肌细胞增强因子2D影响肝癌细胞对索拉菲尼耐药的机制研究

Study on the Mechanism by Which MEF2D Induces the Resistance of Hepatoma Carcinoma Cells to Sorafenib

目的:研究肌细胞增强因子2D(MEF2D)影响肝癌细胞PLC对索拉菲尼耐药的作用机制。方法:建立索拉菲尼肝癌耐药细胞株(PLC-DR3),以过表达Ad-MEF2D载体感染PLC(PLC-Ad MEF2D),CCK-8法检测索拉菲尼处理后PLC、PLC-DR3、PLC-Ad MEF2D增殖能力,并计算索拉菲尼对各细胞的半数抑制浓度(IC50)。采用Western blot法检测PLC、PLC-DR3中MEF2D、细胞外调节蛋白激酶(ERK)、磷酸化ERK(p-ERK)的表达和PLC-Ad MEF2D细胞中MEF2D、ERK、p-ERK、促分裂原活化蛋白激酶(MEK)、磷酸化MEK(p-MEK)、快速发育生长因子同源蛋白4(SPRY4)的表达。采用实时荧光定量聚合酶链式反应法检测PLC、PLC-Ad MEF2D中MEF2D m RNA和SPRY4 m RNA的表达。结果 :索拉菲尼对PLC、PLC-DR3、PLC-Ad MEF2D的IC50分别为(8.23±0.06)、(21.80±0.06)、(19.46±0.063)μmol/L。与PLC比较,PLC-DR3、PLC-Ad MEF2D的IC50明显升高(P<0.001);PLC-DR3中总ERK表达量基本不变,MEF2D、p-ERK的蛋白表达明显增强(P<0.001);PLC-Ad MEF2D中总ERK、MEK表达量基本不变,p-ERK和p-MEK蛋白表达增强(P<0.01),SPRY4蛋白表达明显减弱(P<0.001),SPRY4 m RNA表达水平明显减弱(P<0.001)。结论:MEF2D可能通过抑制SPRY4的表达,激活RAS/ERK通路,从而促进肝癌细胞对索拉菲尼耐药。

OBJECTIVE:To study the mechanism of myocyte enhancer factor 2D(MEF2D)affecting the resistance of hepatoma carcinoma cells PLC to sorafenib.METHODS:The liver cancer drug resistance cell strain(PLC-DR3)of sorafenib were established,over expression Ad-MEF2D carrier infected liver cancer cell PLC(PLC-AdMEF2D)and CCK-8 assay was used to determine the proliferation of PLC,PLC-DR3 and PLC-AdMEF2D,and the half inhibitory concentration(IC50)of sorafenib on each cell was calculated.Western blot assay was used to detect the protein expression of MEF2D,ERK and p-ERK in PLC and PLC-DR3,the protein expression of MEF2D,ERK,p-ERK,MEK,p-MEK and SPRY4 in PLC-AdMEF2D.RT-PCR was adopted to detect the mRNA expression of MEF2D and SPRY4 in PLC and PLC-AdMEF2D.RESULTS:IC50 of sorafenib on PLC,PLC-DR3,PLC-AdMEF2D was(8.23±0.06),(21.80±0.06),(19.46±0.063)μmol/L.Compared with PLC,the IC50 of PLC-DR3 and PLC-AdMEF2D was increased significantly(P<0.001);the total expression of ERK in PLC-DR3 keep stale,while the protein expression of MEF2D and p-ERK was increased significantly(P<0.001);total expression of ERK and MEK in PLC-AdMEF2D kept stable,but the protein expression of p-ERK and p-MEK were increased significantly(P<0.01),the protein expression of SPRY4 was decreased significantly(P<0.001),while mRNA expression of SPRY4 was decreased significantly(P<0.001).CONCLUSIONS:MEF2D can promote the generation and development of drug resistance of hepatoma carcinoma cells to sorafenib by inhibiting expression of SPRY4 and active of RAS/ERK pathway.