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Investigation on the Gene Expression Pattern of Anoikis Resistance Lung Cancer A549 Cell Line and the Expression and Significance of TC-1 Gene

Author: LeiJie
Tutor: WangXiaoPing；ZhangWei；ZhangZhiPei
School: Fourth Military Medical University
Course: Surgery
Keywords: Lung tumors Resistance to anoikis Gene chip TC-1 gene
CLC: R734.2
Type: Master's thesis
Year: 2010
Downloads: 40
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Abstract

Background lung cancer - the most common tumors of the respiratory system, the most common malignancy in the world today, with the characteristics of the high incidence and high transfer rate, high mortality. Most patients first visit Jibei diagnosed with advanced lung cancer, associated with lymph node metastasis or hematogenous metastasis. Once lung cancer cell metastasis on that with the ability of anti-anoikis, that is out of contact with the matrix from apoptosis to survive and the final in the distant suitability organization continue to proliferate, and form metastases. Visible, resistance to anoikis initiating aspects of lung cancer metastasis. Anoikis / anti-anoikis process is a very precise regulation process, gene network and its signaling pathway is tightly regulated process, any gene expression changes in its signaling pathway changes will affect cell anoikis / anti-shedding ability to apoptosis, thus affecting the biological behavior of the cells. The essence of many tumor metastasis genes changed by a mediated anoikis / resistance to anoikis triggered. Anoikis / anti anoikis limited to a single gene or a path to change, there is no comprehensive genome-wide analysis, and thus the lack of systematic, targeted research, the experiment will have lung cancer A549 cells whole genome analysis, comparison anoikis / anti-shedding differences in apoptotic gene expression in both state and pick out the expression of differences in gene further study, a comprehensive analysis of research targeted to further reveal lung cancer cell anoikis / pull-out resistance of the molecular basis of apoptosis. Thyroid cancer related gene 1 (Thyroid Cancer Gene-1 TC-1), also known c8orf4 gene positioned at 8p11.2, widely expressed in the vertebrate, highly expressed in a variety of malignant tissue; encodes a 106 amino acid-containing the inherent disordered protein, the three regions of the COOH terminus of the protein (D44-R53, K58-A64, and D73-T88) is easy to form a tight spiral structure, and enhancing its binding ability with the target protein involved in the regulation of Wnt / β-Catenin pathway and FGFR2 pathway, promote cells to become cancerous and anti-anoikis; involved in the regulation of the NF-KB pathway mediated inflammatory response and the heat shock response. TC-1 gene and lung cancer have not been reported, the experiment and the results of microarray analysis application by RT-PCR and Western Blot Detection of TC-1 gene and the encoded protein in lung cancer A549 cells and anti-anoikis lung expression in A549 cells, and further study of its relations with lung cancer A549 cells resistant to anoikis, laid the theoretical foundation for the future study of the molecular mechanisms of TC-1 lung cancer metastasis. The purpose of screening lung cancer A549 cells resistant to anoikis-related gene, and explore the TC-1 gene expression and meaning. Methods suspension cultures of lung carcinoma A549 cells to develop resistance to anoikis model; using DNA electrophoresis test resistance to anoikis model; adherent culture and suspension culture total RNA preparation of human lung carcinoma A549 cells V2.0 genome-wide oligonucleotide microarray chip (35k human Genome Array); lung cancer A549 cells adherent culture and suspension culture differences in gene; screened genes associated with tumor cell resistance to anoikis by NCBI Pubmed database, and to find out the multiples of differential expression by gene chip found, gene; using reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot (Western blotting) technical determination of expression of the gene expression differences in multiples. The results successfully established lung cancer A549 cell resistance to anoikis model. Good state of cell growth, aggregation lumps and clumps of cells is greater with time, the more closely; extracted DNA agarose gel electrophoresis analysis confirmed its freedom from anoikis, have anti-anoikis ability; successfully prepared lung cancer A549 cells adherent culture and genome-wide state of suspension culture chip - human V2.0 whole genome oligonucleotide microarray chip (35K Human Genome Array), and found that lung cancer adherent state anoikis and suspended state (anti-) differences of 745 genes, these genes are involved in a wide range of functional genes and structural genes, further analysis found 63 genes closely related with tumor cell resistance to anoikis, and filter out the expression differences multiples of most of the genes - TC-1 gene; 3, at the genetic level authentication TC-1 gene expression in lung cancer cells, adherent culture and suspension culture status than high expression of the difference between the two was statistically significant ( P lt; 0.02), and further confirmed at the protein level TC-1 encoded protein in lung cancer cells highly synthetic, and suspension culture state synthesis higher. Conclusions gene chip technology apoptosis-related genes provide a valid method for screening of lung cancer anti-shedding, lung cancer cell resistance to anoikis involving multiple changes in gene expression, which TC-1 gene may be involved in the regulation of lung cancer cell resistance to anoikis process, and behavior related lung cancer cell invasion and metastasis.

Investigation on the Gene Expression Pattern of Anoikis Resistance Lung Cancer A549 Cell Line and the Expression and Significance of TC-1 Gene

Abstract

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