| Laryngeal carcinoma ( LSCC) is common in upper respiratory tract, ac-counts for 1% 8.4% of human malignant tumors, which may relate to smok-ing, drinking and cold weather. There are few data about molecular genetics study in laryngeal cancer at present, although some authors reported that laryn-geal cancer was related to oncogenes ( ras, c - Myc, EGFR, PRAD, Int - 2, etc) and TSGs (p53, Rb, p!6, FHIT, TGM3, CK13, etc), no clear report a-bout gene cloning related to laryngeal carcinoma was reported. It is important for us to clone and locate laryngeal cancer - related genes by taking full advantage of patient resources in our country and participating in human genome project so as to fill the gaps in this field.Following with the rapid development of human genome sequencing and biomformatics, a large number of genomic information and software emerged. It is an important task for functional genomics to discover novel genes and the func-tion of these genes by utilizing these data and tools. It is also an effective strate-gy in gene cloning.In previous study, we found that chromosome region 6q25 was deleted in laryngeal carcinoma with a high frequency. However, there was no report about genes related to laryngeal cancer had been cloned in this region. In this study, MTLC, a putative target of c - Myc in 6q25 , was cloned ( GenBank access num-ber AF527367) by in silico hybridization and molecular methods. We also re-vealed the characteristics of MTLC and the relationship between MTLC and la-ryngeal carcinoma.Materials and methodsTissues and cell lineAll the laiyngeal cancer tissues and matched normal tissues confirmed path-ologically were obtained from the first affiliated hospital of China Medical Uni-versity. Tumor tissue was dissected from the resected specimens. The normal tissue block was taken from the distal resection margin and was apart from canc-er at least 1cm. Laryngeal carcinoma cell line Hep2 was kept in our laboratory.Electronic hybridization and gene predictionAll ESTs expressed in larynx, head and neck or tumor tissues in chromo-some region 6q25 were searched in GenBank database. One of them, AU118145 was used as the electronic probe and electronic hybridization was performed in human genome database. We gained a positive clone(NT023451.10) and ana-lyzed its DNA sequence by software such as GenScan. The results showed that there was a novel gene in this region spanning 21kb and composed of two exons. The peptide coded by this novel gene contains 235 amino acid residues.RT - PCRTotal RNAs were extracted from cancer tissues by TRIZOL reagents, and were reverse - transcripted to the first strand of cDNA using reverse transcriptase system. MTLC cDNA was amplified by PCR. The amplification product was cloned into pMD18 -T vector and confirmed by DNA sequencing.Expression profilingWe performed Northern blot to reveal the expression levels of MTLC in vari-ous tissues. Full length cDNA was used as the probe and labeled by α - 32P -dCTP. Total RNAs were extracted from fetal tissues as templates.Subcellular localizationpMD18 - T vector was digested by BamH I and EcoR I, and then the target fragment was recollected and cloned into pEGFP - C3. Bel7402 cells were trans-fected by expression vector pEGFP - MTLC and empty parental vector with Lipo-fectamin2000. The expression of green fluorescence protein was observed underfluorescence microscope after 36 hours.The effects of MTLC over - expression to Hep2 cell line pMD18 -T vector was digested by BamH I and EcoR I , and then the tar-get fragment was recollected and cloned into pcDNA3.1 vector. Both PCR prod-uct and the expression vector pcDNA3. 1 ?MTLC were confirmed by sequencing to avoid mutation. Hep2 cells in logarithmic phase were transfected with the ex-pression vector or empty parental vector by Lipofectamin2000 and subsequently screened by G418 at a final concentration 5mg/ml. Cells transfected by pcD-NA3. 1 ?MTLC or empty parental vector were plated in 35mm plates. Individual plates were tryp...
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