Regulation of cell cycle checkpoint kinase WEE1 by miR-195 in malignant melanoma
Regulation of cell cycle checkpoint kinase WEE1 by miR-195 in malignant melanoma
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DOI：
10.1038/onc.2012.324
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发表时间：
2013-06-27
期刊：
影响因子：
8
通讯作者：
Kunz, M.
中科院分区：
文献类型：
--
作者：
Bhattacharya, A.;Schmitz, U.;Kunz, M.
Wee1激酶已被描述为G2细胞周期检查点的主要守门人，并参与不同恶性肿瘤的肿瘤进展。在这里，我们使用免疫印迹、实时定量聚合酶链式反应和免疫组织化学方法分析了WEE1在一系列黑色素瘤患者样本和黑色素瘤细胞系中的表达水平。与原发黑色素瘤相比，转移性黑色素瘤患者样本中Wee1的表达显著下调；与低侵袭性黑色素瘤细胞系相比，高侵袭性黑色素瘤细胞系中Wee1表达显著下调。此外，WEE1的表达与WEE1靶向的microRNA miR-195之间存在负相关。进一步的分析表明，用miR-195基因转导黑色素瘤细胞系确实降低了这些细胞中WEE1mRNA和蛋白的表达。报告基因分析证实，miR-195直接靶向WEE1 3‘非翻译区(3’UTR)。在SK-MEL-28黑色素瘤细胞中，miR-195的过表达伴随着WEE1的减少，并显著减少了应激诱导的G2-M细胞周期停滞，WEE1的稳定过表达可以恢复这种作用。此外，miR-195过表达和WEE1基因敲除分别促进了黑色素瘤细胞的增殖。MIR-195的过表达也增强了黑色素瘤细胞的迁移和侵袭力。综上所述，本研究表明WEE1在恶性黑色素瘤中的表达直接受miR-195的调节。MIR-195介导的WEE1在转移灶中的下调可能有助于克服局部组织微环境中应激条件下的细胞周期停滞，从而允许肿瘤细胞不受限制地生长。
WEE1 kinase has been described as a major gate keeper at the G2 cell cycle checkpoint and to be involved in tumour progression in different malignant tumours. Here we analysed the expression levels of WEE1 in a series of melanoma patient samples and melanoma cell lines using immunoblotting, quantitative real-time PCR and immunohistochemistry. WEE1 expression was significantly downregulated in patient samples of metastatic origin as compared with primary melanomas and in melanoma cell lines of high aggressiveness as compared with cell lines of low aggressiveness. Moreover, there was an inverse correlation between the expression of WEE1 and WEE1-targeting microRNA miR-195. Further analyses showed that transfection of melanoma cell lines with miR-195 indeed reduced WEE1 mRNA and protein expression in these cells. Reporter gene analysis confirmed direct targeting of the WEE1 3' untranslated region (3'UTR) by miR-195. Overexpression of miR-195 in SK-Mel-28 melanoma cells was accompanied by WEE1 reduction and significantly reduced stress-induced G2-M cell cycle arrest, which could be restored by stable overexpression of WEE1. Moreover, miR-195 overexpression and WEE1 knockdown, respectively, increased melanoma cell proliferation. miR-195 overexpression also enhanced migration and invasiveness of melanoma cells. Taken together, the present study shows that WEE1 expression in malignant melanoma is directly regulated by miR-195. miR-195-mediated downregulation of WEE1 in metastatic lesions may help to overcome cell cycle arrest under stress conditions in the local tissue microenvironment to allow unrestricted growth of tumour cells.