| Laryngeal carcinoma is a fairly common cancer of the upper aero digestive tract, accounting for 1% ~ 8. 4% of human cancers in our country, yet little is known about the molecular changes in this often disfiguring and fatal disease. The disease is characterized by a relatively poor prognosis and the treatment is generally followed by severe dysfunction. Few protooncogenes and tumor suppressor genes have been found to alter in laryngeal carcinoma. Discovery and study of novel genes related to laryngeal carcinoma helps to understand the molecular mechanisms better in the development of laryngeal carcinoma.This paper consists of two parts. Analysis of nm23 - HI gene in laryngeal carcinoma in the first part and cloning, sequencing and i-dentifying seven novel cDNA fragments related to laryngeal carcinoma are described in the second part.Loss of heterozygosity and microsatellite instability of nm23 - HI were studied in 72 samples of laryngeal carcinoma using 5 microsatellite polymorphism markers in or around the gene, meanwhile the expression was compared between the normal, tumor and the corresponding metastatic lymphonode in 38 samples to investigate the effect of nm23 - HI gene on laryngeal carcinoma. LOH appeared at all the five loci; the highest frequency of 39. 68% was at the D17S1665 polymorphism locus, LOH concerning at least one polymorphism locus reached 76. 39% . Three loci had MI with the highest frequency of 12. 70% ,and the total MI concerning at least one locus was 22.22%. No correlation was found between lymphonode metastasis, clinical stage and differentiation with LOH or MI of nm23 - HI gene, P > 0. 05, but LOH at locus D17S1665 correlated with the differentiation of the tumor, P <0. 05. The difference of nm23 - HI gene expression among tumor, normal tissue around tumor and metastatic lymphonode was not significant, no correlation between mRNA level of nm23 - HI gene and metastasis was found, P >0. 05. So we drew the conclusion that nm23 - HI gene may have important function in the oncogenesis of larynge-al carcinoma, and LOH is the main dysfunction mechanism of nm23 -HI gene.In the process of development, differentiation and metabolism, the differential expression of genes plays a pivotal role. mRNA differential display, established by Liang and Pardee in 1992, is among the most sensitive methods for the isolation of differentially regulated mRNA. In short, mRNA is reverse transcribed into corresponding cDNA, cDNA is amplified by PCR and displayed on 6% PAG gels. With the modification of the method, it becomes more convenient to understand and use. In our study, matched normal and cancerous tissues were differentially displayed and 7 differential cDNA fragments were obtained. They were then purified and cloned into the pGEM - T vector and transformation was performed using JM109 competent cells. Positive clones were found in the plasmids within which fragments were inserted and then sequenced them using T7 and Sp6 primers. RT - PCR was done using the specific primers within the two selected fragments to identify whether they came from laryngeal tissues. Similarity sequences were also analyzed with the help of Blast Similarity Searching System. The sequencing result showed that cDNA fragment Tl to N5. 4 .are 750bp,348 bp,823bp,1162bp, 1083 bp,515bp,H73bp respectively. Sequence comparison revealed that T1,T2, N1 had low similarity with known sequence in human genome and might be novel expression sequence tags to play an important role in the genesis of la-ryngeal carcinoma, while cDNA N2, N3 , N4, N5 were of similarity with known sequence or genes. We also speculated that cDNA Tl and T2 showed positive role in the development of laryngeal cancer as on-cogenes, and the other five showed negative role as tumor suppressor genes. And expression of gene phospholipase A2 and collagen XII decreased in the tumor tissues. Further efforts will be required to isolate the full length of cDNAs and focus on the study of gene's function. |
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