced.3.The sensitivity to the ADM was reduced before and after treating with meldonium which was the competitive inhibitor of OCTN2 protein in HCT15/ADM-shCav1 cells,however the half maximal inhibitory concentration(IC50)was still lower than HCT15/ADM MDR stains.The expression of Cav1 was not affected.Conclusion:1.In the HCT15 colorectal cancer drug-resistant cell line,the expression of OCTN2 transporter is reduced,which is involved in the formation of its drug resistance.2.Cav1 was up-regulated in HCT15/ADM cell lines,and OCTN2 was down-regulated in HCT15/ADM cell lines.There is a negative correlation between the two proteins.3.After Cav1 knocking down,the drug resistance of tumor cells is reversed,and the expression of OCTN2 is up-regulated.4.The drug resistance was reversed by knocking down in HCT15/ADM cell lines through OCTN2(SLC22A5).