Early Cervical Squamous Cell Carcinoma Lymph Node Metastasis Related Est Screening And Full-length Cdna Clone Validation

PURPOSE: Screenin the gene expressed sequences related to early stage cervical cancer, cloning full length of EST, analyzing their biological characters and predicting protein function with bioinformatics. To search and discover novel genes playing regulation in early stage cervical cancer lymph node metastasis.To providing the markers and bases to discuss the mechanism of early stage cervical cancer or diagnosis and therapy in gene level. METHODS: Fifteen differentially expressed ESTs were analyzed. We screened EST sequences searching EST and nr database on NCBI by basic local alignment search tool BLASTN. Then using silicon cloning to obtain the more longer sequences or full length cDNA of the EST of no homologous sequences in database. Analyzed the character of known EST with database just like human genomic project,Unigene. To unknown EST, we analyzed its ORF , searched its full cDNA sequence in DoTS, scanned its exon by Genescan software. At last ,we using reverse transcription PCR(RT-PCR) and Immunohistochemistry (IHC) to identity the mRNA and protein expression levels in early stage squamaceous cervical cancer.RESULTS: Through BLASTN and BLASTX software, the ESTs were compared with GenBank database for homology analysis.Three ESTs have homology with known genes: one is human maternally expressed gene 3(MEG3) of Unigene datebase Hs. and is located at human chromosome 14q32 .It is down-regulated in cervical cancer. The other is huaman MIF gene of Unigene database Hs. and is located at human chromosome ,it is up-regulated in cervical cancer. Another is SNRPN gene of Unigene database Hs. and is located at human chromosome ,it is up-regulated in cervical cancer. One unknown EST is ADCAPE03,it has no homologous sequences in databases.After silicon cloning,its sequence is 1541bp.Nucleic acid sequence analysis showed it contained a 102 integrated ORF,encoding 34 amino acid and was named ADCAPE03. Searching Human gonome project It was located at human chromosome 11q13-14. ADCAPE03 has no homologous with known protein database through BLASTP software. Searching this sequence in Data of Transcripts finding that it had 100% homologous with DT.311034.At last, we examined the mRNA expressions of ADCAPE03,MEG3,SNRPN and MIF in early stage SCC .ADCAPE03,SNRPN and MIF were all up-regulated in early stage SCC; while the expression of MEG3 was down-regulated. The protein expression of MIF was higher in early stage SCC with lymph node metastasis than those without metastasis(P<0.01),and it has correlations with the lymph node metastasis.( r=0.34,P< 0.05).CONCLUSIONS: Gene related to cervical cancer were idengtify using bioinformatics technique. Three known EST were obtained and highly homologous with MEG3,SNRPN,MIF respectively. Through electron expression analysis, they all didn't distributed in cervix and cervical cancers. But, we examinated the mRNA expressions of these genes in early stage SCC surely. Besides, we also obtained one EST ,which has no homologous sequences in database, encoded 34 amino acid. But its exatly sequence need further examined. Other three EST sequences and its homologous protein needed further research in their function analysis in cervical cancer.