Establishment Of RNA Interference Targeting Against Human Telomerase Reverse Transcritase (hTERT) And Research For It's Biological Effects

Telomerase is a ribonucleoprotein complex that maintains and extends telomerases of eukaryotic chromosomes, and regulates cell replicative potential and lifespan. Now, telomerase has become an attractive molecular target toward which to direct cancer therapeutic agents because telomerase activation is observed in almost 90% human malignant cells but not in most normal somatic cells. Human telomerase is composed of an RNA subunit(hTR) that contains the template for telomeric DNA and several protein components that include the catalyst telomerase reverse transcriptase(hTERT) and telomerase-associated protein 1(hTEP1). hTR and hTEP1 are widely expressed in normal human tissues, whereas hTERT is expressed concomitantly with the activation of telomerase during cellular immortalization and plays a pivotal role in carcinogenesis. Recently, more and more researches indicated that through interactions with other molecules, hTERT may exert different functions besides its major role in maintaining the stability of telomere structures. To further study the new biological functions of hTERT and find the related molecules interacting with hTERT, RNA interference (RNAi) technic was applied to inhibit hTERT gene expression in the human hepatocellular cells (HepG2). Biological characters of HepG2 cells such as apoptosis, cell growth rate were observed and several important proteins in apoptosis pathway were assayed for their expression levels to explore the mechanism of apoptosis. The role of p53 in regulating hTERT expression was also investigated. The main contents of this research are as following: 1. Effective siRNA targets screening for human telomerase reverse transcriptase Five double-stranded DNAs that directed synthesize siRNAs targeting coding and non-coding regions of hTERT gene were designed by full length gene targeting technique or selected randomly. All siRNAs were synthesized by T7 transcription system in vitro and transfected to HepG2 cells by Lipofectamine 2000TM. MTT assay, RT-PCR and Western-blot were applied to evaluate the effects of siRNAs on cell growth, mRNA and protein expression level of hTERT gene, respectively. The results showed that siRNAs targeting different hTERT sequences can inhibit HepG2 cells growth in a dose manner and have significantly various inhibitory effects on hTERT gene expression. Two of which have significantly inhibitory effects on hTERT gene expression. The siRNA sequence screened by full length gene targeting technique can significantly inhibits hTERT gene expression and has compatible inhibitory effect with the best siRNA sequence screened by random selection. The results showed that various siRNAs directed at different sites of hTERT have obvious different inhibitory effects and suggested that secondary structure in the target transcript has a major effect on siRNA efficacy. siRNAs and antisense oligonucleic acids may have the same effective target sites. 2. RNA interference targeting against human telomerase reverse transcriptase induces HepG2 cells apoptosis The siRNA screened by full length gene targeting technique was used to treat HepG2 cells for different time with different concentrations. Cells apoptosis was detected by DNA ladder andHoechest staining method. Several proteins in apoptosis pathway including p53, Bcl-2,Bax were assayed for their expression levels by Western-blot to explore the machanism of apoptosis. The results suggested that down-regulating of hTERT expression by RNA interference can effectively induce apoptosis of HepG2 cells. The intrinsic pathway emerging from the mitochondria is primarily involved in hTERT-inhibited induced apoptosis. 3 Expression of siRNA targeting against human telomerase reverse transcriptase for gene silencing by constructing DNA-based plasmid vector hTERT cDNA sequences from nucleotides 3565-3583 and it's corresponding antisense nucleotides followed by five thymines were linked by 9bp nucleotides and cloned to the downstream of the U6 promoter in pPUR/U6 vector, resulting in the RNA interference vector pPUR/U6/hTERT to...