长链非编码RNA DIGIT对血管内皮细胞的作用

Effect of long chain non-coding RNA Divergent to GSC induced by TGF-b family signaling on vascular endothelial cells

目的观察长链非编码RNA DIGIT对血管内皮细胞的作用.方法体外培养人微血管内皮细胞(HMEC-1),通过显微镜采图观察管核比值的变化,实时荧光定量聚合酶链反应(FQ-PCR)和蛋白质印迹法测定血管生成相关因子mRNA和蛋白的表达水平.通过用短发夹RNA(shRNA)靶向的DIGIT转染使HMEC-1中的DIGIT表达沉默,观察DIGIT沉默对细胞活力(锥虫蓝染色法),迁移能力(Trans-Well法),细胞凋亡[膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)凋亡检测法]和管形成能力(显微镜采图法)的影响.结果 HMEC-1培养24h后,管/核的比值随时间增加而增加,血管生成相关因子:内皮细胞生长因子(VEGF),内皮细胞生长因子受体-2(VEGFR2),CD144和内皮型一氧化氮合酶(eNOS)的mRNA水平均显著上调(F=11.813、10.336、13.201、10.125,P<0.01)、蛋白水平随着时间的推移而累积.与对照组比较,DIGIT沉默组(sh-DIGIT)细胞的存活率显著降低[(80.29±4.68)%比(62.50±4.78)%,F=20.386,P<0.01],细胞的迁移能力显著降低[(99.65±3.07)%比(37.53±2.78)%,F=33.573,P <0.01],细胞的管/核比值显著降低(0.691±0.060比0.192±0.020,F=13.471,P<0.01),血管生成相关因子VEGF、VEGFR2、CD144和eNOS的蛋白表达水平下降,凋亡抑制基因B细胞淋巴瘤/白血病-2(bcl-2)蛋白表达下降,凋亡促进蛋白bcl-2相关X蛋白(bax)表达上调,活化半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3和Caspase-9表达水平升高.结论 lncRNA DIGIT在内皮细胞中起促生长,促迁移和促管形成因子的作用.

Objective To observe the effect of lncRNA DIGIT on vascular endothelial cell tube formation. Methods Human microvascular endothelial cells (HMEC-1) were cultured in vitro. The changes of tubes/nucleus ratio were observed by microscope. The expression of mRNA and protein was detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting. The DIGIT expression in human microvascular endothelial HMEC-1 cells was silenced by DIGIT transfection with shRNAs targeting. The effects of DIGIT silencing on cell vitality (Typan Blue Staining Cell Viability Assay Kit), migration ability (Trans-Well method), apoptosis [Annexin V-fluoresceine isothiocyanate (FITC) apoptosis detection method] and tube formation ability (microscope mapping method) were observed. The effects of DIGIT silencing on cell viability, migration, apoptosis and tube formation were then assessed. Results HMEC-1 cells were cultured for 24 h. The ratio of tubes/nucleus was increased with the time prolonged. The mRNA levels of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), CD144, and endothelial nitric oxide synthase (eNOS) were all significantly up-regulated (F=11.813, 10.336, 13.201, 10.125, P<0.01), and all these four angiogenesis-associated proteins accumulated with the prolonged time. Cell viability (80.29±4.68)% vs.(62.50±4.78)%, migration capacity (99.65±3.07)% vs.(37.53±2.78)%, and tubes/nucleus ratio (0.691±0.060) vs.(0.192±0.020) were all significantly reduced in sh-DIGIT group when compared with sh-NC group (F=20.386, 33.573, 13.471, P< 0.01). DIGIT silencing down-regulated the levels of B cell lymphoma/leukemia-2 (bcl-2), VEGF, VEGFR2, CD144 and eNOS, up-regulated bcl-2 associated X protein (bax), and activated cysteinyl aspartate-specific protease (Caspase)-3 and Caspase-9 expression. Conclusion LncRNA DIGIT plays an important role in promoting growth, migration and tubule formation in endothelial cells, and DIGIT may be effective molecular targets for the treatment of atherosclerosis.