Inhibition Of Telomerase Activity By SiRNA Targeted To Human Telomerase Reverse Transcriptase

Telomerase is an attractive molecular target towards cancer therapeutic agents because telomerase activity is expressed in approximately 85% tumors, but undetectable in mormal somatic cells. Telomerase is composed of two major components: the catalytic subunit(hTERT) and the template RNA (hTR). Several proteins are associated with hTERT or hTR and facilitate their folding or assembly. The RNA subunit contains a short template sequence that directs the synthesis of DNA repeats at the ends of chromosomes. hTERT is the most critical component that influences the telomerase activity. hTERT is the limiting component necessary for restora--tion of telomerase activity in these cells. hTERT is expressed in 80%~90% tumors. hTERT seems the idea target of choice as this protein is the rate limi--ting factor for telomerase activity. Inhibition of reverse transcriptases falls into two major classes: antisense oligonucleotides/ribozymes; inhibiting hTERT transcription.Short-interfering RNA (siRNA) has recently been shown to be an effective method for inhibiting the expression of a given gene in human cells. Accordingly, we will construct expressing siRNA vector targeting hTERTand evaluate the ability which inhibit telomerase activity in human Hela cells through siRNA.64 nt oligos were chemically synthesized, and annealed, which is targeted to telomerase hTERT gene. At the same time, pSUPER vector was digested with Bgl II and Hindlll, CIP-treated. Finally, anneled oligos were inserted the downstream of pSUPER's polIII HI promoter to construct siRNA expressing plasmid (pSUP-hTE). Oligos with a scrambled sequence could be used as a nonspecial control, and oligos targeting GAPDH gene as a positive control. Recombinant pSUP-hTE vector was identified by digesting with EcoR I and Hindlll and confirmed the correct insertion by sequencing using T3 primer. The results demonstrated that 64bp had been inserted the expected site. Furthermore, the insertion sequence were exactly correct. So we think that pSUP-hTE expressing RNAi system has been constructed successfully.In order to obtain G418-resistant stable clone, we constructed recombiant enhanced green fluorescent protein(EGFP) expression plasmids pEGFP-hTE, which includs neo gene. pSUP-hTE and pEGFP-Cl plasmid DNA were digested by EcoR I -Xhol, then correct two linearized fragments was ligated, pSUP-hTE's HI promoter and 64bp oligos was located on the flanks of the multiclonal site of pEGFP-Cl. We set up nonspecial control pEGFP-SCR and negative control pEGFP-SUP. Recombiant vector was identified by digested with Xhol I — BamH I . Agarose gel electrophoresis indicated pEGFP-hTE and pEGFP-SCR had 340bp lane, whereas pEGFP-SUP only 280bp and empty plasmid only 50bp. So we think that pEGFP-hTE expressing RNAi system had been constructed successfully. This allowed us to study on telomerase activity inhibition for further in vitro.Transient transfection of HeLa cells using a positive control plasmid pSUP-GAPDH, intended to analyze GAPDH mRNA by RT-PCR technique. The results indicated that GAPDH mRNA had been reduced.Lipofectin and G418-resistant stable transfected of HeLa cells, using a plasmid pEGFP-hTE containing the hTERT gene in both forward and reverse orientations, intended to create a duplex of the hTERT transcripts endogeno--usly. 48h after transfection, the reporter gene expression was observed through fluorescence examination with invert microscope, intended to analyze transfection rate. At the same time, hTERT protein expression, telomerase activity and cell growth activity were analyzed. Here, Fluorescent reverse microscope showed that transfection rate was appoximate 20-30%. Compared with control cells, telomerase hTERT mRNA content, hTERT protein content, and telomerase activity decreased in human cancer cells, but no change in Hela cell growth activity. So RNAi technology can be harnessed to down-regulate telomerase.These data indicate that hTERT is probably a critical component of human telomerase and suggest that RNA interference of the hTERT is an effective target to tumors. Therefore, we could conclude that siRNA targeting hTERT genes may provide a new pathway for cancer biotherapy. We developed the method of synthesizing siRNA, which would provide a new tool for sdudying in mammalian cells and organism.