下调miRNA-199a-5p靶向HIF1α激活JAK/STAT3通路促进乳腺癌细胞增殖与恶性转移

Down-regulated miRNA-199a-5p targeting HIF1αactivates the JAK/STAT3 pathway to promote breast cancer cell proliferation and malignant metastasis

目的:探讨miRNA-199a-5p在乳腺癌MCF-7细胞中的表达及其对细胞增殖与恶性转移的影响。方法:利用RT-qPCR检测miR-199a-5p在乳腺癌细胞(MCF-7细胞)和人正常乳腺上皮细胞(MCF-10A细胞)中的差异表达。过表达或下调miR-199a-5p表达,通过MTT试剂盒检测MCF-7细胞增殖能力,Transwell检测MCF-7细胞的侵袭能力,Western blot检测过表达及下调miR-199a-5p对MCF-7细胞EMT的影响。TargetScan分析miR-199a-5p的作用靶点,双荧光素酶报告基因实验检测miR-199a-5p和HIF1α之间的关系。RT-qPCR检测HIF1α在MCF-7细胞和MCF-10A细胞中的差异表达。HIF1α siRNA质粒转染细胞后,检测下调HIF1α对MCF-7细胞增殖、侵袭和EMT的影响。过表达HIF1α后,Western blot检测细胞内JAK、p-JAK、STAT3和p-STAT3蛋白的表达量。将AG490作用MCF-7细胞后再转染pcDNA-HIF1α质粒,检测MCF-7细胞增殖、侵袭和EMT能力。结果:与MCF-10A细胞相比,MCF-7细胞中miR-199a-5p表达明显降低(P<0.01),HIF1α表达明显上升(P<0.01)。过表达miR-199a-5p抑制了MCF-7细胞增殖、侵袭与EMT,下调miR-199a-5p促进了MCF-7细胞的增殖、侵袭与EMT;miR-199a-5p靶向HIF1α,且负调控HIF1α表达;siRNA下调HIF1α表达后明显抑制了MCF-7细胞增殖、侵袭和EMT;上调HIF1α表达后,激活MCF-7细胞内JAK/STAT3通路,AG490作用MCF-7细胞能明显抑制HIF1α对MCF-7细胞增殖、侵袭和EMT的促进作用。结论:下调miRNA-199a-5p靶向HIF1α激活JAK/STAT3通路促进乳腺癌细胞增殖与恶性转移。

Objective:To investigate the expression of miRNA-199a-5p in breast cancer McF-7 cells and its effect on cell proliferation and malignant metastasis.Methods:The differential expression of miR-199a-5p in breast cancer cells(MCF-7 cells)and human normal breast epithelial cells(MCF-10a cells)was detected by RT-qPCR.The proliferation ability of MCF-7 cells was detected by MTT kit,the invasion ability of MCF-7 cells was detected by Transwell,and the effects of overexpression and down-regulation of miR-199a-5p on EMT of MCF-7 cells were analyzed by Western blot.TargetScan was used to analyze the target of miR-199a-5p,and the relationship between miR-199a-5p and HIF1αwas detected by dual-luciferase reporter gene assay.The differential expression of HIF1αin MCF-7 cells and MCF-10A cells was detected by RT-qPCR.The effects of down-regulation of HIF1αon proliferation,invasion and EMT of MCF-7 cells were detected after transfection with HIF1αsiRNA plasmid.After the overexpression of HIF1α,the expression levels of JAK,p-JAK,STAT3 and p-STAT3 were detected by Western blot.MCF-7 cells were treated with AG490 and then transfected with PCDNA-HIF1αplasmid to detect the proliferation,invasion and EMT of McF-7 cells.Results:Compared with MCF-10A cells,the expression of miR-199a-5p in MCF-7 cells was significantly down-regulated(P<0.01),HIF1αexpression was significantly up-regulated(P<0.01).Overexpression of miR-199a-5p inhibited proliferation、invasion and EMT of MCF-7 cells.Down-regulation of miR-199a-5p promoted proliferation,invasion and EMT of MCF-7 cells.miR-199a-5p targeted HIF1αand negatively regulated HIF1αexpression.The proliferation,invasion and EMT of MCF-7 cells were significantly inhibited after the HIF1αexpression was down-regulated.Upregitating HIF1αexpression activated the JAK/STAT3 pathway in MCF-7 cells.AG490 significantly inhibited the proliferation,invasion and EMT of MCF-7 cells induced by HIF1α.Conclusion:Down-regulated miRNA-199a-5p targeting HIF1αactivates the JAK/STAT3 pathway to promote breast cancer cell proliferation and malignant metastasis.