论文部分内容阅读
目的研究用慢病毒载体法建立乳腺癌α2,3-唾液酸转移酶ST3GalⅢ基因RNA干扰(RNAi)的细胞模型。方法针对ST3GalⅢ基因设计4组短发夹RNA序列,合成DNA与线性化的pGLV3-H1-GFP载体连接,构建慢病毒载体质粒。鉴定后,进行病毒包装和病毒滴度检测;将获得的重组慢病毒pGLV3-H1-GFP-shST3转染人乳腺癌MDA-MB-231细胞后,将转染细胞分为3组:空白组,未转染组(mock cells,M);对照组(转染对照慢病毒,parent cells,P);干扰组,转染慢病毒载体pGLV5-H1-GFP-shST3(shST3-1,2,3,4)。以荧光显微镜观察绿色荧光蛋白表达,经嘌呤霉素抗性筛选;用实时定量PCR、Western blot检测转染后MDA-MB-231细胞中ST3Gal Ⅲ mRNA及蛋白表达,以流式细胞术分析细胞表面α2,3-唾液酸水平。结果成功构建针对ST3GalⅢ基因的RNAi慢病毒载体,病毒悬液滴度均>2×10~8TU·mL~(-1)。各转染细胞的转染效率达90%以上;经嘌呤霉素筛选后,干扰组的ST3Gal Ⅲ mRNA的抑制率分别为42.1%,66.7%,30.1%,80.9%及蛋白表达水平分别下降了35.92%,75.64%,37.12%,81.75%。与对照组比较,干扰组细胞ST3Gal Ⅲ基因的mRNA及蛋白表达显著降低,差异有统计学意义(P<0.05);shST3-4组细胞表面α2,3-唾液酸水平明显降低,差异有统计学意义(P<0.05)。结论成功构建了乳腺癌ST3Gal Ⅲ基因RNAi的细胞模型shST3-4。
Objective To study the cellular model of RNA interference (RNAi) of breast cancer α2,3-sialyltransferase gene ST3GalⅢ by lentiviral vector method. Methods Four short hairpin RNA sequences were designed according to the ST3Gal Ⅲ gene. The synthetic DNA was ligated to the pGLV3-H1-GFP vector and the lentiviral vector was constructed. After identification, the virus was packaged and the virus titer was tested. The recombinant lentivirus pGLV3-H1-GFP-shST3 was transfected into human breast cancer cell line MDA-MB-231 and the cells were divided into 3 groups: The control group (transfected with control lentivirus, parental cells, P) and the interference group were transfected with lentiviral vector pGLV5-H1-GFP-shST3 (shST3-1,2,3, 4). The expression of green fluorescent protein (EGFP) was observed by fluorescence microscopy, and the expression of green fluorescent protein (EGFP) was detected by puromycin resistance. The expression of ST3Gal Ⅲ mRNA and protein in MDA-MB-231 cells was detected by real- α2,3-sialic acid level. Results The RNAi lentiviral vector against ST3Gal Ⅲ gene was successfully constructed. The titer of the virus suspension was> 2 × 10 ~ 8TU · mL -1. The transfection efficiency of all transfected cells was over 90%. After puromycin screening, the inhibitory rates of ST3Gal Ⅲ mRNA in the interference group were 42.1%, 66.7%, 30.1%, 80.9% and 35.92 %, 75.64%, 37.12%, 81.75%. Compared with the control group, mRNA and protein expression of ST3Gal Ⅲ gene in the interference group were significantly decreased (P <0.05), and the level of α2,3-sialic acid in the cell surface of shST3-4 group was significantly lower than that of the control group Significance (P <0.05). Conclusion The cell model shST3-4 of ST3Gal Ⅲ RNAi in breast cancer was successfully constructed.