慢病毒介导的RNA干扰抑制PAR2表达影响结肠癌细胞SW620增殖与迁移

Effects of PAR2 RNA interference by lentivirus on proliferation and migration of colon carcinoma cells SW620

目的构建人蛋白酶激活受体2(protease-activated receptor 2,PAR2)RNA干扰(RNAi)的重组慢病毒表达载体,探讨PAR2在结肠癌细胞SW620增殖与迁移中的作用。方法根据PAR2基因信息,设计4条PAR2 RNAi片段及1条阴性对照片段,western blotting筛选有效干扰片段,于293T细胞进行病毒包装,采用real-time PCR及western blotting检测干扰效率。PAR2激动剂(PAR2-AP)刺激感染后的SW620细胞,通过流式细胞仪、Transwell试验分别检测细胞周期及细胞迁移能力。结果构建的PAR2-RNAi慢病毒颗粒感染SW620细胞后,PAR2 mRNA及蛋白表达均被明显抑制。与对照相比,PAR2-AP不能促进感染后的SW620细胞增殖,细胞各周期比率未见明显差异,迁移试验结果显示穿膜细胞数也未见明显增多。结论成功构建的PAR2-RNAi慢病毒颗粒能显著抑制结肠癌细胞SW620的PAR2表达,进而阻断PAR2-AP对细胞增殖与迁移的促进效应,证明PAR2在SW620细胞增殖与迁移过程中发挥重要作用。

Objective To construct a recombinant lentiviral vector targeting human protease-activated receptor 2（PAR2） and to observe the function of PAR2 in proliferation and migration of colon carcinoma cell SW620.Methods According to the GenBank information of PAR2,four different short hairpin RNAs targeting PAR2 gene and a negative sequence were designed.The effective sequence was selected by Western blotting.The recombinant lentivirus RNAi-PAR2-LV was packaged in 293T cells.The efficiency of RNAi was determined by real-time PCR and Western blotting.Finally,the cell cycles and cell migratory ability of transferred-SW620 cells were examined by FCM and Transwell assay with stimulation of PAR2-AP.Results The recombinant vector was successfully constructed and the expression of PAR2 mRNA and protein on SW620 infected with RNAi-PAR2-LV were effectively suppressed.The FCM and Transwell assays indicated that the infected-SW620 cells,stimulated with PAR2-AP,grew slowly,and the rate of G1 phase and S phase was（68.67±7.2）% and（31.33±3.5）% respectively;the migratory cell number was not increased,comparing to that of control cells.Conclusions The constructed lentiviral RNAi-PAR2-LV may markedly block the expression of PAR2 on SW620 cells and inhibit cell proliferation and cell migration induced by PAR2-AP.The results demonstrate that PAR2 plays an important role in the proliferation and migration of colon cancer cells.