Melatonin ameliorates renal fibroblast-myofibroblast transdifferentiation and renal fibrosis through miR-21-5p regulation

Fibroblast-myofibroblast transdifferentiation (FMT) is widely recognized as the major pathological feature of renal fibrosis. Although melatonin has exerted antifibrogenic activity in many diseases, its role in renal FMT remains unclear. In the present study, the aim was to explore the effect of melatonin on renal FMT and the underlying mechanisms. We established the transforming growth factor (TGF)-β1 stimulated rat renal fibroblast cells (NRK-49F) model in vitro and unilateral ureteral obstruction (UUO) mice model in vivo. We assessed levels of α-smooth muscle actin (α-SMA), col1a1 and fibronectin, STAT3 and AP-1, as well as miR-21-5p and its target genes (Spry1, PTEN, Smurf2 and PDCD4). We found that melatonin reduced the expression of α-SMA, col1a1 and fibronectin, as well as the formation of α-SMA filament in TGF-β1-treated NRK-49F cells. Meanwhile, melatonin inhibited STAT3 phosphorylation, down-regulated miR-21-5p expression, and up-regulated Spry1 and PTEN expression. Moreover, miR-21-5p mimics partially antagonized the anti-fibrotic effect of melatonin. For animal experiments, the results revealed that melatonin remarkably ameliorated UUO-induced renal fibrosis, attenuated the expression of miR-21-5p and pro-fibrotic proteins and elevated Spry1 and PTEN expression. Nevertheless, agomir of miR-21-5p blocked the renoprotective effect of melatonin in UUO mice. These results indicated that melatonin could alleviate TGF-β1-induced renal FMT and UUO-induced renal fibrosis through down-regulation of miR-21-5p. Regulation of miR-21-5p/PTEN and/or miR-21-5p/Spry1 signal might be involved in the anti-fibrotic effect of melatonin in the kidneys of UUO mice.     

Lung CSC-derived exosomal miR-210-3p contributes to a pro-metastatic phenotype in lung cancer by targeting FGFRL1

Lung cancer has the highest mortality rate among human cancers, and the majority of deaths can be attributed to metastatic spread. Lung cancer stem cells (CSCs) are a component of the tumour microenvironment that contributes to this process. Exosomes are small membrane vesicles secreted by all types of cells that mediate cell interactions, including cancer metastasis. Here, we show that lung CSC-derived exosomes promote the migration and invasion of lung cancer cells, up-regulate expression levels of N-cadherin, vimentin, MMP-9 and MMP-1, and down-regulate E-cadherin expression. Moreover, we verified that these exosomes contribute to a pro-metastatic phenotype in lung cancer cells via miR-210-3p transfer. The results of bioinformatics analysis and dual-luciferase reporter assays further indicated that miR-210-3p may bind to fibroblast growth factor receptor-like 1 (FGFRL1); silencing FGFRL1 enhanced the metastatic ability of lung cancer cells, whereas overexpressing FGFRL1 suppressed metastasis. Taken together, our results provide new insights into a potential molecular mechanism whereby lung CSC-derived exosomal miR-210-3p targets FGFRL1 to promote lung cancer metastasis. FGFRL1 may be a promising therapeutic target in lung cancer.     

miR-191 secreted by platelet-derived microvesicles induced apoptosis of renal tubular epithelial cells and participated in renal ischemia-reperfusion injury via inhibiting CBS

In this study, we aimed to reveal the role of miR-191 in apoptosis of renal tubular epithelial cells and in the involvement of renal ischemia-reperfusion injury. Renal transplantation rat model was established. miR-191 and Cystathionine-β-synthase (CBS) were measured by qRT-PCR and Western blot. The regulation of miR-191 on CBS was detected by luciferase reporter assay. We found miR-191 expression in platelets and platelet microvesicles (P-MVs) of patients and model rats was significantly upregulated than that of health and normal rats. Also, mRNA and protein levels of CBS in renal tissues of patients were significantly downregulated than that of health and normal rats. We also found that P-MVs could transfer miR-191 to HK-2 cells. Luciferase reporter assay showed that CBS was a direct target of miR-191. In addition, we proved that P-MVs-secreted miR-191 inhibited CBS expression in HK-2 cells, and P-MVs-secreted miR-191 promoted HK-2 cell apoptosis via CBS. Finally, we verified the trends of CBS expressions, HK-2 cell apoptosis and apoptosis-related proteins in vivo were similar as the trends in vitro. Therefore, CBS was a direct target of miR-191, and miR-191 could transfer to HK-2 cells via P-MVs to decrease the expression of CBS, thus to promote cell apoptosis and renal IR injury.     

Protease‐activated receptor‐1 contributes to renal injury and interstitial fibrosis during chronic obstructive nephropathy

End‐stage renal disease, the final stage of all chronic kidney disorders, is associated with renal fibrosis and inevitably leads to renal failure and death. Transition of tubular epithelial cells (TECs) into mesenchymal fibroblasts constitutes a proposed mechanism underlying the progression of renal fibrosis and here we assessed whether protease‐activated receptor (PAR)‐1, which recently emerged as an inducer of epithelial‐to‐mesenchymal transition (EMT), aggravates renal fibrosis. We show that PAR‐1 activation on TECs reduces the expression of epithelial markers and simultaneously induces mesenchymal marker expression reminiscent of EMT. We next show that kidney damage was reduced in PAR‐1‐deficient mice during unilateral ureter obstruction (UUO) and that PAR‐1‐deficient mice develop a diminished fibrotic response. Importantly, however, we did hardly observe any signs of mesenchymal transition in both wild‐type and PAR‐1‐deficient mice suggesting that diminished fibrosis in PAR‐1‐deficient mice is not due to reduced EMT. Instead, the accumulation of macrophages and fibroblasts was significantly reduced in PAR‐1‐deficient animals which were accompanied by diminished production of MCP‐1 and TGF‐β. Overall, we thus show that PAR‐1 drives EMT of TECs in vitro and aggravates UUO‐induced renal fibrosis although this is likely due to PAR‐1‐dependent pro‐fibrotic cytokine production rather than EMT.     

Extracellular vesicles-derived miR-150-5p secreted by adipose-derived mesenchymal stem cells inhibits CXCL1 expression to attenuate hepatic fibrosis

Hepatic fibrosis (HF) is involved in aggravated wound-healing response as chronic liver injury. Extracellular vesicles (EVs) carrying microRNA (miR) have been reported as therapeutic targets for liver diseases. In this study, we set out to explore whether adipose-derived mesenchymal stem cells (ADMSCs)-derived EVs containing miR-150-5p affect the progression of HF. Carbon tetrachloride (CCl₄) was firstly used to induce HF mouse models in C57BL/6J mice, and activation of hepatic stellate cells (HSCs) was achieved using transforming growth factor β (TGF-β). EVs were then isolated from ADMSCs and co-cultured with HSCs. The relationship between miR-150-5p and CXCL1 was identified using dual luciferase gene reporter assay. Following loss- and gain-function experimentation, HSC proliferation was examined by MTT assay, and levels of fibrosis-, HSC activation- and apoptosis-related genes were determined in vitro. Additionally, pathological scores, collagen volume fraction ( CVF) as well as levels of inflammation- and hepatic injury-associated genes were determined in in vivo. Down-regulated miR-150-5p and elevated CXCL1 expression levels were detected in HF tissues. ADMSCs-derived EVs transferred miR-150-5p to HSCs. CXCL1 was further verified as the downstream target gene of miR-150-5p. Moreover, ADMSCs-EVs containing miR-150-5p markedly inhibited HSC proliferation and activation in vitro. Meanwhile, in vivo experiments also concurred with the aforementioned results as demonstrated by inhibited CVF, reduced inflammatory factor levels and hepatic injury-associated indicators. Both experiments results were could be reversed by CXCL1 over-expression. Collectively, our findings indicate that ADMSCs-derived EVs containing miR-150-5p attenuate HF by inhibiting the CXCL1 expression.     

Neuronal miR-17-5p contributes to interhemispheric cortical connectivity defects induced by prenatal alcohol exposure

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LncRNA lnc-ISG20 promotes renal fibrosis in diabetic nephropathy by inducing AKT phosphorylation through miR-486-5p/NFAT5

Long non-coding RNA (lncRNA) lnc-ISG20 has been found aberrantly up-regulated in the glomerular in the patients with diabetic nephropathy (DN). We aimed to elucidate the function and regulatory mechanism of lncRNA lnc-ISG20 on DN-induced renal fibrosis. Expression patterns of lnc-ISG20 in kidney tissues of DN patients were determined by RT-qPCR. Mouse models of DN were constructed, while MCs were cultured under normal glucose (NG)/high glucose (HG) conditions. The expression patterns of fibrosis marker proteins collagen IV, fibronectin and TGF-β1 were measured with Western blot assay. In addition, the relationship among lnc-ISG20, miR-486-5p, NFAT5 and AKT were analysed using dual-luciferase reporter assay and RNA immunoprecipitation. The effect of lnc-ISG20 and miR-486/NFAT5/p-AKT axis on DN-associated renal fibrosis was also verified by means of rescue experiments. The expression levels of lnc-ISG20 were increased in DN patients, DN mouse kidney tissues and HG-treated MCs. Lnc-ISG20 silencing alleviated HG-induced fibrosis in MCs and delayed renal fibrosis in DN mice. Mechanistically, miR-486-5p was found to be a downstream miRNA of lnc-ISG20, while miR-486-5p inhibited the expression of NFAT5 by binding to its 3'UTR. NFAT5 overexpression aggravated HG-induced fibrosis by stimulating AKT phosphorylation. However, NFAT5 silencing reversed the promotion of in vitro and in vivo fibrosis caused by lnc-ISG20 overexpression. Our collective findings indicate that lnc-ISG20 promotes the renal fibrosis process in DN by activating AKT through the miR-486-5p/NFAT5 axis. High-expression levels of lnc-ISG20 may be a useful indicator for DN.     

Exosomal miR-21-5p derived from cisplatin-resistant SKOV3 ovarian cancer cells promotes glycolysis and inhibits chemosensitivity of its progenitor SKOV3 cells by targeting PDHA1

Ovarian cancer (OC) is a common reason for gynecologic cancer death. Standard treatments of OC consist of surgery and chemotherapy. However, chemoresistance should be considered. Exosomal miR-21-5p has been shown to regulate the chemosensitivity of cancer cells through regulating pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1). However, the role of miR-21-5p/PDHA1 in OC is unclear. The levels of miR-21-5p and PDHA1 in clinical samples and cells were investigated. Exosomes derived from SKOV3/cisplatin (SKOV3/DDP) cells (DDP-Exos) were isolated and used to treat SKOV3 cells to test DDP-Exos effects on SKOV3 cells. Extracellular acidification rate and oxygen consumption rate were tested with a Seahorse analyzer. Cell apoptosis was analyzed by a flow cytometer. PDHA1 was overexpressed and miR-21-5p was silenced in SKOV3 cells to study the underlying mechanism of miR-21-5p in OC. Quantitative real-time PCR and immunoblots were applied to measure gene expression at mRNA and protein levels. The levels of PDHA1 in DDP-resistant SKOV3 or tumor tissues were significantly decreased while the levels of miR-21-5p were remarkably upregulated. miR-21-5p in DDP-Exos was sharply increased compared to that of Exos. Data also indicated that DDP-Exos treatment suppressed the sensitivity of SKOV3 cells to DDP and promoted cell viability and glycolysis of SKOV3 cells through inhibiting PDHA1 by exosomal miR-21-5p. miR-21-5p derived from DDP-resistant SKOV3 OC cells promotes glycolysis and inhibits chemosensitivity of its progenitor SKOV3 cells by targeting PDHA1. Our data highlights the important role of miR-21-5p/PDHA1 axis in OC and sheds light on new therapeutic development.     

LncRNA-ATB regulates epithelial-mesenchymal transition progression in pulmonary fibrosis via sponging miR-29b-2-5p and miR-34c-3p

Dysregulation of non-coding RNAs (ncRNAs) has been proved to play pivotal roles in epithelial-mesenchymal transition (EMT) and fibrosis. We have previously demonstrated the crucial function of long non-coding RNA (lncRNA) ATB in silica-induced pulmonary fibrosis-related EMT progression. However, the underlying molecular mechanism has not been fully elucidated. Here, we verified miR-29b-2-5p and miR-34c-3p as two vital downstream targets of lncRNA-ATB. As opposed to lncRNA-ATB, a significant reduction of both miR-29b-2-5p and miR-34c-3p was observed in lung epithelial cells treated with TGF-β1 and a murine silicosis model. Overexpression miR-29b-2-5p or miR-34c-3p inhibited EMT process and abrogated the pro-fibrotic effects of lncRNA-ATB in vitro. Further, the ectopic expression of miR-29b-2-5p and miR-34c-3p with chemotherapy attenuated silica-induced pulmonary fibrosis in vivo. Mechanistically, TGF-β1-induced lncRNA-ATB accelerated EMT as a sponge of miR-29b-2-5p and miR-34c-3p and shared miRNA response elements with MEKK2 and NOTCH2, thus relieving these two molecules from miRNA-mediated translational repression. Interestingly, the co-transfection of miR-29b-2-5p and miR-34c-3p showed a synergistic suppression effect on EMT in vitro. Furthermore, the co-expression of these two miRNAs by using adeno-associated virus (AAV) better alleviated silica-induced fibrogenesis than single miRNA. Approaches aiming at lncRNA-ATB and its downstream effectors may represent new effective therapeutic strategies in pulmonary fibrosis.     

Pseudogene HSPB1P1 contributes to renal cell carcinoma proliferation and metastasis by targeting miR-296-5p to regulate HMGA1 expression

With the aid of next-generation sequencing technology, pseudogenes have been widely recognized as functional regulators in the development and progression of certain diseases, especially cancer. Our present study aimed to investigate the functions and molecular mechanisms of HSPB1-associated protein 1 pseudogene 1 (HSPB1P1) in renal cell carcinoma (RCC). HSPB1P1 expression at the mRNA levels was determined by quantitative real-time polymerase chain reaction, and its clinical significance was assessed. Cell viability was detected by Cell Counting Kit-8 assay. Cell migration and invasion were detected by transwell assays. The location of HSPB1P1 in RCC cells was detected by subcellular distribution analysis. The direct relationship between HSPB1P1 and miR-296-5p/HMGA1 axis was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Our results identify the elevated expression of HSPB1P1 in RCC tissues and cell lines, which predicted advanced progression and poor prognosis in patients with RCC. Knockdown of HSPB1P1 suppressed cell proliferation, migration, and invasion, and reversed epithelial–mesenchymal transition process in RCC. HSPB1P1 was mostly enriched in the cytoplasm and functioned as a miRNA sponge for miR-296-5p and then regulated high-mobility group A1 expression. In conclusion, our study indicated that HSPB1P1 contributed to RCC progression by targeting the miR-296-5p/HMGA1 axis, and should be considered as a promising biomarker and therapeutic target for clinical applications.     

miR-145-5p attenuates inflammatory response and apoptosis in myocardial ischemia-reperfusion injury by inhibiting (NADPH) oxidase homolog 1

Myocardial ischemia-reperfusion (I/R) injury is a common complication following reperfusion therapy that involves a series of immune or apoptotic reactions. Studies have revealed the potential roles of miRNAs in I/R injury. Herein, we established a myocardial I/R model in rats and a hypoxia/reoxygenation (H/R) model in H9c2 cells and investigated the effect of miR-145-5p on myocardial I/R injury. After 3 h or 24 h of reperfusion, left ventricular end-systolic pressure (LVESP), ejection fraction (EF), and fractional shortening (FS) were obviously decreased, and left ventricular end-diastolic pressure (LVEDP) was increased. Meanwhile, I/R induced an increase in myocardial infarction area. Moreover, a decrease in miR-145-5p and increase in (NADPH) oxidase homolog 1 (NOH-1) were observed following I/R injury. With this in mind, we performed a luciferase reporter assay and demonstrated that miR-145-5p directly bound to NOH-1 3’ untranslated region (UTR). Furthermore, miR-145-5p mimics decreased the levels of tumor necrosis factor (TNF)-α, IL-1β, and IL-6 via oxygen and glucose deprivation/reperfusion (OGD/R) stimulation. Upregulation of miR-145-5p increased cell viability and reduced apoptosis accompanied by downregulation of Bax, cleaved caspase-3, cleaved poly(ADP-ribose) polymerase (PARP) and upregulation of Bcl2. In addition, miR-145-5p overexpression increased superoxide dismutase (SOD) activity and reduced reactive oxygen species (ROS) and malondialdehyde (MDA) content under OGD/R stress. Notably, NOH-1 could significantly abrogate the above effects, suggesting that it is involved in miR-145-5p-regulated I/R injury. In summary, our findings indicated that miR-145-5p/NOH-1 has a protective effect on myocardial I/R injury by inhibiting the inflammatory response and apoptosis.     

PTEN improve renal fibrosis in vitro and in vivo through inhibiting FAK/AKT signaling pathway

Renal fibrosis, the ultimate common pathway of progressive nephropathy, is characterized by excess accumulation and deposition of extracellular matrix (ECM) within the renal interstitium and glomeruli, finally resulting in end-stage kidney failure. TGFβ1 is not only abnormally increased during fibrosis but also involved in ECM induction and accumulation. Based on the bioinformative analyses, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and focal adhesion kinase (FAK) signaling pathway might be involved in TGFβ1 functions on renal fibrosis development. In the present study, fibrosis was induced in HK-2 cells using TGFβ1 and PTEN expression was significantly suppressed by 24 or 48 hours TGFβ1 treatment. PTEN overexpression in HK-2 cells improved TGFβ1-induced fibrosis within α-SMA and E-cadherin. According to the KEGG signaling pathway annotation analyses on microarray profiles (GSE23338 and GSE20247) and immunoblotting validation, FAK signaling might be involved in PTEN functions in TGFβ1-induced fibrosis. PTEN overexpression significantly inhibited TGFβ1- or unilateral ureteral obstruction (UUO)-induced FAK signaling pathway activation both in vitro and in vivo; more importantly, PTEN silence enhanced TGFβ1- or UUO-induced fibrosis, while FAK inhibitor PF567721 significantly reversed the effects of PTEN silence, indicating that PTEN exerted its effects on TGFβ1- and UUO-induced fibrotic development in vitro and in vivo via inhibiting FAK signaling pathway. In summary, these findings indicate that PTEN could improve cellular fibrotic changes and renal fibrosis via inhibiting FAK/AKT signaling pathway. Restoring PTEN expression to target FAK/AKT signaling pathway might be a potent strategy for renal fibrosis treatment.     

Ablation of Gadd45β ameliorates the inflammation and renal fibrosis caused by unilateral ureteral obstruction

The growth arrest and DNA damage‐inducible beta (Gadd45β) protein have been associated with various cellular functions, but its role in progressive renal disease is currently unknown. Here, we examined the effect of Gadd45β deletion on cell proliferation and apoptosis, inflammation, and renal fibrosis in an early chronic kidney disease (CKD) mouse model following unilateral ureteral obstruction (UUO). Wild‐type (WT) and Gadd45β‐knockout (KO) mice underwent either a sham operation or UUO and the kidneys were sampled eight days later. A histological assay revealed that ablation of Gadd45β ameliorated UUO‐induced renal injury. Cell proliferation was higher in Gadd45β KO mouse kidneys, but apoptosis was similar in both genotypes after UUO. Expression of pro‐inflammatory cytokines after UUO was down‐regulated in the kidneys from Gadd45β KO mice, whereas UUO‐mediated immune cell infiltration remained unchanged. The expression of pro‐inflammatory cytokines in response to LPS stimulation decreased in bone marrow‐derived macrophages from Gadd45β KO mice compared with that in WT mice. Importantly, UUO‐induced renal fibrosis was ameliorated in Gadd45β KO mice unlike in WT mice. Gadd45β was involved in TGF‐β signalling pathway regulation in kidney fibroblasts. Our findings demonstrate that Gadd45β plays a crucial role in renal injury and may be a therapeutic target for the treatment of CKD.     

Neural stem cell-derived exosomes suppress neuronal cell apoptosis by activating autophagy via miR-374-5p/STK-4 axis in spinal cord injury

Objectives:To evaluate the roles of MicroRNAs (miRNAs) enclosed in the neuron-derived exosomes in the recovery of the spinal cord injury (SCI) and the mechanism.Methods:The exosomes were isolated from neural stem cells (NSCs) and characterized by transmission electron microscopy (TEM) and NanoSight system (NTA). For in vivo experiments, Basso Mouse Scale, beam walking, and inclined plane tests were used to determine the behavioral symptoms of the SCI mice. For in vitro experiments, H₂O₂ treated HT22 cells were used to simulate SCI cells and cocultured with exosomes to analyze the cell apoptosis using TUNEL assays and flow cytometry. Apoptosis- and autophagy-related protein expression was detected by western blot and the green fluorescent protein (GFP)-LC3 assay was used to detect the level of autophagy. In addition, luciferase assay was performed to assess the relationship between miR-374-5p and SKT-4.Results:Exosomes from NSCs alleviated spinal cord injury by triggering autophagy flux and suppressing apoptosis. Besides, miR-374-5p was highly expressed in these exosomes and was responsible for the decent in injured neural cell apoptosis by activating autophagy flux. The SKT-4 was the target gene regulated by miR-374-5p in this exosomal protective function to SCI cells.Conclusion:The elevated level of miR-374-5p in neuronal exosomes could enhance spinal cord injury recovery by activating autophagy.     

XSSJS inhibits hepatic fibrosis by promoting the miR-29b-3p/VEGFA axis in vitro and in vivo

Hepatic pathological angiogenesis (HPA) is the key event of hepatic fibrosis (HF). Xueshisanjia powder (XSSJS), a Chinese herbal compound, is beneficial for alleviating pathological angiogenesis of hepatic tissue. The present study attempts to reveal the effect and mechanism of XSSJS via regulating miR-29b-3p/VEGFA axis against pathological angiogenesis in HF. In in vitro model, human embryonic kidney 293T cells were transfected with miR-29b-3p mimics, whereby the expression of miR-29b-3p was tested by real-time quantitative polymerase chain reaction (RT-qPCR), ensued by Luciferase assay determining the relationship between miR-29b-3p and vascular endothelial cell growth factor A (VEGFA). In addition, miR-29b-3p mimic transfected into the activated hepatic stellate cell T6 (HSC-T6). The Cell-Counting-Kit 8 (CCK8) and 5-Bromodeoxyuridine (BrdU) staining were first utilized to detect the antiproliferative efficiency of XSSJS following the XSSJS compound serum intervention, and then used to observe the expression of transforming growth factor-β (TGF-β), VEGFA, platelet-derived growth factor (PDGF) via RT-PCR, Western blot (WB), and Immunofluorescence (IF) methods. During the in vivo model, XSSJS with boil-free granules were fed to Wistar rats with liver fibrosis caused by intraperitoneal injection of pig serum followed by the transfection of miR-29b-3p adeno-associated virus (AAV). Hematoxylin–Eosin (HE) staining was used for histopathology assessment. The expression of miR-29b-3p, VEGFA, PDGF, TGF-β have been investigated in liver tissue using RT-PCR, WB, IF. The results verified that XSSJS could up-regulate miR-29b-3p and suppress the expression of VEGFA, PDGA, and TGF-β. In mechanism, miR-29b-3p primarily targeted the 3′UTR of VEGFA. In conclusion, XSSJS could modulate miR-29b-3p/VEGFA axis to inhibit the pathological angiogenesis of HF.     

Bone marrow macrophage‐derived exosomal miR‐143‐5p contributes to insulin resistance in hepatocytes by repressing MKP5

ObjectiveIn this study, we aim to explore the role of bone marrow macrophage‐derived exosomes in hepatic insulin resistance, investigate the substance in exosomes that regulates hepatic insulin signalling pathways, reveal the specific molecular mechanisms involved in hepatic insulin resistance and further explore the role of exosomes in type 2 diabetes.Materials and methodsHigh‐fat diet (HFD)‐fed mice were used as obesity‐induced hepatic insulin resistance model, exosomes were isolated from BMMs which were extracted from HFD‐fed mice by ultracentrifugation. Exosomes were analysed the spectral changes of microRNA expression using a microRNA array. The activation of the insulin signalling pathway and the level of glycogenesis were examined in hepatocytes after transfected with miR‐143‐5p mimics. Luciferase assay and western blot were used to assess the target of miR‐143‐5p.ResultsBMMs from HFD‐fed mice were polarized towards M1, and miR‐143‐5p was significantly upregulated in exosomes of BMMs from HFD‐fed mice. Overexpression of miR‐143‐5p in Hep1‐6 cells led to decreased phosphorylation of AKT and GSK and glycogen synthesis. Dual‐luciferase reporter assay and western blot demonstrated that mitogen‐activated protein kinase phosphatase‐5 (Mkp5, also known as Dusp10) was the target gene of miR‐143‐5p. Moreover, the overexpression of MKP5 could rescue the insulin resistance induced by transfection miR‐143‐5p mimics in Hep1‐6.ConclusionBone marrow macrophage‐derived exosomal miR‐143‐5p induces insulin resistance in hepatocytes through repressing MKP5.     

Circulating miR-23b-3p,miR-145-5p and miR-200b-3p are potential biomarkers to monitor acute pain associated with laminitis in horses

Circulating microRNAs (miRNAs) are emerging as promising biomarkers for several disorders and related pain. In equine practice, acute laminitis is a common disease characterised by intense pain that severely compromises horse welfare. Recently, the Horse Grimace Scale (HGS), a facial expression-based pain coding system, was shown to be a valid welfare indicator to identify pain linked to acute laminitis. The present study aimed to: determine whether miRNAs can be used as biomarkers for acute pain in horses (Equus caballus) affected by laminitis; integrate miRNAs to their target genes and to categorise target genes for biological processes; gather additional evidence on concurrent validity of HGS by investigating how it correlates to miRNAs. Nine horses presenting acute laminitis with no prior treatment were recruited. As control group, nine healthy horses were further included in the experimental design. Samples were collected from horses with laminitis at admission before any treatment (‘pre-treatment’) and 7 days after routine laminitis treatment (‘post-treatment’). The expression levels of nine circulating miRNAs, namely hsa-miR-532-3p, hsa-miR-219-5p, mmu-miR-134-5p, mmu-miR-124a-3p, hsa-miR-200b-3p, hsa-miR-146a-5p, hsa-miR-23b-3p, hsa-miR-145-5p and hsa-miR-181a-5p, were detected and assessed as potential biomarkers of pain by quantitative PCR using TaqMan® probes. The area under the receiver operating curve (AUC) was then used to evaluate the diagnostic performance of miRNAs. Molecular data were integrated with HGS scores assessed by one trained treatment and time point blind veterinarian. The comparative analysis demonstrated that the levels of miR-23b-3p (P=0.029), miR-145-5p (P=0.015) and miR-200b-3p (P=0.023) were significantly higher in pre-treatment and the AUCs were 0.854, 0.859 and 0.841, respectively. MiR-200b-3p decreased after routine laminitis treatment (P=0.043). Combining two miRNAs in a panel, namely miR-145-5p and miR-200b-3p, increased efficiency in distinguishing animals with acute pain from controls. In addition, deregulated miRNAs were positively correlated to HGS scores. Computational target prediction and functional enrichment identified common biological pathways between different miRNAs. In particular, the glutamatergic pathway was affected by all three miRNAs, suggesting a crucial role in the pathogenesis of pain. In conclusion, the dynamic expression of circulating miR-23b-3p, miR-145-5p and miR-200b-3p was detected in horses with acute laminitis and miRNAs can be considered potentially promising pain biomarkers. Further studies are needed in order to assess their relevancy in other painful conditions severely compromising horse welfare. An important implication would be the possibility to use them for the concurrent validation of non-invasive indicators of pain in horses.     

Inflammation-dependent downregulation of miR-194-5p contributes to human intervertebral disc degeneration by targeting CUL4A and CUL4B

Inflammation is one of the major causes of intervertebral disc degeneration (IDD). Emerging evidence has revealed that increase in the levels of pro-inflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α), can activate a variety of signaling pathways, eventually resulting in IDD. Here, we show that the two cullin family genes, CUL4A and CUL4B, but not other cullins, are specifically overexpressed in IDD samples compared with healthy controls, and the CUL4A and CUL4B levels are positively correlated with the severity of IDD. In vitro analyses in human osteoblast cells (hFOB1.19), nucleus pulposus cells (hNPCs), and annulus fibrosus cells (hAFCs) indicated that treatment with IL-6 and TNF-α can increase CUL4A and CUL4B levels. By performing a microRNA-based microarray analysis, we found a set of microRNAs (miRNAs) that were differentially expressed in IDD samples compared with samples from healthy controls. Of these miRNAs, miR-194-5p, was significantly downregulated in IDD samples and could bind to the three prime untranslated regions (3′-UTRs) of both CUL4A and CUL4B, thereby downregulating their expression. The in vitro overexpression or downregulation of miR-194-5p, with a miR-194-5p-mimic or with anti-miR-194-5p, can cause the repression or induction of both CUL4A and CUL4B, respectively. Interestingly, treatment with IL-6 and TNF-α inhibitors in primary hNPCs and hAFCs that were isolated from patients with IDD led to the downregulation of CUL4A and CUL4B. Together, these findings provide insight into how the inflammation-dependent downregulation of miR-194-5p contributes to the pathogenesis of IDD, which may aid in the development of new therapeutic approaches for IDD by directly targeting miR-194-5p or CUL4A and CUL4B.     

Circ_0080425 inhibits cell proliferation and fibrosis in diabetic nephropathy via sponging miR-24-3p and targeting fibroblast growth factor 11

Diabetic nephropathy (DN) is a severe end-stage kidney disease developed from diabetes mellitus. The involvement of circular RNAs (circRNAs) in modulating DN pathogenesis has been implied, but underlying mechanism is still lacking. In this study, we demonstrated that the expression of circ_0080425 correlated with the DN progression, and exerted positive effect on cell proliferation and fibrosis in mesangial cells. Further assessment suggested that circ_0080425 function as sponge harboring miR-24-3p. Moreover, miR-24-3p negatively correlated with the DN progression, and showed an antagonistic effect to circ_0080425on regulating MCs cell proliferation and fibrosis. Bioinformatics analysis predicted fibroblast growth factor 11 (FGF11) acting as direct downstream target of miR-24-3p. Indeed, the expression of FGF11 was significantly activated by circ_0080425 while suppressed by miR-24-3p. Knockdown of FGF11 resulted in a significant reduced cell proliferation rate and fibrosis. In addition, miR-24-3p inhibitor rescued the suppression of si-circ_0080425 on FGF11, suggesting that circ_0080425 competitive binding to miR-24-3p could release FGF11 from miR-24-3p suppression, which subsequently promoted DN progression.In conclusion, we have reported a novel circ_0080425-miR-24-3p-FGF11 axis, and explored the underlying mechanism in regulating DN pathogenesis.