抑制miR-21表达的腺病毒载体构建、病毒包装及其对HepG2细胞的作用  

Construction and package of a recombinant adenoviral vector to inhibit miR-21 expression and its effects on HepG2 cells in vitro

作　　者：张一琳[1] 汤建中[1] 刘宏鹏[1] 张兆波[1] 徐文锦[1] 魏军[1,2] 徐广贤[1,2] 

机构地区：[1]宁夏医科大学检验学院,宁夏银川750004 [2]宁夏医科大学总医院医学实验中心,宁夏银川750004

出　　处：《细胞与分子免疫学杂志》2012年第12期1265-1268,1272,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(31160494;30960275);中国博士后基金项目(20110491554);宁夏自然基金项目(NZ1079);宁夏医科大学"博士学位建设学科"开放课题(KF2010-41)

摘　　要：目的:构建能高效抑制成熟miR-21小分子表达的腺病毒载体,探讨其对肝癌细胞株HepG2的影响与机制。方法:基于miRNA sponge技术,合成一段与miR-21序列完全互补的8个重复片段,克隆至穿梭载体pAdTrack-CMV中,经酶切、测序鉴定正确无误后,与pAdEasy-1腺病毒骨架质粒进行同源重组,转染至293A细胞中,包装成重组腺病毒感染HepG2细胞,通过Hochest33258染色、Western blot法及MTT试验检测细胞的凋亡与增殖水平。结果:经酶切、测序及GFP表达证实,成功构建了携带与miR-21互补的DNA片段的重组腺病毒。经Hochest33258染色、Western blot法及MTT试验证实,该重组腺病毒可以抑制HepG2细胞中miR-21的表达并抑制HepG2细胞的增殖,促进细胞的凋亡。结论:重组包装的抑制miR-21表达的腺病毒可有效的降低miR-21在HepG2细胞中的表达,抑制HepG2细胞的增殖。AIM： To construct the recombinant adenoviral vector which can efficiently inhibit mature miR-21 expression and explore its effects and the underlying mechanisms on hepatoma cell line HepG2. METHODS： Based on miRNA- sponge technology, we synthesized 8 duplicated fragments, fully complementary with the miR-21 sequence and cloned them into a shuttle vector pAdTrack-CMV. The constructed plasmid was sequenced and linearized for homologous recombination with pAdEasy-1 vector in BJ5183 bacteria, then transfected into 293A cells. The recombinant adenovirus was used to challenge HepG2 cells. Mature miR-21 level was detected by real-time PCR. Apoptosis and proliferation of the HepG2 cells were detected by Hochest 33258 staining, Western blotting and MI-I- assay. RESULTS： The restriction enzyme digestion, DNA sequencing and detection of GFP expression demonstrated that recombinant adenoviral vector was constructed successfully. The recombinant adenovirus inhibited the expression of miR-21 in HepG2 cells, and also depressed the proliferation of HepG2 cells and promoted the apoptosis. CONCLUSION： The recombinant adenoviral vector we successfully constructed could efficiently reduce the expression of miR-21 in HepG2 cells and depress the proliferation of HepG2 cells.

关 键 词：MIRNA SPONGE MIR-21 重组腺病毒 

分 类 号：R392.1[医药卫生—免疫学]