microRNA-33a通過靶向HIF-1α抑制黑色素瘤侵襲轉移的分子機制研究
[Abstract]:Objective: To study the expression and significance of miR-33a/ b in human skin melanoma cell line with different metastatic potential. After the expression of miR-33a/ b in different human skin melanoma cell lines, the proliferation, adhesion, movement, apoptosis and epithelial-mesenchymal transition of the cells were observed in vitro. After the expression of miR-33a in A375 cells of melanoma, the size, volume, mass and number of lung metastases were observed at the level of body experiment. To explore the possible mechanisms of miR-33a/ b to influence the malignant biological function of human melanoma cells. Methods: Expression of miR-33a/ b in the skin melanoma cell lines WM451, A375 and SK-MEL-1 with low metastatic invasion force and the skin melanoma cell line WM451, A375 and SK-MEL-1 by the real-time PCR method Determination of the expression of miR-33a/ b in a melanoma cell line, screening of miR-33a/ b high-expression and low-expression melanoma cell line as a further experimental study, Like, constructing a pYr-LVX-pri-miR-33a lentiviral vector and packaging it into a lentivirus, and then infecting A375 cells, establishing a hsa-miR-33a stable over-expression cell line A375-pYr-LVX-pir-miR-33a; constructing a pYr-LVX-miR-33a-sponge lentiviral vector and packaging it into a lentivirus, and then infecting the WM35, WM451 cells, respectively, establishing a stable cell line WM35-pYr-LVX-miR-33a-sponge, which is inhibited by the hsa-miR-33a expression, respectively. In addition, VM451-pYr-LVX-miR-33a-sponge o expressed stable up-regulation/ inhibition cell line as the research object with the 1niR-33a expression, and the cell proliferation ability was detected by MTT method; the cell clone number was detected by the formation of the plate clone; the cell apoptosis rate was detected by flow cytometry; the cell scratch test and the Transwell experiment were used to detect the invasion force of the cell. The expression of E-cadherin and N-cadherin in EMT was detected by the Western Blot method. The expression of miR-33a on cell proliferation, cell apoptosis, invasion and metastasis, EMT and other phenotypes is discussed in a multi-layer manner. The size, volume, mass and the number of lung metastases were observed by subcutaneous and tail injection of A375 and A375-pYr-LVX-pir-miR-33a cells. The expression level of HIF-1 was predicted by a bioinformatics analysis method. The expression level of HIF-1 was detected by the Western Blot method. The direct target group of HIF-1 was further verified by the double-luciferase gene reporting method. Due to the high expression of miR-33a and niR-33b in human skin melanoma cell line WM35 with low metastatic invasion force, the expression levels in human skin melanoma cell lines VM451, A375 and SK-MEL-1 with high metastatic invasion were relatively low (WM35WM451A375SK-MEL-1); upregulating the expression of 1niR-33a in the A375 cell line can inhibit cell proliferation, invasion and metastasis and epithelial-mesenchymal transition; the expression of miR-33a in the WM451 and WM35 cell lines can promote cell proliferation, invasion and metastasis and epithelial-mesenchymal transition; and the effect of the miR-33a on the apoptosis of the cells is not counted. The difference is that the miR-33a can inhibit the formation speed of the planting tumor and reduce the lung transfer range. The number of bioinformatics predictions, HIF-1, is downstream of miR-33a. Target gene. After overexpression of miR-33a in A375, the expression of HIF-1 was decreased; the expression of miR-33a in WM35 and WM451 was inhibited after expression of HIF-1 Up-to-date. The double-luciferase reporter assay further confirmed that the HIF-1 gene was a direct miR-33a Target gene. Conclusion: The expression of miR-33a and miR-33b in human skin melanoma with low metastatic invasion force is higher than that of human skin with high metastatic invasion force. The miR-33a can inhibit the cell proliferation, invasion and metastasis of the human melanoma cell line, regulate the epithelial-mesenchymal transition process, but does not affect the cell apoptosis; and the miR-33a can inhibit the formation of the tumor and the metastatic tumor, and the miR-33a is a melanoma. The tumor suppressor gene. The miR-33a may influence the malignant melanoma cell by directly targeting the HIF-1 antigen. Biological phenotype. Figure 24
【學位授予單位】:中南大學
【學位級別】:博士
【學位授予年份】:2013
【分類號】:R739.5
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