目的: 探讨CD44调控HIF-1α/PLOD2信号通路对肾透明细胞癌（clear cell renal cell carcinoma,ccRCC）上皮-间充质转化（epithelial-mesenchymal transition,EMT）的影响。方法: 通过生物信息学数据库分析CD44在ccRCC组织中的表达;qRT-PCR检测CD44在ccRCC细胞中的表达;蛋白质免疫印迹法分析EMT相关蛋白、转移相关蛋白、HIF-1α和PLOD2的表达;克隆形成实验、transwell实验分别检测细胞增殖、迁移和侵袭。结果: CD44在ccRCC细胞中显著上调。与shNT组相比,sh-CD44组细胞增殖、迁移和侵袭能力显著降低,细胞中E-cadherin蛋白表达量增加,N- cadherin、MMP-2和MMP-9蛋白表达量减少。与Vector组相比,CD44-OE组细胞增殖、迁移和侵袭能力显著增加,细胞中E-cadherin蛋白表达量降低,N- cadherin、MMP-2和MMP-9蛋白表达量增加。此外,与shNT组相比,sh-CD44组细胞中HIF-1α和PLOD2的表达降低;而与Vector组相比,CD44-OE组细胞中HIF-1α和PLOD2的表达增加。sh-CD44组细胞中HIF-1α mRNA水平并未改变,但是VHL与HIF-1α结合增加,促进HIF-1α蛋白降解。与Vector组相比,CD44-OE组细胞中PLOD2的启动子活性增强。与Vector+DMSO组相比,CD44-OE+DMSO组细胞的增殖、迁移和侵袭能力明显提高;而与CD44-OE+DMSO组相比,CD44-OE+PX-478组细胞的增殖、迁移和侵袭能力明显降低。此外,与Vector+DMSO组相比,CD44-OE+DMSO组细胞中E-cadherin蛋白表达量减少,N-cadherin、MMP-2、MMP-9、HIF-1α和PLOD2蛋白表达量增加;而与CD44-OE+DMSO组相比,CD44-OE+PX-478组细胞中E-cadherin蛋白表达量增加,N-cadherin、MMP-2和MMP-9、HIF-1α和PLOD2蛋白表达量减少。结论: CD44在ccRCC细胞增殖和转移中具有促进作用。研究结果提示靶向CD44或HIF-1α/PLOD2途径可能是ccRCC治疗的新靶点。
Abstract
Objective To investigate the effect of CD44 regulation of HIF-1α/PLOD2 signaling pathway on epithelial mesenchymal transformation in clear cell renal cell carcinoma (ccRCC). Methods The expression of CD44 in ccRCC tissues was analyzed by bioinformatics database. The expression of CD44 in ccRCC cells was detected by qRT-PCR. The expressions of epithelial- mesenchymal transition, metastasis-related proteins, and HIF-1α and PLOD2 were analyzed by Western blot. Cell proliferation, migration and invasion were detected by clonal formation assay and transwell assay respectively. Results D44 was significantly upregulated in ccRCC cells. Compared with shNT group, cell proliferation, migration and invasion ability of sh-CD44 group were significantly decreased, the expression of E-cadherin protein was increased, and the expressions of N-cadherin, MMP-2 and MMP-9 protein were decreased. Compared with the Vector group, the proliferation, migration and invasion of cells in the CD44-OE group were significantly enhanced, the expression of E-cadherin protein was decreased, and the expressions of N-cadherin, MMP-2 and MMP-9 protein were increased. In addition, the expressions of HIF-1α and PLOD2 were decreased in sh-CD44 group compared with shNT group. Compared with Vector group, the expressions of HIF-1α and PLOD2 in CD44-OE group were increased. The mRNA level of HIF-1α in the sh-CD44 group did not change, but VHL bound to HIF-1α increased, which promoted the degradation of HIF-1α protein. Compared with the Vector group, the promoter activity of PLOD2 in the CD44-OE group was enhanced. In addition, compared with Vector+DMSO group, the proliferation, migration and invasion of cells in CD44-OE+DMSO group were significantly improved. Compared with the CD44-OE+DMSO group, the proliferation, migration and invasion of cells in the CD44-OE+ PX-478 group were significantly reduced. In addition, compared with Vector+DMSO group, the expression of E-cadherin protein decreased, while the expressions of N-cadherin, MMP-2, MMP-9, HIF-1α and PLOD2 protein were increased in CD44-OE+DMSO group. Compared with CD44-OE+DMSO group, the expression of E-cadherin protein was increased in CD44-OE+PX-478 group, while the expressions of N-cadherin, MMP-2 and MMP-9, HIF-1α and PLOD2 protein were decreased. Conclusion The results of this study indicate the roles of CD44 in promoting the proliferation and metastasis of ccRCC cells. These results suggest that targeting CD44 or HIF-1α/PLOD2 pathways may be new targets for ccRCC therapy.
关键词
CD44 /
HIF-1α/PLOD2信号通路 /
肾透明细胞癌 /
上皮-间充质转化
Key words
CD44 /
HIF-1α/PLOD2 pathway /
clear cell renal cell carcinoma /
epithelial-mesenchymal transition
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基金
宁夏回族自治区自然科学基金项目“PTGFRN通过CD44驱动上皮间充质转换促进肾透明细胞癌进展机理及其治疗靶向价值”（2023AAC03722）
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