MiR‐377 Regulates Inflammation and Angiogenesis in Rats After Cerebral Ischemic Injury

Ischemic stroke is the leading cause of disabilities worldwide. MicroRNA‐377 (miR‐377) plays important roles in ischemic injury. The present study focused on the mechanisms of miR‐377 in protecting ischemic brain injury in rats. Cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in rats. Primary rat microglial cells and brain microvascular endothelial cells (BMECs) were exposed to oxygen‐glucose deprivation (OGD). The concentrations of cytokines (TNF‐α, IL‐1β, IL‐6, IFN‐γ, TGF‐β, MMP2, COX2, and iNOS) in the culture medium were measured by specific ELISA. Tube formation assay was for the in vitro study of angiogenesis. Luciferase reporter assay was performed to confirm whether VEGF and EGR2 were direct targets of miR‐377. The MCAO rats were intracerebroventricular (ICV) injection of miR‐377 inhibitor to assess its protective effects in vivo. MiR‐377 levels were decreased in the rat brain tissues at 1, 3, and 7 d after MCAO. Both microglia cells and BMECs under OGD showed markedly lower expression levels of miR‐377 while higher expression levels of EGR2 and VEGF compared to those under normoxia conditions. Knockdown of miR‐377 inhibited microglial activation and the release of pro‐inflammatory cytokines after OGD. Suppression of miR‐377 promoted the capillary‐like tube formation and cell proliferation and migration of BMECs. The anti‐inflammation effect of EGR2 and the angiogenesis effect of VEGF were regulated by miR‐377 after OGD. Inhibition of miR‐377 decreased cerebral infarct volume and suppressed cerebral inflammation but promoted angiogenesis in MCAO rats. Knockdown of miR‐377 lessened the ischemic brain injury through promoting angiogenesis and suppressing cerebral inflammation. J. Cell. Biochem. 119: 327–337, 2018. © 2017 Wiley Periodicals, Inc.     

MiR‐150 alleviates neuropathic pain via inhibiting toll‐like receptor 5

MicroRNAs (miRNAs) are reported as vital participators in the pathophysiological course of neuropathic pain. However, the underlying mechanisms of the functional roles of miRNAs in neuropathic pain are largely unknown. This study was designed to explore the potential role of miR‐150 in regulating the process of neuropathic pain in a rat model established by chronic sciatic nerve injury (CCI). Overexpression of miR‐150 greatly alleviated neuropathic pain development and reduced inflammatory cytokine expression, including COX‐2, interleukin IL‐6, and tumor necrosis factor (TNF)‐α in CCI rats. By bioinformatic analysis, 3′‐untranslated region (UTR) of Toll‐like receptor (TLR5) was predicted to be a target of miR‐150. TLR5 commonly serves as an important regulator of inflammation. Overexpression of miR‐150 significantly suppressed the expression of TLR5 in vitro and in vivo. Furthermore, upregulation of TLR5 decreased the miR‐150 expression and downregulation of TLR5 increased miR‐150, respectively. Overexpression of TLR5 significantly reversed the miR‐150‐induced suppressive effects on neuropathic pain. In conclusion, our current study indicates that miR‐150 may inhibit neuropathic pain development of CCI rats through inhibiting TLR5‐mediated neuroinflammation. Our findings suggest that miR‐150 may provide a novel therapeutic target for neuropathic pain treatment.     

Role of microRNA‐155 in modifying neuroinflammation and γ‐aminobutyric acid transporters in specific central regions after post‐ischaemic seizures

In the central nervous system, interleukin (IL)‐1β, IL‐6 and tumour necrosis factor (TNF)‐α have a regulatory role in pathophysiological processes of epilepsy. In addition, γ‐aminobutyric acid (GABA) transporter type 1 and type 3 (GAT‐1 and GAT‐3) modulate the levels of extracellular GABA in involvement in the neuroinflammation on epileptogenesis. Thus, in the current report we examined the effects of inhibiting microRNA‐155 (miR‐155) on the levels of IL‐1β, IL‐6 and TNF‐α, and expression of GAT‐1 and GAT‐3 in the parietal cortex, hippocampus and amygdala of rats with nonconvulsive seizure (NCS) following cerebral ischaemia. Real time RT‐PCR, ELISA and Western blot analysis were used to examine the miR‐155, proinflammatory cytokines (PICs) and GAT‐1/GAT‐3 respectively. With induction of NCS, the levels of miR‐155 were amplified in the parietal cortex, hippocampus and amygdala and this was accompanied with increases of IL‐1β, IL‐6 and TNF‐α. In those central areas, expression of GAT‐1 and GAT‐3 was upregulated; and GABA was reduced in rats following NCS. Intracerebroventricular infusion of miR‐155 inhibitor attenuated the elevation of PICs, amplification of GAT‐1 and GAT‐3 and impairment of GABA. Furthermore, inhibition of miR‐155 decreased the number of NCS events following cerebral ischaemia. Inhibition of miR‐155 further improved post‐ischaemia‐evoked NCS by altering neuroinflammation‐GABA signal pathways in the parietal cortex, hippocampus and amygdala. Results suggest the role of miR‐155 in regulating post‐ischaemic seizures via PICs‐GABA mechanisms.     

Role of IL‐15 in spinal cord and sciatic nerve after chronic constriction injury: regulation of macrophage and T‐cell infiltration

The release of inflammatory mediators from immune and glial cells either in the peripheral or CNS may have an important role in the development of physiopathological processes such as neuropathic pain. Microglial, then astrocytic activation in the spinal cord, lead to chronic inflammation, alteration of neuronal physiology and neuropathic pain. Standard experimental models of neuropathic pain include an important peripheral inflammatory component, which involves prominent immune cell activation and infiltration. Among potential immunomodulators, the T‐cell cytokine interleukin‐15 (IL‐15) has a key role in regulating immune cell activation and glial reactivity after CNS injury. Here we show, using the model of chronic constriction of the sciatic nerve (CCI), that IL‐15 is essential for the development of the early inflammatory events in the spinal cord after a peripheral lesion that generates neuropathic pain. IL‐15 expression in the spinal cord was identified in both astroglial and microglial cells and was present during the initial gliotic and inflammatory (NFκB) response to injury. The expression of IL‐15 was also identified as a cue for macrophage and T‐cell activation and infiltration in the sciatic nerve, as shown by intraneural injection of the cytokine and activity blockage approaches. We conclude that the regulation of IL‐15 and hence the initial events following its expression after peripheral nerve injury could have a future therapeutic potential in the reduction of neuroinflammation.     

Antagonizing peroxisome proliferator‐activated receptor γ facilitates M1‐to‐M2 shift of microglia by enhancing autophagy via the LKB1–AMPK signaling pathway

Microglia‐mediated neuroinflammation plays a dual role in various brain diseases due to distinct microglial phenotypes, including deleterious M1 and neuroprotective M2. There is growing evidence that the peroxisome proliferator‐activated receptor γ (PPARγ) agonist rosiglitazone prevents lipopolysaccharide (LPS)‐induced microglial activation. Here, we observed that antagonizing PPARγ promoted LPS‐stimulated changes in polarization from the M1 to the M2 phenotype in primary microglia. PPARγ antagonist T0070907 increased the expression of M2 markers, including CD206, IL‐4, IGF‐1, TGF‐β1, TGF‐β2, TGF‐β3, G‐CSF, and GM‐CSF, and reduced the expression of M1 markers, such as CD86, Cox‐2, iNOS, IL‐1β, IL‐6, TNF‐α, IFN‐γ, and CCL2, thereby inhibiting NFκB–IKKβ activation. Moreover, antagonizing PPARγ promoted microglial autophagy, as indicated by the downregulation of P62 and the upregulation of Beclin1, Atg5, and LC3‐II/LC3‐I, thereby enhancing the formation of autophagosomes and their degradation by lysosomes in microglia. Furthermore, we found that an increase in LKB1–STRAD–MO25 complex formation enhances autophagy. The LKB1 inhibitor radicicol or knocking down LKB1 prevented autophagy improvement and the M1‐to‐M2 phenotype shift by T0070907. Simultaneously, we found that knocking down PPARγ in BV2 microglial cells also activated LKB1–AMPK signaling and inhibited NFκB–IKKβ activation, which are similar to the effects of antagonizing PPARγ. Taken together, our findings demonstrate that antagonizing PPARγ promotes the M1‐to‐M2 phenotypic shift in LPS‐induced microglia, which might be due to improved autophagy via the activation of the LKB1–AMPK signaling pathway.     

Bilobalide alleviates IL‐17‐induced inflammatory injury in ATDC5 cells by downregulation of microRNA‐125a

Ankylosing spondylitis (AS) is a high disability and greatly destructive disease. In this study, we preliminarily studied the function and mechanism of bilobalide (BIL) on interleukin (IL)‐17‐induced inflammatory injury in ATDC5 cells. CCK‐8 and migration assays were used to detect the functions of IL‐7, BIL, and microRNA (miR)‐125a on cell viability and migration. The miR‐125a level was changed by transfection, and tested by real‐time quantitative polymerase chain reaction. Additionally, Western blot tested the levels of inflammatory factors (IL‐6 and tumor necrosis factor‐α), matrix metalloproteinases (MMPs), and pathway‐related proteins. Moreover, the enzyme‐linked immunosorbent assay also was used to detect inflammatory factor levels. IL‐7 was used to construct an inflammatory injury model in ATDC5 cells. Based on this, BIL inhibited IL‐17‐induced cell viability, migration, and expressions of inflammatory factors and MMPs. Furthermore, we found BIL negatively regulated miR‐125a, and the miR‐125a mimic could partly reverse the effects of BIL on IL‐17‐injury. Finally, we showed that BIL inhibited the c‐Jun N‐terminal kinase (JNK) and nuclear factor kappa B (NF‐κB) pathways, and the miR‐125a mimic had the opposite effect. BIL inhibited IL‐17‐induced inflammatory injury in ATDC5 cells by downregulation of miR‐125a via JNK and NF‐κB signaling pathways.     

MicroRNA‐93‐5p expression in tumor tissue and its tumor suppressor function via targeting programmed death ligand‐1 in colorectal cancer

This work aimed to investigate miR‐93‐5p expression in tumor tissue and its in vitro effects in colorectal cancer (CRC) by targeting programmed death ligand‐1 (PD‐L1). MiR‐93‐5p and PD‐L1 expression was detected in CRC and adjacent normal tissues by quantitative real‐time polymerase chain reaction and immunohistochemistry. The correlation between miR‐93‐5p and PD‐L1 was validated by a dual‐luciferase reporter assay. HCT116 and SW480 cells were divided into blank, miR‐NC, miR‐93‐5p mimics, miR‐93‐5p inhibitor, PD‐L1 small interfering RNA (siRNA) and miR‐93‐5p inhibitor + PD‐L1 siRNA groups, and wound‐healing and transwell assays were performed to detect cell migration and invasion, respectively. Protein expression was measured by western blotting. The secretion of cytokines was detected in the CRC cell/T coculture models. MiR‐93‐5p was downregulated in CRC tissues with upregulated PD‐L1. In PD‐L1‐negative patients, miR‐93‐5p expression was increased compared with that in PD‐L1‐positive patients. MiR‐93‐5p and PD‐L1 expression levels were associated with the tumor differentiation, lymphatic metastasis, TNM, Duke's stage, and prognosis of CRC. PD‐L1 siRNA weakened the migration and invasion abilities via decreased expression of matrix metalloproteinase‐1 (MMP‐1), ‐2, and ‐9, and these effects were abolished by the miR‐93‐5p inhibitor. Additionally, anti‐PD‐L1 upregulated the expressions of interleukin‐2 (IL‐2), tumor necrosis factor‐α (TNF‐α), and interferon γ (IFN‐γ) in the coculture of T cells with CRC cells, but downregulated the expressions of IL‐1β, IL‐10, and TGF‐β. However, these changes were partially reversed by miR‐93‐5p inhibition. miR‐93‐5p is expected to be a novel target for CRC treatment since it decreases the migration and invasion, as well as the immune evasion, of CRC cells via targeting PD‐L1.     

Reciprocal regulation of miR‐206 and IL‐6/STAT3 pathway mediates IL6‐induced gefitinib resistance in EGFR‐mutant lung cancer cells

Persistently activated IL‐6/STAT3 pathway promotes acquired resistance to targeted therapy with epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs) in non–small‐cell lung cancer (NSCLC) treatment. miR‐206 has been verified to be dysregulated and plays as a negative regulator in lung cancer. However, whether miR‐206 may overcome IL6‐induced gefitinib resistance in EGFR‐mutant lung cancer remains elusive. In this study, we investigated the role of miR‐206 in IL6‐induced gefitinib‐resistant EGFR‐mutated lung cancer cell lines. We showed that forced miR‐206 expression restored gefitinib sensitivity in IL6‐induced gefitinib‐resistant EGFR‐mutant lung cancer cells by inhibiting IL6/JAK1/STAT3 pathway. Specifically, mechanistic investigations revealed that miR‐206 blocked IL‐6/STAT3 signalling via directly targeting the 3'‐UTR of intracellular IL‐6 messenger RNA. Moreover, IL‐6 induced miR‐206 down‐regulation by reducing the cropping process of primary miR‐206 (pri‐miR‐206) into the Drosha/DGCR8 complex. Taken together, our findings reveal a direct role of miR‐206 in regulating IL‐6/STAT3 pathway and contrarily activated IL‐6/STAT3 signalling mediates the miR‐206 maturation process in gefitinib‐resistant EGFR‐mutant lung cancer cells.     

Overexpression of microRNA‐138 alleviates human coronary artery endothelial cell injury and inflammatory response by inhibiting the PI3K/Akt/eNOS pathway

This study aimed to investigate the role of miR‐138 in human coronary artery endothelial cell (HCAEC) injury and inflammatory response and the involvement of the PI3K/Akt/eNOS signalling pathway. Oxidized low‐density lipoprotein (OX‐LDL)‐induced HCAEC injury models were established and assigned to blank, miR‐138 mimic, miR‐138 inhibitor, LY294002 (an inhibitor of the PI3K/Akt/eNOS pathway), miR‐138 inhibitor + LY294002 and negative control (NC) groups. qRT‐PCR and Western blotting were performed to detect the miR‐138, PI3K, Akt and eNOS levels and the protein expressions of PI3K, Akt, eNOS, p‐Akt, p‐eNOS, Bcl‐2, Bax and caspase‐3. ELISAs were employed to measure the expressions of TNF‐α, IL‐4, IL‐6, IL‐8, IL‐10 and nitric oxide (NO) and the activities of lactate dehydrogenase (LDH) and eNOS. MTT and flow cytometry were performed to assess the proliferation and apoptosis of HCAECs. Compared to the blank group, PI3K, Akt and eNOS were down‐regulated in the miR‐138 mimic and LY294002 groups but were up‐regulated in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups showed decreased concentrations of TNF‐α, IL‐6, IL‐8 and NO and reduced activities of LDH and eNOS, while opposite trends were observed in the miR‐138 inhibitor group. The concentrations of IL‐4 and IL‐10 increased in the miR‐138 mimic and LY294002 groups but decreased in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups had significantly decreased cell proliferation and increased cell apoptosis compared to the blank group. These findings indicate that up‐regulation of miR‐138 alleviates HCAEC injury and inflammatory response by inhibiting the PI3K/Akt/eNOS signalling pathway.     

Down‐regulated lncRNA HOTAIR alleviates polycystic ovaries syndrome in rats by reducing expression of insulin‐like growth factor 1 via microRNA‐130a

It has been found that long noncoding RNA HOTAIR, microRNA‐130a (miR‐130a) and insulin‐like growth factor 1 (IGF1) expression are associated with ovarian cancer, thus, we hypothesised that the HOTAIR/miR‐130a/IGF1 axis might associate with endocrine disorders and biological behaviours of ovarian granulosa cells in rat models of polycystic ovary syndrome (PCOS). PCOS rat models were established by injection of dehydro‐isoandrosterone, followed by treatment of si‐HOTAIR, oe‐HOTAIR, miR‐130a mimics or miR‐130a inhibitors. Serum hormonal levels were determined to evaluate endocrine conditions. The effect of HOTAIR and miR‐130a on activities of isolated ovarian granulosa cells was assessed, as well as the involvement of IGF1.In the ovarian tissues and granulosa cells of PCOS rat models, highly expressed HOTAIR and IGF1 and poorly expressed miR‐130a were identified. In response to oe‐HOTAIR, serum levels of E₂, T and LH were increased and serum levels of FSH were reduced; the proliferation of granulosa cells was reduced and apoptosis was promoted; notably, expression of miR‐130a was reduced while expression of IGF1 was increased. The treatment of si‐HOTAIR reversed the situation. Furthermore, the binding of HOTAIR to miR‐130a and targeting relationship of miR‐130a and IGF1 were confirmed. LncRNA HOTAIR up‐regulates the expression of IGF1 and aggravates the endocrine disorders and granulosa cell apoptosis through competitive binding to miR‐130a in rat models of PCOS. Based on our finding, we predict that competitive binding of HOTAIR to miR‐130a may act as a novel target for the molecular treatment of PCOS.     

The interplay of LncRNA ANRIL and miR‐181b on the inflammation‐relevant coronary artery disease through mediating NF‐κB signalling pathway

This study was designed to investigate whether ANRIL affected the aetiology of coronary artery disease (CAD) by acting on downstream miR‐181b and NF‐κB signalling. Altogether 327 CAD patients diagnosed by angiography were included, and mice models of CAD were established. Human coronary endothelial cells (HCAECs) and human umbilical vein endothelial cells (HUVECs) were also purchased. In addition, shRNA‐ANRIL, shRNA‐NC, pcDNA3.1‐ANRIL, miR‐181b mimic, miR‐181b inhibitor and miR‐NC were transfected into the cells. The lipopolysaccharides (LPS) and pyrrolidine dithiocarbamate (PDTC) were also added to activate or deactivate NF‐κB signalling. Both highly expressed ANRIL and lowly expressed miR‐181b were associated with CAD population aged over 60 years old, with smoking history, with hypertension and hyperlipidemia, with CHOL H 4.34 mmol/L, TG ≥ 1.93 mmol/L and Hcy ≥ 16.8 μmol/L (all P < 0.05). Besides, IL‐6, IL‐8, NF‐κB, TNF‐α, iNOS, ICAM‐1, VCAM‐1 and COX‐2 expressions observed within AD mice models were all beyond those within NC and sham‐operated groups (P < 0.05). Also VEGF and HSP 70 were highly expressed within AD mice models than within NC and sham‐operated mice (P < 0.05). Transfection of either pcDNA‐ANRIL or miR‐181b inhibitor could significantly fortify HCAECs’ viability and put on their survival rate. At the meantime, the inflammatory factors and vascular‐protective parameters were released to a greater level (P < 0.05). Finally, highly expressed ANRIL also notably bring down miR‐181b expression and raise p50/p65 expressions within HCAECs (P < 0.05). The joint role of ANRIL, miR‐181b and NF‐κB signalling could aid in further treating and diagnosing CAD.     

MiR‐643 inhibits lipopolysaccharide‐induced endometritis progression by targeting TRAF6

Endometritis is a prevalent disease with inflammation of uterus endangering women reproductive health. MicroRNAs (miRNAs) play important roles in inflammatory disorders, including endometritis. However, the role and mechanism of miR‐643 in endometritis development remain unclear. This study aimed to investigate the effect of miR‐643 on lipopolysaccharide (LPS)‐induced inflammatory response and clarify the potential mechanism. LPS‐treated human endometrial epithelial cells (HEECs) were cultured to investigate the role of miR‐643 in vitro. The expression levels of miR‐643 and tumor necrosis factor receptor‐associated factor 6 (TRAF6) were measured via quantitative real‐time polymerase chain reaction and western blot, respectively. LPS‐induced inflammatory response was assessed by inflammatory cytokines secretion via enzyme‐linked immunosorbent assay. The activation of nuclear factor‐κB (NF‐κB) pathway was investigated by western blot. The interaction between miR‐643 and TRAF6 was validated by bioinformatics analysis, luciferase reporter assay, and RNA immunoprecipitation. The expression of miR‐643 was decreased and TRAF6 protein level was enhanced in LPS‐treated HEECs. The overexpression of miR‐643 suppressed LPS‐induced secretion of inflammatory cytokines (tumor necrosis factor‐α, interleukin‐1β [IL‐1β], and IL‐6) and activation of NF‐κB pathway. The knockdown of TRAF6 inhibited LPS‐induced inflammatory response in HEECs. TRAF6 was validated as a target of miR‐643 and TRAF6 restoration reversed the effect of miR‐643 on inflammation response in LPS‐treated HEECs. Collectively, miR‐643 attenuated LPS‐induced inflammatory response by targeting TRAF6, indicating a novel avenue for the treatment of endometritis.     

Exosomes from miR‐20b‐3p‐overexpressing stromal cells ameliorate calcium oxalate deposition in rat kidney

Hyperoxaluria‐induced calcium oxalate (CaOx) deposition is the key factor in kidney stone formation, for which adipose‐derived stromal cells (ADSCs) have been used as a therapeutic treatment. Studies revealed that miR‐20b‐3p is down‐regulated in hypercalciuric stone‐forming rat kidney. To investigate whether ADSC‐derived miR‐20b‐3p‐enriched exosomes protect against kidney stones, an ethylene glycol (EG)‐induced hyperoxaluria rat model and an in vitro model of oxalate‐induced NRK‐52E cells were established to explore the protective mechanism of miR‐20b‐3p. The results showed that miR‐20b‐3p levels were decreased following hyperoxaluria in the urine of patients and in kidney tissues from animal models. Furthermore, treatment with miR‐20b‐3p‐enriched exosomes from ADSCs protected EG‐induced hyperoxaluria rats, and cell experiments confirmed that co‐culture with miR‐20b‐3p‐enriched exosomes alleviated oxalate‐induced cell autophagy and the inflammatory response by inhibiting ATG7 and TLR4. In conclusion, ADSC‐derived miR‐20b‐3p‐enriched exosomes protected against kidney stones by suppressing autophagy and inflammatory responses.     

改良CCI 大鼠痛行为学检测与损伤区自发放电活动再观察

目的:应用改良CCI模型研究外周神经损伤后痛觉过敏和自发放电各自特征及相互关系.方法:雄性SD大鼠,随机分为CCI组和Sham组,分别于术前1天和术后1、4、7、9、11、14天测定机械刺激缩足反射阈值和热缩腿反射潜伏期,同时选取术侧机械刺激缩腿反射阈值低于4g或者术侧和健侧热缩腿反射潜伏期差异大于2s的CCI模型大鼠观察术后4-14天损伤区自发放电活动.结果:神经纤维损伤后,机械痛敏和热痛敏随时间表现为逐渐增强,同时在损伤区观察到三类放电模式:整数倍放电阵发放电和周期放电.结论:术后大鼠机械痛阈和热痛阈逐渐降低,机械痛敏的产生和损伤区自发放电活动关系密切,不同的放电模式可能代表不同的传入信息.