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目的构建由人端粒酶反转录酶(h TERT)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h TERT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-h TERT-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c DNA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 h TERT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 a lentiviral vector containing a microRNA-21 (miR-21) sponge inhibitor regulated by human telomerase reverse transcriptase (hTERT) promoter and to investigate the specificity of the recombinant lentiviral vector for telomerase positive tumors Tumor effect and its mechanism. Methods The hTERT promoter sequence was substituted for the CMV promoter upstream of the lentiviral vector RFP. The recombinant lentiviral vector Lenti-h TERT-miR-21-sp was transfected into the telomerase-negative HBE and telomerase positive tumor cells A549 and H1299 were used to observe the expression of RFP. At the same time, the effect of inhibiting the expression of miR-21 on the cell growth and apoptosis was detected in tumor cells. Using c-DNA microarray containing human full-length gene, The expression of differentially expressed genes in human lung cancer cell line A549 was analyzed. Results The lentiviral vector was successfully constructed by restriction enzyme digestion and sequencing. After infected with high titer virus particles, the recombinant virus was found to be highly expressed only in telomerase positive cells and down-regulated by miR (P <0.05). Compared with the control, there were 64 differentially expressed genes in A549 cells that inhibited miR-21 expression, of which 20 Up, 44 down. Conclusion h TERT promoter can strictly guide the expression of miR-21 in the telomerase-positive tumor cells, which can inhibit the growth of tumor cells. The results of the chip suggest that miR-21-induced lung cancer may be a multifactorial Multi-gene results of the joint effect.