miR-21-5p inhibits neuropathic pain development via directly targeting C-C motif ligand 1 and tissue inhibitor of metalloproteinase-3

MicroRNAs (miRNA) play important roles in neuroinflammation and neuropathic pain development; however, the underlying mechanism requires further investigation. The expression of miR-21-5p was remarkably upregulated in chronic constrictive injury (CCI) rat model. A significant alleviated neuropathic pain development and reduced the expression of cytokines was observed in CCI rat after exogenous injection of miR-21-5p mimic. The dual-luciferase analysis revealed that tissue inhibitor of metalloproteinase-3 (TIMP3) and chemokines C-C motif ligand 1 (CCL1) was direct downstream target of miR-21-5p. Moreover, silencing of TIMP3 and CCL1 could rescue mechanical allodynia, thermal hyperalgesia and cytokine release in CCI rat, suggesting that TIMP3 and CCL1 exert their function by mediating neuroinflammation in neuropathic pain development. Therefore, we have identified a novel miR-21-5p–CCL1/TIMP3-cytokine axis in regulation of neuropathic pain development in CCI rat model, which is valuable for enhancing our understanding of neuropathic pain and developing miRNAs as potential therapeutic options in the future.     

Both mature miR-17-5p and passenger strand miR-17-3p target TIMP3 and induce prostate tumor growth and invasion

MicroRNAs (miRNA) precursor (pre-miRNA) molecules can be processed to release a miRNA/miRNA* duplex. In the canonical model of miRNA biogenesis, one strand of the duplex is thought to be the biologically active miRNA, whereas the other strand is thought to be inactive and degraded as a carrier or passenger strand called miRNA* (miRNA star). However, recent studies have revealed that miRNA* strands frequently play roles in the regulatory networks of miRNA target molecules. Our recent study indicated that miR-17 transgenic mice could abundantly express both the mature miR-17-5p and the passenger strand miR-17-3p. Here, we showed that miR-17 enhanced prostate tumor growth and invasion by increasing tumor cell proliferation, colony formation, cell survival and invasion. miRNA target analysis showed that both miR-17-5p and miR-17-3p repressed TIMP metallopeptidase inhibitor 3 (TIMP3) expression. Silencing with small interfering RNA against TIMP3 promoted cell survival and invasion. Ectopic expression of TIMP3 decreased cell invasion and cell survival. Our results demonstrated that mature miRNA can function coordinately with its passenger strand, enhancing the repressive ability of a miRNA by binding the same target. Within an intricate regulatory network, this may be among the mechanisms by which miRNA can augment their regulatory capacity.     

Radiosensitizer EXO-miR-197-3p Inhibits Nasopharyngeal Carcinoma Progression and Radioresistance by Regulating the AKT/mTOR Axis and HSPA5-mediated Autophagy

The biological functions of exosomes and microRNAs (miRs) in nasopharyngeal carcinoma (NPC) remain largely unexplored. Here, miR-197-3p was screened and identified, and whose level was reduced in serum and exosomes of patients with NPC. MiR-197-3p might be a good diagnostic and prognostic indicator. Our data showed that miR-197-3p expression was closely related to radioresistance, apoptosis, proliferation, migration, and survival of NPC. Inhibition of miR-197-3p expression in vitro could promote the proliferation and migration of NPC cells, while promotion of miR-197-3p expression in vivo could significantly inhibit the growth and enhance the radiosensitivity of NPC cells. From the perspective of mechanism, miR-197-3p could inhibit AKT/mTOR phosphorylation activation, inhibit an activated pathway of AKT/mTOR, target Heat Shock 70-kDa Protein 5(HSPA5) related to endoplasmic reticulum homeostasis, inhibit HSPA5-mediated autophagy, and reverse the radioresistance of NPC. Interestingly, exosomal miR-197-3p (EXO-miR-197-3p) reduced the proliferation and migration potential of NPC cells in vitro, and tumor growth and radioresistance of NPC cells in vivo. EXO-miR-197-3p inhibited NPC progression and radioresistance by regulating AKT/mTOR phosphorylation activation and HSPA5-mediated autophagy. In conclusion, our results highlight the potential of EXO-miR-197-3p as an effective radiosensitizer and therapeutic agent for refractory NPC.     

LncRNA TUG1 attenuates ischaemia-reperfusion-induced apoptosis of renal tubular epithelial cells by sponging miR-144-3p via targeting Nrf2

Renal ischaemia/reperfusion (I/R) injury may induce kidney damage and dysfunction, in which oxidative stress and apoptosis play important roles. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) are reported to be closely related to renal I/R, but the specific molecular mechanism is still unclear. The purpose of this research was to explore the regulatory effect of lncRNA TUG1 on oxidative stress and apoptosis in renal I/R injury. This research revealed that in renal I/R injury and hypoxia/reperfusion (H/R) injury in vitro, the expression level of lncRNA TUG1 was upregulated, and oxidative stress levels and apoptosis levels were negatively correlated with the expression level of lncRNA TUG1. Using bioinformatics databases such as TargetScan and microRNA.org, microRNA-144-3p (miR-144-3p) was predicted to be involved in the association between lncRNA TUG1 and Nrf2. This study confirmed that the level of miR-144-3p was significantly reduced following renal I/R injury and H/R injury in vitro, and miR-144-3p was determined to target Nrf2 and inhibit its expression. In addition, lncRNA TUG1 can reduce the inhibitory effect of miR-144-3p on Nrf2 by sponging miR-144-3p. In summary, our research shows that lncRNA TUG1 regulates oxidative stress and apoptosis during renal I/R injury through the miR-144-3p/Nrf2 axis, which may be a new treatment target for renal I/R injury.     

Dysregulation of microRNA-181b and TIMP3 is functionally involved in the pathogenesis of diabetic nephropathy

This study aimed to study the roleof microRNA (miR)-181b and its target TIMP3 in the development of diabetic nephropathy (DMN) via inhibiting the apoptosis of mesangial cells. Real-time polymerase chain reaction (RT-PCR) was adopted to compare the miR-181b expression between subjects with diabetic nephropathy (DN) and normal control. In addition, luciferase assays were utilized to explore the regulatory relationship between TIMP3 and miR-181b. Real-time PCR and densitometry analysis were conducted to measure the levels of TIMP3 mRNA/protein in DMN or in cells treated by miR-181b inhibitors, miR-181b mimics, and TIMP3 siRNA. And the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was adopted to study the effect of miR-181b on cell survival and apoptosis. miR-181b expression was much higher in the DN group, and the results of computational analysis identified TIMP3 as a miR-181b target. The luciferase activity of cells transfected with wild-type TIMP3 and mutant2 TIMP3 was significantly reduced, whereas the luciferase activity of cells transfected with mutant1 TIMP3 was evidently higher. Furthermore, a negative regulatory relationship was established between TIMP3 and miR-181b expression with a correlation efficient of −0.5351. The levels of TIMP3 mRNA/protein expression were apparently increased in the DN group. In addition, the treatment of cells with miR-181b mimics and TIMP3 siRNA remarkably lowered the levels of TIMP3 mRNA/protein, whereas the transfection of cells with miR-181b inhibitors notably elevated the expression of TIMP3 mRNA/protein. miR-181b promoted the survival of cells and inhibited their apoptosis. The miR-181b expression was related to the development of DMN and could be used as a prognosis biomarker of DMN in the patients with DM.     

Upregulation of miRNA-223-3p ameliorates RIP3-mediated necroptosis and inflammatory responses via targeting RIP3 after spinal cord injury

Spinal cord injury (SCI) has been a major burden on the society because of the high rate of disability. Receptor-interacting protein 3 (RIP3)-mediated necroptosis is a newly discovered pathway of programmed cell death and is involved in multiple pathologies of various human diseases. Micro RNAs (miRNAs) have been shown to be a potential target for therapeutic interventions after SCI. The aim of the present study is to explore the potential role of miR-223-3p and possible mechanism in SCI. We found that miR-223-3p was significantly downregulated in spinal neurons after H₂O ₂-induced damage, while RIP3-mediated necroptosis was elevated. Accordingly, RIP3-mediated necroptosis and the inflammatory factor secretion could be significantly inhibited by Nec-1 treatment. In adittion, overexpression of miR-223-3p in spinal neurons protected against H ₂O ₂-induced necroptosis, and ablation of miR-223-3p exhibited the opposite effect. We found that miR-223-3p bound to the 3′-untranslated region of RIP3 mRNA to negatively regulate the expression of RIP3. Moreover, the activated RIP3 reversed the inhibition of RIP3 and MLKL expression and the levels of TNF-α, IL-1β, and lactate dehydrogenase, which were induced by transfection with miR-223-3p in a H ₂O ₂-induced model. Finally, these results indicate that miR-223-3p negatively regulates the RIP3 necroptotic signaling cascades and inflammatory factor secretion, which significantly relieves injury of spinal neurons. The miR-223-3p/RIP3 pathway offers a novel therapeutic target for the protection of spinal neurons after SCI.     

Long noncoding RNA NEAT1 sponges miR-495-3p to enhance myocardial ischemia-reperfusion injury via MAPK6 activation

The biological function of long noncoding RNA NEAT1 has been revealed in a lot of diseases. Nevertheless, it is still not yet clear whether NEAT1 can modulate the process of myocardial ischemia–reperfusion injury (M-I/R). Here, we reported that NEAT1 was able to sponge miR-495-3p to contribute to M-I/R injury through activating mitogen-activated protein kinase 6 (MAPK6). First, elevated expression of NEAT1 was revealed in M-I/R injury mice, meanwhile, lactate dehydrogenase (LDH) and creatine kinase-muscle/brain (CK-MB) were also upregulated in the serum. Meanwhile, as previously reported, miR-495 serves as a tumor suppressor or an oncogenic miRNA in different types of cancer. Currently, we found miR-495-3p was remarkably reduced in M-I/R mice. Additionally, NEAT1 was significantly induced whereas miR-495-3p was greatly reduced by H₂O₂ treatment in H9C2 cells. Moreover, loss of NEAT1 in H9C2 cells could repress the viability and proliferation of cells. For another, overexpression of NEAT1 exhibited an opposite phenomenon. Furthermore, LDH release and caspase-3 activity were obviously triggered by upregulation of NEAT1 while suppressed by NEAT1 knockdown. miR-495-3p was indicated and validated as a target of NEAT1 using the analysis of bioinformatics. Interestingly, we observed that miR-495-3p mimics repressed tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-18 protein expression while their levels were enhanced by the inhibition of miR-495-3p in H9c2 cells. Subsequently, it was manifested that MAPK6 was a target of miR-495-3p, which could exert a lot in the NEAT1/miR-495-3p-mediated M-I/R injury. Overall, our results implied that NEAT1 contributed to M-I/R injury via the modulation of miR-495-3p and MAPK6.     

Protective effects of microRNA-22-3p against retinal pigment epithelial inflammatory damage by targeting NLRP3 inflammasome

NLRP3, as a crucial inflammasome component, plays important roles in age-related macular degeneration. Though some activators of NLRP3 have been studied, microRNAs (miRNAs) which potentially regulate NLRP3 messenger RNA (mRNA) have not been fully explored in retinal pigment epithelial (RPE) cells and retinopathy. In this study, by miRNA microarray profiling and bioinformatic analysis, we identified that four miRNAs, miR-4286, miR-223-3p, miR-365a, miR-22-3p, may target NLRP3 mRNA in RPE inflammatory damage in vivo. Further, real-time polymerase chain reaction verified that only miR-22-3p was significantly decreased, which was associated with NLRP3 upregulation in blue-light-induced retinopathy. Mechanistically, the dual-fluorescent reporter suggested miR-22-3p directly binds NLRP3 mRNA. Moreover, overexpression of miR-22-3p could significantly reduce whereas inhibition miR-22-3p could increase the mRNA and protein expressions of NLRP3, Caspase-1, and mature IL-1β. Collectively, our results indicate that miR-22-3p plays a suppressive role in RPE damage by targeting NLRP3, which provides new insights into the future intervention to retinopathy.     

Circ_0002984 induces proliferation,migration and inflammation response of VSMCs induced by ox-LDL through miR‑326-3p/VAMP3 axis in atherosclerosis

Atherosclerosis can result in multiple cardiovascular diseases. Circular RNAs (CircRNAs) have been reported as significant non-coding RNAs in atherosclerosis progression. Dysfunction of vascular smooth muscle cells (VSMCs) is involved in atherosclerosis. However, up to now, the effect of circ_0002984 in atherosclerosis is still unknown. Currently, we aimed to investigate the function of circ_0002984 in VSMCs incubated by oxidized low-density lipoprotein (ox-LDL). Firstly, our findings indicated that the expression levels of circ_0002984 were significantly up-regulated in the serum of atherosclerosis patients and ox-LDL-incubated VSMCs. Loss of circ_0002984 suppressed VSMC viability, cell cycle distribution and migration capacity. Then, we carried out ELISA assay to determine TNF-α and IL-6 levels. The data implied that lack of circ_0002984 obviously repressed ox-LDL–stimulated VSMC inflammation. Meanwhile, miR-326-3p, which was predicted as a target of circ_0002984, was obviously down-regulated in VSMCs treated by ox-LDL. Additionally, after overexpression circ_0002984 in VSMCs, a decrease in miR-326-3p was observed. Subsequently, miR-326-3p was demonstrated to target vesicle-associated membrane protein 3 (VAMP3). Therefore, we hypothesized that circ_0002984 could modulate expression of VAMP3 through sponging miR-326-3p. Furthermore, we confirmed that up-regulation of miR-326-3p rescued the circ_0002984 overexpressing-mediated effects on VMSC viability, migration and inflammation. Additionally, miR-326-3p inhibitor-mediated functions on VSMCs were reversed by knockdown of VAMP3. In conclusion, circ_0002984 mediated cell proliferation, migration and inflammation through modulating miR-326-3p and VAMP3 in VSMCs, which suggested that circ_0002984 might hold great promise as a therapeutic strategy for atherosclerosis.     

Anti-microRNA-222 (anti-miR-222) and -181B suppress growth of tamoxifen-resistant xenografts in mouse by targeting TIMP3 protein and modulating mitogenic signal

We have shown earlier that miR-221 and -222 are up-regulated in tamoxifen-resistant MCF-7 (OHT(R)) cells and Her2-positive human breast tumors when compared with Her2 negative tumors. In this study, we report markedly enhanced expression of miR-181b in OHT(R) cells and endocrine-resistant tumors. Further, anti-miR-222 or -181b in combination with tamoxifen suppressed growth of tamoxifen-resistant xenografts in mice. Luciferase reporter assay and expression analysis showed that TIMP3, a tissue metalloproteinase inhibitor, is a common target of miR-221/222 and -181b. In situ hybridization and immunohistochemical analysis demonstrated reciprocal relationships between TIMP3 and miR-221/222/181b expression in primary human breast carcinomas. Ectopic expression of TIMP3 inhibited growth of the OHT(R) cells, and its depletion in MCF-7 cells reduced sensitivity to tamoxifen in vitro and in vivo. EGF-induced MAPK and AKT phosphorylation were significantly higher in OHT(R) cells and miR-221/222-overexpressing MCF-7 cells than in control cells, which suggests modulation of mitogenic signaling by TIMP3 and the miRs. On the contrary, phosphoMAPK and phosphoAKT levels were diminished in TIMP3-overexpressing OHT(R) cells and increased in TIMP3-depleted MCF-7 cells. Low levels of estrogen or tamoxifen elicited similar differences in phosphoMAPK levels in these cells. Reduced levels of TIMP3 facilitated growth of tamoxifen-resistant cells by alleviating its inhibitory effect on ADAM10 and ADAM17, which are critical for OHT(R) cell growth. In conclusion, miR-221/222 and -181b facilitate growth factor signaling in tamoxifen-resistant breast cancer by down-regulating TIMP3, and corresponding anti-miRs can be used to render these tumors responsive to tamoxifen.     

The Effect of SPTLC2 on Promoting Neuronal Apoptosis is Alleviated by MiR-124-3p Through TLR4 Signalling Pathway

To investigate the role and mechanism of microRNA-124-3p (miR-124-3p) and serine palmitoyltransferase long chain base subunit 2 (SPTLC2) in neuronal apoptosis induced by mechanical injury. Transient transfection was used to modify the expression of miR-124-3p and SPTLC2. After transfection, neuronal apoptosis was evaluated in an in vitro injury model of primary neurons using TUNEL staining and western blot. The correlation between miR-124-3p and SPTLC2 was identified through a dual luciferase reporter assay in HEK293 cells. A rescue experiment in primary neurons was performed to further confirm the result. To explore the downstream mechanisms, co-immunoprecipitation was performed to identify proteins that interact with SPTLC2 in toll-like receptor 4 (TLR4) signalling pathway. Subsequently, the relative expression levels of TLR4 pathway molecules were measured by western blot. Our results showed that increased miR-124-3p can inhibit neuronal apoptosis, which is opposite to the effect of SPTLC2. In addition, miR-124-3p was proved to negatively regulate SPTLC2 expression and suppress the apoptosis-promoting effect of SPTLC2 via the TLR4 signalling pathway.

     

miR-29b-3p protects cardiomyocytes against endotoxin-induced apoptosis and inflammatory response through targeting FOXO3A

Cardiac dysfunction represents a main component of death induced by sepsis in critical care units. And microRNAs (miRNAs) have been reported as important modulators or biomarkers of sepsis. However, the molecular detail of miRNAs involved in septic cardiac dysfunction remains unclear. Here we showed that endotoxin (lipopolysaccharide, LPS) significantly down-regulated expression of miR-29b-3p in heart. Increased expression of miR-29b-3p by lentivirus improved cardiac function and attenuated damage of cardiac induced by LPS in mice. Furthermore, overexpression or knockdown of miR-29b-3p showed its crucial roles on regulation of apoptosis and production of pro-inflammatory cytokines in NRCMs through directly targeting FOXO3A. miR-29b-3p ameliorates inflammatory damage likely via reducing activation of MAPKs and nuclear-translocation of NF-κB to block LPS-activated NF-κB signaling. Notably, miR-29b is also down-regulated in septic patients' plasma compared with normal subjects, indicating a potential clinical relevance of miR-29b. Taken together, our findings demonstrate that upregulation of miR-29b-3p can attenuate myocardial injury induced by sepsis via regulating FOXO3A, which provide a potential therapy target for interference of septic cardiac dysfunction.     

Identification and Validation of Reference Genes for qPCR Detection of Serum microRNAs in Colorectal Adenocarcinoma Patients

Serum microRNAs (miRNAs) have become a highlighted research hotspot, especially for their great potential as a novel promising non-invasive biomarker in cancer diagnosis. The most frequently used approach for serum miRNAs detection is quantitative real time polymerase chain reaction (qPCR). In order to obtain reliable qPCR data of miRNAs expression, the use of reference genes as endogenous control is undoubtly necessary. However, no systematic evaluation and validation of reference genes for normalizing qPCR analysis of serum miRNAs has been reported in colorectal adenocarcinoma. We firstly profiled pooled serum of colorectal adenocarcinoma, colorectal adenoma and healthy controls and selected a list of 13 miRNAs as candidate reference genes. U6 snRNA (U6) and above-mentioned 13 miRNAs were included in further confirmation by qPCR. As a result, 5 miRNAs (miR-151a-3p, miR-4446-3p, miR-221-3p, miR-93-5p and miR-3184-3p) were not detected in all samples and 2 miRNAs (miR-197-3p and miR-26a-5p) were relatively low with median Cq more than 35, and were excluded from further stability analysis. Then variable stability of other 6 miRNAs (miR-103b, miR-484, miR-16-5p, miR-3615, miR-18a-3p and miR-191-5p) and U6 were evaluated using two algorithms: geNorm and NormFinder which both identified miR-191-5p as the most stably expressed reference gene and selected miR-191-5p and U6 as the most stable pair of reference genes. After validating in an independent large cohorts and selecting miR-92a-3p as target miRNA to evaluate the effect of reference gene, we propose that combination of miR-191-5p and U6 could be used as reference genes for serum microRNAs qPCR data in colorectal adenocarcinoma, colorectal adenoma and healthy controls.     

Silencing of long noncoding RNA MEG3 enhances cerebral protection of dexmedetomidine against hypoxic-ischemic brain damage in neonatal mice by binding to miR-129-5p

Hypoxic-ischemic brain damage (HIBD) is a leading cause of neonatal acute mortality and chronic nervous system injury. Recently, it has been found that long noncoding RNAs (lncRNAs) play a significant role in the neurodevelopment and etiopathogenesis of HIBD. Here, the researchers aimed to determine the role of lncRNA maternally expressed gene (MEG3) in the therapeutic effect of dexmedetomidine (DEX) in neonatal mice with HIBD through the regulation of microRNA-129-5p (miR-129-5p). HIBD models were established in C57/BL6 neonatal mice. Subsequently, the target relationship between MEG3 and miR-129-5p was predicted and verified. The neonatal mice were injected with DEX, ad-shMEG3, and mimics and inhibitors of miR-129-5p to identify roles of MEG3 and miR-129-5p in therapeutic effects of DEX on neuronal apoptosis and injury, cerebral atrophy, and learning and memory ability of neonatal mice with HIBD. MEG3 directly targeted and inhibited the expression of miR-129-5p. Silencing of MEG3 or upregulation of miR-129-5p effectively promoted the therapeutic effect of DEX on neonatal mice with HIBD. Silencing of MEG3 or upregulation of miR-129-5p reduced the neuronal apoptosis rate and degree of cerebral atrophy, and also enhanced the learning and memory ability of HIBD neonatal mice. Collectively, the key findings obtained from the present study support the notion that MEG3 silencing enhances the therapeutic effect of DEX on neonatal mice with HIBD by binding to miR-129-5p.