siRNA表达载体对SW480细胞原癌基因Pokemon的抑制

Inhibition of Proto-oncogene Pokemon Expression by siRNA Expression Vector in SW480 Cells

观察siRNA表达载体对SW480细胞中Pokemon原癌基因的抑制效应,为进一步研究该基因的功能奠定基础。构建针对Pokemon基因的RNAi质粒表达载体,脂质体法转染人结直肠癌SW480细胞系,观察转染效率及细胞表型变化。稳定转染后,实时荧光定量PCR和Western blot检测SW480细胞中Pokemon mRNA及蛋白质的表达情况;MTT法检测siRNA对SW480细胞恶性增殖的影响;流式细胞仪分析细胞凋亡改变。镜下观察阳性转染率约36%,转染表达载体后细胞形态发生了显著变化;Pokemon mRNA及蛋白质的表达受到明显抑制:与阴性对照组相比,表达质粒产生的siRNA对Pokemon mRNA的抑制率在转染后24h和48h分别为34.2%和67.7%;对蛋白的抑制率在48h和72h分别为48.3%和73.6%。MTT法检测细胞生长曲线表明Pokemon抑制可使SW480细胞生长速度明显减慢;流式细胞仪分析显示转染Pokemon siRNA表达质粒后SW480细胞凋亡增加。构建的RNAi表达载体可以有效抑制SW480细胞中Pokemon基因的表达,并对SW480细胞的生长具有明显抑制及诱导凋亡作用。

To investigate the inhibitory effects of siRNA expression vector on proto-oncogene Pokemon expression in SW480 cells and to provide experimental basis for further research about the biological function of Pokemon. siRNA expression vectors were constructed to express a short hairpin RNA against Pokemon. The recombinants were transfected into SW480 cells with liposomc. Cellular morphology and transfection efficiency were observed. The expression of Pokemon were checked by real-time fluorescence quantitative PCR and Western blot. MTT assay was used to detect the effect of siRNA on the growth of SW480 cells and Fluorescence Activated Cell Sorter was applied to study cell apoptosis of SW480 cells. The transfection efficiency was about 36% and the cellular morphology changed greatly at 48h after transfection, siRNA expression vectors could specifically reduce the expression of Pokemon mRNA and protein in SW480 cells. Compared with negative control, the inhibition ratio of Pokemon mRNA expression was 34.2% and 67.7% , in 24th hour and 48thhour, the inhibition ratio of Pokemon protein was 48.3% and 73.6% , in 48th and 72th hour, respectively. Transfection with Pokemon siRNA expression vectors could inhibit cell growth and induce cell apoptosis of SW480 cells, siRNA expression vectors were successfully established that could effectively inhibit the expression of Pokemon in SW480 cells. Pokemon gene silencing could decrease growth and increase apoptosis of SW480 cells.