microRNA-33a通過(guò)靶向HIF-1α抑制黑色素瘤侵襲轉(zhuǎn)移的分子機(jī)制研究
發(fā)布時(shí)間：2019-01-17 11:32
【摘要】：研究目的：探討miR-33a/b在不同轉(zhuǎn)移潛能人皮膚黑色素瘤細(xì)胞系中的表達(dá)特點(diǎn)和意義。干預(yù)不同人皮膚黑色素瘤細(xì)胞系中miR-33a/b表達(dá)后,體外實(shí)驗(yàn)水平觀察細(xì)胞的增殖、粘附、運(yùn)動(dòng)、凋亡和上皮-間質(zhì)轉(zhuǎn)化等腫瘤細(xì)胞惡性生物學(xué)功能的變化。干預(yù)黑色素瘤A375細(xì)胞中miR-33a表達(dá)后,在體實(shí)驗(yàn)水平觀察裸鼠皮下種植瘤的大小、體積、質(zhì)量及肺臟轉(zhuǎn)移灶數(shù)目的變化情況。探討miR-33a/b影響人黑色素瘤細(xì)胞惡性生物學(xué)功能的可能機(jī)制。 研究方法：通過(guò)real-time PCR法檢測(cè)miR-33a/b在低轉(zhuǎn)移侵襲力的皮膚黑色素瘤細(xì)胞株WM35及高轉(zhuǎn)移侵襲力的皮膚黑色素瘤細(xì)胞株WM451、A375和SK-MEL-1中的表達(dá)量。明確miR-33a/b在黑色素瘤細(xì)胞系中的表達(dá)情況,篩選miR-33a/b高表達(dá)和低表達(dá)的黑色素瘤細(xì)胞系作為進(jìn)一步的實(shí)驗(yàn)研究對(duì)象。構(gòu)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(xì)胞,建立hsa-miR-33a穩(wěn)定過(guò)表達(dá)細(xì)胞系A(chǔ)375-pYr-LVX-pir-miR-33a;構(gòu)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W(wǎng)M35、WM451細(xì)胞,建立hsa-miR-33a表達(dá)抑制的穩(wěn)定細(xì)胞系WM35-pYr-LVX-miR-33a-sponge和、VM451-pYr-LVX-miR-33a-sponge o以1niR-33a表達(dá)穩(wěn)定上調(diào)／抑制細(xì)胞系為研究對(duì)象,運(yùn)用MTT法檢測(cè)細(xì)胞增殖能力；平板克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞克隆數(shù)；流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率；細(xì)胞劃痕實(shí)驗(yàn)、Transwell實(shí)驗(yàn)檢測(cè)細(xì)胞遷移侵襲力；、Vestern Blot法檢測(cè)與EMT相關(guān)的E-鈣粘連蛋白(E-Cadherin)、N-鈣粘連蛋白(N-Cadherin)的表達(dá)情況。多層面探討miR-33a的表達(dá)對(duì)細(xì)胞增殖、細(xì)胞凋亡、侵襲轉(zhuǎn)移和EMT等表型的影響。通過(guò)皮下接種及尾靜脈注射A375及A375-pYr-LVX-pir-miR-33a細(xì)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(zhì)量及肺臟轉(zhuǎn)移灶數(shù)目的變化情況。采用生物信息學(xué)分析法預(yù)測(cè)HIF-1α可能為miR-33a的下游靶因；Western Blot法檢測(cè)miR-33a表達(dá)變化時(shí)HIF-1α蛋白的表達(dá)水平；應(yīng)用雙熒光素酶基因報(bào)告法進(jìn)一步驗(yàn)證HIF-1α是否為miR-33a的直接靶基因。 研究結(jié)果：miR-33a與1niR-33b在低轉(zhuǎn)移侵襲力的人皮膚黑色素瘤細(xì)胞株WM35中表達(dá)量均高,而在高轉(zhuǎn)移侵襲力的人皮膚黑色素瘤細(xì)胞株VM451、A375和SK-MEL-1中的表達(dá)量相對(duì)較低(WM35WM451A375 SK-MEL-1);上調(diào)A375細(xì)胞株中1niR-33a的表達(dá)能抑制細(xì)胞增殖、侵襲轉(zhuǎn)移和上皮-間質(zhì)轉(zhuǎn)化；抑制WM451和WM35細(xì)胞株中miR-33a的表達(dá)能促進(jìn)細(xì)胞增殖、侵襲轉(zhuǎn)移和上皮-間質(zhì)轉(zhuǎn)化；miR-33a對(duì)細(xì)胞凋亡的影響無(wú)統(tǒng)計(jì)學(xué)差異。miR-33a能抑制種植瘤的形成速度,減少肺臟轉(zhuǎn)移灶的數(shù)目。生物信息學(xué)預(yù)測(cè)HIF-1α為miR-33a的下游靶基因。過(guò)表達(dá)A375中miR-33a的表達(dá)后,HIF-1α的表達(dá)下降；抑制WM35、WM451中miR-33a的表達(dá)后HIF-1α的表達(dá)上升。雙熒光素酶報(bào)告實(shí)驗(yàn)進(jìn)一步證實(shí)HIF-1α為miR-33a的直接靶基因。 研究結(jié)論：miR-33a與miR-33b在低轉(zhuǎn)移侵襲力的人皮膚黑色素瘤中的表達(dá)量高于高轉(zhuǎn)移侵襲力的人皮膚黑色素瘤。miR-33a能抑制人黑色素瘤細(xì)胞株的細(xì)胞增殖、侵襲轉(zhuǎn)移,調(diào)節(jié)上皮-間質(zhì)轉(zhuǎn)化過(guò)程,但不影響細(xì)胞凋亡；miR-33a能抑制種植瘤及轉(zhuǎn)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(guò)直接靶向調(diào)節(jié)HIF-1α而影響黑色素瘤細(xì)胞的惡性生物學(xué)表型。圖24幅,參考文獻(xiàn)
[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(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R739.5
本文編號(hào)：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(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R739.5
【參考文獻(xiàn)】
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