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人端粒酶反转录酶启动子调控的microRNA-21海绵抑制剂慢病毒载体的构建及功能分析
黄燕燕 续力云 刘晓光*
解剖学报 ›› 2015, Vol. 46 ›› Issue (2) : 208-215.
人端粒酶反转录酶启动子调控的microRNA-21海绵抑制剂慢病毒载体的构建及功能分析
Construction and function analysis of the lentivirus vector containing microRNA-21 sponge inhibitor drived by human telomerase reverse transcriptase promoter
目的 构建由人端粒酶反转录酶（hTERT）启动子调控的microRNA-21(miR-21)海绵抑制剂慢病毒载体，探讨该重组慢病毒载体对端粒酶阳性肿瘤的特异性抑瘤作用及其机制。 方法 将hTERT启动子核心序列取代慢病毒载体RFP上游的CMV启动子；将重组成功的慢病毒载体Lenti-hTERT-miR-21-sp感染端粒酶阴性细胞HBE及端粒酶阳性肿瘤细胞A549、H1299，观察RFP的表达情况；同时在肿瘤细胞中检测抑制miR-21表达后对细胞生长、凋亡的影响；并应用含人类全长基因的cDNA表达谱芯片，对抑制miR-21表达后人肺癌细胞株A549中差异表达基因进行分析。 结果 经酶切及测序法鉴定慢病毒载体构建成功；将包装后获得的高滴度病毒颗粒感染目的细胞后，发现重组病毒只能在端粒酶阳性肿瘤细胞中特异性高表达，且下调miR-21基因表达后，肿瘤细胞的生长能力受到抑制，凋亡率明显上升（P<0.05）；与对照相比，抑制miR-21表达的A549细胞中差异表达的基因共有64条，其中20条上调，44条下调。 结论 hTERT启动子能够严格地引导病毒载体在端粒酶阳性肿瘤细胞中特异性的封闭miR-21的表达，实现抑制肿瘤细胞生长的作用；芯片结果提示，miR-21引发肺癌可能是多因素多基因共同作用的结果。
Objective To construct lentivirus vector containing microRNA-21 (miR-21) sponge inhibitor drived by human telomerase reverse transcriptase (hTERT) promoter, investigate the specific expression level of this recombined vector in telomerase positive cells, and discuss their function in the pathogenesis of cancer. Methods The cytomegalovirus promoter was relaced with hTERT promoter in upstream of RFP on lentivirus vector. The recomhined lentivirus vector was transfected into telomerase positive cells including A549, H1299 and also the telomerase negative cells HBE to investigate the expression status of our recombined vector under fluorescence microscope. The proliferation and apoptosis ability of the lentivirus vector treated A549 were examined by the CCK8 and flow cytometry methods. cDNA microarray was used to identify the differential genes expressed in the A549 cells downregulated expression of miR-21. Results A miR-21 inhibited lentiviral vector (Lenti-hTERT-miR-21-sp) was successfully constructed as confirmed by restrictive enzyme digestion and plasmid sequencing. Promorter function test indicated that this recombinant lentivirus was strictly expression in the telomerase positive tumor cells. Meanwhile, miR-21-decreased expression can induce to impair proliferation ability and increased apoptosis in cancer cells. Sixty four differential expression genes in the mir-21 down-regulated A549 were screened, of which 20 were up regulated and 44 were down regulated. Conclusion hTERT promoter and specifically inhibits miR-21 expression in telomerase positive cells, and suppresses the growth of cancer cells. The experiment of cDNA microarray indicated that lung cancer induced by ectopic expression of miR-21 is a result of coeffecting by polygene and multiple factors.
人端粒酶反转录酶 / 启动子 / microRNA-21海绵抑制剂 / 慢病毒载体的构建 / 实时定量聚合酶链反应 / 基因芯片
Human telomerase reverse transcriptase / Promoter / microRNA-21 sponge inhibitor / Construction of lentivirus vector / Read-time PCR / cDNA microarray
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