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Downregulation of Long Non-coding RNA Nuclear Paraspeckle Assembly Transcript 1 Inhibits MEG-01 Differentiation and Platelet-Like Particles Activity
Frontiers in Genetics ( IF 2.8 ) Pub Date : 2020-09-22 , DOI: 10.3389/fgene.2020.571467
Weihua Bian , Wangping Chen , Xiaoli Jiang , Huiqing Qu , Jing Jiang , Jinfu Yang , Xinyue Liang , Bingrui Zhao , Yeying Sun , Chunxiang Zhang

Platelets are derived from megakaryocytes and play an important role in blood coagulation. By using high throughput sequencing, we have found that the long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) is abundant in platelets (GEO ID: 200097348). However, little is known about its role in regulating megakaryocyte differentiation and platelet activity. This study aims to clarify the effect of NEAT1 on MEG-01 differentiation and platelet-like particle (PLP) activity. NEAT1 in MEG-01 cells was knocked down by siRNA transfection. The adhesion of MEG-01 and PLP to collagen-coated coverslips was observed under a fluorescence microscope. Flow cytometry was used to investigate cell apoptosis, cell cycle, the levels of D41/CD42b on MEG-01 cells and CD62P on PLPs. Quantitative real-time polymerase chain reaction was used to detect NEAT1 and IL-8 expression levels. Western blot was used to measure the protein levels of Bcl-2, Bax, cleaved caspase-3, and IL-8. RNA-binding protein immunoprecipitation was used to detect the interaction of NEAT1 and splicing factor proline/glutamine-rich (SFPQ). Results showed that NEAT1 knockdown decreased the adhesion ability of thrombin-stimulated MEG-01 and PLP. The expression of CD62P on PLPs and CD41/CD42b on MEG-01 cells was inhibited by NEAT1 knockdown. In addition, NEAT1 knockdown inhibited cell apoptosis with increased Bcl2/Bax ratio and decreased cleaved caspase-3, and reduced the percentage of cells in the G0/G1 phase. Meanwhile, NEAT1 knockdown inhibited the expression of IL-8. A strong interaction of NEAT1 and SFPQ, a transcriptional repressor of IL-8, was identified. NEAT1 knockdown reduced the interaction between SFPQ and NEAT1.The results suggest that lncRNA NEAT1 knockdown decreases MEG-01 differentiation, PLP activity, and IL-8 level. The results also indicate that the regulation of NEAT1 on IL-8 may be realized via a direct interaction between NEAT1 and SFPQ.



中文翻译:

下调长的非编码RNA核副斑点大会script本1抑制MEG-01分化和血小板样颗粒活性。

血小板衍生自巨核细胞,在凝血中起重要作用。通过使用高通量测序,我们发现血小板中含有丰富的长非编码RNA(lncRNA)核副斑点装配转录本1(NEAT1)。然而,关于其在调节巨核细胞分化和血小板活性中的作用知之甚少。本研究旨在阐明NEAT1对MEG-01分化和血小板样颗粒(PLP)活性的影响。通过siRNA转染敲除MEG-01细胞中的NEAT1。在荧光显微镜下观察到MEG-01和PLP对胶原包被的盖玻片的粘附。流式细胞仪用于研究细胞凋亡,细胞周期,MEG-01细胞上D41 / CD42b的水平以及PLPs CD62P的水平。实时定量聚合酶链反应用于检测NEAT1和IL-8表达水平。Western印迹用于测量Bcl-2,Bax,裂解的caspase-3和IL-8的蛋白质水平。RNA结合蛋白免疫沉淀用于检测NEAT1和剪接因子脯氨酸/富含谷氨酰胺(SFPQ)的相互作用。结果表明,NEAT1敲低降低了凝血酶刺激的MEG-01和PLP的粘附能力。NEAT1抑制可抑制PLPs上CD62P的表达和MEG-01细胞上CD41 / CD42b的表达。此外,NEAT1敲低抑制细胞凋亡,增加Bcl2 / Bax比,减少caspase-3的裂解,并减少G0 / G1期细胞的百分比。同时,NEAT1敲低抑制IL-8的表达。NEAT1和SFPQ(IL-8的转录阻遏物)之间有很强的相互作用,被确定。NEAT1基因敲低减少了SFPQ和NEAT1之间的相互作用。结果表明,lncRNA NEAT1基因敲低降低了MEG-01分化,PLP活性和IL-8水平。结果还表明,可以实现NEAT1对IL-8的调控通过 NEAT1和SFPQ之间的直接交互。

更新日期:2020-10-17
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