miR-92b在神经胶质瘤裸鼠移植瘤模型中的作用

The role of miR-92b in glioma xenograft model in nude mice

目的建立U251胶质瘤裸鼠皮下移植瘤模型,观察沉默miR-92b对U251神经胶质瘤裸鼠皮下移植瘤生长的影响。方法采用神经胶质瘤U251细胞株建立神经胶质瘤的裸鼠皮下移植瘤动物模型,将shRNA-miR-92b表达的质粒稳定转染U251细胞,筛选稳转细胞采取多点注射的方法注入裸鼠皮下,以阴性质粒和PBS为阴性对照组(shRNA-miR-92b NC组)和空白对照(PBS组);检测裸鼠成瘤体积和重量;real-time PCR检测移植瘤中miR-92b、胰岛素样生长因子结合蛋白-3(IGFBP-3)的表达水平;western blot检测移植瘤中的IGFBP-3、β-链蛋白(β-catenin)和钙黏蛋白(E-cadherin)蛋白水平;免疫组织化学技术检测移植瘤中IGFBP-3、β-catenin和E-cadherin表达水平。结果移植瘤形成过程中裸鼠未出现死亡,全部裸鼠均皮下成瘤,肿瘤体积检测后发现shRNA-miR-92b转入后显著抑制移植瘤的生长(P<0.05);转入shRNA-miR-92b后,移植瘤中的miR-92b表达水平降低,IGFBP-3的表达水平增加(P<0.05);western blot结果显示IGFBP-3和E-cadherin蛋白表达升高,β-catenin蛋白表达降低(P<0.05);免疫组化结果也显示IGFBP-3蛋白表达升高,β-catenin蛋白表达降低(P<0.05)。结论沉默miR-92b表达后可能通过上调IGFBP3、E-cadherin蛋白表达,下调β-catenin蛋白表达,从而抑制神经胶质瘤裸鼠皮下移植瘤的生长。

Objective To establish a U251 glioma xenograft model in nude mice and observe the effect of silent miR-92 b on glioma xenografts in nude mice. Methods Using glioma U251 cell line to establish an glioma xenograft in nude mice,the expression of shRNA-miR-92 b stably plasmid transfected into U251 cells,steady cells screened were injected into nude mice by multiple injection. Negative plasmid as negative control and PBS as blank control. Tumor volume and weight of nude mice; the expression of miR-92 b and IGFBP-3 mRNA in xenograft in nude mice were detected by real-time PCR; the expression levels of IGFBP-3,β-catenin and E-cadherin protein in the xenograft tumor tissues were detected by western blot; the immunohistochemistry was used to detected the expression of IGFBP-3 and β-catenin in tumor tissues. Results In the process of xenograft tumor formation,there was no death nude mice,all the mice were xenograft tumor formation. The tumor volume detection showed that silencing miR-92 b expression can significantly inhibit the growth of tumor volume（ P〈0. 05）. After transfect of shRNA-miR-92 b,the expression of miR-92 b was significantly down-regulated in xenograft tumor tissues and IGFBP-3 mRNA expression was significantly increased（ P〈0. 05）. Western blot showed that the expression of IGFBP-3 and E-cadherin protein were increased,β-catenin protein expression was decreased（ P〈0. 05）. Immunohistochemistry results also showed that IGFBP-3 protein expression were increased,β-catenin protein expression was decreased（ P〈0. 05）. Conclusion Silencing miR-92 b expression may up-regulate the expression of IGFBP-3 and β-catenin protein expression,down-regulate the expression of E-cadherin protein,and inhibit the growth of glioma nude mice xenografts.