The Preliminary Research Of The Early Aberrant Genes On Lung Cancer

Title: The preliminary research of the early aberrant genes onlung cancerCandidate Ph. D. : Pan Shi-YangSupervisor: Professor Zhang Ji-Nan; Professor Zhang Xue-Guangand Professor Tong Ming-QingName of the University: Nanjing Medical UniversityThe protein product of the APC TSG at chromosome 5q21 is an important component of the Wnt signaling pathway, which binds to and inactivates b-catenin. Biallelic inactivation of the gene in familial adenomatous polyposis and most sporadic colorectal tumors promotes tumorigenesis. Inactivation of TSGs may occur via multiple mechanisms, including allelic loss, gene mutation, or by methylation of CpG sites in promoter regions. Two promoters (1A and 1B) of the large APC gene initiate transcription from distinct sites (exons 1A and 1B), and alternative splicing generates multiple transcripts. Aberrant methylation of the 1A promoter occurs in some colorectal and gastrointestinal malignancies and is accompanied by loss of expression of its specific transcript. To determine whether this epigenetic phenomenon occurs in breast and lung cancers, we determined the methylation status of the 1A promoter in lung tumors and cell lines and in nonmalignant tissues. We analyzed gene expression from the APC genetranscription with RT-PCR and by immunohistochemistry methods.1. Lung cancer animal modelOn the purpose to study the mechanism of the oncogenesis and the early aberrant of tumor suppressor genes ( TSGs ) of lung cancer, we successfully developed lung cancer animal model by using direct lung cancer cell injection to the mouse skin and abdomen. The lung cancer cells are derived from clinical lung cancer patients. The lung cancer cell of the model shows triplet karyotype. MDDT (mean diameter doubling time, tumor diameter from 0.5cm to 1.0cm) is 12 days. The tumor tissues from the original lung cancer patient and lung cancer models do not express the proteins of APC and p16. The lung cancer cell from clinical patient has higher ability to form solid tumor in nude mice compared to the lung cancer cell lines, which are collected in ATCC.2. DNA methylation specific detection methodsWe successfully developed methylation specific polymerase chain reaction (MSP), methylation specific sequencing (MSS) and methylation specific microarray (MSM) methods based on chemical modification of DNA sample.3. Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A in non-small cell lung cancer (NSCLC)To determine aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A with respect to its prevalence and the clinicalpathological significance in non-small cell lung cancer (NSCLC), we developed the positive control for methylation specific PCR (MSP) and methylation specific sequencing (MSS) of hypermethylated APC gene ( methAPC), examined the promoter methylation status of the APC using both of the two methods in 17 NSCLC patients and one case of benign. The frequencies of methylation in NSCLCs and corresponding non-neoplastic lung tissues were: 47% (8 of 17) and 18% (3 of 17) for MSP. There were four cases existed methylation in APC gene promoter 1A among the nine MSP negative cases by using the methylation specific sequencing. We approved the total positive detection of methAPC to 71% by using the two methods of MSP and MSS. MSS may be much more sensitive than MSP regarding to the rich CpG sites in APC gene promoter 1A region and the templates which were influenced by heterozygosity changes of meth APC for MSP amplification. Our findings suggest that promoter hypermethylation can be detected in the majority of lung cancer patients with the combined detection methods for methAPC. This approach needs to be evaluated in large early detection and surveillance studies of lung cancer. 4. CpG methylation status of adenomatous polyposis coli (APC) promoter 1A in human lung cancerMethylation specific PCR was used to amplify the DNA template from tissues of 19 cases of lung cancer patient and 13 cases of normal lung of health person. The MSP products were sequenced directly and...