Target Gene Of miR-146a Research Articles

Ghrelin protects against obesity-induced myocardial injury by regulating the lncRNA H19/miR-29a/IGF-1 signalling axis

BackgroundObesity is associated with the impairment of cardiac fitness and consequent ventricular dysfunction and heart failure. Ghrelin has been largely documented to be cardioprotective against ischaemia/reperfusion injury. However, the role of ghrelin in obesity-induced myocardial injury is largely unknown. This study sought to determine the cardiac effect of ghrelin against obesity-induced injury and the underlying mechanisms. MethodsThe effect of ghrelin was evaluated in a mouse model of obesity and a palmitic acid (PA)-treated cardiomyocyte cell line with or without ghrelin transfection. Gene and protein expression levels were determined by real-time PCR and western blot, respectively. Cell apoptosis was measured by flow cytometry analysis. ResultsIn the present study, we found that both a high-fat diet (HFD) and PA treatment caused myocardial injury by increasing apoptosis and the expression of inflammatory cytokines. Overexpression of ghrelin reversed the effects induced by HFD or PA treatment. Knockdown of lncRNA H19 or overexpression of miR-29a abrogated the cardioprotective effects of ghrelin against apoptosis and inflammation. We also found that IGF-1 was a target gene of miR-29a and that H19 regulated IGF-1 expression via miR-29a. Overexpression of IGF-1 partially reversed the apoptosis and inflammation promoting effects of miR-29a. ConclusionsOur findings suggested that ghrelin protected against obesity-induced myocardial injury by regulating the H19/miR-29a/IGF-1 signalling axis, providing further evidence for the clinical application of ghrelin.

The protective effects of microRNA-26a in steroid-induced osteonecrosis of the femoral head by repressing EZH2

ABSTRACT Recently, the role of microRNAs (miRs) in human diseases has been verified. This study was determined to explore the protective effects of microRNA-26a (miR-26a) in steroid-induced osteonecrosis of the femoral head (SONFH) with the involvement of enhancer of zeste homologue 2 (EZH2). Femoral head (FH) samples from SONFH patients and patients with femoral neck fracture were collected, and rat SONFH models were established by Escherichia coli endotoxin combining with large dose steroid pulse assay. The hemorheology, blood lipid, inflammatory factors, and pathologic changes were measured by a series of experiments. Moreover, the detection of osteoblasts, osteoclasts, miR-26a expression, EZH2 expression, osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL), and the apoptosis of osteocytes were conducted. The target relation between miR-26a and EZH2 was clarified by bioinformatics and dual-luciferase reporter gene assay. MiR-26a was poorly expressed, while EZH2 was highly expressed in SONFH, and the elevation of miR-26a could repress EZH2 expression. Elevated miR-26a and reduced EZH2 were able to decelerate the apoptosis of osteocytes, increase osteoblasts, and decrease osteoclasts, resulting in a repression of SONFH progression. Additionally, EZH2 was a target gene of miR-26a. Furthermore, the elevation of EZH2 could reverse the repression of SONFH progression that is induced by elevated miR-26a. We found that up-regulation of miR-26a and knockdown of EZH2 could suppress the development of SONFH, which would contribute to the therapy of SONFH.

CUL4B contributes to cancer stemness by repressing tumor suppressor miR34a in colorectal cancer

Given that colorectal cancer stem cells (CCSCs) play key roles in the tumor dormancy, metastasis, and relapse, targeting CCSCs is a promising strategy in cancer therapy. Here, we aimed to identify the new regulators of CCSCs and found that Cullin 4B (CUL4B), which possesses oncogenic properties in multiple solid tumors, drives the development and metastasis of colon cancer by sustaining cancer stem-like features. Elevated expression of CUL4B was confirmed in colon tumors and was associated with poor overall survival. Inhibition of CUL4B in cancer cell lines and patient-derived tumor organoids led to reduced sphere formation, proliferation and metastasis capacity. Mechanistically, CUL4B coordinates with PRC2 complex to repress miR34a expression, thus upregulates oncogenes including MYCN and NOTCH1, which are targeted by miR34a. Furthermore, we found that elevated CUL4B expression is associated with miR34a downregulation and upregulation of miR34a target genes in colon cancer specimens. Collectively, our findings demonstrate that CUL4B functions to repress miR34a in maintaining cancer stemness in CRC and provides a potential therapeutic target.

MiR-34a Suppression Ameliorates Bone Marrow Mesenchymal Stem Cell Senescence by Targeting Nampt and Regulating NAD <sup>+</sup>-Sirt1 Pathway

Background: Expansion-mediated replicative senescence and age-related natural senescence have adverse effects on MSC regenerative capability and functionality, thus severely impairing the extensive applications of MSC-based therapies. Emerging evidences suggest that miR-34a has been implicated in the process of MSC senescence; however, the molecular mechanisms with regard to how miR-34a influencing MSC senescence remain largely undetermined. Methods: We undertake a systematic study on the functional effects of miR-34a exerting on MSC senescence via gene manipulation. Mechanistically, bioinformatics analysis and dual-luciferase reporter gene assay were applied to decipher the interactions between miR-34a and its potential target Nampt. Findings: miR-34a overexpression in young MSCs resulted in senescence-associated characteristics as displayed by senescence-like morphology, prolonged cell proliferation, declined osteogenic differentiation potency, heightened senescence-associated-β- galactosidase activity, and up-regulated expression levels of the senescence-associated factors. Conversely, miR-34a suppression in replicative senescent and natural senescent MSCs contributed to diminished senescence-related phenotypic features. We identified Nampt as a direct target gene of miR-34a. Additionally, miR-34a repletion resulted in prominent reductions in Nampt expression levels, NAD+ content, NAD+/NADH ratio, and Sirt1 activity, whereas anti-miR-34a treatment exerted the opposite effects. Furthermore, miR-34a-mediated MSC senescence was evidently rescued following the co-treatment with Nampt overexpression. Interpretation: This study identifies a significant role of miR-34a playing in MSC replicative senescence and natural senescence via targeting Nampt and further mediating by NAD+-Sirt1 pathway, possibly carrying great implications for optimal strategies for MSC therapeutic applications. Funding Statement: This study was supported by projects of National Natural Science Foundation of China (Grant number: 81571370), Jilin Province Science and Technology Development Projects (Grant number: 20150414029GH), Science and Technology Projects of the Education Department of Jilin Province (Grant Number: JJKH20190007KJ) and the Fundamental Research Funds for Central Universities, JLU. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Experimental investigations involving animals were permitted by the Institutional Animal Care and the Ethic Committee of Jilin University (permit number: SYXK 2013-0005) and were conducted in compliance with the ethical standards and regulations, the Declaration of Helsinki and national and international guidelines.

MiR-34a Regulates Nasopharyngeal Carcinoma Radiosensitivity by Targeting SIRT1

Background/Aims:Nasopharyngeal carcinoma is a common head and neck cancer in South China and Southeast Asia. Radiotherapy is the standard treatment for nasopharyngeal carcinoma. Accumulating evidence showed that the expression of miR-34a was abnormal in nasopharyngeal carcinoma. Here, this study investigates the effect of miR-34a on radiosensitivity of nasopharyngeal carcinoma cells and explored the underlying mechanisms.Methods:Reverse transcription quantitative polymerase chain reaction was used to analyze the expression of miR-34a in nasopharyngeal carcinoma cell lines and NP69 cells. The effect of miR-34a on radiosensitivity of nasopharyngeal carcinoma (CNE-1 cells) was evaluated by Cell Counting Kit-8, flow cytometry, and Transwell migration assays following transfection with miR-34a mimic. Luciferase reporter assay was used to assess the target genes of miR-34a.Results:In this study, it revealed that miR-34a was downregulated, while silent information regulator 1 was upregulated in nasopharyngeal carcinoma cell lines. The overexpression of miR-34a enhanced radiation-induced proliferation and migration inhibition and apoptosis in CNE-1 cells. Bioinformatics, Luciferase reporter, reverse transcription quantitative polymerase chain reaction, and Western blotting assays indicated that silent information regulator 1 is a direct target of miR-34a in nasopharyngeal carcinoma cells. Knockdown of silent information regulator 1 enhanced radiosensitivity of nasopharyngeal carcinoma cells as evidenced by increasing proliferation and migration inhibition and apoptosis after radiation exposure.Conclusion:In summary, our results indicated that the overexpression of miR-34a enhanced radiosensitivity of nasopharyngeal carcinoma cells by targeting silent information regulator 1. Further studies are warranted to investigate the potential use of miR-34a in the clinical management and treatment prediction of patients with nasopharyngeal carcinoma.

Inhibition of microRNA-29a alleviates hyperoxia-induced bronchopulmonary dysplasia in neonatal mice via upregulation of GAB1

BackgroundThe main features of bronchopulmonary dysplasia (BPD) are alveolar simplification, pulmonary growth arrest, and abnormal lung function. Multiple studies have highlighted microRNA-29 (miR-29) as a potential biomarker for lung diseases and cancers. Upregulation of miR-29a has been known to downregulate GRB2-associated-binding protein 1 (GAB1), which is often highly expressed in the lung. The current study was designed to investigate the potential role of miR-29a in hyperoxia-induced BPD by targeting GAB1 in a neonatal mouse model.MethodsThe expression of miR-29a and GAB1 in lung tissues of neonatal mice with hyperoxia-induced BPD and mouse alveolar epithelial cells (MLE-12) was determined using RT-qPCR and western blot analysis. Subsequently, the relationship between miR-29a and GAB1 was verified using in silico analysis. In order to assess the effects of miR-29a or GAB1 on BPD, the pathological characteristics of alveoli, as well as proliferation and apoptosis of cells were measured through gain- and loss-of-function studies.ResultsUpregulation of miR-29a and downregulation of GAB1 were evident in both lung tissues and MLE-12 cells following BPD modeling. GAB1 was a direct target gene of miR-29a. Inhibition of miR-29a and overexpression of GAB1 were shown to alleviate lung injury, promote cell proliferation and inhibit apoptosis but reduce chord length in lung tissues of neonatal mice following hyperoxia-induced BPD modeling.ConclusionAltogether, down-regulation of miR-29a can potentially elevate GAB1 expression, reducing cell apoptosis and stimulating proliferation, ultimately retarding the development of BPD in mice. This study highlights the potential of a promising new target for preventing BPD.

Effect of Tumor Suppressor MiR-34a Loaded on ZSM-5 Nanozeolite in Hepatocellular Carcinoma: In Vitro and In Vivo Approach

MicroRNA modulation therapy has shown great promise to treat hepatocellular carcinoma (HCC), however Efficient tissue-specific and safe delivery remains a major challenge. We sought to develop an inorganic-organic hybrid vehicle for the systemic delivery of the tumor suppressor miR-34a, and to investigate the efficiency of the delivered miR-34a in the treatment of HCC in vitro and in vivo. In the present study, pEGP-miR cloning and expression vector, expressing miR-34a, was electrostatically bound to polyethyleneimine (PEI), and then loaded onto ZSM-5 zeolite nanoparticles (ZNP). Qualitative and quantitative assessment of the transfection efficiency of miR-34a construct in HepG2 cells was applied by GFP screening and qRT-PCR, respectively. The expression of miR-34a target genes was investigated by qRT-PCR in vitro and in vivo. ZNP/PEI/miR-34a nano-formulation could efficiently deliver into HepG2 cells with low cytotoxicity, indicating good biocompatibility of generated nanozeolite. Furthermore, five injected doses of ZNP/PEI/miR-34a nano-formulation in HCC induced male Balb-c mice, significantly inhibited tumor growth, and demonstrated improved cell structure, in addition to a significant decrease in alphafetoprotein level and liver enzymes activities, as compared to the positive control group. Moreover, injected ZNP/PEI/miR-34a nano-formulation led to a noticeable decrease in the CD44 and c-Myc levels. Results also showed that ZNP/PEI/miR-34a nano-formulation inhibited several target oncogenes including AEG-1, and SOX-9, in vitro and in vivo. Our results suggested that miR-34a is a powerful candidate in HCC treatment and that AEG-1 and SOX-9 are novel oncotargets of miR-34a in HCC. Results also demonstrated that our nano-formulation may serve as a candidate approach for miR-34a restoration for HCC therapy, and generally for safe gene delivery.

Inhibition of microRNA-92a ameliorates lipopolysaccharide-induced endothelial barrier dysfunction by targeting ITGA5 through the PI3K/Akt signaling pathway in human pulmonary microvascular endothelial cells

Overwhelming inflammation and extensive alveolar-endothelial injury are characteristic pathological features of acute respiratory distress syndrome (ARDS)). MicroRNAs are involved in the regulation of a variety of cellular processes including endothelial damage and inflammatory responses. However, little is known about their function and the molecules regulating lung microvascular endothelial injury. Here, we determined the levels of microRNA-92a (miR-92a) in lipopolysaccharide (LPS)-induced human pulmonary microvascular endothelial cells (HPMECs). We found that miR-92a expression was greater in HPMECs treated with LPS than in control cells. Inhibition of miR-92a through transfection with a miR-92a inhibitor significantly increased HPMECs migration, enhanced tube formation, and improved endothelial cell barrier dysfunction. Inhibition of miR-92a ameliorated the inflammatory response by decreasing the release of the proinflammatory factors IL-6 and TNF-α. In addition, integrin α5 (ITGA5) was found to be a target gene of miR-92a in LPS-induced endothelial barrier dysfunction. Western blot analysis showed that inhibition of miR-92a may ameliorate endothelial barrier dysfunction by activating the PI3K/Akt signaling pathway. Together, these results reveal an important role of miR-92a in LPS-induced endothelial barrier dysfunction, and suggest that miR-92a may have potential as a prognostic indicator and a future target for the treatment of acute lung injury (ALI)/ARDS.

Reduced expression of miR-146a in human bronchial epithelial cells alters neutrophil migration

BackgroundThe role of miRNAs in the pathogenesis and determining the phenotypes of asthma is not fully elucidated. miR-146a has been previously shown to suppress inflammatory responses in different cells. In this study, we investigated the functions of miR-146a in human bronchial epithelial cells (HBECs) in association with neutrophilic, eosinophilic, and paucigranulocytic phenotypes of asthma.MethodsBronchial brushing specimens and brochial mucosal biopsy samples were collected from adult patients with asthma and from age- and gender-matched non-asthmatic individuals. The expression of miR-146a in bronchial brushing specimens, bronchial biopsy tissue sections or cultured primary bronchial epithelial cells was analyzed by RT-qPCR or by in situ hybridization. The expression of direct and indirect miR-146a target genes was determined by RT-qPCR or ELISA. The migration of neutrophils was studied by neutrophil chemotaxis assay and flow cytometry. For statistical analysis, unpaired two-way Student’s t test, one-way ANOVA or linear regression analysis were used.ResultsReduced expression of miR-146a was found in bronchial brushing specimens from asthma patients as compared to non-asthmatics and irrespective of the phenotype of asthma. In the same samples, the neutrophil attracting chemokines IL-8 and CXCL1 showed increased expression in patients with neutrophilic asthma and increased IL-33 expression was found in patients with eosinophilic asthma. Linear regression analysis revealed a significant negative association between the expression of miR-146a in bronchial brushings and neutrophil cell counts in bronchoalveolar lavage fluid of patients with asthma. In bronchial biopsy specimens, the level of miR-146a was highest in the epithelium as determined with in situ hybridization. In primary conventional HBEC culture, the expression of miR-146a was induced in response to the stimulation with IL-17A, TNF-α, and IL-4. The mRNA expression and secretion of IL-8 and CXCL1 was inhibited in both stimulated and unstimulated HBECs transfected with miR-146a mimics. Supernatants from HBECs transfected with miR-146a had reduced capability of supporting neutrophil migration in neutrophil chemotaxis assay.ConclusionOur results suggest that decreased level of miR-146a in HBECs from patients with asthma may contribute to the development of neutrophilic phenotype of asthma.

Layer-by-layer assembled PLGA nanoparticles carrying miR-34a cargo inhibit the proliferation and cell cycle progression of triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) accounts for 15-25% of diagnosed breast cancers, and its lack of a clinically defined therapeutic target has caused patients to suffer from earlier relapse and higher mortality rates than patients with other breast cancer subtypes. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of multiple genes through RNA interference to maintain normal tissue function. The tumor suppressor miR-34a is downregulated in TNBC, and its loss-of-expression correlates with worse disease outcomes. Therefore, delivering miR-34a mimics into TNBC cells is a promising strategy to combat disease progression. To achieve this goal, we synthesized layer-by-layer assembled nanoparticles (LbL NPs) comprised of spherical poly(lactic-co-glycolic acid) cores surrounded by alternating layers of poly-L-lysine (PLL) and miR-34a. TNBC cells internalized these LbL NPs to a greater extent than polyplexes comprised of PLL and miRNA, and confocal microscopy showed that LbL NPs delivered a substantial fraction of miR-34a cargo into the cytosol. This yielded robust suppression of the miR-34a target genes CCND-1, Notch-1, Bcl-2, Survivin, and MDR-1, which reduced TNBC cell proliferation and induced cell cycle arrest. These data validate that miR-34a delivery can impair TNBC cell function and support continued investigation of this platform for treatment of TNBC.

Retracted: miR-135a Inhibits the Invasion and Migration of Esophageal Cancer Stem Cells through the Hedgehog Signaling Pathway by Targeting Smo

Cancer stem cells (CSCs) have been reported to be involved in esophageal cancer (EC) development. Hence, we aim to explore whether microRNA-135a (miR-135a) affects EC and its associated mechanism. Cancerous and adjacent tissues from 138 EC patients were collected. The dual-luciferase reporter gene assay and bioinformatics analysis were used to confirm the interaction between nucleotides. A series of mimics or inhibitors of miR-135a or small interfering RNA (siRNA) against Smo were introduced into EC cells. After that, the expression of miR-135a and Hedgehog (Hh) signaling pathway-related genes (Smo, Gli1, Shh, and Gli2) in tissues and cells was measured, accompanied by evaluation of cell viability, apoptosis, invasion, and migration. High expression of Smo, Gli1, Shh, and Gli2 and low expression of miR-135a were observed in EC. Smo was verified to be a target gene of miR-135a. In addition, overexpression of miR-135a or silencing of Smo decreased the expression of Gli1, Gli2, and Shh, thus inhibiting EC cell proliferation, migration, and invasion and promoting apoptosis. Silencing of miR-135a was observed to reverse the inhibitory role of miR-135a in EC. These results suggest that miR-135a inhibited the migration and invasion of EC cells through inhibition of the Smo/Hh axis.

MiR-92a regulates brown adipocytes differentiation, mitochondrial oxidative respiration, and heat generation by targeting SMAD7.

Brown adipocytes are rich in mitochondria and linked to the body's blood fat levels and obesity. MiR-92a is negatively correlated with the activity of brown adipocytes. This study aimed to explore the mechanism of miR-92a on brown adipocytes. The expression of miR-92a in C2C12 cell was detected by a quantitative real-time-polymerase chain reaction (qRT-PCR). C2C12 cells were induced to brown adipocytes. The direct target gene of miR-92a was determined using the dual-luciferase reporter assay. Brown adipocytes were treated with isoprenaline (Iso) and transfected by miR-92a inhibitor and siSMAD7. The expression of heat-producing genes and adipose differentiation genes related to brown adipocytes were detected by qRT-PCR and Western blot analysis. The expression of SMAD7, p-SMAD2, and p-SMAD3 were detected using Western blot analysis. The mitochondrial content was measured by mitotracker fluorescent staining. MiR-92a inhibitor significantly decreased the expression of miR-92a in C2C12 cells. MiR-92a inhibitor could upregulate the expression of Ucp1, Cox7a1, Elovl3, Ppargc1α, PPARγ, and FABP4, and its effect on Ucp1 was increased after the treatment of isoprenaline. Moreover, miR-92a inhibitor increased mitochondrial content, oxygen consumption rate (OCR) and the expression of SMAD7 and suppressed the expressions of p-SMAD2 and p-SMAD3, whereas miR-92a directly targeted SMAD7 to exert its inhibitory effects. SiSMAD7 reversed the effects of the inhibitor on heat-producing genes, mitochondrial content, OCR and the expressions of SMAD7, p-SMAD2, and p-SMAD3 in brown adipocytes. Blocking miR-92a might promote brown adipocytes differentiation, mitochondrial oxidative respiration, and thermogenesis by targeting SMAD7 to inhibit the expressions of p-SMAD2 and p-SMAD3.

The RNA-binding protein SART3 promotes miR-34a biogenesis and G1 cell cycle arrest in lung cancer cells

MicroRNAs (miRNAs or miRs) are small, noncoding RNAs that are implicated in the regulation of most biological processes. Global miRNA biogenesis is altered in many cancers, and RNA-binding proteins play a role in miRNA biogenesis, presenting a promising avenue for targeting miRNA dysregulation in diseases. miR-34a exhibits tumor-suppressive activities by targeting cell cycle regulators CDK4/6 and anti-apoptotic factor BCL-2, among other regulatory pathways such as Wnt, TGF-β, and Notch signaling. Many cancers exhibit down-regulation or loss of miR-34a, and synthetic miR-34a supplementation has been shown to inhibit tumor growth in vivo However, the post-transcriptional mechanisms that cause miR-34a loss in cancer are not entirely understood. Here, using a proteomics-mediated approach in non-small-cell lung cancer (NSCLC) cells, we identified squamous cell carcinoma antigen recognized by T-cells 3 (SART3) as a putative pre-miR-34a-binding protein. SART3 is a spliceosome recycling factor and nuclear RNA-binding protein with no previously reported role in miRNA regulation. We found that SART3 binds pre-miR-34a with higher specificity than pre-let-7d (used as a negative control) and elucidated a new functional role for SART3 in NSCLC cells. SART3 overexpression increased miR-34a levels, down-regulated the miR-34a target genes CDK4/6, and caused a cell cycle arrest in the G1 phase. In vitro binding experiments revealed that the RNA-recognition motifs within the SART3 sequence are responsible for selective pre-miR-34a binding. Our results provide evidence for a significant role of SART3 in miR-34a biogenesis and cell cycle progression in NSCLC cells.

Human Neutrophil Elastase Induces MUC5AC Overexpression in Chronic Rhinosinusitis Through miR-146a.

Background The pathogenesis of chronic rhinosinusitis (CRS) is not yet clear. microRNAs are widely involved in a number of physiological and pathological processes, of which microRNA-146a (miR-146a) plays an important role in innate immunity, inflammatory response, and other pathophysiological processes. Mucins (MUCs) are important components of secreted mucus, of which MUC5AC is the major MUC secreted in the normal airway. Objective This study was performed to examine human neutrophil elastase (HNE)-induced MUC5AC overexpression in CRS via miR-146a. Methods miR-146a, HNE, epidermal growth factor receptor (EGFR), and MUC5AC expression in the sinonasal mucosa were determined using quantitative real-time polymerase chain reaction (qRT-PCR). EGFR, phosphorylated EGFR (pEGFR), and MUC5AC expression were determined in primary cultures of human nasal epithelial cells (HNECs). We examined the expression of miR-146a, MUC5AC, EGFR, and pEGFR by transfecting HNECs with miR-146a mimics and negative control (NC). Moreover, dual-luciferase reporter gene assays were used to validate EGFR as an hsa-miR-146a target gene. Results miR-146a was significantly downregulated, and HNE, EGFR, and MUC5AC were upregulated in CRS patients both with and without nasal polyps. In the in vitro cell experiment, MUC5AC was significantly downregulated after use of an EGFR-specific inhibitor (AG1478). Upon addition of miR-146a inhibitor, miR-146a was downregulated, while MUC5AC was upregulated. MUC5AC was suppressed in normal primary HNECs by miR-146a mimic and pEGFR was downregulated. The results of dual-luciferase reporter assays showed that the luciferase activities were markedly inhibited in the pGL3-EGFR-3′ UTR+miR-146a mimic group compared with the pGL3+ miR-146a mimic group, suggesting that EGFR is a target gene for miR-146a. Conclusion In HNE-induced CRS, miR-146a downregulates the expression of MUC5AC by inhibiting the activation of EGFR, and EGFR is a target gene of miR-146a.

MiR-146a promotes Borna disease virus 1 replication through IRAK1/TRAF6/NF-κB signaling pathway

Background/Aims: Borna disease virus 1 (BoDV-1) is a negative single-stranded RNA virus that is highly neurotropic. BoDV-1 infection can damage the central nervous system and cause inflammation. To survive in host cells, BoDV-1 must evade the host innate immune response. A previous study showed that miR-146a expression increased in neonatal rats infected with BoDV-1. miR-146a is a microRNA suggested to negatively regulate innate immune and inflammatory responses and antiviral pathways. Many groups have reported that its overexpression facilitates viral replication. However, it is unclear whether miR-146a is involved in escape from the host immune response during BoDV-1 infection. Methods: In this study, BoDV-1 was used to infect neonatal rats within 24 h of birth intracranially, as well as to infect human microglial cells (HMC3). miR-146a expression was analyzed by RT-qPCR. The TargetScanHuman database was used to find the target genes of miR-146a. A search of the binding sites of miR-146a and its target gene’s 3′-untranslated region (3′UTR) was also performed using RNAhybrid software. The binding sites of miR-146a and the target gene’s 3′UTR were detected by dual luciferase reporter assays. Overexpression and suppression studies of miR-146a were performed to determine its effect on BoDV-1 replication. The relative protein expression of members of the IRAK1/TRAF6/NF-κB signaling pathway was also evaluated by western blotting in HMC3. Results: After BoDV-1 infection of neurons in vivo and of HMC3 cells, miR-146a expression was significantly upregulated. miR-146a overexpression in HMC3 cells promoted viral replication, while its inhibition inhibited it. Through the TargetScanHuman database, we identified the target genes of anti-inflammatory miR-146a: IRAK1 and TRAF6. We also found that BoDV-1 could inhibit IRAK1 and TRAF6 expression in HMC3 cells. Moreover, we showed that the inhibition of IRAK1 and TRAF6 also led to decreases in the expression of P65 and phosphorylated P65 in the downstream NF-κB pathway. Subsequently, we confirmed the interaction of miR-146a with IRAK1 and TRAF6 by luciferase assay. Conclusion: Our results suggest that miR-146a inhibits the IRAK1/TRAF6/NF-κB signaling pathway to facilitate BoDV-1 survival in host cells.

ZNFX1 anti-sense RNA 1 promotes the tumorigenesis of prostate cancer by regulating c-Myc expression via a regulatory network of competing endogenous RNAs.

ZNFX1 anti-sense RNA 1 (ZFAS1) has been indicated in the tumorigenesis of various human cancers. However, the role of ZFAS1 in prostate cancer (PCa) progression and the underlying mechanisms remain incompletely understood. In the present study, we discovered that ZFAS1 is upregulated in PCa and that ZFAS1 overexpression predicted poor clinical outcomes. ZFAS1 overexpression notably promoted the proliferation, invasion, and epithelial-mesenchymal transition of PCa cells. Furthermore, we not only discovered that miR-27a/15a/16 are targeted by ZFAS1, which binds to their miRNA-response elements, but also revealed their tumor suppressor roles in PCa. We also identified that the Hippo pathway transducer YAP1, as well as its cooperator, TEAD1, are common downstream targets of miR-27a/15a/16. In addition, H3K9 demethylase KDM3A was found to be another target gene of miR-27a. Importantly, YAP1, TEAD1, and KDM3A all act as strong c-Myc inducers in an androgen-independent manner. Taken together, we suggest a regulatory network in which ZFAS1 is capable of enhancing c-Myc expression by inducing the expression of YAP1, TEAD1, and KDM3A through crosstalk with their upstream miRNAs, thereby globally promoting prostate cancer tumorigenesis.

MiR-34a as hub of T cell regulation networks

BackgroundMicro(mi)RNAs are increasingly recognized as central regulators of immune cell function. While it has been predicted that miRNAs have multiple targets, the majority of these predictions still await experimental confirmation. Here, miR-34a, a well-known tumor suppressor, is analyzed for targeting genes involved in immune system processes of leucocytes.MethodsUsing an in-silico approach, we combined miRNA target prediction with GeneTrail2, a web tool for Multi-omics enrichment analysis, to identify miR-34a target genes, which are involved in the immune system process subcategory of Gene Ontology.ResultsOut of the 193 predicted target genes in this subcategory we experimentally tested 22 target genes and confirmed binding of miR-34a to 14 target genes including VAMP2, IKBKE, MYH9, MARCH8, KLRK1, CD11A, TRAFD1, CCR1, PYDC1, PRF1, PIK3R2, PIK3CD, AP1B1, and ADAM10 by dual luciferase assays. By transfecting Jurkat, primary CD4+ and CD8+ T cells with miR-34a, we demonstrated that ectopic expression of miR-34a leads to reduced levels of endogenous VAMP2 and CD11A, which are central to the analyzed subcategories. Functional downstream analysis of miR-34a over-expression in activated CD8+ T cells exhibits a distinct decrease of PRF1 secretion.ConclusionsBy simultaneous targeting of 14 mRNAs miR-34a acts as major hub of T cell regulatory networks suggesting to utilize miR-34a as target of intervention towards a modulation of the immune responsiveness of T-cells in a broad tumor context.

Long non-coding RNA MEG3 promotes fibrosis and inflammatory response in diabetic nephropathy via miR-181a/Egr-1/TLR4 axis.

Long non-coding RNAs (lncRNAs) play vital roles in diabetic nephropathy (DN). This research aimed to study the potential role and underlying molecular mechanisms of long non-coding RNA MEG3 in DN. We found that MEG3 was upregulated in DN in vivo and in vitro and could enhance cell fibrosis and inflammatory response in DN. MEG3 functioned as an endogenous sponge for miR-181a in mesangial cells (MCs) via direct targeting and in an Ago2-dependent manner. MiR-181a inhibition promoted MC fibrosis and inflammatory response. In addition, Egr-1 was confirmed as a target gene of miR-181a. Further investigations verified that MEG3 promotes fibrosis and inflammatory response via the miR-181a/Egr-1/TLR4 axis in vitro and in vivo. These results provide new insights into the regulation between MEG3 and the miR-181a/Egr-1/TLR4 signaling pathway during DN progression.

Silencing microRNA-27a inhibits proliferation and invasion of human osteosarcoma cells through the SFRP1-dependent Wnt/β-catenin signaling pathway.

Osteosarcoma is the most common malignant tumor of bone with a high potential for metastasis. Importantly, microRNA-27a (miR-27a) is involved in the progression of osteosarcoma. The present study aims to discuss the effects of miR-27a and its target gene secreted frizzled related protein 1 (SFRP1) on proliferation and invasion of human osteosarcoma cells via Wnt/β-catenin signaling pathway. The expression of miR-27a and SFRP1 in osteosarcoma tissues and cells was detected, followed by identification of their relations. Subsequently, miR-27a mimic, miR-27a inhibitor, or siRNA against SFRP1 were introduced into cells (HOS and U2OS) to investigate their role in cell proliferation and invasion. The expression of Wnt/β-catenin signaling pathway-related gene was analyzed to further uncover the regulatory mechanism of miR-27a. The osteosarcoma tissues and cells exhibited elevated miR-27 expression and reduced SFRP1 expression. SFRP1 was verified to be a target gene of miR-27a. Meanwhile, silenced miR-27a inhibited proliferation and invasion of human osteosarcoma cells. Finally, silencing miR-27a inhibited the activation of Wnt/β-catenin signaling pathway, evidenced by reduced β-catenin expression. Our study draws a conclusion that silencing miR-27a dampens osteosarcoma progression, which might be achieved through the inactivation of the Wnt/β-catenin signaling pathway by up-regulating SFRP1.

Efflect of miR-34a-Tangeted Regulation of HDACI on Apoptosis of AML Cells

To investigate the regulation of miR-34a on HDAC1 expression and its effect on the apoptosis of acute myeloid leukemia (AML) cells. miR-34a mimics, miR-34a inhibitor and miR-34a scramble were transfected into HL-60 cells. The effects of miR-34a expression levels on proliferation and apoptosis of HL-60 cell were detected by CCK8 assay and flow cytometry respectively. The expression of HDAC1 protein was assessed by Western blot after regulating miR-34a expression, the 3'UTR of HDAC1 was cloned and ligated to construct a dual luciferase reporter vector, and then the dual luciferase reporter assay was applied to verify the target of miR-34a, the expression vector pcDNA3.1-HDAC1 was constructed, the interaction of miR-34a and HDAC1 was analyzed by reversion test. miR-34a over-expression could inhibit the proliferation of HL-60 cells and induce their apoptosis. Bioinformatics analysis indicated that the HDAC1 was a target gene of miR-34a. Western blot indicated that miR-34a overexpression down-regulated the expression of HDAC1. Dual luciferase reporter assay and reversion test showed that miR-34a could act at the 3-UTR of HDAC1 gene to regulate its expression. miR-34a promotes the apoptosis of HL-60 cells via regulating HDAC1 expression.