Downregulation of Long Non-coding RNA Nuclear Paraspeckle Assembly Transcript 1 Inhibits MEG-01 Differentiation and Platelet-Like Particles Activity.
Downregulation of Long Non-coding RNA Nuclear Paraspeckle Assembly Transcript 1 Inhibits MEG-01 Differentiation and Platelet-Like Particles Activity.
复制标题
长非编码 RNA 核副斑组装转录本 1 的下调抑制 MEG-01 分化和血小板样颗粒活性
DOI：
10.3389/fgene.2020.571467
复制
发表时间：
2020
影响因子：
3.7
通讯作者：
Zhang C
中科院分区：
文献类型：
--
作者：
Bian W;Chen W;Jiang X;Qu H;Jiang J;Yang J;Liang X;Zhao B;Sun Y;Zhang C
血小板源自巨核细胞，在血液凝固中发挥重要作用。通过高通量测序，我们发现血小板中富含长非编码RNA（lncRNA）核副斑组装转录物1（NEAT1）（GEO ID：200097348）。然而，对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。 MEG-01 细胞中的 NEAT1 通过 siRNA 转染被敲除。在荧光显微镜下观察 MEG-01 和 PLP 与胶原涂层盖玻片的粘附情况。采用流式细胞术检测细胞凋亡、细胞周期、MEG-01细胞上D41/CD42b和PLP上CD62P的水平。采用实时定量聚合酶链式反应检测NEAT1和IL-8的表达水平。 Western blot用于检测Bcl-2、Bax、cleaved caspase-3和IL-8的蛋白水平。 RNA 结合蛋白免疫沉淀用于检测 NEAT1 和富含脯氨酸/谷氨酰胺剪接因子 (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 敲低可抑制 PLP 上 CD62P 和 MEG-01 细胞上 CD41/CD42b 的表达。此外，NEAT1 敲低可抑制细胞凋亡，增加 Bcl2/Bax 比值，减少 caspase-3 裂解，并降低处于 G0/G1 期的细胞百分比。同时，NEAT1敲低抑制了IL-8的表达。确定了 NEAT1 和 SFPQ（IL-8 的转录抑制因子）之间的强相互作用。 NEAT1 敲低降低了 SFPQ 和 NEAT1 之间的相互作用。结果表明，lncRNA NEAT1 敲低降低了 MEG-01 分化、PLP 活性和 IL-8 水平。结果还表明，NEAT1 对 IL-8 的调节可能是通过 NEAT1 和 SFPQ 之间的直接相互作用来实现的。
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.