敲低miR221／222上调PUMA促进胶质母细胞瘤U251细胞凋亡的体内研究

Inducement of U251 glioblastoma cell apoptosis in vivo through up-regulating PUMA expresion and knocking down miR-221/222

目的探讨敲低miR-221／222表达上瘤瘤块建立裸鼠移植瘤模型，1周后调PUMA对U251人脑胶质母细胞瘤凋亡的影响及其机制。方法5周龄BALB／C裸鼠左侧鼷部皮下接种1mm^3移植胶质接受治疗f转染无义序列组、反义miR-221／222共转染组分别瘤内注射无义对照序列、反义miR-221／222寡核苷酸，对照组注射等量溶剂，每组8只）。治疗开始至28d观察肿瘤的生长情况，观察期结束时取肿瘤组织HE染色检测病理学改变；荧光原位杂交（FISH）、实时定量PCR检测肿瘤组织miR-221、miR-222的表达；TUNEL染色检测肿瘤细胞凋亡，免疫组化染色、Western blotting检测肿瘤组织凋亡相关蛋白PUMA、bax和bcl-2、p53的表达。结果治疗后6—28d反义miR-221／222共转染组移植瘤的体积明显低于对照组和转染无义序列组．差异有统计学意义（P〈05）；与转染无义序列组及对照组比较，反义miR。221／222共转染组肿瘤组织miR-221、miR．222mRNA的表达水平下降；HE染色显示反义miR-221／222共转染组肿瘤组织异型性减低．新生血管数减少；与对照组和转染无义序列组比较，反义miR-221／222共转染组肿瘤组织的凋亡指数较高。差异有统计学意义（P〈0．05）；免疫组化染色检测显示与对照组和转染无义序列组比较．反义miR-221／222共转染组PUMA、bax表达阳性率较高，bcl-2表达阳性率较低，差异有统计学意义（P〈0．05）；Westem blotting检测显示与对照组和转染无义序列组比较．反义miR-221／222共转染组肿瘤组织PUMA和bax蛋白表达升高，bcl-2表达下降，而p53表达无明显变化。结论反义miR-221／222治疗可通过上调PUMA蛋白表达来诱导U251胶质瘤细胞凋亡。

Objective To study the inducement of U251 glioblastoma cell apoptosis in vivo through up-regulating PUMA expresion and knocking down miR-221/222, and explore its mechanism. Methods Nude mouse xenograft models were established in 5-week-old BALB/c nude mice by subcutaneous vaccination of U251 glioblastomas; 1 week later, they were treated with intratumoral injection of lipofectamine-mediated miRNA-221/222 antisense oligonucleotides （Group A）, nonsense sequences （Group B） and controls （Group C）, respectively （n=8）. The tumor growth was monitored until the end of observation period （28 d after the treatment） and pathological changes of the glioblastoma tissues were observed by HE staining at the end of observation. Fluorescence in siVa hybridization （FISH） and real-time PCR were employed to measure the miR-221 and miR-222 expressions. Terminal deoxynucleotidyl transferase-mediated uridine 5＇-triphosphate-biotin nick end labeling （TUNEL） assay was used to detect the apoptosis of glioblastomas. Immunohistochemistry and Western blotting were used to detect the expressions of PUMA, bax, bcl-2 and p53 in removed tumor specimens. Results The volume in Group A was significantly smaller than that of those in group B and group C 6-28 dater treatment （P=-0.006）. The miR-221 and miR-222 mRNA expressions in Group A were significantly decreased as compared with those of those in group B and group C. HE staining indicated that decreased heteromorphism and reduced number of new vessels in Group A were noted as compared with those in group B and group C. The cell apoptotic index in Group A was significantly higher than that in group B and group C （P〈0.05）. Immunohistochemistry showed that the expression levels of PUMA and bax in Group A was significantly up-regulated as compared with those in group B and group C, while the expression of bcl-2 in Group A was significantly down-regulated as compared with that in group B and group C; and no significant changes were noted in the p53 expression. Conclusion By up-regulating PUMA expresion, knocking down miR-221/222 can induce U251 glioma apoptosis in vivo.