| Part I Whole gene expression profiling technology screening Wnt pathway related genes with NSCLC metastasisObjective:This study aims to use the whole gene expression profiling technology to initially screen differentially expressed genes with NSCLC lymph node metastasis, to find transferred genes associated with Wnt pathway and analyze their gene functions, to provide new ideas for the study of invasion and metastasis mechanism and treatment of non-small cell lung cancer.Methods:Tumor tissue was collected from Thoracic Surgery, Qilu Hospital of Shandong University, from April 2014 to October 2014. The fresh tissue was immediately placed in vials after in vitro, and in 10 minutes frozen in liquid nitrogen tank for 2-3 hours, then transferred to-80 ℃ refrigerator frozen. Collect clinical data of the corresponding tumor specimens at the same time. The selected cases had no preoperative radiotherapy and chemotherapy, and pathologically confirmed primary non-small cell lung cancer. Six cases with lymph node metastasis were selected for the experimental group, six cases with no lymph node metastasis as the control group. Extract RNA by Trizol, using the NanoDrop ND-1000 to assess the amount and quality of RNA, RNA integrity was assessed by standard denaturing gel electrophoresis., then the experimental group and the control group were implemented the whole gene expression profiling experiments using 4 X 44K Agilent human gene expression microarray. Use Agilent Feature Extraction software to get the chip chart and the original data. Use GeneSpring GX vl2.1 software to standardize raw data, and GO analysis and Pathway analysis, screen differentially expressed genes associated with Wnt pathway between the experimental group and the control group, and analysis possible biological processes.Results:1. RNA quality control:OD value of the microarray experiment samples revealed that A260/A280 were between 1.8-2.1, agarose gel electrophoresis showed clear bands 28SrRNA and 18SrRNA, good RNA purity and integrity, samples are qualified.2. Cluster analysis:NSCLC metastasis group and non-metastasis group were significantly different, same type of samples can appear in the same cluster, a cluster of samples has same biological properties, suggesting that the test chip has a fixed reproducible, and reliable results.3. Differentially expressed genes:According to fold change 11975 differentially genes are screened,5812 up-regulated and 6163 down-regulated.4. GO analysis:it showed major differences in genes involved in the process of multi-cellular organisms, signal transduction, G protein-coupled receptor activity, extracellular matrix etc; 5. Pathway analysis:it mainly involves MAPK signaling pathways, cell cycle, PI3K-Akt signaling pathway, Wnt pathway, p53 signaling pathway and so on.6. Differential genes screening:Differentially expressed genes with NSCLC metastasis about Wnt pathway are totally of 35, including 18 up-regulated genes,16 down-regulated genes, involved in cell cycle gene, protein kinase C gene, frizzled family receptor gene transcription factor genes. GO analysis showed that these genes involve in protein binding, plasma membrane, intracellular signal transduction, frizzled protein binding, positive regulator of cell proliferation, specific sequences of DNA binding transcription factor activity and other biological processes.Conclusion:1. There were a large number of differentially expressed genes between NSCLC lymph node metastasis and non-metastasis, involved in a variety of function groups such as cell biological processes, extracellular matrix and several signal transduction pathways:MAPK, Wnt, p53 etc.2. The genes screened out associated with Wnt pathway participate in the process of specific sequences of DNA binding transcription factor activity, intracellular signal transduction, positive regulation of cell proliferation etc. But need further verification.Part Ⅱ Wnt pathway related genes verification of NSCLC metastasisObjective:As there is a certain false positive rate of microarray, this study used real-time quantitative polymerase chain reaction (RT-qPCR) technology to detect and analysis target genes, being designed to verify the reliability of microarray and to guide future research directions.Methods:CCND2, PRKCA, NFATC1 were selected from the 35 differentially expressed genes as target genes which are closely related to tumorigenesis and development, continue to apply the above-mentioned 12 cases,6 cases with lymph node metastasis as the experimental group, six cases without lymph node metastasis group, as he control group. RNA extraction methods and quality testing methods are as above, the target genes and housekeeping gene in both groups operate real-time quantitative PCR, and plot diluted concentrations DNA standard curve, calculate the relative amounts of the target genes, use paired t test to detect whether real-time quantitative PCR results are consistent with the microarray results.Results:CCND2, PRKCA, NFATC1 in lymph node metastasis group than those without lymph node metastasis showed upregulation, and the fold changes between the real-time quantitative PCR and microarray was not statistically significant (t= 0.4539, P> 0.05).Conclusion:1. Real-time PCR results of CCND2, PRKCA, NFATC1 were consistent with microarray, the expression microarray technology can high-throughput screen out differentially expressed significant genes.2. CCND2, PRKCA, NFATC1 genes are closely related to the development of human tumors, which have important research value in lung cancer invasion and metastasis. |
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