Abstract: Objective To explore the effect of overexpression of miR-143-3p on thyroid cancer ( TC) cells and its mechanism. Method The expression of miR-143-3p in normal thyroid epithelial cells and different TC cells was analyzed by Real-time PCR. Targetscan website and dual luciferase reporter system verified the targeting relationship between miR-143-3p and TGIF2 3′ UTR; the overexpression efficiency of miR-143-3p and TGIF2 mRNA was detected by Real-time PCR. After successful overexpression, CAL-62 cells were divided into the control group, miR-143-3p mimic group, pcDNA-TGIF2 group and miR-143-3p +TGIF2 group, the contents of inducible nitric oxide synthase ( iNOS) , interleukin ( IL) -1β and IL-6 in cell supernatant were detected by ELISA. The level of superoxide dismutase ( SOD) was detected by the kit, and the production of reactive oxygen species ( ROS) was detected by immunofluorescence. Micro-observation of stem cells into spheroids, and the expressions of TGIF2, p-P65, leucine-rich repeat-containing G protein-coupled receptor 5 ( LGR5 ) and octamer-binding transcription factor 4 ( OCT4) were detected by Western blot. The effect of miR-143-3p overexpression on tumor growth in nude mice was detected. Result The expression of miR-143-3p in TC cells was significantly lower than that in normal TC cells ( P < 0. 05 ) . Targetscan website and dual luciferase reporter system confirmed that miR-143-3p was targeted to TGIF2. In the miR-143-3p mimic group, the expression of miR-143-3p was up-regulated ( P <0. 05) , the expression of TGIF2 mRNA and protein was down-regulated ( P<0. 05) , the contents of iNOS, IL-1β and IL-6 were significantly decreased ( P<0. 05) , the antioxidant capacity and the sphere-forming ability of stem cells were significantly decreased ( P<0. 05) , and the protein expression levels of p-P65, LGR5 and OCT4 were significantly decreased ( P < 0. 05 ) . The results of pcDNA-TGIF2 group were opposite. Overexpression of miR-143-3p can significantly inhibit the pro-cancer effect of pcDNA-TGIF2. The xenograft tumor model in nude mice showed that overexpression of miR-143-3p could reduce tumor weight and volume, and down-regulate the expression of TGIF2 and OCT4 in tumor tissues ( P < 0. 05) . Conclusion miR-143-3p overexpression can inhibit the levels of proinflammatory factors, antioxidant capacity and stem cell-like characteristics of TC cells by down-regulating the expression of TGIF2, and inhibit the formation of tumors in nude mice.

Key words: miR-143-3p, thyroid cancer, pro-inflammatory factors, reactive oxygen species, OCT4

摘要： 目的 探究过表达 miR-143-3p 对甲状腺癌( thyroid cancer,TC) 细胞的影响及其作用机制。 方法 Real-time PCR 分析正常甲状腺上皮细胞和不同 TC 细胞中 miR-143-3p 的表达。 Targetscan 网站和双荧光素酶报告系统验证miR-143-3p 和 TGIF2 3′UTR 的靶向关系;Real-time PCR 检测 miR-143-3p 和 TGIF2 mRNA 的过表达效率;过表达成功后,将 CAL-62 细胞分为对照组、miR-143-3p mimic 组、pcDNA-TGIF2 组和 miR-143-3p+TGIF2 组,ELISA 检测细胞上清液中诱导型一氧化氮合酶( iNOS) 、白细胞介素( IL) -1β 和 IL-6 的含量;试剂盒检测超氧化物歧化酶( SOD) 水平,免疫荧光检测活性氧( ROS)产生;显微观察干细胞成球,Western blot 检测 TGIF2、p-P65、富含亮氨酸重复序列的 G 蛋白偶联受体5 ( LGR5) 和 八聚体结合蛋白4 ( OCT4 ) 蛋 白 表 达。体内构建裸鼠移植瘤, 检测过表达miR-143-3p 对肿瘤生长的影响。 结果 miR-143-3p 在 TC 细胞中的表达明显低于正常甲状腺上皮细胞( P< 0. 05) 。Targetscan 网站和双荧光素酶报告系统证实 miR-143-3p 与 TGIF2 存在靶向关系。 miR-143-3p mimic 组 miR-143-3p的表达上调( P<0. 05) ,TGIF2 mRNA 和蛋白表达下调( P<0. 05) ,iNOS、IL-1β 和 IL-6 含量明显降低( P<0. 05) ,抗氧化能力显著下降 ( P < 0. 05) , 干 细 胞 成 球 能 力 降 低 ( P < 0. 05) , p-P65、 LGR5 和 OCT4 的蛋白表达均显著下调( P<0. 05) 。 pcDNA-TGIF2 组的结果相反,过表达 miR-143-3p 能显著抑制 pcDNA-TGIF2 的促癌作用。 裸鼠移植瘤模型表明过表达 miR-143-3p 能够减小肿瘤的质量和体积,下调瘤组织中 TGIF2 和 OCT4 的表达( P<0. 05) 。 结论过表达 miR-143-3p 能够通过下调 TGIF2 的表达抑制 TC 细胞促炎因子水平、抗氧化能力和干细胞样特性,并抑制裸鼠肿瘤的形成。

关键词: miR-143-3p, 甲状腺癌, 促炎因子, 活性氧, 八聚体结合蛋白 , 4