初步探索miR-27b-3p在普萘洛尔治疗婴幼儿血管瘤过程中的作用

Preliminary exploration of miR-27b-3p in the treatment of infantile hemangioma with propranolol

目的研究miR-27b-3p在普萘洛尔治疗婴幼儿血管瘤(infantile hemangioma,IH)的作用机制。方法用0、15、30、45、60、90μM普萘洛尔处理人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),检测最佳浓度。采用实时PCR检测miR-27b-3p及凋亡因子mRNA表达,通过Western blot法检测凋亡相关蛋白表达,利用双荧光素酶报告检测miR-27b-3p与Apaf-1的靶向关系。结果30μM普萘洛尔的最佳浓度作用后,miR-27b-3p表达下调,而Apaf-1、PARP、Caspase-9和Caspase-3表达上调,与敲除miR-27b-3p后结果相一致。上调miR-27b-3p后,细胞凋亡水平和凋亡因子表达被抑制。targetscan.org基因数据库分析发现miR-27b-3p与Apaf-1mRNA的3’UTR匹配,荧光素酶实验结果显示miR-27b-3p与Apaf-1具有靶向关系。结论miR-27b-3p靶向抑制Apaf-1降低细胞凋亡水平,干扰普萘洛尔疗效。

Objective To explore the role and mechanism of miR-27b-3p in the treatment of infantile hemangioma(IH)with propranolol,and to clarify the cause of poor efficacy of propranolol in children with IH.Methods Human umbilical vein endothelial cells(HUVECs)were used as the research model,and HUVECs were treated with 0,15,30,45,60 or 90μM propranolol to explore the best concentration.RNA interference technology was using to regulate the exPression of miR-27b-3p.The levels of cell proliferation and apoptosis were detected by CCK-8,TUNEL and flow cytometry.Real-time PCR was used to detect the expression of miR-27b-3p and apoptosis pathway-related mRNA,and Western blot was used to detect the expression of apoptosis pathway-related protein.Detection of the targeting relationship between miR-27b-3p and Apaf-1 by double Luciferase report.Results 30μM propranolol has the most significant inhibitory effect on cell activity.After Propranolol treatment,the expression of miR-27b-3p was down-regulated,and the expression of apoptotic factors Apaf-1,PARP,Caspase-9 and Caspase-3 was up-regulated,which was consistent with the increase of apoptosis level and apoptotic factor expression after miR-27b-3p knockout.However,after up-regulation of miR-27b-3p,the level of apoptosis decreased and the expression of apoptotic factors was inhibited.targetscan.org gene database analysis found that miR-27b-3p matched the 3'UTR of Apaf-1mRNA,and luciferase results showed that miR-27b-3p had a targeted relationship with Apaf-1.Conclusion miR-27b-3p target inhibits the expression of Apaf-1,reduces the level of endothelial cell apoptosis,and then interferes with the therapeutic effect of propranolol.