短发夹RNA沉默CTNNB1基因抑制结肠癌Lovo细胞生长的研究

目的 探讨靶向CTNNB1的shRNA对人结肠癌Lovo细胞的基因下调效应和细胞生长的抑制作用.方法 构建靶向CTNNB1的shRNA载体质粒,然后转染入结肠癌Lovo细胞.用逆转录聚合酶链反应(RT-PCR)和蛋白印迹(Western Blot)方法检测CTNNB1的mRNA和蛋白的表达情况.MTT实验评价转染后各组细胞的增殖情况,并用流式细胞仪检测各组细胞的周期分布和凋亡情况.结果靶向CTNNB1的shRNA能够明显下调CTNNB1 mRNA和蛋白质表达(P<0.05),其抑制率分别是40.63%和44.79%.MTT实验提示转染了特异性CTNNB1 shRNA的CTN实验组细胞在转染后呈现随着时间延长而进行性生长抑制的现象.在转染后72 h,CTN组的细胞存活率为45.8%,较空白对照组显著降低.而且该组细胞还显示出明显的G0/G1阻滞和明显增高的凋亡率(P<0.05).结论靶向CTNNB1的特异性shRNA对结肠癌Lovo细胞具有下调CTNNB1基因表达,促进细胞凋亡并且显著抑制细胞增殖的效应.Abstract：Objective To observe the inhibitory effect of CTNNB1 gene expression and cell proliferation of the human colon cancer cell line Lovo. Methods The shRNA plasmid vector against CTNNB1 was constructed and transfected into Lovo cells with LipofectamineTM2000. The down-regulations of CTNNB1 expressions were detected by RT-PCR and western blot analysis. The cell proliferation inhibitions were determined by MTT assay. The effect of these shRNAs on cell cycle distribution and apoptosis were examined by flow cytometry. Results The shRNA vectors targeted against CTNNB1 were successfully constructed and efficiently suppressed the expression of CTNNB1 mRNA and protein(P<0.05). The expression inhibition rates were 40.63% and 44.79% at the mRNA and protein level respectively. The MTT assay indicated that the specific shRNA resulted in significant inhibition of cell growth on the culture plates on a time-dependent manner. At 72 hours post-transfection,the cell viability of CTN group was 45.8%,which was statistical significance when compared with that of blank control group (P<0.05). Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis at 72 hours after transfection. Conclusions The specific shRNAs targeted against CTNNB1 could down-regulate the expression of CTNNB1 gene, increase apoptosis and inhibit the growth of Lovo cells.

ShRNA targeting against CTNNB1 inhibits growth of human colon cancer Lovo cells