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Preparation of Tumor-associated Gene cDNA Microarray and Its Application to the Research of Lung Cancer

Author: FuZhongYan
Tutor: HanJinXiang
School: Shandong University
Course: Biochemistry and Molecular Biology
Keywords: lung caner gene expression profile tumor cell invasion emodin cell apoptosis
CLC: R734.2
Type: PhD thesis
Year: 2007
Downloads: 272
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Lung cancer is one of the most common cancers. Lung carcinogenesis consists of a complicated multistep process that involves interactions among cancer-sensitive molecules and interactions between cancer-sensitive molecules and their surrounding microenvironments. In this study, we investigated the difference of expression profile between lung cancer and the normal tissue, the difference between two clonally-related human giant-cell lung cancer cell lines with different metastatic potential, and the emodin-induced expression profile changes in small cell lung cancer cells.1. Microarray fabricationWe selected the tumor-related genes and purchased them from Research Genetics and Geneservice, respectively. E.coli with inserted genes were cultured with Luria-Bertain culture medium supplemented with ampicillin or chloromycin overnight at 37℃. Clone plasmids were extracted and clone inserts were PCR-amplified in 96-well PCR microtiters. Plasmids and PCR products were electrophoresed on a 1% agarose gel containing 0.5ug/ml ethidium bromide. 1262 nonredundant tumor-related genes were selected as target genes. Five selected genes were sequenced. The PCR products were purified with protocols on line and resuspended in 2 Arrayit spot solution. The purified PCR products were printed on silanated slides with Cartisan PixSys 5500 robot and cDNA were post-processed according to protocol online. Hybridization and scanning were carried out to detect the quality of the microarrays. Electrophoreses results revealed good quality of extracted plasmids and PCR products. Sequencing results showed that the genes were the correct ones. The hybridization and scanning results suggested the quality of the microarrays fabricated this time were good enough to be used in experiments. 2. Expression Profiling of Lung Cancer Tissue Using cDNA MicroarrayTotal RNA from samples was extracted and labeled with fluorescent dye. The labeled probes were hybridized to the microarray and scanned with ScanArray4000 and analyzed by Quantarray3.0. 52 genes were found associated with lung carcinogenesis. They were grouped according to their functions, including transporters, adhesion molecules, cytoskeleton organizers, transcription regulators, genes associated with metabolism, genes associated with immune response, and EST. Among them, 35 genes were positive to lung carcinogenesis and 17 genes were negative to lung carcinogenesis. The screening of human lung cancer expression profile can provide valuable information for revealing the molecular mechanism of the lung carcinogenesis, which is a foundation of detection of lung cancers and treatment of patients with lung cancer.3. Identification of Genes That Promote Invasion in a Lung Cancer ModelHuman giant-cell lung cancer cell lines 95-C and 95-D were derived from the same parent cell line PLA-801. 95-C is of lower potential to metastasis compared with 95-D. In this study, the invasive capacity of 95-C and 95-D cells were characterized and we applied cDNA microarray to these cell lines to identify genes that promote invasion. RT-PCR was used to confirm the results derived from cDNA microarray. Results showed that 95-D cells were more invasive than 95-C cells. Microarray analysis defined 69 genes (of 1262 uniques) that were differentially expressed between 95-C and 95-D cells. These differentially expressed genes were grouped according to their functions, including transporters, adhesion molecules, cytoskeleton organizers, transcription regulators, genes associated with metabolism, genes associated with immune response, and ESTs. The results of RT-PCR were consistent with that obtained from cDNA microarray. We speculate that at least a proportion (if not all) of these differentially expressed genes contribute to the different metastatic capacity between 95-C and 95-D cells. Further exploration of these genes might lead to better understandings of cancer metastasis. 4. Effects of Emodin on Gene Expression Profile in Small Cell Lung Cancer NCI-H446 CellsLung cancers are classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Small cell lung cancer accounts for 13-15% of all lung cancer worldwide. The prognosis for patients with small cell lung cancer is worse, and the treatment is limited by the development of drug resistance. Emodin has been identified to exhibit an anti-cancer effect. But the effect of emodin on small cell lung cancer ramains unclear. In order to investigate the effects of emodin on SCLC, we studied the viability, apoptosis and gene expression profile of SCLC NCI-H446 cells treated with emodin in this report. NCI-H446 cells were treated with emodin and cell viability was determined by MTT assay. Flow cytometric analysis and caspase-3 activity assay were also carried out to monitor the effects of emodin on NCI-H446 cells. The effect of emodin on gene expression profile of NCI-H446 cells was analyzed using cDNA microarray. Semi-quantitative RT-PCR was used to validate the microarray results. Results showed that emodin suppressed viability and induced apoptosis of NCI-H446 cells in a time-dependent manner. Cell cycle changes by emodin were also observed by flow cytometric analysis. Among the 1262 genes, 10 genes were up-regulated and 8 genes were down-regulated more than 2 folds in NCI-H446 cells when compared with the control cells after treatment with emodin for 12 h, while 12 genes were up-regulated and 24 genes were down-regulated after treatment with emodin for 24 h. These genes were involved in metabolism, signal transduction, transcription regulation, cytoskeleton organization, immune response, transport, protein synthesis, cell cycle control, cell adhesion and RNA processing. The RT-PCR results were consistent with those obtained by the microarray. In conclusion, Emodin affected the expression of genes involved in various cellular functions and playing important roles in cell apoptosis, tumor metastasis, and chemotherapy-resistance, which suggests emodin might become an effective chemopreventive or chemotherapeutic agent for small cell lung cancer.

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