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血管内皮细胞覆盖于血管内膜的表面,是血管壁与血液之间的天然屏障,对维持人体生命活动的运行具有重要作用。研究[1-3]显示,血管内皮细胞的激活、增殖、迁移、黏附和凋亡与冠心病、动脉粥样硬化、高血压、脓毒症等诸多心血管疾病的发生和发展密切相关。微小RNA(miRNA)是一类保守的内源性非编码RNA分子,近年研究表明,miRNA表达异常可调控内皮细胞增殖、迁移、黏附等功能,参与血管发育、新血管形成、血管炎症等的调节,与多种心血管疾病病理生理进程有关[4]。有研究[5]指出,在模拟微重力条件下,miR-503-5p可以通过抑制B细胞淋巴瘤2(Bcl-2)表达,在一定程度上诱导人肺微血管内皮细胞凋亡,提示miR-503-5p可能参与调节内皮细胞功能。血管内皮细胞在受到血液中各种危险因子刺激后产生过度的炎症反应导致细胞凋亡。因此,本研究以促炎因子白细胞介素1β(IL-1β)诱导血管内皮细胞,探讨miR-503-5p对IL-1β刺激下血管内皮细胞增殖、迁移、凋亡和黏附的影响和作用机制,以期为临床基于血管内皮细胞预防心血管疾病提供参考。
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与NC组比较,IL-1β组HUVECs在24~72 h细胞活力显著降低,细胞中miR-503-5p表达显著升高;与IL-1β+anti-miR-con组比较,IL-1β+anti-miR-503-5p组HUVECs在24~72 h细胞活力显著升高,细胞中miR-503-5p表达显著降低(P < 0.01)。在同一处理组,培养24、48、72 h细胞活力逐渐升高(P < 0.01)(见表 1)。
分组 miR-503-5p表达 细胞活力(OD490) F P MS组内 24 h 48 h 72 h NC 0.98±0.08 0.33±0.03 0.82±0.05△ 1.53±0.09△□ 854.69 < 0.01 0.004 IL-1β 5.74±0.62* 0.25±0.02* 0.58±0.03*△ 0.85±0.06*△□ 497.57 < 0.01 0.002 IL-1β+anti-miR-con 5.21±0.73* 0.23±0.03* 0.55±0.03*△ 0.87±0.07*△□ 412.66 < 0.01 0.002 IL-1β+anti-miR-503-5p 0.87±0.07*#▲ 0.27±0.03*#▲ 0.69±0.05*#▲△ 1.19±0.10*#▲△□ 427.43 < 0.01 0.005 F 269.42 21.68 79.41 138.94 — — — P < 0.01 < 0.01 < 0.01 < 0.01 — — — MS组内 0.232 0.001 0.002 0.007 — — — q检验:与NC组比较*P < 0.05;与IL-1β组比较#P < 0.05;与IL-1β+anti-miR-con组比较▲P < 0.05;与24 h比较△P < 0.05;与48 h比较□P < 0.05 表 1 转染anti-miR-503-5p对IL-1β诱导内皮细胞增殖的影响(x±s;ni=9)
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与NC组比较,IL-1β组HUVECs在24~72 h时细胞黏附数显著增加(P < 0.01);与IL-1β+anti-miR-con组比较,IL-1β+anti-miR-503-5p组HUVECs在24~72 h时细胞黏附数显著减少(P < 0.01)。同一处理组,培养24、48、72 h内皮细胞黏附数量逐渐增加(P < 0.01)(见表 2)。
分组 24 h 48 h 72 h F P MS组内 NC 31.85±3.68 62.13±3.84△ 93.55±7.63△□ 297.09 < 0.01 28.835 IL-1β 45.72±2.41* 88.26±4.37*△ 145.27±8.74*△□ 665.05 < 0.01 33.764 IL-1β+anti-miR-con 43.23±3.32* 85.44±4.31*△ 157.35±7.62*△□ 1025.43 < 0.01 29.221 IL-1β+anti-miR-503-5p 35.16±3.78*# 69.76±5.23*#△ 119.63±9.16*#△□ 387.80 < 0.01 41.849 F 34.64 70.72 104.69 — — — P < 0.01 < 0.01 < 0.01 — — — MS组内 11.165 19.943 69.144 — — — q检验:与NC组比较*P < 0.05;与IL-1β组比较#P < 0.05;与IL-1β+anti-miR-con组比较▲P < 0.05;与24 h比较△P < 0.05;与48 h比较□P < 0.05 表 2 转染anti-miR-503-5p对IL-1β诱导内皮细胞黏附的影响(x±s;ni=9)
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与NC组比较,IL-1β组HUVECs凋亡率、Cleaved caspase-3蛋白表达增加(P < 0.01);与IL-1β+anti-miR-con组比较,IL-1β+anti-miR-503-5p组HUVECs凋亡率、Cleaved caspase-3蛋白表达显著降低(P < 0.01)(见图 1~2、表 3)。
分组 caspase-3 cleaved caspase-3 细胞凋亡率/% NC 0.41±0.07 0.27±0.04 4.09±0.43 IL-1β 0.37±0.04 0.58±0.07* 15.58±1.76* IL-1β+anti-miR-con 0.38±0.06 0.55±0.06* 14.47±1.65* IL-1β+anti-miR-503-5p 0.42±0.05 0.39±0.05*#▲ 8.43±0.92*#▲ F 1.62 59.88 152.13 P >0.05 < 0.01 < 0.01 MS组内 0.003 0.003 1.713 q检验:与NC组比较*P < 0.05;与IL-1β组比较#P < 0.05;与IL-1β+anti-miR-con组比较▲P < 0.05 表 3 转染anti-miR-503-5p对IL-1β诱导内皮细胞凋亡、caspase-3和Cleaved caspase-3蛋白表达的影响(x±s; ni=9)
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与NC组比较,IL-1β组HUVECs迁移细胞数显著降低(P < 0.01);与IL-1β+anti-miR-con组比较,IL-1β+anti-miR-503-5p组HUVECs迁移细胞数显著升高(P < 0.01)(见表 4)。
分组 细胞迁移数 NC 143.76±11.27 IL-1β 75.27±8.64* IL-1β+anti-miR-con 68.48±7.36*# IL-1β+anti-miR-503-5p 106.65±10.25*#▲ F 118.23 P < 0.01 MS组内 90.224 q检验:与NC组比较*P < 0.05;与IL-1β组比较#P < 0.05;与IL-1β+anti-miR-con组比较▲P < 0.05 表 4 转染anti-miR-503-5p对IL-1β诱导内皮细胞迁移的影响(x±s;ni=9)
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与NC组比较,IL-1β组HUVECs中ICAM-1和VCAM-1蛋白表达显著升高(P < 0.01);与IL-1β+anti-miR-con组比较,IL-1β+anti-miR-503-5p组HUVECs中ICAM-1和VCAM-1蛋白表达显著降低(P < 0.01)(见图 3、表 5)。
分组 ICAM-1 VCAM-1 NC 0.35±0.04 0.42±0.07 IL-1β 0.67±0.05* 0.57±0.04* IL-1β+anti-miR-con 0.63±0.06 0.52±0.06 IL-1β+anti-miR-503-5p 0.42±0.04# 0.46±0.05# F 94.81 12.45 P < 0.01 < 0.01 MS组内 0.002 0.003 q检验:与NC相比较*P < 0.05;与IL-1β+anti-miR-con组比较#P < 0.05 表 5 转染anti-miR-503-5p对IL-1β诱导内皮细胞ICAM-1、VCAM-1的影响(x±s;ni=9)
miR-503-5p对IL-1β诱导的血管内皮细胞增殖、迁移、凋亡和黏附的影响
Effects of miR-503-5p on the proliferation, migration, apoptosis and adhesion of vascular endothelial cells induced by IL-1β
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摘要:
目的探讨miR-503-5p对白细胞介素1β(IL-1β)诱导的血管内皮细胞增殖、迁移、凋亡和黏附的影响和分子机制。 方法将人脐静脉血管内皮细胞(HUVECs)分为NC组、IL-1β组、IL-1β+anti-miR-con组及IL-1β+anti-miR-503-5p组。分别采用噻唑蓝(MTT)法、黏附实验、流式细胞术、Transwell实验检测HUVECs增殖、黏附、凋亡和迁移能力。Western blotting检测细胞间黏附分子1(ICAM-1)、血管细胞黏附分子1(VCAM-1)表达水平。 结果与NC组比较,IL-1β组HUVECs细胞活力、迁移细胞数显著降低,细胞黏附数、凋亡率、ICAM-1和VCAM-1蛋白表达显著升高(P < 0.01)。与IL-1β+anti-miR-con组比较,IL-1β+anti-miR-503-5p组HUVECs细胞活力、迁移细胞数显著升高,细胞黏附数、凋亡率、ICAM-1和VCAM-1蛋白表达显著降低(P < 0.01)。 结论抑制miR-503-5p表达可促进血管内皮细胞增殖和迁移,抑制细胞黏附和凋亡,其机制可能与抑制ICAM-1和VCAM-1表达有关。 Abstract:ObjectiveTo explore the effects of miR-503-5p on the proliferation, migration, apoptosis and adhesion of human umbilical vein endothelial cells(HUVECs) induced by interleukin1β(IL-1β), and its molecular mechanism. MethodsThe HUVECs were divided into the NC group, IL-1β group, IL-1β+anti-miR-con group and IL-1β+anti-miR-503-5p group.The proliferation, adhesion, apoptosis and migration ability of cells were detected using MTT assay, adhesion assay, flow cytometry and Transwell assay, respectively.The expression levels of intercellular adhesion molecule 1(ICAM-1) and vascular cell adhesion molecule 1(VCAM-1) were detected using Western blotting. ResultsCompared with the NC group, the cell viability and migrated cell numbers of HUVECs significantly reduced, and the cell adhesion numbers, apoptosis rate, and ICAM-1 and VCAM-1 protein expression levels significantly increased in the IL-1β group(P < 0.01).Compared with the IL-1β+anti-miR-con group, the cell viability and migrated cell numbers of HUVECs significantly increased, and the cell adhesion numbers, apoptosis rate, and ICAM-1 and VCAM-1 protein expression levels significantly decreased in the IL-1β+anti-miR-503-5p group(P < 0.01). ConclusionsInhibiting the miR-503-5p expression can promote the vascular endothelial cell proliferation and migration, and inhibit vascular adhesion and apoptosis, which may be related to the inhibition of ICAM-1 and VCAM-1 expression. -
Key words:
- miR-503-5p /
- interleukin 1β /
- vascular endothelial cells /
- proliferation /
- apoptosis /
- migration /
- adhesion
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表 1 转染anti-miR-503-5p对IL-1β诱导内皮细胞增殖的影响(x±s;ni=9)
分组 miR-503-5p表达 细胞活力(OD490) F P MS组内 24 h 48 h 72 h NC 0.98±0.08 0.33±0.03 0.82±0.05△ 1.53±0.09△□ 854.69 < 0.01 0.004 IL-1β 5.74±0.62* 0.25±0.02* 0.58±0.03*△ 0.85±0.06*△□ 497.57 < 0.01 0.002 IL-1β+anti-miR-con 5.21±0.73* 0.23±0.03* 0.55±0.03*△ 0.87±0.07*△□ 412.66 < 0.01 0.002 IL-1β+anti-miR-503-5p 0.87±0.07*#▲ 0.27±0.03*#▲ 0.69±0.05*#▲△ 1.19±0.10*#▲△□ 427.43 < 0.01 0.005 F 269.42 21.68 79.41 138.94 — — — P < 0.01 < 0.01 < 0.01 < 0.01 — — — MS组内 0.232 0.001 0.002 0.007 — — — q检验:与NC组比较*P < 0.05;与IL-1β组比较#P < 0.05;与IL-1β+anti-miR-con组比较▲P < 0.05;与24 h比较△P < 0.05;与48 h比较□P < 0.05 表 2 转染anti-miR-503-5p对IL-1β诱导内皮细胞黏附的影响(x±s;ni=9)
分组 24 h 48 h 72 h F P MS组内 NC 31.85±3.68 62.13±3.84△ 93.55±7.63△□ 297.09 < 0.01 28.835 IL-1β 45.72±2.41* 88.26±4.37*△ 145.27±8.74*△□ 665.05 < 0.01 33.764 IL-1β+anti-miR-con 43.23±3.32* 85.44±4.31*△ 157.35±7.62*△□ 1025.43 < 0.01 29.221 IL-1β+anti-miR-503-5p 35.16±3.78*# 69.76±5.23*#△ 119.63±9.16*#△□ 387.80 < 0.01 41.849 F 34.64 70.72 104.69 — — — P < 0.01 < 0.01 < 0.01 — — — MS组内 11.165 19.943 69.144 — — — q检验:与NC组比较*P < 0.05;与IL-1β组比较#P < 0.05;与IL-1β+anti-miR-con组比较▲P < 0.05;与24 h比较△P < 0.05;与48 h比较□P < 0.05 表 3 转染anti-miR-503-5p对IL-1β诱导内皮细胞凋亡、caspase-3和Cleaved caspase-3蛋白表达的影响(x±s; ni=9)
分组 caspase-3 cleaved caspase-3 细胞凋亡率/% NC 0.41±0.07 0.27±0.04 4.09±0.43 IL-1β 0.37±0.04 0.58±0.07* 15.58±1.76* IL-1β+anti-miR-con 0.38±0.06 0.55±0.06* 14.47±1.65* IL-1β+anti-miR-503-5p 0.42±0.05 0.39±0.05*#▲ 8.43±0.92*#▲ F 1.62 59.88 152.13 P >0.05 < 0.01 < 0.01 MS组内 0.003 0.003 1.713 q检验:与NC组比较*P < 0.05;与IL-1β组比较#P < 0.05;与IL-1β+anti-miR-con组比较▲P < 0.05 表 4 转染anti-miR-503-5p对IL-1β诱导内皮细胞迁移的影响(x±s;ni=9)
分组 细胞迁移数 NC 143.76±11.27 IL-1β 75.27±8.64* IL-1β+anti-miR-con 68.48±7.36*# IL-1β+anti-miR-503-5p 106.65±10.25*#▲ F 118.23 P < 0.01 MS组内 90.224 q检验:与NC组比较*P < 0.05;与IL-1β组比较#P < 0.05;与IL-1β+anti-miR-con组比较▲P < 0.05 表 5 转染anti-miR-503-5p对IL-1β诱导内皮细胞ICAM-1、VCAM-1的影响(x±s;ni=9)
分组 ICAM-1 VCAM-1 NC 0.35±0.04 0.42±0.07 IL-1β 0.67±0.05* 0.57±0.04* IL-1β+anti-miR-con 0.63±0.06 0.52±0.06 IL-1β+anti-miR-503-5p 0.42±0.04# 0.46±0.05# F 94.81 12.45 P < 0.01 < 0.01 MS组内 0.002 0.003 q检验:与NC相比较*P < 0.05;与IL-1β+anti-miR-con组比较#P < 0.05 -
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