Kayisaier Kahaer, Abulajiang Tuoheti, Tang Liang, Hasiyeti Waili, Gulibositan Maimaitiaili
Department of Otolaryngology，People's Hospital of Xinjiang Uygur Autonomous Region，Xinjiang，830001，China.
Department of Radiotherapy，People's Hospital of Xinjiang Uygur Autonomous Region.
Lin Chuang Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2020 Feb;34(2):140-145. doi: 10.13201/j.issn.1001-1781.2020.02.010.
To study the effect of miR-340-5p on the proliferation of laryngeal cancer Hep2 cells and explore its intrinsic molecular mechanism, so as to screen potential biomarkers and targets for the diagnosis and treatment of laryngeal cancer. The expression of miR-340-5p in laryngeal cancer tissues, paracancerous tissues, laryngeal cancer cell lines Hep2 and normal bronchial HBE cell lines was quantitatively analyzed by qRT-PCR; The double luciferase reporter vector was constructed to verify whether STAT3 was a potential target gene of microRNA-340-5p; The miR-340-5p mimics/inhibitor was transfected into Hep2 cells by liposome and verified by qRT-PCR; The CCK-8 method and Annexin V/PI method were used to analyze the proliferation and apoptosis of transfected cells; and Western Blot was used to detect the expression of STAT3 and Wnt/β-catenin pathway-related proteins after transfection. The results of qRT-PCR showed that the level of miR-340-5p in laryngeal cancer tissues and Hep2 cells was significantly lower than that in adjacent tissues and HBE cells, and the expression of miR-340-5p was significantly increased or decreased after overexpression or inhibition; Luciferase activity showed that miR-340-5p directly interacted with target gene STAT3 3'-UTR and negatively regulated its expression; Cell proliferation and apoptosis analysis showed that up-regulation of microRNA-340-5p could significantly inhibit the proliferation and induce apoptosis of Hep2 cells in vitro, and vice versa; Western Blot results showed that the levels of STAT3 and β-catenin, c-Myc, TCF-4, CyclinD1 and ROCK1 in Hep2 cells were significantly lower than those in the control group after over-expression of miR-340-5p, and vice versa. The expression of miR-340-5p is abnormally low in laryngeal cancer tissues and Hep2 cells. It can be used as a potential biological target for diagnosis and treatment of laryngeal cancer by targeting STAT3 gene to negatively regulate Wnt/β-catenin signaling pathway.
研究miR-340-5p对喉癌Hep2细胞增殖的影响并探讨其内在分子机制，以筛选喉癌诊断和治疗的潜在生物标志物及靶点。采用qRT-PCR定量分析miR-340-5p在喉癌组织、癌旁组织、喉癌细胞系Hep2及正常支气管HBE细胞系中的表达；构建双荧光素酶报告载体验证STAT3是否为microRNA-340-5p的潜在靶基因；通过脂质体将miR-340-5p模拟物/抑制剂转染至Hep2细胞并经qRT-PCR验证；采用CCK-8法和Annexin V/PI法分析转染后细胞的增殖和凋亡情况；运用Western Blot检测转染后STAT3及Wnt/β-连环蛋白信号通路相关蛋白的表达。qRT-PCR结果显示，喉癌组织及Hep2细胞中miR-340-5p水平显著低于癌旁组织及HBE细胞，过表达或抑制后miR-340-5p表达显著升高或降低；荧光素酶活性显示miR-340-5p直接与靶基因STAT3的3'-UTR相互作用并负向调控其表达；细胞增殖和凋亡分析表明，上调microRNA-340-5p可显著抑制Hep2细胞体外增殖并诱导凋亡，反之亦然；Western Blot结果显示，miR-340-5p过表达后Hep2细胞中STAT3及β-连环蛋白、c-Myc、TCF-4、CyclinD1和ROCK1水平显著低于对照组，反之亦然。miR-340-5p在喉癌组织和Hep2细胞中表达异常低。通过靶向STAT3基因负向调控Wnt/β-连环蛋白信号通路，其可作为喉癌诊断和治疗的潜在生物学靶点。
