Guo Feng, Hou Xinming, Sun Qinghui
Department of Thyroid and Breast Surgery, The Second People's Hospital of Liaocheng, Liaocheng, Shandong 252600, P.R. China.
Oncol Lett. 2018 Nov;16(5):6815-6821. doi: 10.3892/ol.2018.9423. Epub 2018 Sep 7.
MicroRNAs (miRNAs/miRs) are widely studied as key regulators of gene expression and are involved in various diseases by affecting the miRNA-mediated regulatory function. BRAF is an important oncogene in the regulation of cell proliferation and apoptosis. In the present study, reverse transcription-quantitative polymerase chain reaction was used to determine the expression levels of miR-9-5p and BRAF mRNA in patients with papillary thyroid cancer (PTC). Western blotting was used to detect BRAF protein level. A luciferase assay was used to verify the miR-9-5p target site in BRAF. Cell Counting Kit-8 and flow cytometry were used to assess cell proliferation, and apoptosis, respectively. In the present study, it was demonstrated that miR-9-5p is downregulated in malignant PTC. Using bioinformatics analysis, miR-9-5p was predicted to target the human BRAF 3'-untranslated region (3'-UTR). A dual-luciferase assay demonstrated that miR-9-5p downregulated BRAF expression by directly targeting its 3'-UTR. Mutations in the 3'-UTR of BRAF completely abolished its interaction with miR-9-5p. Expression of exogenous miRNA that mimics miR-9-5p miRNA decreased BRAF protein and mRNA levels, while suppression of endogenous miR-9-5p resulted in an increase in BRAF protein, and mRNA levels. Furthermore, regulation of miR-9-5p was observed to suppress the viability of PTC cells by inducing apoptosis. Consistently, downregulation of miR-9-5p promoted proliferation of PTC cells by inhibiting the apoptosis of cells. In conclusion, the present study demonstrated that miR-9-5p may perform an important role in PTC prognosis and therapy.
微小RNA（miRNA/miR）作为基因表达的关键调节因子被广泛研究，并通过影响miRNA介导的调节功能参与各种疾病。BRAF是细胞增殖和凋亡调控中的一个重要癌基因。在本研究中，采用逆转录-定量聚合酶链反应来测定甲状腺乳头状癌（PTC）患者中miR-9-5p和BRAF mRNA的表达水平。采用蛋白质免疫印迹法检测BRAF蛋白水平。采用荧光素酶报告基因检测法验证BRAF中的miR-9-5p靶位点。分别使用细胞计数试剂盒-8和流式细胞术评估细胞增殖和凋亡。在本研究中，结果表明miR-9-5p在恶性PTC中表达下调。通过生物信息学分析预测miR-9-5p靶向人BRAF 3'-非翻译区（3'-UTR）。双荧光素酶报告基因检测表明，miR-9-5p通过直接靶向其3'-UTR下调BRAF表达。BRAF 3'-UTR中的突变完全消除了其与miR-9-5p的相互作用。模拟miR-9-5p的外源性miRNA的表达降低了BRAF蛋白和mRNA水平，而内源性miR-9-5p的抑制导致BRAF蛋白和mRNA水平升高。此外，观察到miR-9-5p的调节通过诱导凋亡抑制PTC细胞的活力。同样，miR-9-5p的下调通过抑制细胞凋亡促进PTC细胞增殖。总之，本研究表明miR-9-5p可能在PTC的预后和治疗中发挥重要作用。
