Inhibition Of Androgen Receptor And Human Telomerase Reverse Transcriptase (hTERT) Gene Expression In Prostate Cancer Cells By ShRNA Vector

Background and objective: Prostate cancer (PCa) is the most common malignancy and the second most frequent cause of cancer-related death of men in western countries. The incidence and mortality rate of prostate cancer in China has been dramatically increasing, although they are still much lower than those of western countries. Early-stage prostate cancers are usually androgen depend and reponse to androgen ablation therapy well. Unfourntally, prostate cancers eventually become androgen independent, rendering anti-androgen therapy ineffective and mortality. There is still no effective strategy to treate the patients with androgen independent prostate cancer. The gene therapy is considered to be the most potential method for androgen independent prostate cancer. Androgen receptors(AR) not only play an important role in the development of prostate cancer, but also are highly expressed and amplified in advanced and androgen independent prostate cancer cells. Direct targeting androgen receptor is the new strategy for the gene therapy of prostate cancer. hTERT gene, which was highly expressed in tissues and culture cells of prostate cancer, has not been researched well. RNA interference (RNAi) is a new gene blocking technology that silences target gene at post-transcriptional level induced by the small interference RNA (siRNA). RNAi has been demonstrated great prospect in gene functional research and gene therapy areas. We utilized shRNA vector to inhibit hTERT and AR gene expression in prostate cancer cells, and investigate its anti-cancer effects.Experiment part one: RNAi targeting human telomerase reverse transcriptase gene in prostate cancer cells and its anti-cancer effectsPurpose: To evaluate the inhibition of hTERT gene expression in prostate cancer cells using shRNA vector and its anti-cancer effects.Methods: Small interference RNA homologous to hTERT gene was designed, Pgenesil-hTERT-shRNA vector and general negtive vector Pgenesil-HK-shRNA were constructed and transfected into prostate cancer PC-3, DU145, and LNCaP cells. The changes of prostate cancer cells proliferation, apoptosis and hTERT gene expression in both siRNA treatment groups and control groups were determined by MTT, flow cytometry, real-time fluorescent quantitative reverse transcription polymerase chain reaction(FQ-PCR) and Western-Blot.Result: The rate of transfected cells in all groups was more than 60%. The standard curve of hTERT genemRNA expression was established, and the correlation coefficient was 0.998 between the amount of template cDNA and the intensity of fluorescence signal. The results of FQ-PCR indicated that hTERT mRNA level of cells transfected by vector Pgenesil-hTERT-shRNA was much lower than that of Pgenesil-HK-shRNA and control group. Western-Blot showed the protein expression of hTERT gene in cells transfected by vector Pgenesil-hTERT-shRNA was weaker than that of Pgenesil-HK-shRNA and control group. Telomerase activity of control group cells and cells transfected by vector Pgenesil-HK-shRNA was significantly higher than that of Pgenesil-hTERT-shRNA group. The inhibition rate of cell growth of Pgenesil-hTERT-shRNA group was significantly higher than that of Pgenesil-HK-shRNA group.Conclusion: The expression of hTERT gene in both androgen dependent and independent prostate cancer cells can be significantly inhibited by hTERT-shRNA, resulting in down regulation of telomerase activity and obvious inhibition of cell growth.Experiment part two: Inhibition of androgen receptor gene expression in prostate cancer cells using shRNA vector.Purpose: To evaluate the inhibition of AR gene expression in LNCaP cells by shRNA vector and its anti-cancer effects.Methods: Two small interference RNAs homologous to AR gene were designed, Pgenesil-ARl-shRNA, Pgenesil-AR2-shRNA and Pgenesil-HK-shRNA vectors were constructed and transfected into prostate cancer LNCaP cells. The changes of prostate cancer cells proliferation and AR gene expression in both siRNA treatment groups and control groups were determined by MTT, flow cytometry, FQ-PCR and Weste...