1.siRNA Targeting CSB And Its Effects On Cell Proliferation And Radiosensitivity 2.Abnormal Promoter Methylation Of P14 And P16 Genes In The α Particle Transformed Cells

Purpose: To construct cellular model of depressed expression of CSB gene by small interfering RNA and investigate of depressing CSB expression on cell growth and radiosensitivity.Methods: According to the siRNA designing regulation proposed by Tuschl and bioinformatic analysis, siRNAs targeting CSB gene were designed based on the gene sequence and mRNA secondary structure of CSB. CSB targeting siRNA plasmids were constructed by inserting the siRNA fragments into the cloning vector pSilencer?.1. Plasmids transfection of HeLa cells was mediated by Lipofectamine 2000. Non-specific siRNA plasmid transfected cells were taken as control. Gene expression was detected by semi-quantitative RT-PCR and Western blot. Cell proliferation was mainly demonstrated by cell growth curves. The apoptosis was assayed by FCM and Fluorescence Dyeing, the radiosensitivity was analyzed by colony forming ability.Results: Two CSB targeting siRNA plasmids were constructed. CSB gene expression detected by RT-PCR was decreased to 44.5% and 53.5% of the control in HeLa cells transfected with 52-siRNA and 107-siRNA respectively, and CSB protein was also remarkably depressed. siRNAs targeting CSB could slow down the proliferation of HeLa cell, and increased the sensitivity of HeLa cell to UV radiation. The cell survival after 5J/m2 UV irradiation was 29.1+4.5% (52-siRNA group) and 44.8?.7% (107-siRNA group) , significant lower than that of control (60.8 + 5.0%). The cell survival of siRNA groups after 10J/m2 UV irradiation was also significant lower than that of control. The spontaneous apoptosis increased in the siRNA targeting CSB cells. The data of FCM as well as Fluorescence Dyeing assays demonstrated that siRNA targeting CSB could obviously increased apoptosis induced by UV irradiation.Conclusion: siRNA targeting CSB can effectively inhibit its expression in mRNAlevel as well as protein level. Depressed expression of CSB could enhance the spontaneous apoptosis level, decrease cell proliferation, and increase the cellular sensitivity to UV radiation. These results further sulgest that CSB gene is a potential target for cancertheray.