microRNA-33a通过靶向HIF-1α抑制黑色素瘤侵袭转移的分子机制研究

发布时间：2019-01-17 11:32

【摘要】：研究目的：探讨miR-33a/b在不同转移潜能人皮肤黑色素瘤细胞系中的表达特点和意义。干预不同人皮肤黑色素瘤细胞系中miR-33a/b表达后,体外实验水平观察细胞的增殖、粘附、运动、凋亡和上皮-间质转化等肿瘤细胞恶性生物学功能的变化。干预黑色素瘤A375细胞中miR-33a表达后,在体实验水平观察裸鼠皮下种植瘤的大小、体积、质量及肺脏转移灶数目的变化情况。探讨miR-33a/b影响人黑色素瘤细胞恶性生物学功能的可能机制。研究方法：通过real-time PCR法检测miR-33a/b在低转移侵袭力的皮肤黑色素瘤细胞株WM35及高转移侵袭力的皮肤黑色素瘤细胞株WM451、A375和SK-MEL-1中的表达量。明确miR-33a/b在黑色素瘤细胞系中的表达情况,筛选miR-33a/b高表达和低表达的黑色素瘤细胞系作为进一步的实验研究对象。构建pYr-LVX-pri-miR-33a慢病毒载体,并将其包装成慢病毒,然后感染A375细胞,建立hsa-miR-33a稳定过表达细胞系A375-pYr-LVX-pir-miR-33a;构建pYr-LVX-miR-33a-sponge慢病毒载体,并将其包装成慢病毒,然后分别感染WM35、WM451细胞,建立hsa-miR-33a表达抑制的稳定细胞系WM35-pYr-LVX-miR-33a-sponge和、VM451-pYr-LVX-miR-33a-sponge o以1niR-33a表达稳定上调／抑制细胞系为研究对象,运用MTT法检测细胞增殖能力；平板克隆形成实验检测细胞克隆数；流式细胞术检测细胞凋亡率；细胞划痕实验、Transwell实验检测细胞迁移侵袭力；、Vestern Blot法检测与EMT相关的E-钙粘连蛋白(E-Cadherin)、N-钙粘连蛋白(N-Cadherin)的表达情况。多层面探讨miR-33a的表达对细胞增殖、细胞凋亡、侵袭转移和EMT等表型的影响。通过皮下接种及尾静脉注射A375及A375-pYr-LVX-pir-miR-33a细胞建立裸鼠种植瘤及移植瘤模型,观察裸鼠皮下种植瘤的大小、体积、质量及肺脏转移灶数目的变化情况。采用生物信息学分析法预测HIF-1α可能为miR-33a的下游靶因；Western Blot法检测miR-33a表达变化时HIF-1α蛋白的表达水平；应用双荧光素酶基因报告法进一步验证HIF-1α是否为miR-33a的直接靶基因。研究结果：miR-33a与1niR-33b在低转移侵袭力的人皮肤黑色素瘤细胞株WM35中表达量均高,而在高转移侵袭力的人皮肤黑色素瘤细胞株VM451、A375和SK-MEL-1中的表达量相对较低(WM35WM451A375 SK-MEL-1);上调A375细胞株中1niR-33a的表达能抑制细胞增殖、侵袭转移和上皮-间质转化；抑制WM451和WM35细胞株中miR-33a的表达能促进细胞增殖、侵袭转移和上皮-间质转化；miR-33a对细胞凋亡的影响无统计学差异。miR-33a能抑制种植瘤的形成速度,减少肺脏转移灶的数目。生物信息学预测HIF-1α为miR-33a的下游靶基因。过表达A375中miR-33a的表达后,HIF-1α的表达下降；抑制WM35、WM451中miR-33a的表达后HIF-1α的表达上升。双荧光素酶报告实验进一步证实HIF-1α为miR-33a的直接靶基因。研究结论：miR-33a与miR-33b在低转移侵袭力的人皮肤黑色素瘤中的表达量高于高转移侵袭力的人皮肤黑色素瘤。miR-33a能抑制人黑色素瘤细胞株的细胞增殖、侵袭转移,调节上皮-间质转化过程,但不影响细胞凋亡；miR-33a能抑制种植瘤及转移瘤的形成,提示miR-33a为黑色素瘤的抑癌基因。miR-33a可能通过直接靶向调节HIF-1α而影响黑色素瘤细胞的恶性生物学表型。图24幅,参考文献
[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
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.5

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