| Objective: To investigate the impact of MiR-181 a on multidrug resistance of Hepatocellular cell line HepG2/ADM and multi-drug resistance revearsal induced by Oxymatrine,and to explore its relative molecular mechanism.Methods:(1)Methyl Thiazolyl Tetrazolium(MTT)assay was used to determine the HepG2/ADM cells’ drug sensitivities to adriamycin,cisplatin and 5-fluorouracil.(2)The transient transfection technique in vitro was used to transfect agomir and antagomir of miR-181 a to up-regulate or down-regulate the expressions of mi R-181 a in cells,and to determine the changes of sensitivities to chemotherapy.Flow cytometry(FCM),qPCR and Western blot were used to explore the molecular mechanism of mi R-181a’s impact on the chemotherapy sensitivity.(3)The Target prediction software microRNA.org was applied to predict the target of miR-181 a and to check the influence of miR-181 a on the expressions of predict target.(4)MTT assay was used to test the influence of Oxymatrine on HepG2/ADM cells’ proliferation and sensitivities to chemotherapeutic drugs.qPCR and Western blot were used to explore possible mechanisms of Oxymatrine’s reversal of chemo-resistance in HCC.Results:(1)Compared with sensitive cells,HepG2/ADM appeared multidrug resistant to adriamycin,cisplatin and 5-fluorouracil.The resistance index were 6.71,8.03 and 2.69,respectively.So these cells were chosen for the following study.(2)The level of miR-181 a in chemoresistant cells line HepG2/ADM was significantly higher than those in chemosensitive cells line HepG2(P<0.001).The resistance to chemotherapeutics were increased aftermiR-181 a overexpression(P<0.05),While they were decreased after miR-181 a knockdown(P<0.05).There were significant difference in the chemotherapeutics IC50 between the transfected group and control group(P<0.05).(3)Studies on the mechanism of reversing multidrug resistance by miR-181a: After the down-regulation of miR-181 a expressions,FCM revealed that the apoptosis rates of anti-miR-181 a group were16.4% ? 2.52%,which were higher than negative group(6.17% ? 1.01%),but lower than adriamycin group(35.73%?3.91%).And the combined group(adriamycin and anti-miR-181a)were 58.17% ? 5.52%.The difference between the groups were statistically significant(F=141.576,P<0.001).The expressions of cleaved-caspase-3 and cleaved-caspase-9 were increased after knockdown of miR-181 a by Western blot,While their were decreased after miR-181 a overexpression.(4)Bim could be the taget of miR-181 a according to information prediction software.The expressions of Bim mRNA and protein in HepG2 cells were higher than those in the HepG2/ADM cells(P<0.05),But there were no significant difference after transfected with mi R-181a(P>0.05).This indicated that Bim might not be the target of miR-181 a.(5)Oxymatrine could inhibit proliferation of HepG2/ADM cells in a dose-dependent manner in vitro.After HepG2/ADM cells were treated with nontoxic oxymatrine at the concentration of 0.5 mg/ml for 48 h,the IC50 of adriamycin,cisplatin and5-fluorouracil were decreased(P<0.05),and the rever fold were 2.42,4.6 and 1.29,respectively.It indicated that Oxymatrine could rever the multidrug of Hepatocellular cell in partly.QPCR result showed that the relative expression of miR-181 a was 0.6±0.15 after treated with Oxymatrine,and it was lower than control group(P<0.05),But the Bim mRNA expression did not change,which was 1.94±0.93 times than the control group,and there was no significant difference(P=0.221).And the Bim protein and cleaved-caspase-9 were increased(P<0.05),what’s more,the cleaved-caspase-3 was detected by Western blot.Conclusions:(1)The expression of miR-181 a in HepG2/ADM cells was higher than HepG2 cells.(2)Overexpressions of miR-181 a could enhance the multidrug resistance ofHepatocellular carinoma cell,and the sensitivities to chemotherapeutics could be enhanced after down-regulations of miR-181 a expressions.MiR-181 a could induce resistance to chemotherapeutics in Hepatocellular carinoma cells via to regulate the cell apoptosis.(3)Oxymatrine can partially reverse multidrug-resistance of HepG2/ADM cells,and this mechanism might be based on suppression of miR-181 a expression,enhanced Bim expression,activation of the caspase-9 and-3-mediated mitochondrial apoptotic pathway. |
PDF Full Text Request