| Objective: To explore the effect of LncRNA H19 on multidrug resistance in chemotherapy and the new mechanism of related pathways and oxymatrine in reversing the drug resistance of liver cancer.Methods:(1)Methyl Thiazolyl Tetrazolium(MTT)assay was used to detect the sensitivity of HepG2/ADM to 5-Fu,ADM and CDDP.(2)Using lentiviral construction and plasmid transfection technology to inhibit and enhance the expression of H19 in cells,detect the sensitivity of chemotherapeutic drugs in HepG2/ADM and HepG2 cells,and detect the H19 before and after transfection by qPCR.The expression of P-gp,ZEB1 and EC protein before and after transfection was detected by Western blot,and the changes of apoptosis before and after transfection were detected by flow cytometry.The effect of H19 on the tolerance of chemotherapeutic drugs in liver cancer was explored path.(3)Using the target gene screening software to predict the target gene that can bind to H19,and verify the effect of H19 on the expression of target gene at the protein level and molecular level.(4)The expression of H19 was detected by qPCR and the expression of P-gp,ZEB1 and EC protein was detected by Western blot.The possible molecular mechanism of oxymatrine reversing chemotherapy resistance of liver cancer was explored.Results:(1)qPCR results indicated that the expression of H19 in HepG2/ADM cells was 4.582±0.217,and the relative expression of HepG2 cells was 3.352±0.209.The above NC group were: 4.386±0.236,2.950±0.109,low expression group.The score was 0.871±0.143,and the high expression group was 4.852±0.212.The difference between the two groups was statistically significant(P<0.001).After transfection of H19,the high expression group had IC50 for the chemotherapy drugs 5-Fu,ADM and CDDP.The IC50 values of the chemotherapeutic drugs were 19.58±3.01,2.47±0.91,and 9.64±1.74,respectively.The IC50 values of the chemotherapy groups in the low-expression group were 25.90±7.01,2.12±0.39,and 4.67±1.85,respectively.The difference was statistically significant(P<0.05).(2)H19 reversed drug resistance mechanism: After inhibiting the expression of H19,the apoptosis rate by flow cytometry showed that the apoptosis rate of the low expression group was 20.79±2.22%,which was higher than that of the negative control group(4.16±0.23)%,the apoptotic rate from low to high was in the blank control group <negative control group<low expression progenitor.Statistical analysis showed that the difference between the low expression group and the blank group and the negative group was statistically significant(F =165.425,P<0.001),the difference between LSD and other LSD was statistically significant(P<0.01).Western blot analysis showed that the expression of ZEB1 protein was decreased and the expression of EC protein was increased in the low expression group(P<0.05),while the expression in the high expression group was opposite to that in the low expression group,and the expression of ZEB1 protein was higher than that of the control group.The protein was lower than the control group(P<0.05).(3)Western blot analysis showed that P-gp and ZEB1 were higher in HepG2/ADM than in parental cells(P<0.05).(4)qPCR results showed that there was no significant change in the relative expression of H19 in HepG2/ADM+OMT group compared with HepG2/ADM group,which was not statistically significant(P=0.550).Western blot analysis showed that the expression of P-gp and ZEB1 protein in HepG2/ADM+OMT group decreased and the expression of EC protein increased(P<0.05).Conclusion: 1.The expression of H19 in HepG2/ADM cells and HepG2 cells was different.The expression of H19 in HepG2/ADM cells was significantly higher than that in HepG2 cells.The change of expression could regulate the chemoresistance of tumor cells..2.In HepG2/ADM and HepG2,down-regulation of H19 expression can inhibit EMT and promote apoptosis of hepatoma cells to increase the sensitivity of chemotherapy.3.In HepG2/ADM and HepG2,H19 can act on P-gp,ZEB1,and EC,and regulate the chemoresistance of liver cancer cells by EMT.4.Oxymatrine can partially reverse the multidrug resistance of hepatocellular carcinoma cells to chemotherapeutic drugs.The mechanism is not through the action of H19,but may affect the expression of ZEB1 and EC,thereby weakening the epithelial-mesenchymal transition and inhibiting P-gp expression reverses chemotherapy resistance in liver cancer cells. |
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