Downregulation of Long Non-coding RNA Nuclear Paraspeckle Assembly Transcript 1 Inhibits MEG-01 Differentiation and Platelet-Like Particles Activity

Weihua Bian,Jing Jiang,Yeying Sun,Wangping Chen,Xinyue Liang,Chunxiang Zhang,Huiqing Qu,Bingrui Zhao,Xiaoli Jiang,Jinfu Yang

doi:10.3389/fgene.2020.571467

Abstract

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.

Highlights

Platelets are nucleus-free fragments produced by maturemega karyocytes (Escobar et al, 2020), which play vital roles in hemostasis, thrombosis, and inflammation (Sim et al, 2016; van der Poll and Parker, 2020)
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)
The number of thrombin-stimulated platelet-like particle (PLP) that adhered on collagen-coated coverslips was markedly reduced (Figures 1F,G). These results suggested that NEAT1 knockdown decreased the adhesion of MEG-01 cells and PLPs

Summary

Introduction

Platelets are nucleus-free fragments produced by maturemega karyocytes (Escobar et al, 2020), which play vital roles in hemostasis, thrombosis, and inflammation (Sim et al, 2016; van der Poll and Parker, 2020). As they do not have nuclei, platelets cannot be cultured in vitro. MEG-01 and PLP formation have been used as in vitro models for megakaryocyte and platelet related research (Kirschbaum et al, 2015; Montenont et al, 2016; Yang et al, 2016)

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