LncRNA PVT1 promotes neuroinflammation after intracerebral hemorrhage by regulating the miR-128-3p/TXNIP axis

Intracerebral hemorrhage (ICH) has significant morbidity and mortality. TXNIP and the competing endogenous RNA (ceRNA) regulatory mechanism involved in long non-coding RNA (lncRNA) play roles in ICH. We probed the upstream microRNAs (miRNAs)/lncRNAs that regulated TXNIP expression in the ceRNA mechanism. ICH mouse model was established, and ICH secondary injury was simulated in BV2 microglia by hemin treatment. TXNIP was silenced 48 h before ICH modeling. The ICH mouse brain water content (BWC) and brain lesion volume after ICH were recorded. Neuronal apoptosis and neurological deficits were evaluated by double staining of NeuN and TUNEL/modified Garcia/corner turn/forelimb placement tests. Iba1 + microglia number and tumor necrosis factor-α (TNF-α)/interleukin-1β (IL-1β)/IL-10/TXNIP/PVT1/miR-128-3p levels were assessed by immunohistochemistry, Western blot, ELISA, and RT-qPCR. Cell viability/death of BV2 cells conditioned medium-treated neuron HT22 cells were assessed by CCK-8/LDH assays. miRNA that had a targeted binding relationship with TXNIP was screened. The targeted bindings of miR-128-3p to TXNIP/PVT1 to miR-128-3p were verified by dual-luciferase reporter gene assay. TXNIP knockdown reduced post-ICH microglial activation/release of pro-inflammatory factors/brain edema/brain lesion volume/neurological deficits in mice and increased releases of anti-inflammatory factors. TXNIP/PVT1 knockdown inhibited hemin-induced inflammatory responses in BV2 cells and protected <i>in vitro</i> co-cultured HT22 cells. PVT1 was a sponge of miR-128-3p to repress TXNIP expression. miR-128-3p knockdown diminished PVT1 knockdown-inhibited hemin-induced BV2 cell inflammatory responses/neurotoxicity. PVT1 silencing reduced hemin-induced neuroinflammation and had a protective effect on neurons by increasing the targeted inhibition of TXNIP by miR-128-3p.

脑出血（Intracerebral hemorrhage, ICH）具有显著的发病率与死亡率。硫氧还蛋白相互作用蛋白（TXNIP）以及长链非编码RNA（long non-coding RNA, lncRNA）参与的竞争内源RNA（competing endogenous RNA, ceRNA）调控机制在ICH进程中发挥关键作用。本研究针对ceRNA调控网络中调控TXNIP表达的上游微RNA（microRNAs, miRNAs）及lncRNA展开系统性探究。研究团队构建了ICH小鼠模型，并采用血红素处理BV2小胶质细胞以模拟ICH继发性损伤。在ICH建模前48小时，对TXNIP进行基因沉默干预。记录ICH小鼠的脑含水量（brain water content, BWC）与脑损伤体积。通过NeuN与TUNEL双重染色、改良Garcia神经功能评分、转角实验及前肢放置实验，评估神经元凋亡情况与神经功能缺损程度。通过免疫组织化学、蛋白质印迹（Western blot）、酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）与实时定量聚合酶链反应（real-time quantitative polymerase chain reaction, RT-qPCR），检测Iba1阳性小胶质细胞数量，以及肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）、白细胞介素-1β（interleukin-1β, IL-1β）、IL-10、TXNIP、浆细胞瘤变体转录本1（PVT1）、miR-128-3p的表达水平。采用BV2细胞条件培养基处理HT22神经元细胞后，通过CCK-8实验与乳酸脱氢酶（lactate dehydrogenase, LDH）释放实验，评估细胞活力与细胞死亡水平。筛选出与TXNIP存在靶向结合关系的miRNA。通过双荧光素酶报告基因实验，验证miR-128-3p与TXNIP、PVT1与miR-128-3p之间的靶向结合关系。实验结果表明：敲低TXNIP可减轻ICH小鼠的小胶质细胞活化、促炎因子释放、脑水肿、脑损伤体积与神经功能缺损，并上调抗炎因子的分泌；敲低TXNIP或PVT1可抑制血红素诱导的BV2细胞炎症反应，并对体外（in vitro）共培养的HT22细胞发挥保护作用；PVT1可作为miR-128-3p的分子海绵，通过竞争性结合miR-128-3p解除其对TXNIP的表达抑制；敲低miR-128-3p可逆转PVT1敲低对血红素诱导的BV2细胞炎症反应与神经毒性的抑制效应；敲低PVT1可减轻血红素诱导的神经炎症，并通过增强miR-128-3p对TXNIP的靶向抑制作用，发挥神经元保护功能。