Tian Yuan, Fu Shuang, Qiu Guang-Bin, Xu Zhen-Ming, Liu Ning, Zhang Xiao-Wen, Chen Sheng, Wang Ye, Sun Kai-Lai, Fu Wei-Neng
Department of Medical Genetics, China Medical University, Shenyang 110001, People's Republic of China.
BMC Cancer. 2014 Sep 18;14:678. doi: 10.1186/1471-2407-14-678.
miRNA-27a has been confirmed as an important regulator in carcinogenesis and other pathological processes. Whether and how it plays a role in the laryngeal carcinoma is unknown.
Mature miRNA-27a expression in laryngeal cancer was detected by qRT-PCR. Gain-of-function studies using mature miR-27a were performed to investigate cell proliferation and apoptosis in the Hep2 cells. In silico database analysis and luciferase reporter assay were applied to predict and validate the direct target, respectively. Loss-of-function assays were performed to investigate the functional significance of the miR-27a target gene. qRT-PCR and Western blot were used to evaluate mRNA and protein levels of the target, respectively.
miR-27a was significantly up-regulated in the laryngeal tumor tissues compared to the adjacent non-tumor tissues. In silico database analysis result revealed that PLK2 is a potential target of miR-27a. luciferase reporter assay result showed the direct inhibition of miR-27a on PLK2-3'UTR. In the cases with miR-27a up-regulation, PLK2 protein expression level was significantly lower in cancer tissues than that in the adjacent non-tumor tissues, which showed a negative correlation with miR-27a expression level. Both miR-27a and knockdown of PLK2 caused the increase of the cell viability and colony formation and inhibition of the late apoptosis in the Hep2 cell lines. Moreover, miR-27a but not PLK2 also repressed the early apoptosis in the Hep2 cells. Additionally, no alteration of the Hep2 cell cycle induced by miR-27a was detected.
miR-27a acts as an oncogene in laryngeal squamous cell carcinoma through down-regulation of PLK2 and may provide a novel clue into the potential mechanism of LSCC oncogenesis or serve as a useful biomarker in diagnosis and therapy in laryngeal cancer.
miRNA-27a已被确认为致癌作用及其他病理过程中的重要调节因子。其是否以及如何在喉癌中发挥作用尚不清楚。
采用qRT-PCR检测喉癌中成熟miRNA-27a的表达。利用成熟miR-27a进行功能获得性研究，以研究Hep2细胞中的细胞增殖和凋亡情况。分别应用计算机数据库分析和荧光素酶报告基因检测来预测和验证直接靶点。进行功能缺失实验以研究miR-27a靶基因的功能意义。分别采用qRT-PCR和蛋白质印迹法评估靶点的mRNA和蛋白质水平。
与相邻的非肿瘤组织相比，miR-27a在喉肿瘤组织中显著上调。计算机数据库分析结果显示PLK2是miR-27a的潜在靶点。荧光素酶报告基因检测结果显示miR-27a对PLK2-3'UTR有直接抑制作用。在miR-27a上调的病例中，癌组织中PLK2蛋白表达水平显著低于相邻的非肿瘤组织，且与miR-27a表达水平呈负相关。miR-27a和PLK2基因敲低均导致Hep2细胞系中细胞活力和集落形成增加，并抑制晚期凋亡。此外，miR-27a而非PLK2也抑制了Hep2细胞的早期凋亡。另外，未检测到miR-27a诱导的Hep2细胞周期改变。
miR-27a通过下调PLK2在喉鳞状细胞癌中发挥癌基因作用，可能为喉鳞状细胞癌发生的潜在机制提供新线索，或作为喉癌诊断和治疗中的有用生物标志物。
