关键词: 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

Abstract:

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.

Key words: Human telomerase reverse transcriptase, Promoter, microRNA-21 sponge inhibitor, Construction of lentivirus vector, Read-time PCR, cDNA microarray