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
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篇名: 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
TITLE:
摘要: 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-AdMEF2D),CCK-8法检测索拉菲尼处理后PLC、PLC-DR3、PLC-AdMEF2D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-AdMEF2D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-AdMEF2D中MEF2D mRNA和SPRY4 mRNA的表达。结果:索拉菲尼对PLC、PLC-DR3、PLC-AdMEF2D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-AdMEF2D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-AdMEF2D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 mRNA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。
ABSTRACT: 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.
期刊: 2018年第29卷第17期
作者: 马清霞,王园园,马海龙,李培宇,杨月成,杨志宏,刘佳,姜国辉
AUTHORS: MA Qingxia,WANG Yuanyuan,MA Hailong,LI Peiyu,YANG Yuecheng,YANG Zhihong,LIU Jia,JIANG Guohui
关键字: 索拉菲尼;肝癌细胞;耐药机制;肌细胞增强因子2D
KEYWORDS: Sorafenib; Hepatoma carcinoma cells; Resistant mechanism; MEF2D
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