| BackgroundColorectal cancer (CRC) is one of the most common malignancies that is the third-highest mortality in malignancies around the world. In western countries, colorectal cancer is the second cause of cancer-related death, only next to lung cancer. With the improvement of living standards, the eating habit has changed, including high-protein, high-fat, low-cellulose and low-roughage and so on, which made the mortality of colorectal carcinoma rapidly increase, especially in the urban and developed rural area of China. Therefore, it is a focal problem for colorectal cancer to be concerned and settled urgently.It is has been found that the process of occurrence and development involved in a number of related genes in colorectal cancer now. Changes in these genes affect the biological behavior of colorectal epithelial cells. It has shown that AP-2αis considered to be a tumor suppressor gene in the majority of tumors. As a sequence-special DNA-binding protein, AP-2αparticipates in tumor proliferation, invasion and metastasis through regulating the expression product of target gene. Recent studies indicate that cancer-related genes, which include p21WAF/CIP, transforming growth factor-α, c-Kit, HER-2/neu, MCAM/MUC18, c-myc, VEGF, MMP-2, KAI1, KiSS-1, ER-βand so on, have all binding sites of AP-2αand cis-regulatory sequences. So far, it has been reported that ER-βis an AP-2-regulated gene, but it hasn't been carried out on the interaction between AP-2αand ER-βand the research of malignant proliferation and invasion capacity in colorectal cancer at home and abroad. This is the problem what this experiment needs to solve. The ultimate goals of this project are to clarify possible role and significance of AP-2αin colorectal cancer, and the regulation mechanism on ER-βexpression. Furthermore, it is expected to provide the new clues for revealing the molecular mechanism in the process of colorectal cancer and the new ideas for colorectal cancer in development of genetic diagnosis and treatment technologies.Objective①To explore the inhibitory effect of AP-2αon proliferation and invasive growth abilities of SW620 colorectal cancer cells in vitro②To explore the possible interaction mechanism between AP-2αand ER-βMethods①LipofectamineTM 2000 Reagent was used to mediate the transfection of pcDNA3.1(+)-AP-2αand pcDNA3.1(+) into SW620 colon cancer cells. To examine the mRNA and protein level of AP-2αin the cells of each group, Real- time PCR analysis and Western Blot analysis were performed. The DNA-binding activity of the exogenous AP-2αprotein in the transfected cells was analyzed by Electrophoretic mobility shift assay (EMSA).②Proliferative activities of the cells of each group were evaluated by MTT assay 1,2,3,4 or 5d after the transfection. Anchorage-independent growth assay were performed to detect the effect of AP-2αon the malignant property of SW620 cells. Flow cytometry was used to analyze the cell cycle and the rates of apoptosis. To evaluate the role of AP-2αin SW620 invasion potential in vitro, the method of metrical gel adhesion assay, Transwell invasion assay, Transwell migration assay and scrape-wound-migration assay was performed.③To determine whether AP-2αhas direct interaction with the ER-βpromoter derived from the genomic DNA of SW620 cells, oligonucleotides containing a putative AP-2αbinding site were synthesized, and EMSAs were performed. To further assess the effect of the AP-2αprotein on the ER-βexpression in SW620 colon cancer cells, the mRNA and protein level of endogenous ER-βin the AP-2α-transfected cells were detected by the use of Real time-PCR,Western Blot and Immunofluorescence cytochemistry analysis respectively.Results①Real time-PCR analysis detected high level of mRNA in the AP-2α-transfected SW620 cells but only residual levels in neo-transfected cells. Expression of AP-2αwas observed in the AP-2α-transfected SW620 cells as compared with the neo-transfected cells. EMSA results revealed high DNA-binding activity of AP-2αin the AP-2α-transfected SW620 cells.②MTT results displayed that the overexpression of AP-2αinhibited the proliferation of SW620 cells. The results of flow cytometry showed that the cell cycle was arrested in G1 and the rate of apoptosis increased. Expression of AP-2αmarkedly reduced the adhesion,invasive and migration abilities of SW620 cells compared with the neo-transfected cells.③EMSAs in our study showed that AP-2αre-expressed in SW620 colon cancer cells binds directly to the oligonucleotides which containing putative AP-2αbinding sites. Transfection of AP-2αgene into SW620 colon cancer cells enhanced the ER-βmRNA and expression of protein.ConclusionThe expression of ER-βis probably regulated by AP-2αand that forced over-expression of AP-2αsuppresses the growth and invasion potential of SW620 colon cancer cells, possibly through down-regulating ER-βgene expression.
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