CAJ | 학술논문

目的探讨敲低miR-221/222表达上调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1 mm3移植胶质瘤瘤块建立裸鼠移植瘤模型,1周后接受治疗(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28 d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6～28 d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＜0.05)；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-222 mRNA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); Western 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.
목적 탐토고저miR-221/222표체상조PUMA대U251인뇌효질모세포류조망적영향급기궤제. 방법 5주령BALB/c라서좌측혜부피하접충1 mm3이식효질류류괴건립라서이식류모형,1주후접수치료(전염무의서렬조、반의miR-221/222공전염조분별류내주사무의대조서렬、반의miR-221/222과핵감산,대조조주사등량용제,매조8지).치료개시지28 d관찰종류적생장정황,관찰기결속시취종류조직HE염색검측병이학개변；형광원위잡교(FISH)、실시정량PCR검측종류조직miR-221、miR-222적표체;TUNEL염색검측종류세포조망,면역조화염색、Western blotting검측종류조직조망상관단백PUMA、bax화bcl-2、p53적표체. 결과 치료후6～28 d반의miR-221/222공전염조이식류적체적명현저우대조조화전염무의서렬조,차이유통계학의의(P＜0.05)；여전염무의서렬조급대조조비교,반의miR-221/222공전염조종류조직miR-221、miR-222 mRNA적표체수평하강；HE염색현시반의miR-221/222공전염조종류조직이형성감저,신생혈관수감소;여대조조화전염무의서렬조비교,반의miR-221/222공전염조종류조직적조망지수교고,차이유통계학의의(P＜0.05);면역조화염색검측현시여대조조화전염무의서렬조비교,반의miR-221/222공전염조PUMA、bax표체양성솔교고,bcl-2표체양성솔교저,차이유통계학의의(P＜0.05); Western blotting검측현시여대조조화전염무의서렬조비교,반의miR-221/222공전염조종류조직PUMA화bax단백표체승고,bcl-2표체하강,이p53표체무명현변화. 결론 반의miR-221/222치료가통과상조PUMA단백표체래유도U251효질류세포조망.
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 lipofcctamine-mediated miRNA-221/222 antisense oligonucleotides (GroupA), 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 situ 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 Westem 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.