南方医科大学学报2017，Vol.37Issue(12)：1603-1608,6.DOI:10.3969/j.issn.1673-4254.2017.12.07
shRNA-PAX6慢病毒载体构建及其对胶质瘤U251细胞增殖的影响
Construction of a lentiviral vector carrying short-hairpin RNA targeting PAX6 and its effect on proliferation of glioma U251 cells in vitro
摘要
Abstract
Objective To construct a lentiviral vector for delivering short hairpin RNA(shRNA)targeting PA X 6 and investigate its effect on the proliferation of glioma U251 cells in vitro.Methods Two small interfering RNA sequences targeting PAX6 gene were designed based on the reported sequence of PA X 6 and annealed to form a double-stranded chain, which was inserted into a lentiviral vector to construct the recombinant lentiviral vector shRNA-PA X 6.The recombinant vector was infected into U251 cells, and the expression of PA X 6 mRNA and protein in the cells was detected by real-time PCR and Western blotting, respectively.The changes in the proliferation of U251 cells after the infection was assessed using MTT assay.Results Double enzyme digestion of the lentiviral vector pLKD-CMV-G&NR-U6-shRNA yielded an 8208-bp fragment, and colony PCR and sequencing analysis confirmed successful construction of the lentiviral vector shRNA-PA X 6. Infection of the cells with shRNA-PAX6 caused a significant reduction of the expressions of PAX6 mRNA and protein(P<0.05)and resulted in obviously increased proliferation of U251 cells (P<0.05). Conclusion We successfully constructed the recombinant vector shRNA-PA X 6 for silencing PAX6 gene.PAX6 gene silencing results in increased proliferation of U251 cells in vitro.关键词
PAX6基因/胶质瘤细胞/细胞增殖/RNA干扰/慢病毒载体Key words
PA X 6 gene/glioma cells/proliferation/RNA interference/lentivirus vector引用本文复制引用
廖晓红,尹蔚兰,王芳,邬力祥,黄柏胜..shRNA-PAX6慢病毒载体构建及其对胶质瘤U251细胞增殖的影响[J].南方医科大学学报,2017,37(12):1603-1608,6.基金项目
国家自然科学基金(81472160) （81472160）
湖南省自然科学基金(2015JJ2157)Supported by National Natural Science Foundation of China(81472160). （2015JJ2157）
