蜂毒素对人肝癌细胞生长的抑制作用及其机制探讨

目的：研究蜂毒素对人肝癌细胞( HepG2、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HepG2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、4 mg/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 HepG2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 HepG2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。

Inhibitory effect of melittin on growth of human hepatocellular carcinoma cells

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.