慢病毒载体法建立乳腺癌ST3Gal Ⅲ基因RNAi细胞模型

Establishment of breast cancer cells with stable expression of ST3Gal Ⅲ gene RNA interference via lentivirus vector

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 establish of breast cancer cells with stable expression of of 2, 3 - sialyltransferase III（ ST3Gal III ） gene RNA interference via lentivirus vector. Methods Four short hairpin RNA （shRNA） sequences targeting ST3Gal III gene were designated, connected the synthesizd the DNA chains with linear pGLV3 - H1 - GFP carriers, and then lentivirus plasmid were constructed. Lentiviral vectors were packaged after identifying the sequences and the virus titer were measured. The recombinant lentiviral vector pGLV3 - H1 - GFP - shST3 was transfected into human breast cancer MDA - MB - 231cells, and transfected cells were divided into three groups ： blank group （ mock cells, M）, control group（parent cells, P）, RNA interfere groups（ shST3 -1, 2,3,4）. Then the expression of ST3Gal m in transfected MDA - MB -231 cells was detected by real time -PCR and Western blotting. The content of α 2,3 -sialic acids was analyzed by flow cytometry. Results The recombinant vectors of RNAi lentiviral vector targeting the ST3 Gal m gene were successfully constructed and all the virus titers were more than 2 × 10^8 TU · mL^-1. The transfection efficiency was detected over 90% by observing green fluorescence protein after the recombinant vectors transfected into MDA -MB -231cells. The inhibitory rate of ST3Gal III mRNA expression in shST3 -1,2,3,4 were 42. 1%, 66.7%, 30. 1%, 80. 9% respectively with significantly （P 〈0. 05）. The lower expression of ST3Gal III protein in shST3 - 1,2,3,4 were 35.92% ,75.64% ,37.12% , 81.75% respectively, compared with control group, were significantly decreased （P 〈 0.05 ）. The contents of α 2,3 - sialic acids was reduced in shST3 - 4 with significantly （ P 〈 0. 05 ）. Conclusion The shST3 - 4 cells model which could interfere ST3Gal III gene expression in MDA- MB -231 cells was successfully constructed.