SMART Library Screening New Gene C20orf108 Involved In Mitochondrial Apoptosis Pathway Of Tumor Cell

[Objective] establish SMART library to find the new target of apoptosis-related genes in tumor cells.[Methods] Trichostatin A was added to cell lines MCF-7 and 0,4,8,12,18,24,36h later cells were collected. Total RNA was obtained and mRNA was separated and reverse transcripted into cDNA, cDNA was inserted into plasmid pCEP4 to form the SMART library,40μg of the antisense cDNA library was transfected into HeLa cells with empty vector as control.48h after the transfection, TSA (150nmol/L) and together with HYB (200μg/L) were used for screening and 12d later, the surviving colonies were tested for the inserting genes.[Results] 250nmol/L TSA can induce apoptosis of MCF-7 and Hela cells.90% cDNA, reverse transcripted from MCF-7 mRNA, was successfully inserted. The mean size of inserts was approximately 1.5kb and the total volume was about 2×105. The cDNA library was transfected into Hela cells. Hirt DNA was extracted from the obtained positive clones after TSA and HYB selection.76 effective colonies were identified after restriction enzyme cutting.[Conclusion] 1.Establishing the model of TSA inducing tumor cell apoptosis.2. Obtaining 76 effective colonies by SMART and biological information analysis.3. Further mechanical research was done about Liv-1,Smad4 and Ubiquitin B screened from the library. [Methods]C20orf108 was screened using SMART, its siRNA was synthesized and the sense expressing plasmid was established. PCR was used to detect C20orf108 in several cell lines. The effect of C20orf108 on cell apoptosis was tested by flow cytometry. The location was identified by Westen-blotting and fluorescent immunoassay. The co-acting protein was detected using immunoprecipitation.[Results]Down regulation of C20orf108 expression could decrease HDACi induced apoptosis in MCF-7and HeLa cell lines. Up regulation of C20orf108 expression could increase cell apoptosis and the effect depended on mitochondrion apoptosis pathway. C20orf108 was located in mitochondrion.[Conclusion]1. C20orfl08 was one of the new genes regulating cell apoptosis. C20orf108 was different expressed in tumor cells and normal cells and was involved in the mitochondrion apoptosis pathway.2. Further detail research was done on how C20orf108 participated in the mitochondrion apoptosis pathway...