Construction Of Eukaryotic Expression Plasmid Experssing SiRNA Targeting ATM Gene And The Regulation By RNA Interference Based Knockdown Of ATM On Radiotherapy Sensitivity Of Cervical Carcinoma

The ATM gene (ataxia-telangiectasia mutated ATM) is a kind of important gene founded in present years, which plays a key role in initiating cellular repair responses to DNA damage. The protein encoded by this gene belongs to the PI3 kinase family. This protein is an important cell cycle checkpoint kinase that phosphorylates; thus, it functions as a regulator of a wide variety of downstream proteins, including tumor suppressor proteins p53 and BRCA1, checkpoint kinase CHK2, checkpoint proteins RAD 17 and RAD9, and DNA repair protein NBS1. The phosphorylation of these proteins is thought to be necessary for activing cell cycle checkpoint signaling pathways, initiating cell response to DNA damage and keeping genome stability. It also shows ATM can resist apoptosis. As a result, ATM plays an important role in cellular responses to DNA damage. In addition, study also shows ATM can protect cells from radiation, the inactivation of ATM gene in tumor cells will make them lost protection from ATM, improve their radiosensitivity. In a word, the inhibition and even knock-down of ATM gene is useful for tumor gene therapy.RNA interference (RNAi) represents a phenomenon of double-stranded RNA (dsRNA) mediated post-transcriptional gene silencing (PTGS). During this process, siRNAs is the mediator, which is made up of 19-23 bp small RNA fragments. By joining an effector complex termed RISC (RNA-induced silencing complex), siRNA binds to the cellular RNA with homologous sequences, and contributes to the degradation of the corresponding RNA. At present, RNAi has been widely applied in the research of gene function and gene therapy as an efficient tool for specific gene silencing. As a result, ATM gene is an promising target for tumor therapy, and knockdown of special genes by RNA interference also shows favorable application foreground.AIM: Construct eukaryotic expression plasmid experssing siRNA targeting ATM gene and transfect it into cervical carcinomal SiHa and HeLa cells, to study the inhibitory effect of ATM protein by RNA interference, and its relationship to the sensitivity of radiotherapy in cervical carcinoma.METHODS: (1) Hairpin siRNA templates were designed based on ATM gene sequence and mRNA structure and were cloned into eukaryotic expression plasmid pSuppressorNeo, then confirmation them by restrict endonuclease digestion and DNA sequencing. (2) SiHa and HeLa cells were transfected by pSup-ATM with electroporation, the non-transfected cells and non-specific siRNA transfected cells were taken as controls. Inhibitory effect of ATM mRNA and protein expression were detected by semi-quantitative RT-PCR and immunofluorescency respectively in instantaneous transfected cells. (3) Stabilized transfected cervical carcinomal cells were constructed by selection with G418, inhibitory effect of ATM mRNA was detected by semi-quantitative RT-PCR, inhibitory effect of ATM protein was detected by FCM and Western-blot. (4) Observed the effect of knockdown of ATM on proliferation ability of SiHa cell by MTT assay. (5) Compared the changes of sensitivity to radiotherapy and chemotherapy by FCM, immunofluorescency and clone formation assay, so as to...