Overexpression Of miR-125b Research Articles

Effect of miR-27b on the proliferation and apoptosis of diffuse large b-cell lymphoma cells by targeting the regulation of MET/PI3K/AKT pathway

BackgroundThis study aimed to explore the regulation of miR-27b expression on MET/PI3K/AKT pathway, and to explain its effect on biological functions of DLBCL cells.MethodsThe expressions of miR-27b and MET gene in DLBCL cells and normal human B cell lines were determined by qRT-PCR. miR-27b expression in DLBCL cell line Toledo was over-expressed with the cell transfection method. The proliferation of DLBCL cells was determined by MTT. And the invasiveness of DLBCL cells was determined by Transwell. The level of apoptosis in DLBCL cells was determined by ELISA. miR-27b targeting of MET was verified by dual- luciferase reporter assay. The activation of MET/PI3K/AKT pathway and the expression of downstream related proteins were determined by Western blot.ResultsThe results showed that miR-27b was poorly expressed in DLBCL cell lines compared with normal human B cell lines, and was associated with its high proliferation, high invasiveness and low apoptosis level. High miR-27b expression can reduce the proliferation and increase the apoptosis level in DLBCL cells. By examining the effect of miR-27b over-expression on the MET/PI3K/AKT pathway, it was found that miR-27b can inhibit the proliferation and invasiveness and promote the apoptosis of DLBCL cells by targeting the inhibition of MET expression and the activation of PI3K/AKT pathway.ConclusionmiR-27b can inhibit the proliferation and invasiveness of DLBCL cells and promote the apoptosis of the cells by targeting MET/PI3K/AKT pathway.

MicroRNA-10 Family Promotes the Epithelial-to-Mesenchymal Transition in Renal Fibrosis by the PTEN/Akt Pathway.

Renal fibrosis (RF) is a common reason for renal failure, and epithelial-mesenchymal transition (EMT) is a vital mechanism that promotes the development of RF. It is known that microRNA-10 (miR-10) plays an important role in cancer EMT; however, whether it takes part in the EMT process of RF remains unclear. Therefore, we established an in vivo model of unilateral ureteral obstruction (UUO), and an in vitro model using TGF-β1, to investigate whether and how miR-10a and miR-10b take part in the EMT of RF. In addition, the combinatorial effects of miR-10a and miR-10b were assessed. We discovered that miR-10a and miR-10b are overexpressed in UUO mice, and miR-10a, miR-10b, and miRs-10a/10b knockout attenuated RF and EMT in UUO-treated mouse kidneys. Moreover, miR-10a and miR-10b overexpression combinatorially promoted RF and EMT in TGF-β1-treated HK-2 cells. Inhibiting miR-10a and miR-10b attenuated RF and EMT induced by TGF-β1. Mechanistically, miR-10a and miR-10b suppressed PTEN expression by binding to its mRNA3'-UTR and promoting the Akt pathway. Moreover, PTEN overexpression reduced miR-10a and miR-10b effects on Akt phosphorylation (p-Akt), RF, and EMT in HK-2 cells treated with TGF-β1. Taken together, miR-10a and miR-10b act combinatorially to negatively regulate PTEN, thereby activating the Akt pathway and promoting the EMT process, which exacerbates RF progression.

MiR-125b Promotes the Proliferation, Migration and Invasion of Oral Squamous Cell Carcinoma (OSCC)

miR-125b is involved in several tumors. However, miR-125b’s role in oral squamous cell carcinoma (OSCC) is unclear. Tumor tissues and oral normal mucosa tissues of OSCC patients were collected to measure miR-125b level. Oral cancer Tca8113 cells were separated into control group, miR-125b inhibitor group, and miR-125b mimics group, followed by measuring miR-125b level by real time PCR, cell survival, migration and invasion, PI3K/mTOR signaling protein level by Western blot. miR-125b was upregulated in OSCC tumor tissues and related to clinical/TNM stage, metastasis and overall survival (P < 0.05). miR-125b overexpression significantly promoted tumor cell behaviors and increased PI3K/mTOR phosphorylation (P < 0.05); while inhibiting miR-125b expression significantly inhibited tumor cell biological behaviors, and decreased PI3K/mTOR phosphorylation (P < 0.05). miR-125b level is increased in OSCC tumor tissues, which is related to clinicopathological characteristics. miR-125b overexpression promotes OSCC cell behaviors by regulating PI3K/mTOR signaling.

Milk Exosomal miR-27b Worsen Endoplasmic Reticulum Stress Mediated Colorectal Cancer Cell Death.

The relationship between dietary constituents and the onset and prevention of colorectal cancer (CRC) is constantly growing. Recently, the antineoplastic profiles of milk and whey from Mediterranean buffalo (Bubalus bubalis) have been brought to attention. However, to date, compared to cow milk, the potential health benefits of buffalo milk exosome-miRNA are still little explored. In the present study, we profiled the exosomal miRNA from buffalo milk and investigated the possible anticancer effects in CRC cells, HCT116, and HT-29. Results indicated that buffalo milk exosomes contained higher levels of miR-27b, miR-15b, and miR-148a compared to cow milk. Mimic miR-27b transfection in CRC cells induced higher cytotoxic effects (p < 0.01) compared to miR-15b and miR-148a. Moreover, miR-27b overexpression in HCT116 and HT-29 cells (miR-27b+) induced apoptosis, mitochondrial reactive oxygen species (ROS), and lysosome accumulation. Exposure of miR-27b+ cells to the bioactive 3kDa milk extract aggravated the apoptosis rate (p < 0.01), mitochondrial stress (p < 0.01), and advanced endoplasmic reticulum (ER) stress (p < 0.01), via PERK/IRE1/XBP1 and CHOP protein modulation (p < 0.01). Moreover, GSK2606414, the ER-inhibitor (ER-i), decreased the apoptosis phenomenon and XBP1 and CHOP modulation in miR-27b+ cells treated with milk (p < 0.01 vs. miR-27b++Milk), suggesting the ER stress as a cell-death-aggravating mechanism. These results support the in vitro anticancer activity of 3kDa milk extract and unveil the contribution of miR-27b in the promising beneficial effect of buffalo milk in CRC prevention.

Long non-coding RNA NCK1-AS1 functions as a ceRNA to regulate cell viability and invasion in esophageal squamous cell carcinoma via microRNA-133b/ENPEP axis

ABSTRACT This study is designed to explore the role of long non-coding RNAs (lncRNAs) NCK1-AS1 in proliferative and invasive activities of esophageal squamous cell carcinoma (ESCC) cells by binding to microRNA-133b (miR-133b) to regulate ENPEP. Differentially expressed lncRNAs, miRs, genes and their targeting relationships were screened on ESCC-related gene expression datasets GSE17351 and GSE6188. The targeting relationships among NCK1-AS1, miR-133b, and ENPEP were verified using functional assays. Loss- and gain- of function assays were carried out to examine the roles of NCK1-AS1, miR-133b, and ENPEP in ESCC cell proliferative, invasive, migrative and apoptotic abilities as well as tumorigenesis in vivo. Elevated NCK1-AS1 and ENPEP but reduced miR-133b expression were found in ESCC. NCK1-AS1 knockdown or miR-133b overexpression inhibited the malignant properties of ESCC cells as well as tumorigenesis in vivo. NCK1-AS1 regulated the ENPEP expression by competitively binding to miR-133b. ENPEP overexpression reversed inhibition of NCK1-AS1 knockdown on the function of ESCC cells. This study provides evidence that silencing NCK1-AS1 inhibits expression of ENPEP by sponging miR-133b, thereby suppressing ESCC.

MiRNA29B Induces Fetal Hemoglobin through Targeting DNMT3 and MYB in Vitro and In Vivo in Preclinical Townes Sickle Cell Mice

Introduction: Therapies aimed at inducing fetal hemoglobin (HbF) expression is an effective approach for ameliorating the clinical severity of sickle cell disease (SCD) in adults and children, however drug treatment options are limited. DNA methyltransferase (DNMT) inhibitors tested in clinical trials induce HbF expression by producing γ-globin gene promoter DNA hypomethylation. Alternative approaches to increase HbF involve development of small non-coding microRNAs (miR) to silence major repressors of γ-globin gene transcription. Studies from our group demonstrated that miR29b inhibits DNA methylation through binding the 3' UTR of DNMT3 genes and is associated with high HbF levels in SCD patients. Studies performed in KU812 cells showed concomitant DNMT3 silencing along with ү-globin gene activation and HbF induction. Moreover, miR29b decreased expression of the γ-globin repressor protein MYB in normal adult CD34+ stem cells. Therefore, we tested the hypothesis that miR29b activates γ-globin and HbF expression via DNA hypomethylation and MYB gene silencing in normal erythroid progenitors and in Townes SCD mice. Methods: Human KU812 were cultured and normal CD34+ cells (n=3) were differentiated using a two-phase erythropoiesis culturing system. KU812 cells were co-transfected with the MYB luciferase reporter plasmid and the effects of miR29b (50 nM and 100 nM) overexpression or negative Scrambled (Scr) mimic on luciferase activity was quantified after 48 hours. The effect of MYB knockdown on ү-globin activation and HbF induction in KU812 cells using a retroviral vector encoding a MYB shRNA was also assessed. Subsequent studies were conducted in CD34 cells electroporated with miR29b mimic (50nM and 100nM) or negative Scrambled (Scr) control on day 8 and collected after 24 hours. Erythroid differentiation was assessed using Giemsa staining, and flow cytometry was used to measure the percentage of HbF positive cells (F-cells). Real-time PCR quantified expression of miR29b, γ-globin, and β-globin gene expression, and Western blot measured DNMT, HbF and HbA protein. In vivo studies were performed in Townes SCD mice (6 per group), treated by 28-day continuous infusion using subcutaneous mini-osmotic pumps. The treatment groups consisted of miR29b (2, 3, and 4 mg/kg/day) and corresponding doses of Scr control. Blood samples were collected and processed for complete blood count (CBC) with differential and reticulocytes percent by flow cytometry. Gene expression was determined by real-time PCR. Results: Co-transfection of miR29b and a luciferase reporter plasmid containing the MYB 3'-UTR into KU812 cells resulted in a >90% decrease in luciferase activity at both miR29b concentrations. MYB knockdown by shRNA significantly increased the ү/ү+β ratio and miR29b expression in KU812 cells and normal CD34 cells. We observed efficient erythroid differentiation at day 7, day 12, and day 14 of cell culture assessed by Giemsa stain. Treatment with miR29b increased the ү/ү+β mRNA ratio by 2.5-fold (p<0.01) and F-cell levels increased from ~6.0% (Scr) to 15% with miR29b (100 nM); we observed lower DNMT3 mRNA and protein levels in cells transfected with 100 nM miR29b. Analysis of HbF induction by miR29b revealed >85% increase in F-cells by flow cytometry in normal erythroid progenitors as compared to Scr cells. Subsequent in vivo studies were conducted with Townes SCD mice treated with miR29b for 28 days; treatment was well tolerated with good weight gain and no deaths. There was minimal effects on hematopoiesis with a decrease in total white blood count and increase in neutrophils. Hemoglobin, reticulocyte and platelet counts remained stable. By week 4, we observed a dose-dependent 2.6-fold, 2.8-fold, and 3.0-fold (p<0.05) increase in F-cells at the respective miR29b doses compared to Scr control. We also observed a significant decrease in the percent of sickle cells on peripheral smear (p<0.05) mediated by miR29b with 20% inhibition (p<0.05) at week 4. MiR29b significantly decreased DNMT3 mRNA expression at week 2 and MYB mRNA at week 4 with a concomitant increase in the ү/ү+β mRNA ratio at weeks 2 and 4. Conclusions: Our findings support the ability of miR-29b to induce HbF in sickle erythroid progenitors without significant toxicity in vitro and SCD mice in vivo. This research highlights a novel miRNA-based epigenetic approach to induce HbF supporting discovery of new drugs to expand treatment options for SCD.

Lactate promotes vascular smooth muscle cell switch to a synthetic phenotype by inhibiting miR-23b expression.

Recent research indicates that lactate promotes the switching of vascular smooth muscle cells (VSMCs) to a synthetic phenotype, which has been implicated in various vascular diseases. This study aimed to investigate the effects of lactate on the VSMC phenotype switch and the underlying mechanism. The CCK-8 method was used to assess cell viability. The microRNAs and mRNAs levels were evaluated using quantitative PCR. Targets of microRNA were predicted using online tools and confirmed using a luciferase reporter assay. We found that lactate promoted the switch of VSMCs to a synthetic phenotype, as evidenced by an increase in VSMC proliferation, mitochondrial activity, migration, and synthesis but a decrease in VSMC apoptosis. Lactate inhibited miR-23b expression in VSMCs, and miR-23b inhibited VSMC's switch to the synthetic phenotype. Lactate modulated the VSMC phenotype through downregulation of miR-23b expression, suggesting that overexpression of miR-23b using a miR-23b mimic attenuated the effects of lactate on VSMC phenotype modulation. Moreover, we discovered that SMAD family member 3 (SMAD3) was the target of miR-23b in regulating VSMC phenotype. Further findings suggested that lactate promotes VSMC switch to synthetic phenotype by targeting SMAD3 and downregulating miR-23b. These findings suggest that correcting the dysregulation of miR-23b/SMAD3 or lactate metabolism is a potential treatment for vascular diseases.

IL-6/Stat3 suppresses osteogenic differentiation in ossification of the posterior longitudinal ligament via miR-135b-mediated BMPER reduction.

Interleukin-6 (IL-6) has been reported to induce osteogenic differentiation of mesenchymal stem cells for increasing bone regeneration, while the role of IL-6 in osteogenic differentiation during ossification of the posterior longitudinal ligament (OPLL) remains to be determined. The current study aims to explore the downstream mechanism of IL-6 in cyclic tensile strain (CTS)-stimulated OPLL, which involves bioinformatically identified microRNA-135b (miR-135b). Initially, we clinically collected posterior longitudinal ligament (PLL) and ossified PLL tissues, from which ossified PLL cells were isolated, respectively. The obtained data revealed a greater osteogenic property of ossified PLL than non-ossified PLL cells. The effect of regulatory axis comprising IL-6, Stat3, miR-135b, and BMPER on osteogenic differentiation of CTS-stimulated ossified PLL cells was examined with gain- and loss-of-function experiments. BMPER was confirmed as a target gene to miR-135b. Knockdown of BMPER or overexpression of miR-135b inhibited the osteogenic differentiation of CTS-induced ossification in PLL cells. Besides, IL-6 promoted the post-transcriptional process to mature miR-135b via Stat3 phosphorylation. In conclusion, IL-6 inhibited CTS-induced osteogenic differentiation by inducing miR-135b-mediated inhibition of BMPER through Stat3 activation.

Integrated analysis of miRNAs and mRNA profiling reveals the potential roles of miRNAs in sheep hair follicle development

BackgroundMerino sheep exhibit high wool production and excellent wool quality. The fleece of Merino sheep is predominantly composed of wool fibers grown from hair follicles (HFs). The HF is a complex biological system involved in a dynamic process governed by gene regulation, and gene expression is regulated by microRNAs (miRNAs). miRNA inhibits posttranscriptional gene expression by specifically binding to target messenger RNA (mRNA) and plays an important role in regulating gene expression, the cell cycle and biological development sequences. The purpose of this study was to examine mRNA and miRNA binding to identify key miRNAs and target genes related to HF development. This will provide new and important insights into fundamental mechanisms that regulate cellular activity and cell fate decisions within and outside of the skin.ResultsWe analyzed miRNA data in skin tissues collected from 18 Merino sheep on four embryonic days (E65, E85, E105 and E135) and two postnatal days (D7 and D30) and identified 87 differentially expressed miRNAs (DE-miRNAs). These six stages were further divided into two longer developmental stages based on heatmap cluster analysis, and the results showed that DE-mRNAs in Stage A were closely related to HF morphogenesis. A coanalysis of Stage A DE-mRNAs and DE-miRNAs revealed that 9 DE-miRNAs and 17 DE-mRNAs presented targeting relationships in Stage A. We found that miR-23b and miR-133 could target and regulate ACVR1B and WNT10A. In dermal fibroblasts, the overexpression of miR-133 significantly reduced the mRNA and protein expression levels of ACVR1B. The overexpression of miR-23b significantly reduced the mRNA and protein expression levels of WNT10A.ConclusionThis study provides a new reference for understanding the molecular basis of HF development and lays a foundation for further improving sheep HF breeding. miRNAs and target genes related to hair follicular development were found, which provided a theoretical basis for molecular breeding for the culture of fine-wool sheep.

Molecular Classification of Colorectal Cancer by microRNA Profiling: Correlation with the Consensus Molecular Subtypes (CMS) and Validation of miR-30b Targets.

Colorectal cancer consensus molecular subtypes (CMSs) are widely accepted and constitutes the basis for patient stratification to improve clinical practice. We aimed to find whether miRNAs could reproduce molecular subtypes, and to identify miRNA targets associated to the High-stroma/CMS4 subtype. The expression of 939 miRNAs was analyzed in tumors classified in CMS. TALASSO was used to find gene-miRNA interactions. A miR-mRNA regulatory network was constructed using Cytoscape. Candidate gene-miR interactions were validated in 293T cells. Hierarchical-Clustering identified three miRNA tumor subtypes (miR-LS; miR-MI; and miR-HS) which were significantly associated (p &lt; 0.001) to the reported mRNA subtypes. miR-LS correlated with the low-stroma/CMS2; miR-MI with the mucinous-MSI/CMS1 and miR-HS with high-stroma/CMS4. MicroRNA tumor subtypes and association to CMSs were validated with TCGA datasets. TALASSO identified 1462 interactions (p &lt; 0.05) out of 21,615 found between 176 miRs and 788 genes. Based on the regulatory network, 88 miR-mRNA interactions were selected as candidates. This network was functionally validated for the pair miR-30b/SLC6A6. We found that miR-30b overexpression silenced 3'-UTR-SLC6A6-driven luciferase expression in 293T-cells; mutation of the target sequence in the 3'-UTR-SLC6A6 prevented the miR-30b inhibitory effect. In conclusion CRC subtype classification using a miR-signature might facilitate a real-time analysis of the disease course and treatment response.

MicroRNA-19b Plays a Key Role in 5-Fluorouracil Resistance and Predicts Tumor Progression in Locally Advanced Rectal Cancer Patients.

The standard clinical management of locally advanced rectal cancer (LARC) patients includes neoadjuvant 5-fluorouracil (5-FU)-based chemoradiotherapy (CRT) followed by mesorectal excision. MicroRNA (miR)-19b expression levels in LARC biopsies obtained from initial colonoscopy have recently been identified as independent predictors of both patient outcome and pathological response to preoperative CRT in this disease. Moreover, it has been discovered that this miR increases its expression in 5-FU resistant colon cancer cells after 5-FU exposure. Despite the fact that these observations suggest a functional role of miR-19b modulating 5-FU response of LARC cells, this issue still remains to be clarified. Here, we show that downregulation of miR-19b enhances the antitumor effects of 5-FU treatment. Moreover, ectopic miR-19b modulation was able to restore sensitivity to 5-FU treatment using an acquired resistant model to this compound. Notably, we also evaluated the potential clinical impact of miR-19b as a predictive marker of disease progression after tumor surgery resection in LARC patients, observing that miR-19b overexpression significantly anticipates patient recurrence in our cohort (p = 0.002). Altogether, our findings demonstrate the functional role of miR-19b in the progressively decreasing sensitivity to 5-FU treatment and its potential usefulness as a therapeutic target to overcome 5-FU resistance, as well as its clinical impact as predictor of tumor progression and relapse.

The long non-sacoding RNA TMEM147-AS1/miR-133b/ZNF587 axis regulates the Warburg effect and promotes prostatic carcinoma invasion and proliferation.

The Warburg effect is a characteristic tumor cell behavior regarded as one of the cancer hallmarks and promotes tumor progression by promoting glucose uptake and lactate production. Long non-coding RNAs (lncRNAs) had been reported to emerge as a vital function in cancer development. The present research is designed to investigate the underlying molecular mechanism of lncRNA TMEM147 antisense RNA 1 (TMEM147-AS1) on aerobic glycolysis in prostatic carcinoma. lncRNA TMEM147-AS1, miR-133b and ZNF587 levels in prostatic carcinoma tissues and cells were detected by a polymerase chain reaction or western blot assays. Cell viability or invasion was determined by Edu (i.e. 5-ethynyl-2'-deoxyuridine), MTT (i.e. 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) or transwell assays. Hematoxylin and eosin and immunohistochemical staining were applied for histopathological examination. Tumor xenograft model was employed to investigate tumor growth in vivo. The combinative relationship between TMEM147-AS1 or ZNF587 and miR-133b was confirmed by a luciferase reporter assay. TMEM147-AS1 and ZNF587 were up-regulated in prostatic carcinoma tissues and cells. Knockdown of TMEM147-AS1 or ZNF587 within prostate cancer cells significantly restrained cell viability, invasion and aerobic glycolysis in vitro and suppressed the neoplasia of prostatic carcinoma in vivo. miR-133b was directly targeted in both TMEM147-AS1 and ZNF587. Overexpression of miR-133b restrained prostate cancer cell viability, invasion and aerobic glycolysis. TMEM147-AS1 competitively targeted miR-133b, therefore counteracting miR-133b-mediated repression on ZNF587. TMEM147-AS1 plays a tumor-promoting action in prostatic carcinoma aerobic glycolysis via affecting the miR-133b/ZNF587 axis, therefore regulating prostatic carcinoma cells invasion and proliferation. These outcomes implied that TMEM147-AS1 could be an effective treatment strategy for further study of prostatic carcinoma.

Suppression of lncRNA Snhg1 inhibits high glucose-induced inflammation and proliferation in mouse mesangial cells

Diabetic nephropathy (DN) is the direct cause of end-stage renal disease, and nephritic inflammation plays a role in its growth and advancement. Aberrant expression of long non-coding RNAs (lncRNAs) correlates with many diseases, including DN. In this study, we investigated whether lncRNA small nucleolar RNA host gene 1 (Snhg1) was mechanistically involved in inflammation and mesangial cell (MC) proliferation in DN. We found that Snhg1 was significantly upregulated in DN renal tissues and high glucose (HG)-treated MCs. Overexpression of Snhg1 promoted inflammatory cytokine expression in MCs and MC proliferation under low-glucose conditions; meanwhile, Snhg1 knockdown suppressed inflammatory cytokine production and MC proliferation under HG conditions. Mechanistically, Snhg1 was found to directly bind miR-27b, thereby preventing the miRNA from binding its target KDM6B mRNA. Furthermore, miR-27b overexpression recapitulated the inhibitory effects of Snhg1 knockdown, whereas restoration of Snhg1 expression attenuated the function of miR-27b in MCs under HG conditions. Taken together, these results indicate that suppression of Snhg1 inhibited HG-induced inflammation and proliferation of MCs by regulating the miR-27b/KDM6B axis.

BMP15 Modulates the H19/miR-26b/SMAD1 Axis Influences Yak Granulosa Cell Proliferation, Autophagy, and Apoptosis.

Bone morphogenetic protein 15 (BMP15) regulates the growth and development of follicles. In particular, the long non-coding RNA H19 plays an important role in mammalian reproduction. However, the function and regulatory mechanism of the interaction of BMP15 with H19 in yak granulosa cell (GC) proliferation, autophagy, and apoptosis are poorly understood. In our study, quantitative reverse-transcription-polymerase chain reaction analysis showed that H19 were highly expressed in yak healthy follicles. H19 was induced by BMP15 protein in yak GCs. In addition, we confirmed that overexpression of H19 promoted yak GC proliferation and autophagy and inhibited apoptosis. Bioinformatic analysis and luciferase reporter assays demonstrated that H19 directly binds to miR-26b, and SMAD1 was identified as a target of miR-26b. miR-26b overexpression inhibited GC proliferation and autophagy and promoted apoptosis through decreased SMAD1 expression, which was attenuated by H19 overexpression. RNA immunoprecipitation-quantitative polymerase chain reaction and dual-luciferase assays showed that miR-26b was sponged by H19 to preserve SMAD1 expression. Furthermore, SMAD1 mRNA expression was induced and miR-26b expression was reduced after yak GCs were treated with BMP15 protein. In conclusion, our results demonstrated that the H19/miR-26b/SMAD1 axis responds to BMP15 to regulate yack GC proliferation, autophagy, and apoptosis.

MicroRNA-26b regulates BMSC osteogenic differentiation of TMJ subchondral bone through β-catenin in osteoarthritis.

Temporomandibular joint osteoarthritis (TMJ-OA) is a degenerative disease of the joint. The early manifestations of TMJ-OA are abnormal remodeling of condylar subchondral bone. In bone tissue, bone marrow mesenchymal stem cells (BMSCs) and osteoblasts play important roles in the differentiation and maturation of most hematopoietic cells. MicroRNA-26b (miR-26b) is upregulated during the osteogenesis of BMSCs, and miR-26b overexpression leads to the activation of β-catenin and the enhancement of osteogenesis and cartilage formation. However, the pathologic mechanism remains unclear. In the present study, we used a rat model with OA-like changes in the TMJ induced by experimental unilateral anterior crossbite (UAC) and found that the level of miR-26b was markedly lower in BMSCs from the subchondral bones of UAC rats than in those from sham control rats. MiR-26b overexpression by agomiR-26b increased condylar subchondral bone osteogenesis in UAC rats. Notably, although agomiR-26b primarily affected miR-26b levels in the subchondral bone (but not in cartilage or the synovium), the overexpression of miR-26b in BMSCs in UAC rats largely rescued OA-like cartilage degradation, while the inhibition of miR-26b in BMSCs exacerbated cartilage degradation in UAC rats. We measured the expression levels of β-catenin and related osteogenic and osteoclastic factors after using miR-26b mimics and inhibitors in vivo. Moreover, BMSCs were treated with the β-catenin blocker Wnt-C59 and then transfected with miR-26b mimics or inhibitors. Then, we examined the expression of β-catenin as the direct target of miR-26b. The results of the present study indicate that miR-26b may modulate subchondral bone loss induced by abnormal occlusion and influence the osteogenic differentiation of subchondral BMSCs through β-catenin in the context of TMJ-OA progression.

CXCR6 Mediates Pressure Overload-Induced Aortic Stiffness by Increasing Macrophage Recruitment and Reducing Exosome-miRNA29b.

Aortic stiffness is an independent risk factor for aortic diseases such as aortic dissection which commonly occurred with aging and hypertension. Chemokine receptor CXCR6 is critically involved in vascular inflammation and remodeling. Here, we investigated whether and how CXCR6 plays a role in aortic stiffness caused by pressure overload. CXCR6-/- and WT mice underwent transverse aortic constriction (TAC) surgery for 8weeks. CXCR6 deficiency significantly improved TAC-induced aortic remodeling and endothelial dysfunction by decreasing CD11c+ macrophage infiltration, suppressing VCAM-1 and ICAM-1, reducing collagen deposition, and downregulating MMP12 and osteopontin in the aorta. Consistently, blocking the CXCL16/CXCR6 axis also reduced aortic accumulation of CD11c+ macrophages and vascular stiffness but without affecting the release of TNF-α and IL-6 from the aorta. Furthermore, pressure overload inhibited aortic release of exosomes, which could be reversed by suppressing CXCR6 or CXCL16. Inhibition of exosome release by GW4869 significantly aggravated TAC-induced aortic calcification and stiffness. By exosomal microRNA microarray analysis, we found that microRNA-29b was significantly reduced in aortic endothelial cells (AECs) receiving TAC. Intriguingly, blocking the CXCL16/CXCR6 axis restored the expression of miR-29b in AECs. Finally, overexpression of miR-29b significantly increased eNOS and reduced MMPs and collagen in AECs. By contrast, antagonizing miR-29b in vivo further enhanced TAC-induced expressions of MMP12 and osteopontin, aggravated aortic fibrosis, calcification, and stiffness. Our study demonstrated a key role of the CXCL16/CXCR6 axis in macrophage recruitment and macrophage-mediated aortic stiffness under pressure overload through an exosome-miRNAs-dependent manner.

The microRNA-10b-Bim axis promotes cancer progression through activating autophagy in oral squamous cell carcinoma

Autophagy is related to many cellular mechanisms and dysregulation of autophagy involves the pathological process in cancer. miR-10b activates autophagy, which promotes invasion and migration of OSCC. Its functional role in the mechanism of OSCC to autophagy remains to be unclear. Overexpression of miR-10b was followed by enhanced OSCC invasion and migration and activated autophagic protein, such as LC3II/ATG5. MiR-10b attracted Bim directly according to the Bio-informatics analyses and double luciferases reporter assays. Functional experiments further revealed that miR-10b could promote invasion and migration in vitro. In addition, miR-10b induced autophagy via inhibiting Bim in invasion and migration of OSCC. Notably, animal experiments confirmed that miR-10b-Bim promoted proliferation and autophagy in OSCC. In addition, this study provides a theoretical support for regulating the mechanism of OSCC by inducing autophagy with miR-10b-Bim as a target.

MicroRNA-29b Suppresses Inflammation and Protects Blood-Brain Barrier Integrity in Ischemic Stroke.

Objectives Following cerebral ischemia, microRNA- (miR-) 29b in circulating blood is downregulated. This study investigates the underlying mechanism and implications of miR-29b in leukocyte induction. Methods miR-29b from stroke patients and rats with middle cerebral artery occlusion (MCAO) were assessed using real-time polymerase chain reaction (PCR). miR-29b agomir was used to increase miR-29b expression in leukocytes via intravenous injection. C1q and tumor necrosis factor (C1QTNF) 6, interleukin- (IL-) 1β, zonula occludens- (ZO-) 1, occludin, and ischemic outcomes were assessed in MCAO rats. Additionally, hCMEC/D3 cells were subjected to oxygen–glucose deprivation (OGD) and cocultured with HL-60 cells. Results miR-29b levels in neutrophils were found to be significantly lower in stroke patients compared with healthy controls, which may indicate its high diagnostic sensitivity and specificity for stroke. Moreover, miR-29b levels in leukocytes showed a negative correlation with National Institute of Health Stroke Scale (NIHSS) scores and C1QTNF6 levels. In MCAO rats, miR-29b overexpression reduced brain infarct volume and brain edema, decreasing IL-1β levels in leukocytes and in the brain 24 hours poststroke. miR-29b attenuated IL-1β expression via C1QTNF6 inhibition, leading to decreased blood-brain barrier (BBB) disruption and leukocyte infiltration. Moreover, miR-29b overexpression in HL-60 cells downregulated OGD-induced hCMEC/D3 cell apoptosis and increased ZO-1 and occludin levels in vitro. Conclusion Leukocytic miR-29b attenuates inflammatory response by augmenting BBB integrity through C1QTNF6, suggesting a novel miR-29b-based therapeutic therapy for ischemic stroke.

Blood-Based Biomarkers in Afp Normal/Stable Hepatocellular Carcinoma: Diagnostic and Prognostic Relevance of Mir-10b for Patients on Liver Transplant List

As a diagnostic criteria of hepatocellular carcinoma (HCC), the exact threshold of alpha-fetoprotein (AFP) is controversial. In additional, not all HCC tumors are AFP positive or secrete elevated amounts of AFP into the serum. However, the diagnosis of HCC is quite important on the liver transplant list. Therefore, the purpose of this study was to investigate the expression of circulating micro RNAs (miRNAs) in AFP-stable HCC patients. Thus, we aimed to determine a diagnostic biomarker in these patients. Sixteen miRNAs were evaluated using a real-time quantitative reverse transcription polymerase chain reaction system in AFP-stable HCC and AFP-trending HCC patients. In our study, 46.7% (n=28) of the patients diagnosed with HCC had stable/normal AFP levels. We detected that high expression of miR-24, miR-10b and the low expression of miR-143 were independently and significantly associated with HCC in AFP-stable compared with AFP trending (P < .05). Additionally, we demonstrated that the overexpression of miR-10b was associated with poor disease-free survival in HCC (P=0.001). Although more clinical validations are needed for the diagnosis of HCC, our current results indicate that the coexistence of high expression of miR-10b and miR-24 may help clinicians adjust in the diagnosis of HCC in patients who are on the liver transplant list but awaiting biopsy for the diagnosis of HCC.

Overexpression of mir-135b and mir-210 in mesenchymal stromal cells for the enrichment of extracellular vesicles with angiogenic factors.

Extracellular vesicles (EVs) are known as molecular carriers involved in cell communication and the regulation of (patho)physiological processes. miRNAs and growth factors are the main contents of EVs which make them a good candidate for the treatment of diseases caused by ischemia, but the low production of EVs by a cell producer and a significant variation of the molecular contents in EVs according to the cell source are the main limitations of their widespread use. Here, we show how to improve the therapeutic properties of mesenchymal stromal cell (MSC)-derived EVs (MSC-EVs) by modifying MSCs to enrich these EVs with specific angiomiRs (miR-135b or miR-210) using lentiviral vectors carrying miR-135b or miR-210. MSCs were obtained from the mouse bone marrow and transduced with a corresponding lentivector to overexpress miR-135b or miR-210. The EVs were then isolated by ultracentrifugation and characterized using a flow cytometer and a nanoparticle tracking analyzer. The levels of 20 genes in the MSCs and 12 microRNAs in both MSCs and EVs were assessed by RT‒qPCR. The proangiogenic activity of EVs was subsequently assessed in human umbilical vein endothelial cells (HUVECs). The results confirmed the overexpression of the respective microRNA in modified MSCs. Moreover, miR-135b overexpression upregulated miR-210-5p and follistatin, whereas the overexpression of miR-210 downregulated miR-221 and upregulated miR-296. The tube formation assay showed that EVs from MSCs overexpressing miR-210-5p (EVmiR210) significantly promoted tubular structure formation in HUVECs. A significant increase in angiogenic proteins (PGF, endothelin 1, and artemin) and genes (VEGF, activin A, and IGFBP1) in HUVECs treated with VEmiR210 justifies the better tubular structure formation of these cells compared with that of EVmiR135b-treated HUVECs, which showed upregulated expression of only artemin. Collectively, our results show that the EV cargo can be modified by lentiviral vectors to enrich specific miRNAs to achieve a specific angiogenic potential.