Yang Long, Zhao Guangning, Wang Fan, Li Chunchang, Wang Xiangzhong
Cell Physiol Biochem. 2018;49(3):920-931. doi: 10.1159/000493224. Epub 2018 Sep 5.
BACKGROUND/AIMS: miR-146a has recently been shown to promote cell proliferation, migration, and invasion in many cancers, but the role of miR-146a in clear cell renal cell carcinoma (ccRCC) remains unclear.
Reverse transcription quantitative PCR (RT-qPCR) was performed to investigate the mRNA expression of miR-146a and CADM2 in ccRCC tissues. The luciferase reporter assay, Western blotting, and ChIP assay were carried out to explore the promoter and the transcription factor of miR-146a. Moreover, the effect of miR-146a and CADM2 on ccRCC cells was explored using methyl thiazolyl tetrazolium, colony formation, and migration and invasion assays. The luciferase reporter assay, RT-qPCR, western blotting, and immunofluorescence assay were carried out to investigate whether CADM2 is directly regulated by miR-146a. A tumor xenograft model and immunohistochemical staining were used to examine the carcinogenic effect of miR-146a and CADM2 in vivo.
miR-146a has been shown to promote cell proliferation, migration, and invasion. Here, we found that miR-146a is highly expressed in ccRCC tissues, whereas CADM2 is down-regulated. Hypoxia can induce the expression of miR-146a by stimulating its promoter. In addition, we demonstrated that miR-146a promoted and CADM2 inhibited proliferation, migration, and invasion of ccRCC cells. The 3' untranslated region (UTR) luciferase reporter assay identified that miR-146a targeted the 3' UTR of CADM2 and negatively regulated its expression. Ectopic expression of CADM2 counteracted the promoting effect of miR-146a on cell proliferation, migration, invasion, and the epithelial-mesenchymal transition process.
Together, the finding of down-regulation of CADM2 by miR-146a can provide new insights into ccRCC pathogenesis and might contribute to the development of novel therapeutic strategies.
背景/目的：最近有研究表明，miR-146a在多种癌症中可促进细胞增殖、迁移和侵袭，但miR-146a在肾透明细胞癌（ccRCC）中的作用仍不清楚。
采用逆转录定量PCR（RT-qPCR）检测ccRCC组织中miR-146a和CADM2的mRNA表达。进行荧光素酶报告基因检测、蛋白质免疫印迹法和染色质免疫沉淀检测，以探究miR-146a的启动子和转录因子。此外，采用甲基噻唑基四氮唑、集落形成以及迁移和侵袭实验，探究miR-146a和CADM2对ccRCC细胞的影响。进行荧光素酶报告基因检测、RT-qPCR、蛋白质免疫印迹法和免疫荧光检测，以研究CADM2是否受miR-146a直接调控。采用肿瘤异种移植模型和免疫组织化学染色，在体内检测miR-146a和CADM2的致癌作用。
已证实miR-146a可促进细胞增殖、迁移和侵袭。在此，我们发现miR-146a在ccRCC组织中高表达，而CADM2表达下调。缺氧可通过刺激其启动子诱导miR-146a表达。此外，我们证明miR-146a促进而CADM2抑制ccRCC细胞的增殖、迁移和侵袭。3'非翻译区（UTR）荧光素酶报告基因检测确定miR-146a靶向CADM2的3'UTR并负向调节其表达。CADM2的异位表达抵消了miR-146a对细胞增殖、迁移、侵袭及上皮-间质转化过程的促进作用。
总之，miR-146a下调CADM2这一发现可为ccRCC发病机制提供新见解，并可能有助于开发新的治疗策略。
