Construction Of Human Telomerase Antisense RNA And It's Effect On Human Hepatoma Cells

Telomeres are specialized ribonucleoprotein structures at the ends of eukaryotic linear chromosomes. They are composed of repetitive nucleotide sequences and associated proteins that protect chromosomes from degradation and recombination. They are essential for chromosome stability. In human somatic cells, telomeres shorten by 30 to 200 bp with each cell division. When telomeres were shortened to some threshold value, cell senescence or apoptosis was triggered. The mechanism is that telomerase is activated so that telomeres are maintained at a certain length in tumors, keeping cell growing. Telomerase is a ribonucleoprotein complex that adds TTAGGG repeats onto the 3'ends of chromosomal DNA termini, thereby compensating for incomplete replication. Human telomerase reverse transcripase (hTERT) is a key component of telomerase catalytic activity. High telomerase activity has been demonstrated in 85 to 90%of all human tumors according to statistics. Telomerase plays an important role in tumor cell proliferation. A relationship between telomerase and tumor cells has been established. The activation of telomerase may be an important path of cell immortalization and cancerization. Therefore, telomerase will be a new target for tumor gene treatment. We constructed the antisense recombinant human telomerase expression vector by molecular clone technique and study the effect of human telomerase antisense RNA on hepatocelluar carcinoma cells. Telomerase hTERT gene fragment was amplified from PLNCX-hTERT plasmid. The hTERT fragment was subcloned into eukaryotic expression plasmid pcDNA3.1/Myc-His in reverse direction after it was sequenced to confirm the correction of gene. The plasmid pcDNA3.1-hTERT (as) was transfected by lipofectamine into hepatocellular carcinoma cells. An inducible cell line that can express telomerase RNA was gotten by the selection using 400 mg/l G418 solution. Methods of MTT ,TRAP-ELISA,flow cytometry and real-time quantitative PCR were used to investigate the proliferation, telomerase activity, apoptosis and hTERT mRNA expression of human hepatoma cells SMMC-7721. The results are as follows: 1. The effect of human telomerase antisense RNA on hepatoma cell proliferation was studied by counting the number of cells and MTT assay. As compared with SMMC-7721 control cells and cells transfected with pcDNA3.1/Myc-His void vector, the proliferation rate was significantly decreased in SMMC-7721 cells transfected with antisense RNA. 2. The effect of human telomerase antisense RNA on the telomerase activity of hepatom cells SMMC-7721 was studied by TRAP-ELISA method. As compared with control cells and pcDNA3.1/Myc-His void vecter transfected cells, telomerase activity was decreased significantly in antisense RNA transfected cells. 3. The effect of human telomerase antisense RNA on the apoptosis and cell cycle of hepatoma cells was studied by flow cytometry. As compared with the control cells and pcDNA3.1/Myc-His void vecter transfected cells, apoptosis was induced in the antisense RNA transfected SMMC-7721 cells; the number of S period cells was significantly increased in the antisense RNA transfected SMMC-7721 cells, the number of G0/G1 period cells was significantly decreased in the antisense RNA transfected SMMC-7721 cells. 4. The effect of human telomerase antisense RNA on hTERT mRNA expression in hepatoma cells SMMC-7721 was investigated by real-time quantitative PCR. As compared with the control cells and pcDNA3.1/Myc-His void vector transfected cells, hTERT mRNA expression did not changed significantly in antisense RNA transfected cells. In conclusion, telomerase hTERT antisense RNA can effectively inhibit the growth of hepatoma cells SMMC-7721 and induce the apoptosis of hepatoma cells. This study provided basic researches for hepatoma-treatment by a gene therapeutic approach.