蜂毒素对人肝癌细胞生长的抑制作用及其机制探讨

Inhibitory effect of melittin on growth of human hepatocellular carcinoma cells

目的研究蜂毒素对人肝癌细胞(Hep G2、SMMC-7721)生长的抑制作用及其机制。方法采用MTT法检测蜂毒素对Hep G2和SMMC-7721细胞增殖的抑制作用,实时定量PCR检测细胞中microRNA-203(mir-203)以及PIK3CA mRNA的表达变化,Western blot法检测磷脂酰肌醇3-激酶(PI3K)、磷酸化蛋白激酶B(P-AKT)的表达变化,瞬时转染人mir-203 mimics过表达mir-203后应用实时定量PCR和Western blot检测mir-203对PI3K/AKT信号通路的作用。结果与正常组比较,蜂毒素处理组细胞增殖明显受到抑制,并呈浓度依赖性;实时定量PCR结果显示蜂毒素(1、2、4mg/L)可以明显升高mir-203的表达,PIK3CA mRNA的表达无明显改变;Western blot法结果显示蜂毒素(1、2、4 mg/L)可以降低PI3K以及P-AKT蛋白的表达。在Hep G2和SMMC-7721细胞中过表达mir-203后,与正常组及转染阴性对照组比较,PIK3CA mRNA的表达无明显改变但PI3K以及P-AKT的蛋白表达降低。结论蜂毒素能抑制Hep G2和SMMC-7721细胞的增殖,其机制可能是通过上调mir-203的表达,进而在转录后水平靶向抑制PI3K的表达,抑制PI3K/AKT信号通路的活化。

Objective To investigate the inhibitory effect and its mechanisms of melittin on the growth of human hepatocellular carcinoma cells （HepG2,SMMC-7721）. Methods The inhibition rate of growth of cells treated with melittin was measured using MTT assay. Relative levels of microRNA-203（mir-203）and PIK3CA mRNA in HepG2 and SMMC-7721 cells were determined with a SYBR Green using quantitative real-time PCR detection system. The relative protein expressions of PI3K,P-AKT and AKT were observed by Western blot. miR-203 mimics was trans-fected into HepG2 and SMMC-7721 cells to enhance miR-203 expression. The effect of up-regulation of miR-203 on PI3 K/AKT signaling pathway of HepG2 and SMMC-7721 cells was evaluated using Western blot and quantitative real-time PCR. Results Compared with the control group,melittin had obvious inhibitory effect on the growth of human hepatocellular carcinoma cells in a dose-and-time dependent manner. After the treatment with melittin at dif-ferent concentrations for 24 h, the results showed that melittin（1,2,4 mg/L） could up-regulate the expression of mir-203 . Relative levels of PIK3 CA mRNA had not noticeably altered but the relative protein expressions of PI3 K and P-AKT were obviously increased. Over-expression of mir-203 significantly inhibited the expression of PI3K and P-AKT in the protein level. Conclusion Proliferation activity of HepG2, SMMC-7721 cells is inhibited by melittin which may be related to the up-regulation of mir-203 , which inhibited the expression of PI3 K in the protein level and the inhibitory effect on PI3K/AKT signaling transduction.