An experimental study on colorectal carcinoma cell biological characteristics bv RNA interference technology of human telomerase reverse transcriptase gene silence

Objective To observe the bionomics effects of short hairpin RNA (shRNA) fragment of human telomerase reverse transcriptase (hTERT) gene on human SW480 tumor cell line.Methods The human colorectal carcinoma cell line SW480 was transfected with recombinant eukaryotic expression plasmid pGPU6/GFP/Neo-hTERT shRNA (hTERT-shRNA) by LipofectamineTM 2000,and cells were divided by four groups with 3 repetitive holes in each group.The hTERT mRNA and protein expression levels in SW480 cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry respectively,and the telomerase activity was detected by PCR-TRAP-ELISA. The effects of shRNA on cell cycle were analyzed by flow cytometry (FCM).terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) and transmission electron microscopy were used to observe the changes in SW480 cells apoptosis and mitochondrial membrane potential (MMP).Results Forty-eight h after transfection,hTERT mRNA relative expression was 0.32 ±0.03,hTERT protein average gray scale was 209.60 ±17.10,A450-630 nm value of telomerase was 2.242 ±0.284 in hTERT-shRNA group,which were reduced remarkably as compared with other three groups (P ＜0.05,P ＜0.01 ).The cells number at phase G0/G1 was (49.37 ± 1.63 )％,the proliferation index was (50.60 ± 1.57 )％,and the apoptotic index was 22.2％ in hTERT-shRNA group,which were increased obviously as compared with other three groups (P ＜ 0.05,P ＜ 0.01 ).Under the transmission electron microscopy,apoptotic cells were found,the cellular volume got smaller,and the protrusion and minovillus were reduced,some even disappeared.The fluorescent intensity of Rhodamine 123 was 719.99 ± 17.08,and MMP was reduced markedly in hTERT-shRNA group as compared with control groups (P ＜ 0.01 ).Conclusion RNA interference (RNAi) silencing hTERT gene can effectively inhibit SW480 cell growth,reduce the activity of telomerase and induce tumor cells apoptosis. Key words: RNA interference;  Human telomerase reverse transcriptase;  Gene silence; Colorectal cancer