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

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. Key words: Divergent to GSC; Atherosclerosis; Vascular endothelial cell; Long non-coding RNA