RNA Interference Directed By Small Interference RNA Of HTERT Expressed In Prostate Carcinoma Cell ALVA-41

Objective The knowledge and application of RNA interference technology have been developed greatly since Fire discovered it in 1998. RNA interference (RNAi) is the process of highly sequence-specific post transcriptional gene silencing mechanism , induced by endogenous or extragenous double-stranded RNA (dsRNA) . Twenty one nucleotide small interfering RNAs ( siRNA) can specifically induces potent silencing effect of gene expression in mammalian cells. This remarkable study provides a new tool for the treatment of many human diseases including AIDS , hepatitis and tumors , etc. Telomerase expresses in 80%-90% of maliganancy but dose not express in the most of norm cells. Telomerase may be a universal mark of human tumors. The highly expression of human telomerase in prostate carcinoma cell indcates that telomerase be applicated in the diagnosis of prostate carcinoma, and telomerase inhibitor be applicated in the therapy of prostate carcinoma. This study is to investigate the inhibitory effect of siRNA human telomerase transcriptase (hTERT) on activity of telomerase and proliferation. It may open a new approach to the use of siRNA as a new tool to study gene function in cancer cell lines, and may be developed to be a new gene therapeutic agent for cancer.Methods (1)A plasmid including U6 promoter and siRNA of hTERT was designed and constructed, and identified by electrophoresis and sequence analysis;(2) The reconstructed plasmid was transfected into ALVA-41 cell line. The telomerase activity was tested by telomerase repeat amplification protocol ELISA (TRAP-ELISA);(3) hTERT expression was assessed by Western blot;(4) Cell calculating assay was used to assay the cell proliferation activity seven days later after transfected. Results (l)mU6-hTERT-siRNA was synthesized successfully and identified by electrophoresis and sequence analysis. The target sequences location was right. mU6-hTERT-siRNA plasmid, mU6 plasmid were transfected into ALVA-41 cells respectively. Measure the effects of RNA interference and contrast with the blank control group;(2) Western blot show that the mU6-hTERT-siRNA showed obvious interfering effect;(3) Telomerase repeat amplification protocol ELISA showed that mU6-hTERT-siRNA could down regulate the expression of hTERT protein;(4) Cellcalculating assay showed that mU6-hTERT-siRNA inhibit telomerase activity andproliferation of ALVA-41 cells.Conclusion (1) ) mU6-hTERT-siRNA was synthesized successfully;(2)siRNA ofhTERT can inhibit the expression of human telomerase and proliferation of ALVA-41cells;(3)RNA interference which target hTERT may provide one new way for tumorsgene therapy.