小分子干扰RNA特异性抑制人胰腺癌细胞株PANC-1突变型K-ras基因表达的探讨

目的:研究小分子干扰RNA(small interfering RNA,siRNA)对人胰腺癌细胞株PANC-1中突变型K-ras基因表达的抑制作用.方法:构建真核表达载体pSilencer3.1-K-rasv12,转染PANC-1细胞后应用RT-PCR及Western blot检测突变型K-ras基因mRNA及蛋白质表达变化;噻唑蓝(MTT)测定细胞生长曲线;流式细胞仪测定细胞凋亡率.结果:测序证实siRNA真核表达载体构建成功.RT-PCR光密度比值结果空载体组、阴性对照组、实验组分别为:95.3%±2.5%、97.6%±2.8%、40.1%±3.1%,差异有统计学意义(P＜0.05);Western blot灰度比值结果空载体组、阴性对照组、实验组分别为:96.1%±2.2%、98.5%±2.0%、36.5%±3.2%,差异有统计学意义(P＜0.05);实验组细胞生长受到明显抑制,凋亡率较对照组明显升高(P＜0.05).结论:pSilencer3.1-K-rasv12能有效抑制突变型K-ras基因在人胰腺癌细胞株PANC-1中的表达,抑制细胞生长,诱导细胞凋亡,为肿瘤的生物学治疗提供了新的方法.

The Study of Specific Inhibition of Mutant K-ras Gene Expression by Short Interfering RNAs in Human Pancreatic Carcinoma Cell PANC-1

Objective: To study the specific inhibition effect of short interfering RNAs(siRNA) on the mutant K-ras gene expression in PANC-1 cells. Methods: One pair of 63bp reverse repeated sequence targeting mutant K-rasv12 mRNA were synthesized and inserted into plasmid pSilencer3.1 eukaryotic expression vector. After transfection into PANC-1 cells, the expression of K-ras gene was detected using RT-PCR and Western blot techniques. The effect of cell proliferation was studied by MTT test. Flow cytometry was used to detect the apoptosis of transfected cells. Results: The recombinant plasmid pSilencer3.1-K-rasv12 was successfully constructed by the sequencing. The introduction of pSilencer3.1-K-rasv12 was showed to efficiently and specifically inhibit the expression of K-rasv12 gene according to the results of RT-PCR and Western blot, and there was a significant difference between the siRNA transfected group and the control groups (P<0.05). The inhibitory action on cell proliferation was confirmed by MTT test. The apoptotic rate of the pSilencer3.1-K-rasv12 group was significantly higher than that of the control groups (P<0.05). Conclusion: The siRNA targeting mutant K-ras mRNA can specially suppress the expression of mutant K-ras gene in PANC-1 cells and it provides a new method and material to the biological therapy of cancer.