普萘洛尔通过长链非编码RNA核旁丛组装转录本1靶向微小RNA-194-5p调控血管瘤内皮细胞增殖  

Propranolol regulates the proliferation of hemangioma endothelial cells by targeting microRNA-194-5p with long non-coding RNA nuclear paraspeckle assembly transcript 1

作　　者：王慧明 徐伟立[1] 胡志刚[2] 安雯婷[3] 李萌[1] 马亚贞[1] 孙驰[1] Wang Huiming;Xu Weili;Hu Zhigang;An Wenting;Li Meng;Ma Yazhen;Sun Chi(Department of Pediatric Surgery,the Second Hospital of Hebei Medical University,Shijiazhuang 050004,China;Department of General Surgery,the Second Hospital of Hebei Medical University,Shijiazhuang 050004,China;Department of Central Laboratory,the Second Hospital of Hebei Medical University,Shijiazhuang 050004,China)

机构地区：[1]河北医科大学第二医院小儿外科,石家庄050004 [2]河北医科大学第二医院普外科,石家庄050004 [3]河北医科大学第二医院中心实验室,石家庄050004

出　　处：《中华实验外科杂志》2024年第8期1716-1720,共5页Chinese Journal of Experimental Surgery

基　　金：河北省自然科学基金(H2020206258)。

摘　　要：目的探讨普萘洛尔通过长链非编码RNA核旁丛组装转录本1(lncRNA NEAT1)靶向微小RNA(miR)-194-5p调控血管瘤内皮细胞(HemECs)增殖的作用。方法收集2021年3月至2022年8月在河北医科大学第二医院小儿外科手术切除的血管瘤标本20例,根据Mulliken标准分为增生期组和消退期组。采用实时定量反转录聚合酶链反应(RT-qPCR)方法检测两组瘤体中NEAT1和miR-194-5p水平。制备HemECs,细胞计数试剂盒(CCK-8)试验检测敲低和过表达NEAT1对HemECs增殖影响。双荧光素酶酶报告基因实验及RT-qPCR验证miR-194-5p与NEAT1之间的相互作用。不同浓度普萘洛尔给药后,CCK-8试验检测细胞活性,5-乙炔基-2’脱氧尿嘧啶核苷(EdU)法检测细胞增殖,RT-qPCR方法检测NEAT1和miR-194-5p水平。两组间比较采用独立样本t检验进行分析,多组间比较采用单因素方差分析和重复测量方差分析,采用Spearman分析确定各指标间相关性。结果RT-qPCR结果显示增生期血管瘤NEAT1表达高于退化期血管瘤(17.43±5.38比8.82±3.70,t=4.176,P<0.01);而增生期血管瘤miR-194-5p表达低于退化期血管瘤(0.28±0.11比0.41±0.11,t=2.692,P<0.01)。CKK-8检测结果显示,si-NEAT1组细胞活性低于si-NC组(0.86±0.05比1.11±0.03,t=7.794,P<0.01);OE-NEAT1组细胞活性高于OE-NC组(1.33±0.01比1.11±0.05,t=8.072,P<0.05)。Si-NEAT1组miR-194-5p表达水平高于si-NC组(2.88±0.33比1.14±0.47,t=5.206,P<0.01);OE-NEAT1组miR-194-5p表达水平低于OE-NC组(0.43±0.06比1.24±0.11,t=10.830,P<0.01)。双荧光素酶报告基因实验结果显示miR-194-5p和NEAT1-WT共转染组荧光素酶活性高于miR-NC和NEAT1-WT共转染组(0.35±0.05比0.65±0.04,t=5.273,P<0.01)。OE-NEAT1+普萘洛尔组细胞EdU染色阳性细胞率显著低于OE-NEAT1组和OE-NC组(35.97±1.09比69.66±2.85、51.89±1.09,F=65.100,P<0.01),OE-NEAT1+普萘洛尔组NEAT1、miR-194-5p表达量均与OE-NEAT1组比较,差异有统计学意义(1.63±0.30比3.94±0.50,t=6.851,P<0.01;0.86±0.26比0ObjectiveTo investigate the role of propranolol on the proliferation of hemangioma-derived endothelial cells(HemECs)by targeting microRNA(miR)-194-5p expression with long non-coding RNA nuclear paraspeckle assembly transcript 1(lncRNA NEAT1).MethodsTotally,20 cases of hemangioma specimens were surgically removed and collected in the Department of Pediatric Surgery of the Second Hospital of Hebei Medical University from March 2021 to August 2022.The specimens were collected and divided into proliferative phase and involuting phase according to Mulliken criteria.The levels of NEAT1 and miR-194-5p in tumors were detected by real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR).HemECs were prepared and the effects of knockdown and overexpression of NEAT1 on the proliferation of HemECs were determined by cell counting kit-8(CCK-8)assay.The interaction between miR-194-5p and NEAT1 was verified by dual-luciferase assay.After different concentrations of propranolol were administered,the cell activity was detected by CCK-8 assay,cell proliferation was detected by 5-Ethynyl-2′-deoxyuridine(EdU)assay,and NEAT1 and miR-194-5p were detected by RT-qPCR.The independent samples t-test was used for comparison between the two groups.The one-way ANOVA and repeated-measures ANOVA were used for comparisons between multiple groups,and Spearman’s analysis was used to determine the correlation between the indicators.ResultsRT-qPCR showed that the expression of NEAT1 in proliferative hemangiomas was higher than that in involuting hemangiomas(17.43±5.38 vs.8.82±3.70,t=4.176,P<0.01).Contrarily,the expression of miR-194-5p was decreased(0.28±0.11 vs.0.41±0.11,t=2.692,P<0.01).CKK-8 assay showed that the cell activity of si-NEAT1 group was lower than that of the si-NC group(0.86±0.05 vs.1.11±0.03,t=7.794,P<0.01);whereas the cell activity of OE-NEAT1 group was higher than that of the OE-NC group(1.33±0.01 vs.1.11±0.05,t=8.072,P<0.05).The expression level of miR-194-5p in si-NEAT1 group was higher than that

关 键 词：普萘洛尔 长链非编码RNA 微小RNA 血管瘤内皮细胞 增殖 

分 类 号：R732.2[医药卫生—肿瘤]