lncRNA NEAT1通过miR-377-3p/Wnt通路影响ox-LDL诱导的人血管平滑肌细胞增殖、侵袭迁移  

lncRNA NEAT1 affects the proliferation, invasion and migration of ox-LDL-induced human vascular smooth muscle cells through miR-377-3p/Wnt pathway

作　　者：袁咏梅 程晓丹 孙家安 常东歌 何莹莹 刘畅 YUAN Yongmei;CHENG Xiaodan;SUN Jiaan;CHANG Dongge;HE Yingying;LIU Chang(Department of Emergency,Zhengzhou Central Hospital Affiliated to Zhengzhou University,Zhengzhou 450007,China)

机构地区：[1]郑州大学附属郑州中心医院急诊科,郑州450007

出　　处：《上海交通大学学报（医学版）》2022年第11期1534-1541,共8页Journal of Shanghai Jiao tong University:Medical Science

基　　金：2020年度河南省高等学校重点科研项目(20B320024)。

摘　　要：目的·研究长链非编码RNA (long non-coding RNA,lncRNA)核旁斑组装转录本1 (nuclear paraspeckle assembly transcript 1,NEAT1)通过微RNA-377-3p (microRNA-377-3p,miR-377-3p)调控Wnt通路对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导人血管平滑肌细胞(human vascular smooth muscle cell,HVSMC)增殖、侵袭迁移的影响。方法·以100μg/mL的ox-LDL处理体外培养的HVSMC。将HVSMC随机分为对照组、模型组、lncRNA NEAT1siRNA组、miR-377-3p抑制组、转染+抑制组(转染NEAT1 siRNA和miR-377-3p抑制剂)、转染阴性对照组(NEAT1 siRNA阴性对照)。除对照组外,其余各组以100μg/mL的ox-LDL诱导建立动脉粥样硬化细胞模型,分组转染处理后,通过细胞计数试剂盒8 (cell counting kit-8,CCK-8)测定各组细胞活力;通过流式细胞实验测定各组细胞凋亡率;通过Transwell实验评测各组细胞迁移侵袭情况;通过免疫印迹实验评测各组细胞增值细胞标志蛋白[增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)]及上皮-间充质转化(epithelial-mesenchymal transition,EMT)标志蛋白[E-cadherin、vimentin]的表达;通过qRT-PCR实验测定各组细胞NEAT1、miR-377-3p及无翅型MMTV整合位点家族成员5A (wingless-related MMTV integration site 5A, WNT5A) mRNA表达。结果·与对照组相比,模型组NEAT1 mRNA表达升高,miR-377-3p mRNA表达降低,细胞凋亡率降低,细胞活力、迁移侵袭细胞数升高,PCNA、vimentin蛋白表达蛋白升高,E-cadherin表达降低,WNT5A mRNA表达升高(均P=0.000)。干扰NEAT1后,细胞凋亡率升高,细胞活力、迁移侵袭细胞数降低,PCNA、vimentin蛋白表达降低,E-cadherin蛋白表达升高,WNT5A mRNA表达降低(均P=0.000);抑制miR-377-3p后上述各项指标均呈现相反的表达趋势(均P<0.05)。此外,抑制miR-377-3p可逆转NEAT1干扰对细胞增殖、侵袭迁移和凋亡的调控作用(均P=0.000)。结论·下调NEAT1表达,可能通过与miR-377-3p竞争性结合,升高miR-377-3p表达,�Objective·To study the effects of long non-coding RNA(lncRNA) nuclear paraspeckle assembly transcript 1(NEAT1on the proliferation, invasion and migration of oxidized low-density lipoprotein(ox-LDL)-induced human vascular smooth muscle cells(HVSMCs) through regulating micro RNA-377-3p(mi R-377-3p)/Wnt pathway. Methods·HVSMCs cultured in vitro were treated with 100 μg/m L ox-LDL. HVSMCs were randomly divided into control group, model group, NEAT1 siRNA group, miR-377-3p inhibitor group, transfection+inhibitor(NEAT1 siRNA+miR-377-3p inhibitor) group, and transfection-negative control(NEAT1siRNA-negative control) group. Except for the control group, the atherosclerosis cell models were induced with 100 μg/mL ox-LDL in other groups. After grouping and transfection, the cell viability of each group was determined by using cell counting kit-8(CCK-8) experiment;the apoptosis rate of each group was measured by flow cytometry;the migration and invasion of cells in each group were evaluated by the Transwell experiment;the expression of cell proliferation markers such as proliferating cell nuclear antigen(PCNA), and epithelial-mesenchymal transition(EMT) markers, such as E-cadherin and vimentin in each group, was determined by immunoblotting experiment;the expression of NEAT1, miR-377-3p and wingless-related MMTV integration site 5A(WNT5A)mRNA in each group was determined by qRT-PCR experiment. Results·Compared with the control group, the mRNA expression of NEAT1 in the model group increased, the mRNA expression of miR-377-3p decreased, the apoptosis rate decreased, the cell viability, and the number of migrating and invasive cells increased, the protein expression of PCNA and Vimentin increased, the expression of E-cadherin decreased, and WNT5A mRNA expression increased(all P=0.000). After NEAT1 was interfered, the apoptosis rate increased, the cell viability, and the number of migrating and invasive cells decreased, the protein expression of PCNA and Vimentin decreased, the expression of E-cadherin increased, and WNT5

关 键 词：长链非编码RNA 核旁斑组装转录本1 微RNA-377-3p 人血管平滑肌细胞 增殖 侵袭迁移 

分 类 号：R543.5[医药卫生—心血管疾病]