Cloning And Preliminary Studies Of Laryngeal Carcinoma Related Gene Promoter

Laryngeal Carcinoma is a common malignant tumor,which leads to death of a lot of people.However,the mechanisms underlying the suggested laryngeal carcinoma carcinogenesis has not been investigated.Studies suggested that the LCRG1(Laryngeal Carcinoma Related Gene 1) gene is significantly down regulated in laryngeal carcinomas. It suggests that there have the relationgship between the down expression of LCRG1 gene and the tumour generation of Laryngeal epithelium. But still now, the transcription regulatory mechanisms of its promoter have not been investigated. In this study, we have engaged a molecular dissection of the promoter of the LCRG1 gene and investigated its transcriptional regulatory initially.Methods(1) Applying the methods of 5′RACE to determining the transcriptional start site of the LCRG1 gene;(2) LCRG1 promoter and its possible binding sites of transcription factors were predicted and analyzed by bioinformatical methods;(3) Using molecular dissection to construct, based upon the luciferase reporter gene, a set of the 5′, or 3′deletion mutants from the full-length upstream region;(4) Via the transient transfection/dual reporter assay, the promoter activities of the above constructs were vigorously tested;(5) Via the analyses co-transfection,the proteins binding specific to the cis-elments within the key region with significant functional consequence was determined.Results(1) The base of most of 450bp and 600bp product after the 5'race adaptor is A. So the transcriptional start site is A and labelling the A with +1. This transcriptional start site located before the translation site 284bp;(2) There was five possible promoter regions predicted by the promoter on-line predicton software. They were all located in the upstream regions of LCRG1 gene transcript. Many possible transcription factor binding sites such as Sp1, zf9, E2F1 were predicted by MnInspector, a transcription factors analysis web system;(3) The minimal promoter of LCRG1 gene was defined to a fragment from-169 to +127 position relative to the transcription start site;(4) The exogenous transcription factors Sp1 can up-regulation the promoter activity of the LCRG1 gene.Conclusions(1) The transcriptional start site of LCRG1 gene is A and labelling the A with +1.this transcriptional start site located before the translation site 284bp;(2) The minimal promoter of LCRG1 gene was defined to a fragment from-169 to +127 position relative to the transcription start site;(3) The exogenous transcription factors Sp1 can up-regulation the promoter activity of the LCRG1 gene.