人GINS2基因RNA干扰慢病毒载体的构建及其在上皮性卵巢癌中的表达

Construction of a lentiviral vector for RNA interference of human GINS2 gene and its stable expression in epithelial ovarian cancer cells

目的构建人GINS2基因RNA干扰慢病毒表达载体,并建立稳定干扰GINS2基因表达的上皮性卵巢癌细胞系,为研究GINS2蛋白在上皮性卵巢癌发生发展中的作用奠定基础。方法针对GINS2基因序列,按照RNAi序列的设计原则,设计、合成GINS2基因特异性小分子干扰siRNA(small interference RNA,siRNA)靶点,与慢病毒构建重组形成shRNA表达载体,利用PCR和测序鉴定获取正确克隆。将筛选出的最有效重组载体与慢病毒包装质粒共转染293T细胞并测定病毒滴度。随后将其感染上皮性卵巢癌SKOV3细胞株细胞,采用Real-time PCR 检测靶基因的沉默效率。结果慢病毒RNA干扰载体经酶切和测序鉴定证实准确连接测序正确,且包装出来的病毒纯化后滴度达5×10^8TU/mL。RT-qPCR、Western blot结果显示感染后GINS2 mRNA及蛋白水平显著下降。结论成功构建了人GINS2基因特异性的慢病毒干扰载体,并获得了GINS2基因稳定干扰的上皮性卵巢癌SKOV3细胞系。

Objective To investigate the role of GINS2 in the pathogenesis and progress of epithelial ovarian cancer via constructing a lentiviral vector for RNA interference (RNAi) of human GINS2 gene and assessing the gene-silencing effect of the vector in human epithelial ovarian cancer cells. Methods According to the design principle of RNAi sequence,siRNA sequence targeting GINS2 gene were designed,synthesized,annealed and inserted into the lentiviral vector.The correct cloning were confirmed by PCR and sequencing.The most effective recombinant lentiviral vector was screened and recombinant plasmid and a lentivirus packing mix were co-transfected into 293T cells to obtain packaged lentivirus particles.Viral titer was determined.The silencing efficiency of target gene in SKOV3 cells was detected by Real-time PCR. Results DNA sequencing showed that the shRNA sequence was successfully inserted into the lentivirus vector.The recombinant lentiviral vector harboring shRNA targeting the GINS2 gene was successfully transfected into 293T cells.The recombinant lentivirus harvested from 293T cells had a titer of 5×10^8TU/ml.After silencing GINS2 in SKOV3 cells,the expression in GINS2 mRNA and protein levels decreased significantly. Conclusion A lentiviral vector targeting human GINS2 gene,capable of stable GINS2 gene knockdown in SKOV3 cells,has been successfully constructed,which provides a basis for further study of the study of the relationship between human epithelial ovarian cancer and GINS2 protein.