人RNA结合蛋白MSI1 RNA干扰慢病毒载体的构建及对人脑胶质瘤细胞生物学活性的影响

Construction of lentiviral vector harboring short hairpin RNA of RNA binding protein MSI1 and its effects on biological behaviors of human glioma cells

目的 构建针对MSI1基因的小分子RNA干扰慢病毒表达载体,检测其在人脑胶质瘤U-87MG细胞中的表达效率及对细胞生物学行为的影响.方法 构建针对MSI1短发卡RNA（shRNA）的慢病毒载体pGCL-绿色荧光蛋白（GFP）-MSI1,用293T工具细胞包装后,转染人脑胶质瘤U-87MG细胞,实验分为3组：（1）空白组;（2）阴性对照组;（3）pGCL-GFP-MSI组.荧光显微镜下检测转染72 h后pGCL-GFP-MSI1的转染效率;Western blot检测U-87MG细胞内MSI1蛋白和肿瘤干细胞标记蛋白HES1的表达;噻唑蓝（MTT）法检测pGCL-GFP-MSI1转染后U-87MG细胞1-7 d的增殖活性;平板克隆形成实验检测U-87 MG细胞的克隆形成能力;Transwell实验检测U-87MG细胞体外侵袭能力;流式细胞仪（FCM）检测pGCL-GFP-MSI1转染后对U-87MG细胞凋亡的影响.结果 成功构建pGCL-GFP-MSI1慢病毒载体,并成功转染U-87MG细胞,荧光显微镜显示转染效率达80％以上;Western blot结果显示pGCL-GFP-MSI1抑制U-87MG细胞中MSI1蛋白（31.64％）和HES1蛋白（37.25％）的表达（P＜0.01）;MTT结果显示转染pGCL-GFP-MSI1后第3天起U-87MG细胞的增殖活性明显下降（P＜0.01）;平板克隆形成实验结果显示转染pGCL-GFP-MSI1后U-87MG细胞的克隆形成数明显下降（27.24±2.52,P＜0.0l）;Transwell实验结果显示pGCL-GFP-MSI1转染后U-87MG细胞的侵袭细胞数明显下降（18.24 ±3.52,P＜0.01）;FCM结果表明pGCL-GFP-MSI1转染后明显提高U-87MG细胞的凋亡率（22.81％,P＜0.01）.结论 MSIl-小干扰RNA（siRNA）慢病毒载体能特异性下调人脑胶质瘤U-87MG细胞中MSI1的表达,通过降低肿瘤干细胞信号通路中相关因子的活性来抑制人脑胶质瘤U-87MG细胞的增殖活性及侵袭能力,并促进细胞的凋亡。

Objective To construct a lentiviral vector carrying short hairpin RNA （shRNA） of human MSI1 gene,and to study the relationship between the expression of MSI1 and the biological behaviors of human glioma U-87MG cells.Methods A lentiviral vector-mediated MSI1 shRNA （pGCL-GFP-MSI1） was constructed and transfected into the packaging cells 293T.The lentiviral vector was used to transfect into the human glioma U-87MG cells,and blank control group,negative group and pGCL-GFP-MSI1 group wer set up.The infection efficiency was evaluated.Fluorescence microscopy was used to detect the transfection efficiency after 72 h.The expression of MSI1 and cancer stem cells marker （HES1）in U-87MG cells was detected by Western blotting.Methyl thiazol tetrazolium （MTT） assay was used to observe the proliferation behaviors of U-87MG cells 1 to 7 days after transfection.Colony formation assay was used to observe the colony formation of U-87MG cells.Transwell assay was used to observe the invasive behaviors.Flow cytometry was used to observe the apoptosis.Results Lentiviral vector-mediated MSI1 shRNA was correctly constructed.Immunofluorescence assays demonstrated that the transfection efficiency was above 80%.Western blotting analyses demonstrated that pGCL-GFP-MSI1 could significantly inhibit the expression of MSI1 （31.64%） and HES1 （37.25%） in U-87MG cells （P 〈 0.01）.MTT results showed that pGCL-GFP-MSI1 could significantly inhibited the proliferation of U-87MG cells.Colony formation assays showed that pGCL-GFP-MSI1 could decrease the colony formation number in U-87MG cells （27.24 ± 2.52,P 〈 0.01）.Transwell assays showed that pGCL-GFP-MSI1 could reduce the invasive cell number to （18.24 ±3.52,P 〈0.01）.FCM results showed that the apoptosis was increased significantly in U-87MG cells （22.81%,P 〈 0.01）.Conclusion pGCL-GFP-MSI1 could significantly inhibit the expression of MSI1 in human glioma U-87MG cells.It could suppress the growth and invasion,and induce apoptosis of human glioma U-87MG cells by regulating the cancer stem cells factors in its signal path.