1、The Molecular Mechanism Of The Up-regulated Expression Of DLK1 Gene In Lung Cancer 2、Study On Heterogeneity Of EGFR/KRAS Mutation In Multifocal Lung Adenocarcinoma

This master degree project is composed of two parts of research work.Chapter I. The molecular mechanism of the up-regulated expression of DLK1 gene in lung cancerLung cancer is the leading cause of cancer-related morbidity and mortality throughout the world, and has been attached much importance to prevention, treatment, and research. According to the global expression profiling and bioinformatic analysis on squamous cell carcinomas of lung, performed in this laboratory previously, that DLKl gene was associated with metastasis. The preliminary results revealed that the DLK1 could promote cell invasion of lung cancer, and that DLK1 expression was up-regulated in non-small cell lung cancers (NSCLC) either on mRNA level or protein level.In this study, we continue to explore the mechanism of DLK1’s up-regulated expression of the DLK1 in NSCLC. Imprinting status of the gene, transcription factors, and interacting proteins of DLK1 protein were considered.To start with, using methylation specific PCR (MSP), we determined the methylation status at DLKl imprinting control elements. Neither of the five human lung cancer cell lines nor the 30 NSCLC tumor samples involved in this study showed loss of imprinting. Besides, identical methylation status was found in the tumor tissues, the adjacentnormal tissues, and the corresponding white blood cells of the NSCLC patients investigated. These results suggest that the over-expressionof DLK1 in NSCLC is not caused by loss of imprinting (LOI).HEY1 was guessed as a candidate transcription factor of the DLK1. Human lung cancer cell lines A549 and H1299 were taken to perform the interplay of DLK1 and HEY1 in vitro; and the cells that over-expressed exogenous DLK1 or in that the endogenous DLK1 was knocked down were generated. However the results suggest that HEY1 could not influence DLK1 expression, and HEY1 might not work as a transcription factor of DLK1. Combined with previous research data in this laboratory, conclusion can be draw that DLK1 expression was up-regulated in NSCLCs may be due, at least in part, to DNA hypomethylation in promoter region of the gene.For the sake of provide clues of influence may DLK1 be on Notch passway, co-immunoprecipitationand immunofluorescence co-localization were performed to identify DLK1 interacting proteins. Finally it was detected that NCOR1 was interacting with DLK1 in vitro. Meanwhile, NCOR1 could influence Notch and NF-κB passway, which lead to the conjecture that this interaction may participate in the cross talk of Notch and NF-κB passway.In conclusion, this study reveals that LOI and HEY1 is not responsible for the up-regulated expression of DLK1. The protein-protein interaction between DLK1 and NCOR1 may participate in the cross talk of Notch and NF-κB passway.Chapter II. Study on heterogeneity of EGFR / KRAS mutation in multifocal lung adenocarcinomaSignificant advances in the understanding the molecular targeted therapy of cancer have allowed the development of EGFR-targeting agents for the improved treatment of NSCLC. Therefor, an emerging issue concerning the tyrosine kinase inhibitors (TKIs) of EGFR for NSCLC treatment is to identify the mutation status of EGFR and KRAS genes, for selecting patients who will most likely benefit from these agents.Based on the whole-genome/exome sequencing performed on 15 intrapulmonary tumor of 6 multifocal lung adenocarcinomaspatients, paired tumors from a single patient showed extensive discrepancyin EGFR / KRAS mutations between each other. On the other hand, for multifocal lung adenocarcinoma patients, EGFR / KRAS mutation was generally examined on the largest tumor or the one containing the most tumor cells, and thus could omit part of tumors harboring the EGFR mutation which may cause loss of opportunity for EGFR-TKIs therapy.A total 58 cases of multifocal lung adenocarcinoma, including 129 tumors, was recruited for this study. The EGFR / KRAS mutational status of each tumor was tested by Sanger’s DNA sequencing. The result reveals that, among 129 intrapulmonary tumors, EGFR mutation was detected in 59 (58.6%) tumors, while KRAS mutation was detected in 7(8.6%) tumors; the main type of EGFR mutation was exon 19del, exon 21 p.L858R, and that of KRAS is p.G 12A. There were 21 (55.3%) cases showing mutational heterogeneity, and 6 tumors wereharboring 2 different types of EGFR mutation; whereas none of the specimens contain both EGFR and KRAS mutation at the same tumor.The current finding suggest that EGFR and KRAS mutational heterogeneity is widely existed in multifocal lung adenocarcinomas. Therefore, reliable and exhaustive examination for EGFR/KRASmutation should be applied to provide more individualized therapy choices for the patients.