关键词: 端粒酶卡侯体蛋白1, RNA干扰, 基因沉默

Abstract:

Objective To investigate the inhibitory effects of telomerase Cajal body protein 1(TCAB1)-shRNA on oral squamous cell carcinoma Cal27 and to explore a novel cancer gene therapy approach by depletion of TCAB1 using RNA interference. Methods Four recombinant lentiviruses, called shTCAB1-A–D, which contain oligonucleotide(67 bp) targeting TCAB1 and green fluorescent protein EGFP Puro, were constructed based on different sites in the TCAB1 gene. A series of experiments were then carried out to demonstrate the silencing effects of the shTCAB1 lentiviruses on Cal27 cells. Semiquantitative analysis of TCAB1-mRNA expression levels was performed. TCAB1 protein expression level was analyzed using Western blot. Cell proliferation viability was tested by methye thiazolye telrazlium assay. Transwell invasion assay was used to evaluate cell invasiveness. Results Polymerase chain reaction and DNA sequencing demonstrated that the shTCAB1 lentiviral vectors were constructed successfully. The titers of the viruses ranged from 1.5×108 U•mL-1 to 3×108 U•mL-1. Compared with the blank control group, shTCAB1-A–D exhibited pronounced inhibitory effects, which significantly reduced both TCAB1 mRNA levels(48.7% to 62.0%) and protein levels(45.6% to 77.5%) in Cal27 cells. shTCAB1-C, the most effective inhibition group, caused slower growth of tumor cells and significant suppression of invasion potency(67.5%), (P<0.05). Conclusion The lentiviral vector shTCAB1 could effectively express siRNA against TCAB1 in Cal27 cells, inducing post-transcriptional gene silencing.

Key words: telomerase Cajal body protein 1, RNA interference, gene silencing