POSTER NO: 422

Screening and Identifying Novel Genes Related To Laryngeal Carcinoma by cDNA-Representative Difference Analysis

Guang He, Kailai Sun, Weineng Fu, Zhen Zhao
Department of Medical Genetics, China Medical University, No.92 North 2nd Road Heping District, Shenyang

Laryngeal Carcinoma is a common cancer of the upper respiratory tract making for 1%-8.4% of total human cancers. The incidence of the disease is high in Northeast of China and increases year after year. The curative effect and prognosis of operation is relatively poor, and is generally followed by severe dysfunction and recrudescence. Discovery novel related gene of laryngeal carcinoma will contribute to learn the molecular mechanisms and provide us target gene for gene diagnosis and gene therapy. cDNA-Representative Difference Analysis (cDNA-RDA) is a reproductive and highly efficient method for the identification of novel genes. In our study, matched normal and cancerous tissues (as Tester and Driver respectively) were used to perform three round of subtractive hybridizations at different ratio 1 : 100, 1 :400 , 1 : 80,000 ,and in the last subtractive hybridization(Dp3), we obtained four differential cDNA fragments (Dp3-1,2,3,4). The sequencing results showed that Dp3-1,Dp3-2, Dp3-3 and Dp3-4 was 343bp,280bp, 205bp and 153bp respectively consistent with the results of purification and enzyme digestion. The following Northern Blot, showed that they were all overexpressed in 8 cases of laryngeal normal tissues as for the matched cancerous tissues the expressions were dramatically decreased. Sequence comparison revealed a highly significant homology of Dp3-1,Dp3-2 and Dp3-3 with transglutaminase 3, cytokeratin 13 and keratin 4, and 95% homology of Dp3-4 with human fibronectin. The immunohistochemistry result of cytokeratin13 suggests that it can act as markers of differentiation in the diagnosis and prognosis of squamous cell laryngeal carcinoma. In addition, they maybe contribute to the suppression of tumor invasion and metastasis. Fibronectin is potentially concerned with metastasis. LOH analysis of cytokeratin 13 and transglutaminase 3 gene confirmed their deletion. From these findings, we drew the conclusion that these fragments we had cloned played an important role in the genesis of laryngeal carcinoma, and we also speculated that cytokeratin 13 and TGM3 gene might be act as tumor suppressor genes. Further efforts will be required to verify this hypothesis using PCR-SSCP to detect mutation and deletion of these genes, and then focus on their structures and functions.