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
發(fā)布時間：2019-01-17 11:32
【摘要】：研究目的：探討miR-33a/b在不同轉移潛能人皮膚黑色素瘤細胞系中的表達特點和意義。干預不同人皮膚黑色素瘤細胞系中miR-33a/b表達后,體外實驗水平觀察細胞的增殖、粘附、運動、凋亡和上皮-間質轉化等腫瘤細胞惡性生物學功能的變化。干預黑色素瘤A375細胞中miR-33a表達后,在體實驗水平觀察裸鼠皮下種植瘤的大小、體積、質量及肺臟轉移灶數(shù)目的變化情況。探討miR-33a/b影響人黑色素瘤細胞惡性生物學功能的可能機制。 研究方法：通過real-time PCR法檢測miR-33a/b在低轉移侵襲力的皮膚黑色素瘤細胞株WM35及高轉移侵襲力的皮膚黑色素瘤細胞株WM451、A375和SK-MEL-1中的表達量。明確miR-33a/b在黑色素瘤細胞系中的表達情況,篩選miR-33a/b高表達和低表達的黑色素瘤細胞系作為進一步的實驗研究對象。構建pYr-LVX-pri-miR-33a慢病毒載體,并將其包裝成慢病毒,然后感染A375細胞,建立hsa-miR-33a穩(wěn)定過表達細胞系A375-pYr-LVX-pir-miR-33a;構建pYr-LVX-miR-33a-sponge慢病毒載體,并將其包裝成慢病毒,然后分別感染WM35、WM451細胞,建立hsa-miR-33a表達抑制的穩(wěn)定細胞系WM35-pYr-LVX-miR-33a-sponge和、VM451-pYr-LVX-miR-33a-sponge o以1niR-33a表達穩(wěn)定上調／抑制細胞系為研究對象,運用MTT法檢測細胞增殖能力；平板克隆形成實驗檢測細胞克隆數(shù)；流式細胞術檢測細胞凋亡率；細胞劃痕實驗、Transwell實驗檢測細胞遷移侵襲力；、Vestern Blot法檢測與EMT相關的E-鈣粘連蛋白(E-Cadherin)、N-鈣粘連蛋白(N-Cadherin)的表達情況。多層面探討miR-33a的表達對細胞增殖、細胞凋亡、侵襲轉移和EMT等表型的影響。通過皮下接種及尾靜脈注射A375及A375-pYr-LVX-pir-miR-33a細胞建立裸鼠種植瘤及移植瘤模型,觀察裸鼠皮下種植瘤的大小、體積、質量及肺臟轉移灶數(shù)目的變化情況。采用生物信息學分析法預測HIF-1α可能為miR-33a的下游靶因；Western Blot法檢測miR-33a表達變化時HIF-1α蛋白的表達水平；應用雙熒光素酶基因報告法進一步驗證HIF-1α是否為miR-33a的直接靶基因。 研究結果：miR-33a與1niR-33b在低轉移侵襲力的人皮膚黑色素瘤細胞株WM35中表達量均高,而在高轉移侵襲力的人皮膚黑色素瘤細胞株VM451、A375和SK-MEL-1中的表達量相對較低(WM35WM451A375 SK-MEL-1);上調A375細胞株中1niR-33a的表達能抑制細胞增殖、侵襲轉移和上皮-間質轉化；抑制WM451和WM35細胞株中miR-33a的表達能促進細胞增殖、侵襲轉移和上皮-間質轉化；miR-33a對細胞凋亡的影響無統(tǒng)計學差異。miR-33a能抑制種植瘤的形成速度,減少肺臟轉移灶的數(shù)目。生物信息學預測HIF-1α為miR-33a的下游靶基因。過表達A375中miR-33a的表達后,HIF-1α的表達下降；抑制WM35、WM451中miR-33a的表達后HIF-1α的表達上升。雙熒光素酶報告實驗進一步證實HIF-1α為miR-33a的直接靶基因。 研究結論：miR-33a與miR-33b在低轉移侵襲力的人皮膚黑色素瘤中的表達量高于高轉移侵襲力的人皮膚黑色素瘤。miR-33a能抑制人黑色素瘤細胞株的細胞增殖、侵襲轉移,調節(jié)上皮-間質轉化過程,但不影響細胞凋亡；miR-33a能抑制種植瘤及轉移瘤的形成,提示miR-33a為黑色素瘤的抑癌基因。miR-33a可能通過直接靶向調節(jié)HIF-1α而影響黑色素瘤細胞的惡性生物學表型。圖24幅,參考文獻
[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
本文編號：2410021
[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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