过表达中期因子增强SMMC 7721细胞对5-Fu耐药及可能机制研究

Overexpression of midkine enhancing resistance of SMMC 7721 cells to 5-Fu and its possible mechanism

目的探讨过表达中期因子(MK)增强SMMC 7721细胞对5-氟尿嘧啶(5-Fu)耐药及其可能的机制。方法将SMMC 7721细胞转染pIRES2-EGFP-MK重组质粒过表达,采用实时荧光定量PCR(qPCR)和蛋白质印迹法(WB)检测MK mRNA和蛋白的表达。5-Fu处理后,采用MTT法检测SMMC 7721细胞对5-Fu的耐药。采用WB法检测磷酸化磷脂酰肌醇3-激酶(p-PI3K)、磷酸化蛋白激酶B(p-Akt)和NF-κB及Bcl-2、survivin、caspase-3的表达。结果将SMMC 7721细胞转染pIRES2-EGFP-MK重组质粒过表达后,MK mRNA和蛋白的表达增加,ConC组细胞对5-Fu的IC50显著高于Con-A组、Con-B组[(27.36±4.31)mg/L vs (4.57±0.34)mg/L,(4.96±0.46)mg/L],差异具有统计学意义(P<0.05)。此外,与Con-A组、Con-B组比较,Con-C组细胞p-PI3K(0.66±0.04 vs 0.35±0.03,0.57±0.03)、p-Akt (0.31±0.02 vs 0.17±0.02、0.25±0.02)、NF-κB (0.63±0.05 vs 0.27±0.02,0.53±0.04)、Bcl-2(0.42±0.04 vs 0.13±0.01, 0.38±0.04)、survivin(0.58±0.04 vs 0.18±0.02,0.51±0.04)呈现高表达,caspase-3(0.41±0.04vs 0.88±0.06,0.43±0.03)呈现低表达(P<0.05)。结论过表达MK可增强SMMC 7721细胞对5-Fu耐药,其机制可能与激活PI3K/AKT信号通路有关。

Objective To investigate the effect of overexpression of midkine(MK) on the resistance of SMMC 7721 cells to 5-fluorouracil(5-Fu) and its possible mechanism. Methods SMMC 7721 cells were transfected into pIRES2-EGFPMK recombinant plasmid for overexpression. Real-time quantitative PCR(qPCR) and Western blot(WB) were used to detect the expression of MK mRNA and protein. After 5-Fu treatment, the resistance of SMMC 7721 cells to 5-Fu was detected by MTT assay. The expression of phosphorylated phosphatidylinositol 3-kinase(p-PI3 K), phosphorylated protein kinase B(p-Akt) and NF-κB, and Bcl-2, survivin and caspase-3 were detected by WB assay. Results After overexpression of SMMC 7721 cells transfected with pIRES2-EGFP-MK recombinant plasmid, the expression of MK mRNA and protein increased, and the IC50 of 5-Fu in Con-C group increased significantly than that in the Con-A group and Con-B group[(27.36±4.31) mg/L vs 4.57±0.34] mg/L,(4.96±0.46) mg/L], and the difference was statistically significant(P<0.05).In addition, compared with the Con-A group and Con-B group, p-PI3 K(0.66±0.04 vs 0.35±0.03, 0.57±0.03), p-Akt(0.31±0.02 vs 0.17±0.02, 0.25±0.02), NF-κB(0.63±0.05 vs 0.27±0.02, 0.53±0.04), Bcl-2(0.42±0.04 vs 0.13±0.01,0.38±0.04), survivin(0.58±0.04 vs 0.18±0.02, 0.51±0.04) showed high expression, caspase-3(0.41±0.04 vs 0.88±0.06,0.43±0.03) showed low expression in the Con-C group(P<0.05). Conclusion Overexpression of MK can enhance the resistance of SMMC 7721 cells to 5-Fu, and its mechanism may be related to the activation of PI3 K/AKT signaling pathway.