Li T K, Yin K, Chen Z, Bao Y, Zhang S X
Department of Stomatology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei, China.
Department of Stomatology, Xingtai People's Hospital of Hebei Medical University, Xingtai, Hebei, China.
Genet Mol Res. 2017 Mar 8;16(1):gmr-16-01-gmr.16019327. doi: 10.4238/gmr16019327.
Ras association domain family member 5 (RASSF5), a member of the Ras association domain family, induces cell apoptosis by phosphorylating FOXO3a, which triggers target gene BIM (pro-apoptotic factor) activation. MiR-214 is overexpressed in oral cancer tissue, indicating its possible involvement in oral cancer pathogenesis. Bioinformatics analysis has revealed a complimentary sequence between miR-214 and the 3'-UTR of RASSF5 mRNA. However, whether miR-124 regulates RASSF5 in oral cancer remains poorly understood. We aimed to investigate the role of miR-214 in RASSF5 expression regulation in oral cancer. Tumor and paracarcinoma tissues were obtained from 48 oral cancer patients to examine miR-214 and RASSF5 expression. The relationship between miR-214 and RASSF5 was investigated by dual luciferase reporter gene assay. Oral cancer KB cells were cultured in vitro and divided into inhibitor NC, miR-214 inhibitor, Scramble-pMD18, RASSF5-pMD18, and miR-214 inhibitor + RASSF5-pMD18 groups. Caspase 3 activity, cell apoptosis, and total protein expression were measured by spectrophotometry, flow cytometry, and western blot, respectively. MiR-214 expression was significantly increased, while that of RASSF5 decreased in oral cancer tumor tissues compared to paracarcinoma tissues. Luciferase assay showed that miR-214 suppressed RASSF5 expression by targeting its 3'-UTR. Down-regulation of miR-214 and/or enhancement of RASSF5 expression markedly increased FOXO3a phosphorylation, BIM expression, caspase 3 activity, and apoptosis. In conclusion, miR-214 expression was elevated and RASSF5 was down-regulated in oral cancer. Moreover, miR-214 regulated KB cell apoptosis through targeted inhibition of RASSF5 expression, FOXO3a phosphorylation, and BIM expression, suggesting its possible application as a novel therapeutic oral cancer target.
Ras关联结构域家族成员5（RASSF5）是Ras关联结构域家族的一员，通过磷酸化FOXO3a诱导细胞凋亡，进而触发靶基因BIM（促凋亡因子）的激活。MiR-214在口腔癌组织中过表达，表明其可能参与口腔癌的发病机制。生物信息学分析揭示了miR-214与RASSF5 mRNA的3'-UTR之间存在互补序列。然而，miR-214在口腔癌中是否调节RASSF5仍知之甚少。我们旨在研究miR-214在口腔癌中RASSF5表达调控中的作用。从48例口腔癌患者中获取肿瘤组织和癌旁组织，检测miR-214和RASSF5的表达。通过双荧光素酶报告基因检测法研究miR-214与RASSF5之间的关系。体外培养口腔癌KB细胞，分为抑制剂阴性对照、miR-214抑制剂、乱序-pMD18、RASSF5-pMD18以及miR-214抑制剂+RASSF5-pMD18组。分别采用分光光度法、流式细胞术和蛋白质印迹法检测Caspase 3活性、细胞凋亡及总蛋白表达。与癌旁组织相比，口腔癌肿瘤组织中miR-214表达显著升高，而RASSF5表达降低。荧光素酶检测表明，miR-214通过靶向RASSF5的3'-UTR抑制其表达。miR-214的下调和/或RASSF5表达的增强显著增加了FOXO3a的磷酸化、BIM的表达、Caspase 3活性及细胞凋亡。总之，口腔癌中miR-214表达升高而RASSF5下调。此外，miR-214通过靶向抑制RASSF5表达、FOXO3a磷酸化和BIM表达来调节KB细胞凋亡，提示其可能作为口腔癌新的治疗靶点。
