MicroRNA expression profile of human periodontal ligament cells under the influence of Porphyromonas gingivalis LPS

Periodontitis is a chronic inflammatory disease which is caused by bacterial infection and leads to the destruction of periodontal tissues and resorption of alveolar bone. Thus, special attention should be paid to the mechanism under lipopolysaccharide (LPS)‐induced periodontitis because LPS is the major cause of periodontitis. However, to date, miRNA expression in the LPS‐induced periodontitis has not been well characterized. In this study, we investigated miRNA expression patterns in LPS‐treated periodontal ligament cells (PDLCs). Through miRNA array and differential analysis, 22 up‐regulated miRNAs and 28 down‐regulated miRNAs in LPS‐treated PDLCs were identified. Seven randomly selected up‐regulated (miR‐21‐5p, 498, 548a‐5p) and down‐regulated (miR‐495‐3p, 539‐5p, 34c‐3p and 7a‐2‐3p) miRNAs were examined by qRT‐PCR, and the results proved the accuracy of the miRNA array. Moreover, targets of these deregulated miRNAs were analysed using the miRWalk database. Database for Annotation, Visualization and Integration Discovery software were performed to analyse the Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes pathway of differential expression miRNAs, and the results shown that Toll‐like receptor signalling pathway, cAMP signalling pathway, transforming growth factor‐beta signalling pathway, mitogen‐activated protein kinase (MAPK) signalling pathway and other pathways were involved in the molecular mechanisms underlying LPS‐induced periodontitis. In conclusion, this study provides clues for enhancing our understanding of the mechanisms and roles of miRNAs as key regulators of LPS‐induced periodontitis.     

Gastrodin relieves inflammation injury induced by lipopolysaccharides in MRC‐5 cells by up‐regulation of miR‐103

The beneficial function of gastrodin towards many inflammatory diseases has been identified. This study designed to see the influence of gastrodin in a cell model of chronic obstructive pulmonary disease (COPD). MRC‐5 cells were treated by LPS, before which gastrodin was administrated. The effects of gastrodin were evaluated by conducting CCK‐8, FITC‐PI double staining, Western blot, qRT‐PCR and ELISA. Besides this, the downstream effector and signalling were studied to decode how gastrodin exerted its function. And dual‐luciferase assay was used to detect the targeting link between miR‐103 and lipoprotein receptor‐related protein 1 (LRP1). LPS induced apoptosis and the release of MCP‐1, IL‐6 and TNF‐α in MRC‐5 cells. Pre‐treating MRC‐5 cells with gastrodin attenuated LPS‐induced cell damage. Meanwhile, p38/JNK and NF‐κB pathways induced by LPS were repressed by gastrodin. miR‐103 expression was elevated by gastrodin. Further, the protective functions of gastrodin were attenuated by miR‐103 silencing. And LRP1 was a target of miR‐103 and negatively regulated by miR‐103. The in vitro data illustrated the protective function of gastrodin in LPS‐injured MRC‐5 cells. Gastrodin exerted its function possibly by up‐regulating miR‐103 and modulating p38/JNK and NF‐κB pathways.     

Identification of C‐terminal Hsp70‐interacting protein as a mediator of tumour necrosis factor action in osteoblast differentiation by targeting osterix for degradation

In patients with inflammatory arthritis, tumour necrosis factor (TNF)‐α are overproduced in inflamed joints. This leads to local erosion of cartilage and bone, periarticular osteopenia, as well as osteoporosis. But less is known regarding the molecular mechanisms that mediate the effect of TNF‐α on osteoblast function. The purpose of this study was to test that C terminus of Hsc70‐interacting protein (CHIP) has a specific role in suppressing the osteogenic activity of osteoblasts under inflammatory conditions. C2C12, MC3T3‐E1 and HEK293T cell lines were cultured and cotransfected with related plasmids. After transfection, the cells were cultured further in the presence or absence of murine TNF‐α and subjected to real time RT‐PCR, Western blot, Ubiquitination assay, Co‐immunoprecipitation, Luciferase reporter assay, Small interfering RNAs and Mineralization assay. The expression levels of TNF‐α‐induced CHIP and Osx were examined by RT‐PCR and Western blot analysis. Co‐immunoprecipitation and ubiquitination assays revealed ubiquitinated Osx, confirmed that CHIP indeed interacted with Osx and identified K55 and K386 residues as the ubiquitination sites in Osx, Luciferase reporter assay and Small interfering RNAs examined whether TNF‐α target the bone morphogenetic protein signalling through CHIP. We established stable cell lines with the overexpression of HA‐CHIP, Mineralization assay and CHIP siRNA demonstrated the important roles of CHIP on osteoblast function in conditions in which TNF‐α is overexpressed. We found that the K55 and K386 residues are ubiquitination site(s) in Osx, and that TNF‐α inhibits osteoblast differentiation by promoting Osx degradation through up‐regulation of E3 ubiquitin ligase CHIP in osteoblast. Thus, CHIP targets Osx for ubiquitination and degradation in osteoblasts after chronic exposure to TNF‐α, and inhibition of CHIP expression in osteoblasts may be a new mechanism to limit inflammation‐mediated osteoporosis by promoting their differentiation into osteoblasts.     

miR‐212 downregulation contributes to the protective effect of exercise against non‐alcoholic fatty liver via targeting FGF‐21

Non‐alcoholic fatty liver disease (NAFLD) is associated with obesity and lifestyle, while exercise is beneficial for NAFLD. Dysregulated microRNAs (miRs) control the pathogenesis of NAFLD. However, whether exercise could prevent NAFLD via targeting microRNA is unknown. In this study, normal or high‐fat diet (HF) mice were either subjected to a 16‐week running program or kept sedentary. Exercise attenuated liver steatosis in HF mice. MicroRNA array and qRT‐PCR demonstrated that miR‐212 was overexpressed in HF liver, while reduced by exercise. Next, we investigated the role of miR‐212 in lipogenesis using HepG2 cells with/without long‐chain fatty acid treatment (±FFA). FFA increased miR‐212 in HepG2 cells. Moreover, miR‐212 promoted lipogenesis in HepG2 cells (±FFA). Fibroblast growth factor (FGF)‐21, a key regulator for lipid metabolism, was negatively regulated by miR‐212 at protein level in HepG2 cells. Meanwhile, FFA downregulated FGF‐21 both at mRNA and protein levels in HepG2 cells. Also, FGF‐21 protein level was reduced in HF liver, while reversed by exercise in vivo. Furthermore, siRNA‐FGF‐21 abolished the lipogenesis‐reducing effect of miR‐212 inhibitor in HepG2 cells (±FFA), validating FGF‐21 as a target gene of miR‐212. These data link the benefit of exercise and miR‐212 downregulation in preventing NAFLD via targeting FGF‐21.     

MiR‐101 promotes pain hypersensitivity in rats with chronic constriction injury via the MKP‐1 mediated MAPK pathway

This study was performed to characterize the effect of microRNA‐101 (miR‐101) on the pain hypersensitivity in CCI rat models with the involvement of mitogen‐activated protein kinase phosphatase 1 (MKP‐1) in spinal cord microglial cells. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in the developed CCI models were determined to assess the hypersensitivity of rats to mechanical stimulation and thermal pain. To assess inflammation, the levels of interleukin (IL)‐1β, IL‐6 and tumour necrosis factor‐α (TNF‐α) in the spinal dorsal horns of CCI rats and lipopolysaccharide (LPS)‐activated microglial cells were examined. miR‐101 and MKP‐1 gain‐ and loss‐of‐function experiments were conducted in in vivo and in vitro settings to examine the roles of miR‐101 and MKP‐1 in CCI hypersensitivity and inflammation. The results showed that miR‐101 was highly expressed in the spinal dorsal horn and microglial cells of CCI rat models. Furthermore, overexpression of miR‐101 promoted the pain hypersensitivity in CCI rat models by reducing MWT and TWL. The overexpression of miR‐101 also promoted inflammation in LPS‐exposed microglial cells, as indicated by increased levels of IL‐1β, IL‐6 and TNF‐α. MiR‐101 was shown to target MKP‐1, inhibiting its expression. Moreover, miR‐101 promoted pain hypersensitivity in CCI rat models by inhibiting MKP‐1 expression and activating the mitogen‐activated protein kinase (MAPK) signalling pathway. Taken together, miR‐101 could potentially promote hypersensitivity and inflammatory response of microglial cells and aggravate neuropathic pain in CCI rat models by inhibiting MKP‐1 in the MAPK signalling pathway.     

MicroRNA and mRNA expression profiling in metastatic melanoma reveal associations with BRAF mutation and patient prognosis

The role of microRNAs (miRNAs) in melanoma is unclear. We examined global miRNA expression profiles in fresh‐frozen metastatic melanomas in relation to clinical outcome and BRAF mutation, with validation in independent cohorts of tumours and sera. We integrated miRNA and mRNA information from the same samples and elucidated networks associated with outcome and mutation. Associations with prognosis were replicated for miR‐150‐5p, miR‐142‐3p and miR‐142‐5p. Co‐analysis of miRNA and mRNA uncovered a network associated with poor prognosis (PP) that paradoxically favoured expression of miRNAs opposing tumorigenesis. These miRNAs are likely part of an autoregulatory response to oncogenic drivers, rather than drivers themselves. Robust association of miR‐150‐5p and the miR‐142 duplex with good prognosis and earlier stage metastatic melanoma supports their potential as biomarkers. miRNAs overexpressed in association with PP in an autoregulatory fashion will not be suitable therapeutic targets.     

Alcohol‐mediated miR‐34a modulates hepatocyte growth and apoptosis

MicroRNAs (miRs) have been recently shown to be heavily involved in the development of alcoholic liver disease (ALD) and suggested as a potential therapeutic target in ALD. The miR‐34a was consistently reported to be significantly elevated in several ALD rodent models, but it remains unclear how miR‐34a modulates the cellular behaviours of hepatocytes in ALD development and progression. This study aims to characterize alcohol‐induced miR‐34a impact on hepatocytes growth and apoptosis. The miRNA array was performed to assess changes in miRNA after chronic alcohol feeding. Liver and blood samples were used to examine ALD progression. The miR‐34a was overexpressed in human hepatocytes to evaluate its impact on cell growth and apoptosis. Real‐time quantitative PCR and Western blot were used to determine the growth and apoptosis molecular signalling pathways associated with miR‐34a. Alcohol feeding significantly promoted fatty liver progression, serum ALT levels, apoptosis and miR‐34a expression in rat liver. Overexpression of miR‐34a in human hepatocytes suppressed cell growth signallings, including c‐Met, cyclin D1 and cyclin‐dependent kinase 6 (CDK6). The miR‐34a might also inhibit the expression of sirtuin 1 (Sirt1) and its target, B‐cell lymphoma 2. Interestingly, the expression of miR‐34a reverses the suppressive effects of ethanol on cell growth. But, miR‐34a promotes hepatocyte senescence and apoptosis. Although the miR‐34a‐mediated down‐regulation of cell growth‐associated genes may contribute to cell growth retardation, other miR‐34a targets, such as Sirt1, may reverse this phenotype. Future studies will be needed to clarify the role of miR‐34a in ALD progression.     

miR‐34b‐5p inhibition attenuates lung inflammation and apoptosis in an LPS‐induced acute lung injury mouse model by targeting progranulin

Inflammation and apoptosis play important roles in the initiation and progression of acute lung injury (ALI). Our previous study has shown that progranulin (PGRN) exerts lung protective effects during LPS‐induced ALI. Here, we have investigated the potential roles of PGRN‐targeting microRNAs (miRNAs) in regulating inflammation and apoptosis in ALI and have highlighted the important role of PGRN. LPS‐induced lung injury and the protective roles of PGRN in ALI were first confirmed. The function of miR‐34b‐5p in ALI was determined by transfection of a miR‐34b‐5p mimic or inhibitor in intro and in vivo. The PGRN level gradually increased and subsequently significantly decreased, reaching its lowest value by 24 hr; PGRN was still elevated compared to the control. The change was accompanied by a release of inflammatory mediators and accumulation of inflammatory cells in the lungs. Using bioinformatics analysis and RT‐PCR, we demonstrated that, among 12 putative miRNAs, the kinetics of the miR‐34b‐5p levels were closely associated with PGRN expression in the lung homogenates. The gain‐ and loss‐of‐function analysis, dual‐luciferase reporter assays, and rescue experiments confirmed that PGRN was the functional target of miR‐34b‐5p. Intravenous injection of miR‐34b‐5p antagomir in vivo significantly inhibited miR‐34b‐5p up‐regulation, reduced inflammatory cytokine release, decreased alveolar epithelial cell apoptosis, attenuated lung inflammation, and improved survival by targeting PGRN during ALI. miR‐34b‐5p knockdown attenuates lung inflammation and apoptosis in an LPS‐induced ALI mouse model by targeting PGRN. This study shows that miR‐34b‐5p and PGRN may be potential targets for ALI treatments.     

miR‐148a suppresses inflammation in lipopolysaccharide‐induced endometritis

Endometritis is a postnatal reproductive disorder disease, which leads to great economic losses for the modern dairy industry. Emerging evidence indicates that microRNAs (miRNAs) play a pivotal role in a variety of diseases and have been identified as critical regulators of the innate immune response. Recent miRNome profile analysis revealed an altered expression level of miR‐148a in cows with endometritis. Therefore, the present study aims to investigate the regulatory role of miR‐148a in the innate immune response involved in endometritis and estimate its potential therapeutic value. Here, we found that miR‐148a expression in lipopolysaccharide (LPS)‐stimulated endometrial epithelial cells was significantly decreased . Our results also showed that overexpression of miR‐148a using agomiR markedly reduced the production of pro‐inflammatory cytokines, such as IL‐1β and TNF‐α. Moreover, overexpression of miR‐148a also suppressed NF‐κB p65 activation by targeting the TLR4‐mediated pathway. Subsequently, we further verified that miR‐148a repressed TLR4 expression by binding to the 3′‐UTR of TLR4 mRNA. Additionally, an experimental mouse endometritis model was employed to evaluate the therapeutic value of miR‐148a. In vivo studies suggested that up‐regulation of miR‐148a alleviated the inflammatory conditions in the uterus as evidenced by H&E staining, qPCR and Western blot assays, while inhibition of miR‐148a had inverse effects. Collectively, pharmacologic stabilization of miR‐148a represents a novel therapy for endometritis and other inflammation‐related diseases.     

Arachidonic acid stimulates TNFα production in Kupffer cells via a reactive oxygen species-pERK1/2-Egr1-dependent mechanism

Kupffer cells are a key source of mediators of alcohol-induced liver damage such as reactive oxygen species, chemokines, growth factors, and eicosanoids. Since diets rich in polyunsaturated fatty acids are a requirement for the development of alcoholic liver disease, we hypothesized that polyunsaturated fatty acids could synergize with ethanol to promote Kupffer cell activation and TNFα production, hence, contributing to liver injury. Primary Kupffer cells from control and from ethanol-fed rats incubated with arachidonic acid showed similar proliferation rates than nontreated cells; however, arachidonic acid induced phenotypic changes, lipid peroxidation, hydroperoxides, and superoxide radical generation. Similar effects occurred in human Kupffer cells. These events were greater in Kupffer cells from ethanol-fed rats, and antioxidants and inhibitors of arachidonic acid metabolism prevented them. Arachidonic acid treatment increased NADPH oxidase activity. Inhibitors of NADPH oxidase and of arachidonic acid metabolism partially prevented the increase in oxidant stress. Upon arachidonic acid stimulation, there was a rapid and sustained increase in TNFα, which was greater in Kupffer cells from ethanol-fed rats than in Kupffer cells from control rats. Arachidonic acid induced ERK1/2 phosphorylation and nuclear translocation of early growth response-1 (Egr1), and ethanol synergized with arachidonic acid to promote this effect. PD98059, a mitogen extracellular kinase 1/2 inhibitor, and curcumin, an Egr1 inhibitor, blocked the arachidonic acid-mediated upregulation of TNFα in Kupffer cells. This study unveils the mechanism whereby arachidonic acid and ethanol increase TNFα production in Kupffer cells, thus contributing to alcoholic liver disease.     

Salvianic acid A alleviates chronic alcoholic liver disease by inhibiting HMGB1 translocation via down‐regulating BRD4

Alcoholic liver disease (ALD) is the major cause of chronic liver disease and a global health concern. ALD pathogenesis is initiated with liver steatosis, and ALD can progress to steatohepatitis, fibrosis, cirrhosis and even hepatocellular carcinoma. Salvianic acid A (SAA) is a phenolic acid component of Danshen, a Chinese herbal medicine with possible hepatoprotective properties. The purpose of this study was to investigate the effect of SAA on chronic alcoholic liver injury and its molecular mechanism. We found that SAA significantly inhibited alcohol‐induced liver injury and ameliorated ethanol‐induced hepatic inflammation. These protective effects of SAA were likely carried out through its suppression of the BRD4/HMGB1 signalling pathway, because SAA treatment largely diminished alcohol‐induced BRD4 expression and HMGB1 nuclear translocation and release. Importantly, BRD4 knockdown prevented ethanol‐induced HMGB1 release and inflammatory cytokine production in AML‐12 cells. Similarly, alcohol‐induced pro‐inflammatory cytokines were blocked by HMGB1 siRNA. Collectively, our results reveal that activation of the BRD4/HMGB1 pathway is involved in ALD pathogenesis. Therefore, manipulation of the BRD4/HMGB1 pathway through strategies such as SAA treatment holds great therapeutic potential for chronic alcoholic liver disease therapy.     

MicroRNAs in alcoholic liver disease: Recent advances and future applications

Alcoholic liver disease (ALD) is characterized by hepatocyte damage, inflammatory cell activation, and increased intestinal permeability leading to the clinical manifestations of alcoholic hepatitis. Selected members of the family of microRNAs (miRNAs) are affected by alcohol, resulting in an abnormal miRNA profile in the liver and circulation in ALD. Increasing evidence suggests that miRNAs that regulate inflammation, lipid metabolism and promote cancer are affected by excessive alcohol administration in mouse models of ALD. This communication highlights recent findings in miRNA expression and functions as they relate to the pathogenesis of ALD. The cell-specific distribution of miRNAs, as well as the significance of circulating extracellular miRNAs, is discussed as potential biomarkers. Finally, the prospects of miRNA-based therapies are evaluated in ALD.     

Activation of TRAIL‐DR5 pathway promotes sensorineural degeneration in the inner ear

Tumor necrosis factor (TNF) family cytokines are important mediators of inflammation. Elevated levels of serum TNF‐α are associated with human sensorineural hearing loss via poorly understood mechanisms. We demonstrate, for the first time, expression of TNF‐related apoptosis‐inducing ligand (TRAIL) and its signaling death receptor 5 (DR5) in the murine inner ear and show that exogenous TRAIL can trigger hair cell and neuronal degeneration, which can be partly prevented with DR5‐blocking antibodies.     

MiR‐802 causes nephropathy by suppressing NF‐κB‐repressing factor in obese mice and human

Obesity is associated with significant microvascular complications including renal injuries and may induce end‐stage renal disease. Emerging studies have demonstrated microRNAs (miRNAs) are potential mediators in the pathophysiological process of nephropathy. The present study aimed to investigate the role of miR‐802 in obesity‐related nephropathy and potential molecular mechanisms. Through utilizing obese mouse model and human subjects, we explored the therapeutic benefits and clinical application of miR‐802 in protecting against nephropathy. Renal miR‐802 level was positively correlated with functional parameters, including blood urea nitrogen and creatinine in obese mice. Specific silencing of renal miR‐802 improved high fat diet (HFD)‐induced renal dysfunction, structural disorders and fibrosis. The up‐regulated inflammatory response and infiltrated macrophages were also significantly decreased in miR‐802 inhibitor‐treated obese mice. Mechanistically, miR‐802 directly bond to 3?‐UTR of NF‐κB‐repressing factor (NRF) and suppressed its expression. In clinical study, the circulating miR‐802 level was significantly increased in obese subjects, and positively correlated with plasma creatinine level but negatively correlated with creatinine clearance. Taken together, our findings provided evidence that miR‐802/NRF signalling was an important pathway in mediating obesity‐related nephropathy. It is a possible useful clinical approach of treating miR‐802 inhibitor to combat nephropathy.     

Recent insights into the role of the innate immune system in the development of alcoholic liver disease

The innate immune system is responsible for the rapid, initial response of the organism to potentially dangerous stresses, including pathogens, tissue injury, and malignancy. Pattern-recognition receptors of the toll-like receptor (TLR) family expressed by macrophages provide a first line of defense against microbial invasion. Activation of these receptors results in a stimulus-specific expression of genes required to control the infection, including the production of inflammatory cytokines and chemokines, followed by the recruitment of neutrophils to the site of infection. The early stages in the development of alcoholic liver disease (ALD) follow a pattern characteristic of an innate immune response. Kupffer cells, the resident macrophages in the liver, are activated in response to bacterial endotoxins (lipopolysaccharide, LPS), leading to the production of inflammatory and fibrogenic cytokines, reactive oxygen species, as well as the recruitment of neutrophils to the liver. One mechanism by which chronic ethanol can turn the highly regulated innate immune response into a pathway of disease is by disrupting the signal transduction cascades mediating the innate immune response. Recent studies have identified specific modules in the TLR-4 signaling cascade that are disrupted after chronic ethanol exposure, including CD14 and the mitogen-activated protein kinase family members, ERK1/2 and p38. Enhanced activation of these TLR-4 dependent signaling pathways after chronic ethanol likely contributes to the development of alcoholic liver disease.