摘要
目的:研究hsa-miR-146b-3p对内皮细胞的影响及其在动脉粥样硬化中的潜在作用和机制。方法:通过慢病毒构建EA.hy926内皮细胞干预模型,将细胞分为4组,(1)过表达组:OE_hsa-miR-146b-3p组和OE_control组;(2)沉默组:SH_hsa-miR-146b-3p组和SH_control组。采用CCK-8实验检测细胞的增殖能力,细胞划痕及Transwell实验检测迁移能力,细胞周期和凋亡检测分别探讨细胞的分布特征和凋亡率;通过转录组测序(RNA-seq测序)和双荧光素酶实验验证hsa-miR-146b-3p的下游靶点;通过实时荧光定量PCR(RT-qPCR)和蛋白质免疫印迹实验(western blotting)检测靶基因的表达量。结果:与对照组比较,过表达hsa-miR-146b-3p显著促进EA.hy926细胞增殖(P<0.01)、迁移(P<0.0001)及S期细胞比例(P<0.01),并升高晚期凋亡率和总凋亡率(P<0.05);沉默组则呈现增殖抑制(P<0.05)、迁移能力减弱(P<0.0001)并降低S期比例(P<0.05),同时减少早期凋亡率(P<0.05)。RNA-seq测序联合双荧光素酶实验证实hsa-miR-146b-3p调控PI3K/Akt通路上的CREB3L3(P<0.001)。RT-qPCR和western blotting结果显示,hsa-miR-146b-3p过表达导致CREB3L3表达水平下降。结论:Hsa-miR-146b-3p可能通过下调CREB3L3的表达水平及其所在的PI3K/Akt通路影响内皮细胞的功能并促进动脉粥样硬化的发生发展。
Objective:To explore the effects of hsa-miR-146b-3p on endothelial cells and the possible roles and mechanisms of hsa-miR-146b-3p in the development of atherosclerosis.Methods:Lentivirus was used to construct an endothelial cell intervention model in EA.hy926 cells.The cells were categorized into two distinct groups:overexpression groups,including the OE_hsa-miR-146b-3p group and the OE_control group;and silencing groups,consisting of the SH_hsa-miR-146b-3p group and the SH_control group.The cell counting kit-8(CCK-8)assay was used to detect the proliferation ability of cells,while scratching and Transwell assays were employed to assess the migration ability.Cell cycle and apoptosis assays were respectively conducted to explore the distribution characteristics and apoptosis rate of cells.The downstream targets of hsa-miR-146b-3p were verified by RNA sequencing(RNA-seq)and dual-luciferase assay.Finally,the expression levels of target genes were detected by reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and western blotting.Results:Compared with the control group,overexpression of hsa-miR-146b-3p significantly promoted the proliferation(P<0.01),migration(P<0.0001)of EA.hy926 cells and the cell proportion in the S phase(P<0.01),and increased the late apoptosis rate and total apoptosis rate(P<0.05).The silencing groups showed inhibited proliferation(P<0.05),weakened migration(P<0.0001),a decreased proportion of cells in the S phase(P<0.05),and reduced early-stage apoptosis(P<0.05).RNA-seq combined with dual-luciferase assay confirmed that hsa-miR-146b-3p regulated CREB3L3 via the PI3K/Akt pathway(P<0.001).RT-qPCR and western blotting results showed that overexpression of hsa-miR-146b-3p led to a decrease in CREB3L3 expression level.Conclusion:Hsa-miR-146b-3p may influence the function of endothelial cells and promote the development of atherosclerosis by downregulating the expression level of the CREB3L3 gene and the PI3K/Akt pathway.
作者
周世繁
张书铭
陈宁园
黄玲
宋重阳
潘尚领
彭均华
ZHOU Shifan;ZHANG Shuming;CHEN Ningyuan;HUANG Ling;SONG Chongyang;PAN Shangling;PENG Junhua(School of Basic Medical Sciences,Guangxi Medical University,Nanning 530021,China)
出处
《广西医科大学学报》
2025年第3期342-350,共9页
Journal of Guangxi Medical University
基金
国家自然科学基金地区项目(No.32060188)。
