miR-143-3p通过调节TGIF2的表达在甲状腺癌中的作用研究

Effects of miR-143-3p in Thyroid Cancer by Regulating the Expression of TGIF2

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。

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.