| Part Ⅰ Bioinformatics analysis of miRNA-mRNA expression profiles in non-small cell lung cancerObjectiveIn this part,we used GEO database to select miRNA expression chip data set,with R language tools processing data.We also screened out the different miRNA between patients with non-small cell lung cancer and normal people for follow-up study.Methods1、The miRNA expression chip data sets GSE17681 and GSE29250 were downloaded to process the data with R language tools.The target gene of miRNA were predicted through databases such as Targetscan and Starbase v2.0.2、n addition,mRNA expression microarray dataset GSE21933 was selected,and after processing the data,the differential mRNAs were screened out and intersected with the predicted miRNA target genes to obtain the differential genes.3、Bioinformatics was used to analyze differential genes and to identify the core functional genes that affect the occurrence and development of NSCLC。Go and KEGG wasused to analyze biological functions,processes and action pathways.Results1、GSE17681 screened a total of 49 differential miRNAs which including 37 up-regulated miRNAs and 12 down-regulated miRNAs.GSE29250 screened a total of 47 miRNAs which including 27 up-regulated miRNAs and 20 down-regulated miRNAs.The two chips of differential miRNAs intersected to obtain a common differential miRNA of miR-1294,and the results showed that the expression of miR-1294 was down-regulated in tumor tissues.2、The target genes of miR-1294 were predicted by online database,of which 3749 were predicted by Targetscan 7.2,2773 were predicted by Starbase v2.0,and 1466 were common target genes.GSE21933 chip in GEO database was used to analyze the differential mRNA of NSCLC,including 1378 up-regulated mRNA and 1513 down-regulated mRNA.Because the expression of miR-1294 is decreased in NSCLC tumor tissue,the mRNA related to miR-1294 should be up-regulated,and then cross with the predicted mRNA,114 differential mRNA related to miR-1294 were obtained.3、The functional enrichment analysis of differential mRNA showed that the biological process focused on cell changes,including cell cycle,intracellular pressure changes,intracellular hypoxia,negative regulation of cell differentiation,as well as stem cell differentiation,protein-mediated mitochondrial membrane apoptosis,etc.KEGG signaling pathways were focused on cancer-related pathways,including pancreatic cancer,bladder cancer,NSCLC pathways,etc.,and also included Hippo,p53 and PI3K-Akt signaling pathways.4、Using string database to construct PPI network targeting miR-1294 mRNA,through the mode plug-in analysis of cycloscape software,a total of 10 key genes were screened,including VEGFA,ccnf,TPX2,mki67,PRM2,ywhag,ube2n,cenpn,SLC2A1,troap genes.Conclusion1、By using bioinformatics analysis method,this part screened the miRNA difference between normal control and NSCLC patients,and selected miR-1294 as the target miRNA for follow-up study.2、The key functions and signal pathways of miR-1294 targeted mRNA in NSCLC tumor cells were elucidated.Part Ⅱ Expression level and clinical significance of miR-1294 in NSCLC cell lines and patients with non-small cell lung cancerObjectiveIn this part,the results of bioinformatics analysis were verified by clinical pathological experiment data to clarify the expression level and clinical significance of miR-1294 in NSCLC patients.Methods1、In this study,we collected the clinical information,tumor tissue samples and normal tissue samples of NSCLC patients in People’s Hospital of Henan Province.2、qPCR was used to detect the differential expression of miR-1294 in NSCLC tumor tissues and normal tissues adjacent to cancer.The expression of miR-1294 in normal lung tissue cell line and three NSCLC cell lines h1650,A549,95D was detected.3、Combined with the clinical information of NSCLC patients,the relationship between the expression level of miR-1294 and the clinical characteristics of NSCLC patients was analyzed.Results1、miR-1294 was lowly expressed in tumor tissues and tumor cell lines of NSCLC patients,and the lowest expression value was found in A549 cell lines.The difference was statistically significant.2、miR-1294 low expression was associated with smoking,lymph node metastasis,late TNM stage and poor tumor differentiation.miRNA-1294 low expression was associated with relatively short overall survival in patients.Conclusion1、In NSCLC tissues and cell lines,miR-1294 expression levels were significantly down-regulated.2、miR-1294 plays an important role in the clinical features and development of NSCLC,and it can be used as a reliable indicator for prognosis evaluation of NSCLC patients.Part Ⅲ The biological function of miR-1294 in non-small cell lung cancer cell A549ObjectiveTo study the biological function of miR-1294 in non-small cell lung cancer cell line A549.Methods1、Using gene transfection technology,miR-1294mimics were transfected into NSCLC tumor cells A549,and the expression level of miR-1294 was up-regulated.2、The cell proliferation ability was detected by MTT cell proliferation assay and plate cloning assay,the cell migration ability was detected by scratch assay,and the cell invasion ability was detected by Transwell assay to investigate whether miR-1294 plays a role in regulating NSCLC cell proliferation,migration and invasion.3、Flow cytometry was used to detect whether miR-1294 played a role in regulating the apoptosis of lung adenocarcinoma cell line A549.4、A tumor-bearing nude mouse model was established,and the tumor growth curve was measured to observe the effect of miR-1294mimics transfected cells on the growth of tumor-bearing nude mice.Results1、Overexpression of miR-1294 significantly inhibited the proliferation,migration and invasion ability of NSCLC tumor cells A549.2、Overexpression of miR-1294 can promote the apoptotic capacity of lung adenocarcinoma cell line A549.3、Overexpression of miR-1294 can inhibit tumor formation and inhibit tumor proliferation and growth in nude mice bearing tumors.Conclusion1、miR-1294 acts as a tumor suppressor gene in NSCLC cells.2、Up-regulating miR-1294 can inhibit the proliferation,migration and invasion of lung adenocarcinoma cells.Part Ⅳ miR-1294 inhibits VEGFA to regulate proliferation,apoptosis and invasion of NSCLC cellsObjectiveTo study the effect of miR-1294 on the biological function of non-small cell lung cancer.Methods1、The double luciferase experiment was performed to verify whether VEGFA is a direct target gene of miR-1294 and to detect the difference in VEGFA protein expression in NSCLC cells before and after transfection.2、Real-time quantitative PCR was used to detect the VEGFA content in tumor tissues and adjacent tissues,and the correlation between VEGFA and miR-1294 was analyzed.3、The changes of cellular secretion of VEGFA after transfection with miR-1294mimics and miR-1294inhibitors were detected by Western blot.4、Western blot was used to detect the expression of VEGFA protein after co-transfection of A549 with pcDNA-VEGFA plasmid and miR-1294mimics.5、The proliferation assay of MTT cells,flow analysis technique and Transwell assay were used to further verify the effect of VEGFA on the proliferation,apoptosis and invasion of miR-1294 on lung adenocarcinoma cells.Results1、miR-1294 directly targets VEGFA.2、VEGFA expression was up-regulated in patients with lung adenocarcinoma,and was negatively correlated with miR-1294 expression.3、In cells overexpressing miR-1294,the abundance of VEGFA protein decreased;in cells with miR-1294 expression suppressed,the abundance of VEGFA protein increased,and the difference was statistically significant.4、pcDNA-VEGFA plasmid transfection can replenish miR-1294 mimics caused by VEGFA down-regulation.5、The results of cell proliferation,apoptosis assay and cell invasion assay showed that up-regulation of VEGFA protein level could back-compensate the effect of miR-1294 on tumor cell proliferation,apoptosis and invasion.Conclusion1、miR-1294 directly targets VEGFA and is negatively correlated with VEGFA expression.2、miR-1294 can regulate the expression level of VEGFA,and then affect the biological function of lung adenocarcinoma cells. |
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