脑靶向肽Angiopep2包载si-WDR4通过磷脂酰肌醇3激酶/蛋白激酶B信号抑制胶质母细胞瘤增殖  

Si-WDR4 encapsulated by the brain-targeting peptide Angiopep2 inhibits glioblastoma proliferation via phosphatidylinositol 3 kinase/protein kinase B signaling

作　　者：钱彩华[1] 笪明珍 李云涛 聂小虎 Qian Caihua;Da Mingzhen;Li Yuntao;Nie Xiaohu(Department of Nursing,Huzhou Central Hospital,Huzhou 313000,China;Department of Neurosurgery,Huzhou Central Hospital Affiliated to Huzhou Normal University,Huzhou 313000,China;Huzhou Key Laboratory of Basic and Clinical Translation of Neural Regulation,Huzhou 313000,China)

机构地区：[1]湖州市中心医院护理部,湖州313000 [2]湖州师范学院附属湖州市中心医院神经外科,湖州313000 [3]湖州市神经调控基础与临床转化重点实验室,湖州313000

出　　处：《中华实验外科杂志》2024年第9期1956-1959,共4页Chinese Journal of Experimental Surgery

基　　金：浙江省自然科学基金(LTGY24H160002)。

摘　　要：目的 探究脑靶向肽Angiopep2包载si-WDR4对胶质母细胞瘤的影响及其潜在机制.方法 制备Angiopep2/si-RNA,并检测其包封率和摄取率;在体外,分别对U87细胞进行磷酸盐缓冲液(PBS),Angiopep2/si-NC,si-WDR4和Angiopep2/si-WDR4处理,通过实时荧光定量聚合酶链反应(RT-qPCR)检测WDR4的mRNA含量,通过蛋白质印迹法(Western blot)检测WDR4、磷脂酰肌醇3激酶(PI3K)和蛋白激酶B(Akt)的蛋白表达水平,通过细胞计数试剂盒(CCK-8)实验检测U87细胞活力,通过5-乙炔基-2'脱氧尿嘧啶核苷(EdU)染色检测U87细胞的增殖;在体内,通过U87-Luc细胞构建异种原位移植瘤模型,7 d后,分别通过尾静脉注射PBS、Angiopep2/si-NC、si-WDR4和Angiopep2/si-WDR4.第28天,通过小动物活体成像检测肿瘤双荧光素酶活性,通过苏木精-伊红(HE)染色检测肿瘤大小.计量资料比较采用t检验.结果 Angiopep2对si-WDR4具有良好的结合能力,在N/P比为3∶1时形成完整复合物,并能够促进肿瘤细胞对si-RNA的摄取;在体外,与Angiopep2/si-NC 比较,Angiopep2/si-WDR4 处理能降低 U87 细胞内 WDR4 的 mRNA(0.21±0.14,t=4.13,P<0.05)和蛋白水平(0.42±0.17,t=3.53,P<0.05),减少 PI3K(0.34±0.19,t=5.17,P<0.05)和 Akt(0.24±0.17,4.26,P<0.05)的表达,降低 U87 细胞活性(52.00±11.33,t=4.59,P<0.05),减少 EdU 阳性细胞数(23.00±7.28,t=12.12,P<0.05);在体内,Angiopep2/si-WDR4 处理后裸鼠脑组织双荧光素酶活性降低(10.02±2.06,t=6.14,P<0.05),肿瘤相对体积减小(0.38±0.17,t=7.42,P<0.05).结论 Angiopep2/si-WDR4能促进肿瘤细胞对si-WDR4的摄取,抑制胶质母细胞瘤的增殖.Objective To investigate the effect of brain-targeted peptide,Angiopep2 encapsulated with si-WDR4 on glioblastoma and its potential mechanism.Methods Angiopep2/si-RNA was prepared,and its encapsulation efficiency and uptake rate were detected.In vitro,U87 cells were treated with phos-phate buffer saline(PBS),Angiopep2/si-NC,si-WDR4 and Angiopep2/si-WDR4,respectively.The mRNA expression level of WDR4 was detected by RT-qPCR,the protein expression levels of WDR4,phos-phatidylinositol 3 kinase(PI3K),and protein kinase B(Akt)were detected by Western blotting,the via-bility of U87 cells was detected by cell counting kit-8(CCK-8)assay,and the proliferation of U87 cells was detected by 5-Ethynyl-2'-deoxyuridine(EdU)staining.In vivo,a xenograft tumor model was estab-lished by U87-Luc cells.After 10 days,PBS,Angiopep2/si-NC,si-WDR4,and Angiopep2/si-WDR4 were injected through the tail vein,respectively.On the 28th day,the dual-luciferase activity of the tumor was detected by small animal in vivo imaging,and the tumor size was detected by hematoxylin and eosin(HE)staining.The quantitative data were compared using t test.Results Angiopep2 had a good binding ability to si-WDR4,forming a complete complex at an N/P ratio of 3∶1,and could promote the uptake of si-RNA by tumor cells;in vitro,compared with Angiopep2/si-NC,Angiopep2/si-WDR4 treatment could reduce the mRNA(0.21±0.14,t=4.13,P<0.05)and protein levels(0.42±0.17,t=3.53,P<0.05)of WDR4 in U87 cells,reduce the expression of PI3K(0.34±0.19,t=5.17,P<0.05)and Akt(0.24±0.17,4.26,P<0.05),decrease the activity of U87 cells(52.00±11.33,t=4.59,P<0.05),and decline the number of EdU-positive cells(23.00±7.28,t=12.12,P<0.05);in vivo,the dual luciferase activity in the brain tissue of nude mice treated with Angiopep2/si-WDR4 was declined(10.02±2.06,t=6.14,P<0.05).The relative volume of tumors was reduced(0.38±0.17,t=7.42,P<0.05).Conclusion Angiopep2/si-WDR4 can promote the uptake of si-WDR4 by tumor cells and in-hibit the proliferation of glioblastoma.

关 键 词：胶质母细胞瘤 Angiopep2/si-WDR4 细细胞增殖 磷脂酰肌醇3激酶/蛋白激B信号通路 

分 类 号：R739.41[医药卫生—肿瘤]