miR-125a-5p靶向MEIS2基因对垂体瘤AtT20细胞增殖、凋亡的影响

目的探讨微小RNA-125a-5p(miR-125a-5p)是否可靶向调控MEIS2基因表达并分析其对垂体瘤AtT20细胞增殖及凋亡的影响。方法采用实时荧光定量PCR(qRT-PCR)检测AtT20细胞及小鼠正常垂体细胞(NPC)中miR-125a-5p及MEIS2 mRNA表达量。采用瞬时转染技术分别将miR-125a-5p mimic质粒、anti-miR-125a-5p抑制剂及si-MEIS2质粒转染至AtT20细胞。双荧光素酶报告基因检测miR-125a-5p与MEIS2的靶基因关系,同时共转染miR-125a-5p mimic与pcDNA-MEIS2验证miR-125a-5p是否通过靶向MEIS2表达发挥作用。MTT法检测各组细胞增殖能力;流式细胞术检测各组细胞凋亡率;蛋白免疫印迹(Western blotting)检测各组细胞MEIS2蛋白表达。结果 qRT-PCR检测结果显示,与NPC比较,miR-125a-5p在AtT20细胞中表达水平显著降低,MEIS2 mRNA表达水平显著升高;与miR-NC组比较,miR-125a-5p组miR-125a-5p表达水平显著升高。W...     

MicroRNA-146a-5p attenuates neuropathic pain via suppressing TRAF6 signaling in the spinal cord

Glia-mediated neuroinflammation plays an important role in the pathogenesis of neuropathic pain. Our recent study demonstrated that TNF receptor associated factor-6 (TRAF6) is expressed in spinal astrocytes and contributes to the maintenance of spinal nerve ligation (SNL)-induced neuropathic pain. MicroRNA (miR)-146a is a key regulator of the innate immune response and was shown to target TRAF6 and reduce inflammation. In this study, we found that in cultured astrocytes, TNF-α, IL-1β, or lipopolysaccharide (LPS) induced rapid TRAF6 upregulation and delayed miR-146a-5p upregulation. In addition, miR-146a-5p mimic blocked LPS-induced TRAF6 upregulation, as well as LPS-induced c-Jun N-terminal kinase (JNK) activation and chemokine CCL2 expression in astrocytes. Notably, LPS incubation with astrocytes enhanced the DNA binding activity of AP-1 to the promoters of mir-146a and ccl2. TRAF6 siRNA or JNK inhibitor SP600125 significantly reduced LPS-induced miR-146a-5p increase in astrocytes. In vivo, intrathecal injection of TNF-α or LPS increased spinal TRAF6 expression. Pretreatment with miR-146a-5p mimic alleviated TNF-α- or LPS-induced mechanical allodynia and reduced TRAF6 expression. Finally, SNL induced miR-146a-5p upregulation in the spinal cord at 10 and 21 days. Intrathecal injection of miR-146a-5p mimic attenuated SNL-induced mechanical allodynia and decreased spinal TRAF6 expression. Taken together, the results suggest that (1) miR-146a-5p attenuates neuropathic pain partly through inhibition of TRAF6 and its downstream JNK/CCL2 signaling, (2) miR-146a-5p is increased by the activation of TRAF6/JNK pathway. Hence, miR-146a-5p may be a novel treatment for chronic neuropathic pain.     

miR-30a-5p inhibition promotes interaction of Fas+ endothelial cells and FasL+ microglia to decrease pathological neovascularization and promote physiological angiogenesis

Ischemia-induced angiogenesis contributes to various neuronal and retinal diseases, and often results in neurodegeneration and visual impairment. Current treatments involve the use of anti-VEGF agents but are not successful in all cases. In this study we determined that miR-30a-5p is another important mediator of retinal angiogenesis. Using a rodent model of ischemic retinopathy, we show that inhibiting miR-30a-5p reduces neovascularization and promotes tissue repair, through modulation of microglial and endothelial cell cross-talk. miR-30a-5p inhibition results in increased expression of the death receptor Fas and CCL2, to decrease endothelial cell survival and promote microglial migration and phagocytic function in focal regions of ischemic injury. Our data suggest that miR-30a-5p inhibition accelerates tissue repair by enhancing FasL–Fas crosstalk between microglia and endothelial cells, to promote endothelial cell apoptosis and removal of dead endothelial cells. Finally, we found that miR-30a levels were increased in the vitreous of patients with proliferative diabetic retinopathy. Our study identifies a role for miR-30a in the pathogenesis of neovascular retinal disease by modulating microglial and endothelial cell function, and suggests it may be a therapeutic target to treat ischemia-mediated conditions.     

Clinical and preclinical evaluation of miR-144-5p as a key target for major depressive disorder

Background

Neuronal abnormalities are closely associated with major depressive disorder (MDD). Available evidence suggests a role for microRNAs (miRNAs) in regulating the expression of genes involved in MDD. Hence, miRNAs that can be potential therapeutic targets need to be identified.

Methods

A mouse model of chronic unpredictable stress (CUS) was used to evaluate the function of miRNAs in MDD. miR-144-5p was screened from the hippocampi of CUS mice based on sequencing results. Adenovirus-associated vectors were used to overexpress or knockdown miR-144-5p in mice. BpV(pic) and LY294002 were used to determine the relationship between miR-144-5p target genes PTEN and TLR4 in neuronal impairment caused by miR-144-5p deficiency. Western blotting, immunofluorescence, ELISA immunosorbent assay, and Golgi staining were used to detect neuronal abnormalities. Serum samples from healthy individuals and patients with MDD were used to detect miR-144-5p levels in the serum and serum exosomes using qRT-PCR.

Results

miR-144-5p expression was significantly decreased within the hippocampal dentate gyrus (DG) of CUS mice. Upregulation of miR-144-5p in the DG ameliorated depression-like behavior in CUS mice and attenuated neuronal abnormalities by directly targeting PTEN and TLR4 expression. Furthermore, miR-144-5p knockdown in normal mice led to depression-like behavior via inducing neuronal abnormalities, including abnormal neurogenesis, neuronal apoptosis, altered synaptic plasticity, and neuroinflammation. miR-144-5p deficiency-mediated neuronal impairment was mediated by PI3K/Akt/FoxO1 signaling. Furthermore, miR-144-5p levels were downregulated in the sera of patients with MDD and associated with depressive symptoms. Consistently, serum exosome-derived miR-144-5p levels were decreased in patients with MDD.

Conclusion

miR-144-5p plays a vital role in regulating neuronal abnormalities in depression. Our findings provide translational evidence that miR-144-5p is a new potential therapeutic target for MDD.     

垂体腺瘤组织miR-26b-5p表达与术后复发的关系

目的 探讨微小核糖核酸-26b-5p（miR-26b-5p）与垂体腺瘤术后复发的关系。方法 收集2018年1月~2020年1月手术切除的124例垂体腺瘤组织,采用qRT-PCR检测miR-26b-5p相对表达水平。术后随访2年,利用多因素logistic回归模型分析垂体腺瘤术后复发的危险因素,绘制ROC曲线分析miR-26b-5p评估垂体腺瘤术后复发的效果。结果 124例中,术后复发34例,复发率为27.42%（34/124）。复发垂体腺瘤miR-26b-5p相对表达量（1.25±0.43）明显低于非复发垂体腺瘤[（1.87±0.49）;P<0.001]。多因素logistic回归分析显示,miR-26b-5p低表达是术后复发的独立危险因素（OR=1.232;95% CI 1.058~3.524;P<0.001）。ROC曲线分析显示,miR-26b-5p表达水平评估垂体腺瘤术后复发的曲线下面积为0.841（95% CI 0.767~0.915;P<0.001）,最佳截断值为1.518,敏感度为84.80%,特异度为81.00%。结论 复发垂体腺瘤的miR-26b-5p表达水平明显下降,miR-26b-5p表达变化与术后复发密切相关,对评估垂体腺瘤术后复发有一定的价值。     

miR-27a-3p在高血压性脑出血患者外周血中呈下调表达

目的探讨脑出血患者外周血血清miR-27a-3p的表达情况。方法选取31例脑出血患者(脑出血组)和11例体检健康对照者(对照组),收集临床资料,qRT-PCR法测定外周血血清中miR-27a-3p的表达水平。结果与健康对照组比较,脑出血组血清miR-27a-3p表达呈显著下调(miR-27a-3p相对表达量为0.29),脑出血组血清miR-27a-3p表达水平与血肿体积呈明显线性负相关(相关系数R=--0.502,P=0.004)。结论在脑出血患者外周血血清miR-27a-3p表达水平呈下调趋势,其表达量与血肿体积呈线性负相关关系,可能成为脑出血患者血肿体积的预测指标。     

miR-384-5p ameliorates neuropathic pain by targeting SCN3A in a rat model of chronic constriction injury

ABSTRACT

Objective: To explore the potential regulation mechanisms of miR-384-5p in Neuropathic pain (NP).

Methods: Rat model of chronic constriction injury (CCI) was established to induce NP in vivo. NP levels were assessed using Withdrawal Threshold (PWT) and Paw Withdrawal Latency (PWL). qPCR and Western blotting were used to determine the relative expression of miR-384-5p and SCN3A. The inflammation response in spinal microglia cells was determined by ELISA assay. Immunofluorescence assay was used to demonstrate the co-localization of miR-384-5p with SCN3A in rat dorsal root ganglions (DRGs). The target genes of miR-384-5p were verified by dual-luciferase report assays.

Results: In the current study, the miR-384-5p expression level was significantly downregulated in CCI rats when comparing to the sham group. In addition, miR-384-5p agomir significantly repressed mechanical allodynia and heat hyperalgesia in CCI rats. Meanwhile, the current study indicated miR‐384‐5p could decrease inflammation progress in spinal microglia cells incubated in lipopolysaccharide. Consistently, overexpression of miR-384-5p obviously depressed inflammation cytokine levels in CCI rats. Dual-luciferase reporter assays indicated that SCN3A is a target gene of miR-384-5p.

Conclusion: miR-384-5p is a negative regulator in the development of neuropathic pain by regulating SCN3A, indicating that miR-384-5p might be a promising therapeutic target in the treatment of neuropathic pain.

Abbreviations: CCI: Chronic constriction injury; ZEB1: Zinc finger E box binding protein-1; MAPK6: Mitogen-activated protein kinase 6; COX-2: cyclooxygenase-2.     

黄连碱上调miR-146a-5p后通过PI3K/AKT通路减轻由MPP+诱导的帕金森病细胞模型损伤

目的探讨黄连碱对1-甲基-4-苯基吡啶离子(MPP+)诱导的帕金森病(PD)细胞损伤的影响及其机制。方法用0.3 mmol/L的MPP+处理SK-N-SH细胞作为PD细胞模型,记为MPP+组,以正常培养的细胞作为空白对照组。用浓度分别为10μmol/L、20μmol/L、40μmol/L的黄连碱预处理4h后再用0.3 mmol/L的MPP+处理作为不同浓度黄连碱处理组。将miR-con、miR-146a-5p转染至SK-N-SH细胞后再用0.3 mmol/L的MPP+处理记为MPP++miR-con组、MPP++miR-146a-5p组;将anti-miR-con、anti-miR-146a-5p转染至SK-N-SH细胞后用20μmol/L的黄连碱预处理4h及0.3 mmol/L的MPP+处理记为MPP++Cop+anti-miR-con组、MPP++Cop+anti-miR-146a-5p组。四甲基偶氮唑盐比色法(MTT)检测细胞存活率;Western blotting实验检测活化的半胱氨酸天冬氨酸蛋白酶-3(caspase-3)、细胞周期蛋白D1(Cyclin D1)、磷酸化蛋白激酶B(p-AKT)、磷酸化磷脂酰肌醇3激酶(p-PI3K)蛋白表达水平;流式细胞术检测细胞凋亡;实时荧光定量PCR(RT-qPCR)检测miR-146a-5p表达水平。结果与空白对照组比较,MPP+处理后SK-N-SH细胞存活率显著降低,活化caspase-3表达水平显著升高,细胞凋亡率显著升高,CyclinD1、miR-146a-5p表达水平显著降低,差异均有统计学意义(P<0.05)。黄连碱处理及miR-146a-5p过表达后MPP+诱导的SK-N-SH细胞中细胞存活率显著升高,活化caspase-3表达水平显著降低,细胞凋亡率显著降低,CyclinD1、miR-146a-5p表达水平显著升高,差异均有统计学意义(P<0.05)。低表达miR-146a-5p逆转了黄连碱对SK-N-SH细胞增殖促进和凋亡抑制的作用。黄连碱处理后MPP+诱导的SK-N-SH细胞中p-AKT、p-PI3K表达水平显著升高,低表达miR-146a-5p逆转了黄连碱对p-AKT、p-PI3K表达水平的促进作用。结论黄连碱可促进细胞存活,抑制MPP+诱导的细胞凋亡,其机制可能与miR-146a-5p及PI3K/AKT信号通路有关。     

Up-regulation of Sirt1/miR-149-5p signaling may play a role in resveratrol induced protection against ischemia via p53 in rat brain

Micro-RNA(miRNA) are well studied small noncoding RNA, which plays a diverse role in the regulation of vital elements in cell survival and apoptosis. However, the functional significance of miRNAs after the pathogenesis of ischemic stroke remains unclear. The present study is designed to investigate the regulatory role of miR-149-5p on Sirtuin-1/p53 axis during ischemic-reperfusion-induced injury. Middle cerebral artery occlusion (MCAO) was performed by nylon monofilament for 60 min. Resveratrol was administered via intraperitoneal (IP) route, 30 min before the MCAO. Our study demonstrated that the miR-149-5p levels were markedly decreased at 24 h after ischemic-reperfusion (I/R) injury. Further, we observed decreased p53 protein expression and increased miR-149-5p activity on sirtuin1 (Sirt1) activation with resveratrol after 24 h following MCAO. Moreover, immunohistochemistry studies found that resveratrol treatment significantly decreased the immunoreactivity of p53 and caspase-3 on activation of Sirt1/miR149-5p axis. In conclusion, our findings suggest that miR-149-5p could play a regulatory role in neuronal cell death via Sirt1/p53 axis, which offers a new target for novel therapeutic interventions during acute ischemic stroke.     

MiR‐20b‐5p relieves neuropathic pain by targeting Akt3 in a chronic constriction injury rat model

Neuropathic pain is caused by somatosensory nervous system disorder which happens in patients with different diseases. Akt3 regulates innate immune function and plays a role in neuropathic pain pathogenesis in rats. MiR‐20b‐5p is a microRNA which has been suggested to inhibit Akt3 expression through directly targeting Akt3 mRNA. This research focused on miR‐20b‐5p function in neuropathic pain by Akt3 expression inhibition. Chronic constriction injury (CCI) was employed to induce neuropathic pain in rats. Paw withdrawal thresholds and paw withdrawal latency were examined to show neuropathic pain development. Expression levels of relative genes or microRNA were checked using qRT‐PCR and western blot. Inflammation cytokine levels were measured by enzyme‐linked immunosorbent assay kits. In CCI rat model, miR‐20b‐5p level was declined and Akt3 mRNA level was upregulated. MiR‐20b‐5p mimics suppressed the enhanced neuropathic pain, neuroinflammation, and Akt3 expression. MiR‐20b‐5p directly targeted Akt3 mRNA and downregulated the Akt3 expression in rat primary microglial cells. MiR‐20b‐5p inhibitory function in neuropathic pain was suppressed by the upregulation of Akt3 expression. This research illustrated that miR‐20b‐5p alleviated neuropathic pain through the inhibition of Akt3 expression in CCI rat model.     

Genetic deletion of endothelial microRNA-15a/16-1 promotes cerebral angiogenesis and neurological recovery in ischemic stroke through Src signaling pathway

Cerebral angiogenesis is tightly controlled by specific microRNAs (miRs), including the miR-15a/16-1 cluster. Recently, we reported that endothelium-specific conditional knockout of the miR-15a/16-1 cluster (EC-miR-15a/16-1 cKO) promotes post-stroke angiogenesis and improves long-term neurological recovery by increasing protein levels of VEGFA, FGF2, and their respective receptors VEGFR2 and FGFR1. Herein, we further investigated the underlying signaling mechanism of these pro-angiogenic factors after ischemic stroke using a selective Src family inhibitor AZD0530. EC-miR-15a/16-1 cKO and age- and sex-matched wild-type littermate (WT) mice were subjected to 1 h middle cerebral artery occlusion (MCAO) and 28d reperfusion. AZD0530 was administered daily by oral gavage to both genotypes of mice 3-21d after MCAO. Compared to WT, AZD0530 administration exacerbated spatial cognitive impairments and brain atrophy in EC-miR-15a/16-1 cKO mice following MCAO. AZD0530 also attenuated long-term recovery of blood flow and inhibited the formation of new microvessels, including functional vessels with blood circulation, in the penumbra of stroked cKO mice. Moreover, AZD0530 blocked the Src signaling pathway by downregulating phospho-Src and its downstream mediators (p-Stat3, p-Akt, p-FAK, p-p44/42 MAPK, p-p38 MAPK) in post-ischemic brains. Collectively, our data demonstrated that endothelium-targeted deletion of the miR-15a/16-1 cluster promotes post-stroke angiogenesis and improves long-term neurological recovery via activating Src signaling pathway.     

Plasma Levels of miR-125b-5p and miR-206 in Acute Ischemic Stroke Patients After Recanalization Treatment: A Prospective Observational Study

Introduction: Multiple microRNAs (miRNAs) participate in the response to hypoxic/ischemic and ischemia-reperfusion events. However, the expression of these miRNAs in circulation from patients with acute ischemic stroke (AIS) receiving recanalization treatment has not been examined, and whether they are associated with the severity and outcome of stroke is still unknown. Materials and methods: In this prospective cohort study, plasma levels of miR-125b-5p, miR-15a-3p, miR-15a-5p, and miR-206 were measured at 24 hours after thrombolysis with or without endovascular treatment in 94 patients with AIS, as determined by qRT-PCR. Stroke severity was assessed based on National Institutes of Health Stroke Scale (NIHSS) score and infarct lesion. Intracranial haemorrhage (ICH) was recorded. An unfavorable outcome was defined as a modified Rankin Scale score greater than 2 at day 90 after stroke. Results: miR-125b-5p and miR-206 levels were correlated with NIHSS scores (P = .014 and P = .002) and cerebral infarction volumes (P = .025 and P = .030). miR-125b-5p levels were significantly higher in patients with an unfavorable outcome than in patients with a favorable outcome (P = .002) and showed good diagnostic accuracy in discriminating the presence of an unfavorable outcome (area under the curve .735, 95% confidence interval .623-.829, P < .001). No association was found between different miRNAs and ICH. Conclusions: In AIS patients after thrombolysis with or without endovascular treatment, miR-125b-5p is a novel prognostic biomarker highly associated with an unfavorable outcome. miR-125b-5p and miR-206 levels are associated with stroke severity.