| Lung cancer is one of the most common malignancies with high morbidity and mortality around the world. Tumor invasion and metastasis is strongly associated with the overall survival of the cancer patients. Previous investigations in this laboratory had found that DLKl (Delta-like1homolog) was one of metastasis-associated genes, according to the global expression profiling on lung squamous cell carcinomas and the bioinformatic analysis. Preliminary results revealed that DLK1could promote lung cancer cell invasion in vitro, but the mechanism still remained to be explored. The aims of present study are to discover the role(s) of DLK1in non-small cell lung cancers (NSCLCs) and to explore the molecular mechanisms.We first examined the expressive status of DLK1protein in the tumor tissues of NSCLC with immunohistochemical (IHC) analysis. IHC staining was applied on formalin fixed paraffin embedded sections derived from204cases of NSCLCs, including102squamous cell carcinomas (SCCs) and102adenocarcinomas (ADCs). The expression of DLK1was significantly higher in the tumor tissues in comparing with that in the corresponding adjacent normal tissues. Moreover, the level of the DLK1were associated with the tumor size and stage in patients with SCCs according to the clinical characters.Meanwhile, we investigated the molecular mechanism of DLK1on promoting lung cancer cells invasion through classic cell biologic methods. Human lung cancer cell lines H1299and A549were taken for in vitro investigations, and the cells that over-express exogenous DLK1or in that the endogenous DLK1was knocked down were generated. Over-expression of DLK1promoted invasion of the lung cancer cells, based on the results of Transwell assay. On the other hand, over-expressed DLK1was associated with up-regulation of MMP9and could also increase NOTCH1expression. Together with the up-regulated expression of the effector gene HES1, the data indicate that DLK1could activate Notch signaling pathway. On the contrary, knocking down DLK1with siRNA could down-regulate expression of MMP9, NOTCH1and HES1in the lung cancer cells. To discover the mechanism for abnormal expression of DLK1in NSCLCs, we compared DNA copy number of DLK1gene in the tumor tissues with their paired adjacent non-cancerous tissues by realtime PCR. Using methylation specific PCR (MSP), we determined the methylation status at DLK1imprinting control elements. The results suggested that the causation of DLK1over-expression in lung cancers is neither gene amplification nor loss of imprint. In vitro,5-aza-dC treatment increased DLK1expression in lung cancer cells, indicating that expression of the gene was regulated by DNA methylation. Results obtained by bisulfite sequencing showed that CpG island within the promoter region was hypo-methylated in the tumor samples with over-expressed DLK1.By the IHC analysis described above, nucleus staining of DLK1was observed. Then we confirmed the nuclear translocation of DLK1by Western blot and immunofluorecence approach. Co-immunoprecipitation/mass spectrum (Co-IP/MS) assay was performed to identify DLK1interacting proteins in order to provide clues on the mechanism and function of DLK1nuclear translocation. A total of12proteins with high confidence were selected for further identification; and those candidate proteins participate in various cell signaling pathways, including Notch signaling and NF-κB signaling.In conclusions, this study reveal that DLK1was over-expressed in the tumor tissues of NSCLC patients. In vitro the DLK1could promote cancer cell invasion by up-regulating MMP9expression via activating Notch signaling pathway. DNA hypo-methylation on DLK1promoter was the primary cause for its aberrant expression in lung cancer. In addition, DLK1has nuclear translocation in lung cancer cells, which indicates an undiscovered function of this gene. |
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