The Molecular Mechanism And Biological Bioinformatics Research Of DAL-1 Regulating NSCLC Epithelial-mesenchymal Transition

Background:Lung cancer is currently the world’s highest incidence and mortality of malignant tumors, including Non-small cell lung cancer(NSCLC). Invasion and metastasis are the main causes of death, so the research on the molecular mechanism of NSCLC invasion and metastasis is the key to be performed. Objective:This study focused on the role of DAL-1 gene in NSCLC cell migration and invasion, the possible mechanism of the gene in NSCLC, the main cause of inactivation and its possible therapeutic approach. We look forward to finding a new target for clinical treatment of NSCLC and providing experimental basis. Methods:According to the sequence of human DAL-mRNA, 3 specific shRNA DAL-1 expressionplasmids(pGPU6/GFP/Neo-sh710, sh1436, sh1329) and negative control plasmid(shNC) were designed and synthesized. The NCI-H460 cell line was transfected with recombinant plasmid, and the expression of DAL-1 gene was detected by Western blot and RT-PCR, respectively. MTT assay was used to detect the DAL-1 gene silencing cell proliferation activity. Transwell chamber was used to detect cell migration and invasion ability of NSCLC cell.Based on the open source R language programming platform, Bioconductor was used as a biological computing environment, and a variety of software packages were used to construct a data analysis and data mining system for microarray data. The original data GSE33532 of Affymetrix oligonucleotide microarray was published in the public database GEO. The data was analyzed, including the acquisition, pretreatment, quality detection, quality detection, differential expression gene screening, GO analysis, pathway analysis, gene expression regulatory network, molecular interaction network analysis and visualization. Results:1. The effect of DAL-1 gene on NCI-H460 cells.The pGPU6/GFP/Neo-DAL-1sh RNA plasmid and the negative control plasmid pGPU6/GFP/Neo-shNC were constructed, and the NCI-H460-sh1329 cell lines with stable silencing of DAL-l were established. Compared with negative and empty plasmid control, the DAL-l inhibition rate of sh1329 transfected cells was 88%, and the Western blot assay was got similar results. There was no significant difference between in NCI-H460-shNC and NCI-H460/ET transfection groups.2. The effect of silencing DAL-1 gene on the biological behavior of NCI-H460 cells.Compared to the negative control group(NCI-H460-shNC) and NCI-H460/ET cells, NCI-H460-sh1329 had more invasion rate with significant difference(P<0.05).3. The deletion or overexpression of DAL-1/4.1B changed the expression of EMT markers.Silencing of DAL-1/4.1B RNA changed the expression of EMT markers, including E-cadherin andβ-catenin, and the overexpression of DAL-1/4.1B had the opposite effect.4. TGF-β induced DAL-1/4.1B expression.The expression of DAL-1/4.1B at protein and mRNA level was increased after TGF-β induction. 4.1B DAL-1/ deficient cells were weaken on EMT, migration and invasion induced by TGF-β.5. Bioinformation of NSCLC genome in GSE3353249 Differential expression genes were selected from the GSE33532 data set, and the vascular endothelial contration pathway was the key regulatory units of NSCLC. Conclusion:The shRNA pGPU6/GFP/Neo-DAL-1 plasmid and the negative control plasmid pGPU6/GFP/Neo-shNC were constructed successfully, and the NCI-H460-sh1329 cell lines with stable silencing DAL-1 were established. After silencing of DAL-1 gene, the proliferation, migration and invasion of NCI-H460 cells were increased, which indicated that DAL-1 could inhibit the proliferation, migration and invasion of NSCLC cells. Vascular endothelial contraction pathway might regulate NSCLC. Innovations:1. DAL-1 siRNA sequences were designed to construct pGPU6/GFP /Neo-DAL-1 shRNA expression vector. The DAL-1 expression of NCI-H460 cells could be inhibited effectively by pGPU6/GFP/Neo-DAL-1 NCI-H460, and the DAL-1 inhibition cells line was established to further study;2. RNAi technique is used to analyze the effect of DAL-1 on the proliferation, migration and invasion of NCI-H460 cells, and the relationship between DAL-1 and NSCLC is discussed, which provides an experimental basis for further study of the development on non small cell lung cancer.3. Using bioinformatics method to analyze the gene data of GSE33532, and KEGG were used to get the key regulatory units which contain vascular endothelial contraction pathway, providing a basis for further study on the molecular mechanism of NSCLC.