Mechanism On Inhibiting PC-3Prostate Cancer Cells Proliferation By Specific SiRNA Targeting Human Telomerase Reverse Transcriptase

Prostate cancer is one of the malignant tumors that occur in male prostate tissue, which is resulted from prostate gland bubble abnormal cellular sprawling growth. The risk of prostate cancer has obvious geographic and racial differences. In Europe, United States and other developed countries and regions, it is the most common cancer in men with the mortality rate ranking second; In Asia, its incidence is lower than in the western world, but is rapidly increasing in recent years. Current treatments for prostate cancer are radiation therapy, chemotherapy, and surgical operations, and patient survival rates have increased significantly, but the treatments are with low selectivity and poor target ability. Therefore, with the rapid development of molecular biology, gene targeting treatment for prostate cancer has drawn more and more attention.Telomerase is an enzyme that is responsible for the extension of telomeres, and is the basic nucleoprotein reverse transcriptase, which can be added to telomere DNA chromosomal end eukaryotic cells. The activities of telomerase in normal human body tissues are suppressed, while in the tumors reactivated and may participate in malignant transformation. As the human telomerase reverse transcriptase(hTERT) is an important protein component of telomerase, its mRNA level is of close association with the telomerase activity and is the limiting step to adjust the telomerase activity, and specific inhibition hTERT can reduce the expression of telomerase activity leading to inhibition and death of cells, thus hTERT can be the ideal target for gene therapy.Among all the gene therapy approaches for cancers, RNA interference (RNAi) technology is commonly utilized for hTERT gene silence, The design of siRNA sequence and its synthesis are the keys. In this study, according to the RNAi principle, small interfering RNA was designed to target hTERT. hTERT-highly-expressed prostate cancer PC-3cells were chosen as the research model.Most effective siRNA sequence was identified after in vitro transfection of prostate cancer PC-3cells based on the inhibitory effect on cell proliferation and hTERT mRNA down-regulation. Protein expression and cell apoptosis were further investigated to elucidate the possible mechanism.In this study, liposome carrier LipofectaminTM2000was first used, and additionally carbon nanotube (CNT, Carbon Nanotube) was also chosen as the carrier of siRNA.Due to the poor water-solubility of carbon nanotubes, the negatively charged single-walled carbon nanotubes(SWNT) were first subject to surface modification with positively charged DOTAP via the electrostatic interactions, which greatly improved the water-solubility of carbon nanotubes. The functionalized carbon nanotubes showed high efficiency to load siRNA and transfer siRNA into PC-3cells resulting in significant tumor cell growth inhibition.A new approach for the treatment of prostate cancer was explored in this study as outlined below:1Screening of the effective siRNA sequence for targeting hTERTBoth the mRNA sequence(AFO15950) of hTERT gene in GenBank and the designed hTERT-targeting siRNA through software were used for BLASTTM analysis in GenBank, eliminating the related gene homological sequences to assure the specificity of the designed sequences.The hTERT cDNA sequences with a GC content of at least50%were selected, and four hTERT-siRNA were designed, and double chain siRNA was synthesized. With LipofectaminTM2000mediated, they were tranfected into prostate cancer PC-3cells. SRB (Sulforhodamine B colorimetric assay) and RT-PCR method were applied to evaluate the inhibition toward cells and hTERT mRNA expression so as to screen out the best sequence.2PC-3cell apoptosis induced by suppression of hTERT expression with RNA interferenceThe most effective sequences was used for the further mechanistic study. The siRNA sequences was transfected into the prostate cancer PC-3cells with LipofectaminTM2000, and then PI single staining method was used to examine the influence on the cell cycle, and Annexin V-FITC/PI double staining method was used to detect the induced cell apoptosis. In addition, Western blot was performed to detect the change of hTERT protein to explore the mechanism by which apoptosis was caused.3Inhibition of PC-3cell proliferation with SWNT-DOTAP as a novel gene carrierIn this section, SWNT-DOTAP was synthesized, and its potential, particle size, loading ratio with siRNA and its toxicity toward prostate cancer PC-3cells were all tested. Then the most effective siRNA was transfected into prostate cancer PC-3cells with SWNT-DOTAP to study its growth inhibition effect.Conclusion:1:Four hTERT siRNA sequences were designed and synthesized, and the most effective sequence was identified, which is hTERT-siRNA-2.2:With LipofectaminTM2000siRNA-2was transfected into PC-3cells and it was found that the expression of hTERT mRNA and protein was significantly down-regulated with an inhibition rate of85%and65%, respectively, which confirmed the specificity of siRNA to the hTERT in PC-3cells.3:Transfection of siRNA-2into PC-3cells also inhibited tumor cell growth and induced cell apoptosis at a rate of44%and14%, respectively, which suggests its in vitro anti-prostate cancer effects.4:A novel gene carrier SWNT-DOTAP has been successfully developed, which showed little toxic effect on PC-3cells. Transfection of siRNA-2into PC-3cells with SWNT-DOTAP significantly inhibited the proliferation of PC-3cells with an inhibition rate of40%, which is comparable to that of LipofectaminTM2000, suggesting that this new gene carrier may be used for cancer gene therapy with siRNA.