| Cancer is a common disease of a serious threat to human health which may lead to die , in recent years one of the important issues covered in the medical field.The incidence rate of oral cancer is the sixth of morbidity of cancer all over the world. With the advance in molecular techniques, the study on the pathogenetc mechanism and the relationship among tumor, oncogene and tumor suppressor genes at molecular level has become one of the main developing directions in gene therapy on tumor. Mutation, deactivation and deregulated expression of oncogenes and tumour suppressor genes may be involved in the pathogenesis of oral cancer. Deleted in oral cancer-1(DOC-1) is a novel putative oral tumor suppressor genes identified and isolated from the hamster oral cancer model in 1995 and p12 protein is a function of its encoded protein with tumor growth inhibition. As a new candidate tumor suppressor gene has been concerned by medical researchers. The DOC-1 gene is located on chromosome 12q24 and its full length cDNA is 1627bp. Translation of the DOC-1 cDNA predicts a protein of 115 amino acids residues with a molecular weight of 12.4kDa. DOC-1 induces apoptosis in malignant hamster oral keratinocytes. Recently, p12 has been show to be an S-phase regulator through two important cellular partners: CDK2 and DNA polymerase-α-primase. p12 is a CDK2-associated protein that negatively regulates its kinase activity. p12 suppresses tumor growth through reduction of the expression level of CDK2 activity, which to suppress the Rb activities. Ectopic expression of p12 in squamous carcinoma cells reversed the malignant phenotype of these cells, in part due an ability of p12 to bind to both DNA polymerase-α-primase and to cyclin-dependent kinase 2 (CDK2), thereby inhibiting their activities. The expression of p12 in squamous cell carcinoma of human cavity is significantly degraded or even deletion, but the mechanisms and levels of the absence have not been reported.In order to predict the physico-chemical property,secondary structure and the functional motifs of p12 , we use some databases and softwares of bioinformatics.Screening the proteins which interact with p12 by yeast two hybridization to find related factors which induce the deletion of p12 in oral cancer , so we can know the mechanism of DOC-1 in oral cancer and also lays a important foundation for further study the function of DOC-1 gene.Objective: To Construct cDNA Library from human tongue squamous cell carcinoma Tca-8113 cells and prediction of p12 related factors by bioinformatics, screen new proteins by yeast two-hybrid system for further study on function and modulation of doc-1 and its protein p12doc-1 in Squamous Cell Carcinoma of the human oral cavity.Methods: 1.The relative data of bioinformatics and software were used to predict some structure and functional site of DOC-1 gene,which encoded p12; Further more, to find out the relative factors. To acquire the base pair sequence of DOC-1 and amino acids sequence of p12 from GenBank database. Obtaining the extron and intron information of DOC-1 through Spidey in http://www.ncbi.nlm.nih.gov/IEB/Research/Ostell/Spidey. ExPASy in http://www.expasy.org/tools/# proteome was used to predict physico-chemical property of p12 protein. Finding out the functional motifs of p12 by using the tools which in http://myhits.isb-sib.ch/cgi-bin/ motif-scan and http://www.compbio.dundee.ac.uk/-www-jpred. To predict and analysis the secondary structure (αhelix,βfold,βturn and andom coil, etc) of p12 by using Predictprotein in http://www.expasy.org. To identify the protein that encoded by this gene whether has transmembrane structures in http://www.cbs.dtu.dk/ services/TMHMM. 2. The cDNA library of human tongue squamous cell carcinoma, Tca-8113 cells is constructed. Extraction the total RNA of Tca-8113 cells by using Trizol methods, then separating and purifying the mRNA by mRNA Mini Kit and the cDNA library of human tongue squamous cell carcinoma, Tca-8113 cells is constructed by Library Construction Kit and cDNA Synthesis Kit.Result: Gain the informations about extron, some motifs which exist possiblely, physico-chemical property and secondary structure;The recombination rate was 95% and the titre of the primary library was 5.4×105pfu/ml.The titre of amplified library was 7.8×107 pfu/ml.The length of most cDNA inserted in the library was 600-1500bp.Conclusion: Successfully predict the physico-chemical property, secondary structure and some motifs which exist possiblely. Human tongue squamous cell carcinoma Tca-8113 cells is successfully constructed which provide subject for screening related genes and proteins of human tongue squamous cell carcinoma Tca-8113 cells.
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