Attenuation Of Telomerase Activity By A Hammerhead Ribozyme Targeting The Template Region Of Ovarian Cancer

Ovarian cancer is the most common malignant tumor of female reproductive organ, although at present, the diagnosis and treatment methods have progressed in some extent, but the mortality rate hasn't decreased obviously, the 5-year survival rate is still less than 30%. So, the therapy of ovarian cancer has become a puzzled problem. Telomere is a protein-DNA structure in the end of eukaryotic chromosomes, it can protect and stable the end of chromosome. In all normal somatic cells, each cycle of cell division and DNA replication, the repetitive telomere sequences lose increasingly which leading to cells aging. Telomerase is a ribonucleoprotein polymerase that synthesizes tolemeric DNA onto chromosomal ends using a segment of its RNA moiety as a template. Most maligmant tumor cells express telornerase activity to prevent telomere shortening and contributes to chromosomal stability which is related to immortalization of the cells. Therefore, it has been proposed that anticancer agents based on telomerase inhibition may potentially provide effective therapy. To explore the effects of human telomerase RNA specific 5 hammerhead ribozyme on ovarian carcinoma cell and to further discuss the possibility of clinical application of ribozymes, we conducted the following experiments. 1 Constructions of ribozyme vector: Hammerhead ribozyme directed against template element of the human telomerase RNA component were designed and synthesized according to telomerase eDNA. After gene recombination, a vitro transcription vector and a mammalian expression vector for ribozyme pcDNA3-RZ were constructed. For production of ribozyme substrates, we cloned a portion of the human telomerase RNA component containing a telomeric template element using reverse transcriptase-PCR(RT-PCR). The PCR product of expected size was cloned in pGEM-T Easy vector according to the manufacturer s instructions. All the vectors were verified by DNA sequencing. 2 Activity identification of the telomerase RNA ribozyme: ribozyme and target were transcribed in vitro by T7 phage RNA polymerase and tested for in vitro cleavage activity. After 6%denature PAGE and autoradiography, results were analyzed. Cleavage efficiency of ribozyme was 68%. 3 Effects of telomerase RNA ribozyme on HO-8910: RNA dot hybridization proved that pcDNA3-RZ had been successful transfected into HO-89l0 cells By LipofectAMlNE. We observed the propagation of the cells and detected their telomerase activity by TRAP-Hyb Kit. After transfection, the propagation of the cells was inhibited and the telomerase activity was descended. Cells in G1 phase were significantly increased observed under flowcytometry, and cells in S phase were decreased by 30%(P