NAMPT enhances LOX expression and promotes metastasis in human chondrosarcoma cells by inhibiting miR-26b-5p synthesis.

Chondrosarcoma is a malignant bone tumor that emerges from abnormalities in cartilaginous tissue and is related with lung metastases. Nicotinamide phosphoribosyltransferase (NAMPT) is an adipocytokine reported to enhance tumor metastasis. Our results from clinical samples and the Gene Expression Omnibus data set reveal that NAMPT levels are markedly higher in chondrosarcoma patients than in normal individuals. NAMPT stimulation significantly increased lysyl oxidase (LOX) production in chondrosarcoma cells. Additionally, NAMPT increased LOX-dependent cell migration and invasion in chondrosarcoma by suppressing miR-26b-5p generation through the c-Src and Akt signaling pathways. Overexpression of NAMPT promoted chondrosarcoma metastasis to the lung in vivo. Furthermore, knockdown of LOX counteracted NAMPT-facilitated metastasis. Thus, the NAMPT/LOX axis presents a novel target for treating the metastasis of chondrosarcoma.

IL-2RG as a possible immunotherapeutic target in CRC predicting poor prognosis and regulated by miR-7-5p and miR-26b-5p.

Despite advances in treatment strategies, colorectal cancer (CRC) continues to cause significant morbidity and mortality, with mounting evidence a close link between immune system dysfunctions issued. Interleukin-2 receptor gamma (IL-2RG) plays a pivotal role as a common subunit receptor in the IL-2 family cytokines and activates the JAK-STAT pathway. This study delves into the role of Interleukin-2 receptor gamma (IL-2RG) within the tumor microenvironment and investigates potential microRNAs (miRNAs) that directly inhibit IL-2RG, aiming to discern their impact on CRC clinical outcomes. Bioinformatics analysis revealed a significant upregulation of IL-2RG mRNA in TCGA-COAD samples and showed strong correlations with the infiltration of various lymphocytes. Single-cell analysis corroborated these findings, highlighting IL-2RG expression in critical immune cell subsets. To explore miRNA involvement in IL-2RG dysregulation, mRNA was isolated from the tumor tissues and lymphocytes of 258 CRC patients and 30 healthy controls, and IL-2RG was cloned into the pcDNA3.1/CT-GFP-TOPO vector. Human embryonic kidney cell lines (HEK-293T) were transfected with this construct. Our research involved a comprehensive analysis of miRPathDB, miRWalk, and Targetscan databases to identify the miRNAs associated with the 3' UTR of human IL-2RG. The human microRNA (miRNA) molecules, hsa-miR-7-5p and hsa-miR-26b-5p, have been identified as potent suppressors of IL-2RG expression in CRC patients. Specifically, the downregulation of hsa-miR-7-5p and hsa-miR-26b-5p has been shown to result in the upregulation of IL-2RG mRNA expression in these patients. Prognostic evaluation of IL-2RG, hsa-miR-7-5p, and hsa-miR-26b-5p, using TCGA-COAD data and patient samples, established that higher IL-2RG expression and lower expression of both miRNAs were associated with poorer outcomes. Additionally, this study identified several long non-coding RNAs (LncRNAs), such as ZFAS1, SOX21-AS1, SNHG11, SNHG16, SNHG1, DLX6-AS1, GAS5, SNHG6, and MALAT1, which may act as competing endogenous RNA molecules for IL2RG by sequestering shared hsa-miR-7-5p and hsa-miR-26b-5p. In summary, this investigation underscores the potential utility of IL-2RG, hsa-miR-7-5p, and hsa-miR-26b-5p as serum and tissue biomarkers for predicting CRC patient prognosis while also offering promise as targets for immunotherapy in CRC management.

MiR-26b-5p/TET3 regulates the osteogenic differentiation of human bone mesenchymal stem cells and bone reconstruction in female rats with calvarial defects.

BACKGROUND: MicroRNAs (miRNAs) play critical roles in the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs), but the mechanism by which miRNAs indirectly modulate osteogenesis remains unclear. Here, we explored the mechanism by which miRNAs indirectly modulate gene expression through histone demethylases to promote bone regeneration. METHODS AND RESULTS: Bioinformatics analysis was performed on hBMSCs after 7 days of osteogenic induction. The differentially expressed miRNAs were screened, and potential target mRNAs were identified. To determine the bioactivity and stemness of hBMSCs and their potential for bone repair, we performed wound healing, Cell Counting Kit-8 (CCK-8), real-time reverse transcription quantitative polymerase chain reaction (RT‒qPCR), alkaline phosphatase activity, alizarin red S (ARS) staining and radiological and histological analyses on SD rats with calvarial bone defects. Additionally, a dual-luciferase reporter assay was utilized to investigate the interaction between miR-26b-5p and ten-eleven translocation 3 (TET3) in human embryonic kidney 293T cells. The in vitro and in vivo results suggested that miR-26b-5p effectively promoted the migration, proliferation and osteogenic differentiation of hBMSCs, as well as the bone reconstruction of calvarial defects in SD rats. Mechanistically, miR-26b-5p bound to the 3' untranslated region of TET3 mRNA to mediate gene silencing. CONCLUSIONS: MiR-26b-5p downregulated the expression of TET3 to increase the osteogenic differentiation of hBMSCs and bone repair in rat calvarial defects. MiR-26b-5p/TET3 crosstalk might be useful in large-scale critical bone defects.

M2 macrophage-derived exosomal miR-26b-5p regulates macrophage polarization and chondrocyte hypertrophy by targeting TLR3 and COL10A1 to alleviate osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent chronic musculoskeletal diseases among the elderly population. In this study, macrophage-derived exosomes were isolated and identified. Exosomes were subjected to microRNA (miRNA) sequencing and bioinformatic analysis, and differentially expressed miRNAs were verified. miR-26b-5p target genes were confirmed through target-site mutation combined with a dual-luciferase reporter assay. The effects of miR-26b-5p on macrophage polarization and chondrocyte hypertrophy were assessed in vitro. miR-26b-5p agomir was applied to mice with OA induced by anterior cruciate ligament transection (ACLT). The therapeutic effects of miR-26b-5p were evaluated via pain behavior experiments and histological observations. In vitro, miR-26b-5p repolarized M1 macrophages to an anti-inflammatory M2 type by targeting the TLR3 signaling pathway. miR-26b-5p could target COL10A1, further inhibiting chondrocyte hypertrophy induced by M1 macrophage-conditioned medium (M1-CM). In vivo, miR-26b-5p agomir ameliorated gait abnormalities and mechanical allodynia in OA mice. miR-26b-5p treatment attenuated synovitis and cartilage degeneration, thereby delaying OA progression. In conclusion, M2 macrophage-derived exosomal miR-26b-5p could protect articular cartilage and ameliorate gait abnormalities in OA mice by targeting TLR3 and COL10A1. miR-26b-5p further affected macrophage polarization and chondrocyte hypertrophy. Thus, this exosomal miR-26b-5p-based strategy might be a potential method for OA treatment.

Interplay of miR-542, miR-126, miR-143 and miR-26b with PI3K-Akt is a Diagnostic Signal and Putative Regulatory Target in HPV-Positive Cervical Cancer.

Human papillomavirus accounts for 99.7% of all cervical cancer cases worldwide. The viral oncoproteins alter normal cell signaling and gene expression, resulting in loss of cell cycle control and cancer development. Also, microRNAs (miRNAs) have been reported to play a critical role in cervical carcinogenesis. Especially these are not only appropriate targets for therapeutic intervention in cervical cancer but also early diagnostic signals. The given study tries to improve the sparse knowledge on miRNAs and their role in this physiological context. Deregulated miRNAs were identified by analyzing the raw data of the well-founded GSE20592 dataset including 16 tumor/normal pairs of human cervical tissue samples. The dataset was quantified by a conservative strategy based on HTSeq and Salmon, followed by target prediction via TargetScan and miRDB. The comprehensive pathway analysis of all factors was performed using DAVID. The theoretical results were subject of a stringent experimental validation in a well-characterized clinical cohort of 30 tumor/normal pairs of cervical samples. The top 31 miRNAs and their 140 primary target genes were closely intertwined with the PI3K-Akt signaling pathway. MiR-21-3p and miR-1-3p showed a prominent regulatory role while miR-542, miR-126, miR-143, and miR-26b are directly targeting both PI3K and AKT. This study provides insights into the regulation of PI3K-Akt signaling as an important inducer of cervical cancer and identified miR-542, miR-126, miR-143, and miR-26b as promising inhibitors of the PI3K-Akt action.

Loss of miR-26b-5p promotes gastric cancer progression via miR-26b-5p-PDE4B/CDK8-STAT3 feedback loop.

BACKGROUND: Chronic inflammation is a well-known risk factor for the development of gastric cancer (GC). Nevertheless, the molecular mechanisms underlying inflammation-related GC progression are incompletely defined. METHODS: Bioinformatic analysis was performed based on data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), and the expression of miR-26b-5p in GC cells and tissues was validated by quantitative real-time PCR (qRT-PCR). Cell proliferation was examined through Cell Counting Kit-8 (CCK8), 5-Ethynyl-2'-deoxyuridine (EdU), colony formation, flow cytometry, and tumor xenografts. Correlation between miR-26b-5p and Cyclin dependent kinase 8 (CDK8) or Phosphodiesterase 4B (PDE4B) was analyzed by dual-luciferase reporter assays, qRT-PCR, and Western blot. The effect of miR-26b-5p on the Signal transducer and activator of transcription 3 (STAT3) pathway was investigated using Western blot, immunofluorescence (IF), and immunohistochemistry (IHC). The impact of STAT3 on miR-26b-5p was determined by dual-luciferase reporter assays and qRT-PCR. RESULTS: The expression of miR-26b-5p was significantly downregulated in Helicobacter Pylori (H. pylori)-infected GC cells. The decreased expression of miR-26b-5p was also detected in GC cells and tissues compared to normal gastric epithelium cells (GES1) and normal adjacent gastric tissues. The low expression of miR-26b-5p promoted GC proliferation in vitro and in vivo and was related to the poor outcome of GC patients. In terms of mechanism, miR-26b-5p directly targeted PDE4B and CDK8, resulting in decreased phosphorylation and nuclear translocation of STAT3, which was associated with the regulation of GC proliferation by miR-26b-5p. Notably, miR-26b-5p was transcriptionally suppressed by STAT3, thus forming the miR-26b-5p-PDE4B/CDK8-STAT3 positive feedback loop. CONCLUSION: The newly identified miR-26b-5p-PDE4B/CDK8-STAT3 feedback loop plays an important role in inflammation-related GC progression and may serve as a promising therapeutic target for GC.

Astragaloside reduces toxic effect of periodontal ligament fibroblasts induced by lipopolysaccharide.

Periodontitis is a non-specific and chronic disease which is highly prevalent, resulting in inflammation and destruction of periodontal tissues. This study aims to explore the effect and mechanism of astragaloside on periodontitis. We used CCK-8, Western Blot, qPCR and flow cytometry to analyze cell viability, related protein and mRNA expression, and cell apoptosis. We found that AST could promote cell proliferation and reduce apoptosis induced by LPS. Besides, AST could alleviate the increased expression of COX-2 and ICAM-1 induced by LPS. MiR-26b-3P specifically targeted the 3' UTR of ICAM-1. These results indicate that AST reduces toxic effect of human periodontal ligament cells through regulating miR-26b-3P/ICAM-1, thus highlighting its protective role in periodontitis.

Targeting of PFKFB3 with miR-206 but not mir-26b inhibits ovarian cancer cell proliferation and migration involving FAK downregulation.

Few studies explored the role of microRNAs (miRNAs) in the post-transcriptional regulation of glycolytic proteins and downstream effectors in ovarian cancer cells. We recently showed that the functional activation of the cytoskeletal regulator FAK in endothelial cells is fostered by the glycolytic enhancer 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). We tested the hypothesis that miR-206 and mir-26b, emerging onco-suppressors targeting PFKFB3 in estrogen-dependent tumors, would regulate proliferation and migration of serous epithelial ovarian cancer (EOC) cells via common glycolytic proteins, i.e., GLUT1 and PFKFB3, and downstream FAK. PFKFB3 was overexpressed in SKOV3, and its pharmacological inhibition with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) significantly reduced cell proliferation and motility. Both miR-206 and miR-26b directly targeted PFKFB3 as evaluated by a luciferase reporter assay. However, endogenous levels of miR-26b were higher than those of miR-206, which was barely detectable in SKOV3 as well as OVCAR5 and CAOV3 cells. Accordingly, only the anti-miR-26b inhibitor concentration-dependently increased PFKFB3 levels. While miR-206 overexpression impaired proliferation and migration by downregulating PFKFB3 levels, the decreased PFKFB3 protein levels related to miR-26 overexpression had no functional consequences in all EOC cell lines. Finally, consistent with the migration outcome, exogenous miR-206 and miR-26b induced opposite effects on the levels of total FAK and of its phosphorylated form at Tyr576/577. 3PO did not prevent miR-26b-induced SKOV3 migration. Overall, these results support the inverse relation between endogenous miRNA levels and their tumor-suppressive effects and suggest that restoring miR-206 expression represents a potential dual anti-PFKFB3/FAK strategy to control ovarian cancer progression.

Plasma exosome miRNA-26b-3p derived from idiopathic short stature impairs longitudinal bone growth via the AKAP2/ERK1/2 axis.

BACKGROUND: Currently, the etiology of idiopathic short stature (ISS) is still unclear. The poor understanding of the molecular mechanisms of ISS has largely restricted this strategy towards safe and effective clinical therapies. METHODS: The plasma exosomes of ISS children were co-cultured with normal human chondrocytes. The differential expression of exosome miRNA between ISS and normal children was identified via high-throughput microRNA sequencing and bioinformatics analysis. Immunohistochemistry, In situ hybridization, RT-qPCR, western blotting, luciferase expression, and gene overexpression and knockdown were performed to reveal the key signaling pathways that exosome miRNA of aberrant expression in ISS children impairs longitudinal bone growth. RESULTS: Chondrocytes proliferation and endochondral ossification were suppressed after coculture of ISS plasma exosomes with human normal chondrocytes. High-throughput microRNA sequencing and RT-qPCR confirmed that plasma exosome miR-26b-3p was upregulated in ISS children. Meanwhile, exosome miRNA-26b-3p showed a high specificity and sensitivity in discriminating ISS from normal children. The rescue experiment showed that downregulation of miR-26b-3p obviously improved the repression of chondrocyte proliferation and endochondral ossification caused by ISS exosomes. Subsequently, miR-26b-3p overexpression inhibited chondrocyte proliferation and endochondral ossification once again. In situ hybridization confirmed the colocalization of miR-26b-3p with AKAP2 in chondrocytes. In vitro and in vivo assay revealed exosome miRNA-26b-3p impairs longitudinal bone growth via the AKAP2 /ERK1/2 axis. CONCLUSIONS: This study is the first to confirm that miR-26b-3p overexpression in ISS plasma exosomes leads to disorders in proliferation and endochondral ossification of growth plate cartilage via inhibition of AKAP2/ERK1/2 axis, thereby inducing ISS. This study provides a new research direction for the etiology and pathology of ISS and a new idea for the biological treatment of ISS.

Circulating miR-26b-5p and miR-451a as diagnostic biomarkers in medullary thyroid carcinoma patients.

PURPOSE/METHODS: The determination of tumour biomarkers is paramount to advancing personalized medicine, more so in rare tumours like medullary thyroid carcinoma (MTC), whose diagnosis is still challenging. The aim of this study was to identify non-invasive circulating biomarkers in MTC. To achieve this goal, paired MTC tissue and plasma extracellular vesicle samples were collected from multiple centres and microRNA (miRNA) expression levels were evaluated. RESULTS: The samples from a discovery cohort of 23 MTC patients were analysed using miRNA arrays. Lasso logistic regression analysis resulted in the identification of a set of circulating miRNAs as diagnostic biomarkers. Among them, miR-26b-5p and miR-451a, were highly expressed and their expression decreased during follow-up in disease-free patients in the discovery cohort. Circulating miR-26b-5p and miR-451a were validated using droplet digital PCR in a second independent cohort of 12 MTC patients. CONCLUSION: This study allowed the identification and validation of a signature of two circulating miRNAs, miR-26b-5p and miR-451a, in two independent cohorts reporting a significant diagnostic performance for MTC. The results of this study offer advancements in molecular diagnosis of MTC proposing a novel non-invasive tool to use in precision medicine.

LncRNA LINC00205 stimulates osteoporosis and contributes to spinal fracture through the regulation of the miR-26b-5p/KMT2C axis.

BACKGROUND: Osteoporosis (OP) is a common bone disease marked by decreased bone strength. Increasing evidence suggests that long non-coding RNA (lncRNAs) play important roles in the occurrence and progression of OP. This study aimed to investigate the role and mechanism of LINC00205 in the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and OP. METHODS: Bone tissue samples were obtained from healthy controls and patients with osteoporosis with a spinal fracture (OP-Frx) or without a spinal fracture (OP-no-Frx). HMSCs were cultured and induced to undergo osteogenic differentiation. The expression of LINC00205, lysine (K)-specific methyltransferase 2C (KMT2C), and miR-26b-5p in bone tissues and cells was evaluated using western blotting and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The effects of LINC00205, miR-26b-5p, and KMT2C on calcium deposition, alkaline phosphatase (ALP) activity, and mRNA levels of the osteogenic differentiation marker genes [ALP, osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2)] were investigated using alizarin red S staining, an ALP activity assay, and qRT-PCR, respectively. Dual-luciferase reporter assay was performed to ascertain the binding relationship between miR-26b-5p and LINC00205/KMT2C. RESULTS: LINC00205 and KMT2C were upregulated in patients with OP-Frx and OP-no-Frx, whereas miR-26b-5p was downregulated. Furthermore, LINC00205 and KMT2C expression decreased, whereas that of miR-26b-5p increased over time from day 7 to 21 of the osteogenic differentiation of hMSCs. The knockdown of LINC00205 and KMT2C significantly increased ALP activity, calcium deposition, and the expression of RUNX2, ALP, and OCN. In contrast, the inhibition of miR-26b-5p yielded the opposite result. These data suggest that LINC00205 inhibits the osteogenic differentiation of hMSCs by modulating the miR-26b-5p/KMT2C signaling axis. CONCLUSION: LINC00205 promotes OP and is involved in spinal fractures. LINC00205 is also a potential negative regulator of the osteogenic differentiation of hMSCs.

Lysosome-related exosome secretion mediated by miR-26b / Rab31 pathway was associated with the proliferation and migration of MCF-7 cells treated with BPA.

Bisphenol A (BPA), one of the typical environmental endocrine disruptors (EEDs), can promote the proliferation and migration of cancer cells, but the mechanism of which remains largely unclear. Exosome secretion plays an important role in the stress response of cells to environmental stimuli. This study was designed to explore whether exosome secretion was involved in the toxic effect of BPA on the proliferation and migration of MCF-7 cells, and the related mechanism. Our data shows that the IC50 value of MCF-7 exposure to BPA was about 65.82 µM. The exposure of MCF-7 to 10 µM BPA resulted in a decreased miR-26b expression and the activation of miR-26b/Rab-31 pathway, consequently, the number and activity of lysosomes decreased, the secretion of exosomes increased, cell proliferation and migration were enhanced obviously. Interestingly, miR-26b mimic up-regulated the number and activity of lysosomes via miR-26b/miR-31 pathway, exosome secretion was down-regulated, cell proliferation and migration decreased. Further, when GW4869 was used to directly inhibit the exosome secretion of MCF-7 treated with BPA, their proliferation and migration were down-regulated. Herein, we concluded that the stimulating effect of BPA on the proliferation and migration of MCF-7 cells was associated with the lysosome - related exosome secretion via miR-26b / Rab31 pathway.

Fatty Acid Desaturation Is Suppressed in Mir-26a/b Knockout Goat Mammary Epithelial Cells by Upregulating INSIG1.

MicroRNA-26 (miR-26a and miR-26b) plays a critical role in lipid metabolism, but its endogenous regulatory mechanism in fatty acid metabolism is not clear in goat mammary epithelial cells (GMECs). GMECs with the simultaneous knockout of miR-26a and miR-26b were obtained using the CRISPR/Cas9 system with four sgRNAs. In knockout GMECs, the contents of triglyceride, cholesterol, lipid droplets, and unsaturated fatty acid (UFA) were significantly reduced, and the expression of genes related to fatty acid metabolism was decreased, but the expression level of miR-26 target insulin-induced gene 1 (INSIG1) was significantly increased. Interestingly, the content of UFA in miR-26a and miR-26b simultaneous knockout GMECs was significantly lower than that in wild-type GMECs and miR-26a- and miR-26b-alone knockout cells. After decreasing INSIG1 expression in knockout cells, the contents of triglycerides, cholesterol, lipid droplets, and UFAs were restored, respectively. Our studies demonstrate that the knockout of miR-26a/b suppressed fatty acid desaturation by upregulating the target INSIG1. This provides reference methods and data for studying the functions of miRNA families and using miRNAs to regulate mammary fatty acid synthesis.

CDK14/ß-catenin/TCF4/miR-26b positive feedback regulation modulating pancreatic cancer cell phenotypes in vitro and tumor growth in mice model in vivo.

INTRODUCTION: Chemotherapy and radiotherapy have been reported to be basically ineffective for pancreatic ductal adenocarcinoma patients; thus, gene therapy might provide a novel approach. CDK14, a new oncogenic member of the CDK family involved in the pancreatic cancer cell response to gemcitabine treatment, has been reported to be regulated by microRNAs. In the present study, we aimed to investigate whether miR-26b regulated CDK14 expression to affect the phenotype of pancreatic cancer cells. METHODS: Overexpression or knockdown of CDK14 or miR-26b was generated in pancreatic cancer cell lines and the function of CDK14 and miR-26b on cell phenotype and the Wnt signaling pathway was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-ethynyl-2'-deoxyuridine and transwell assays, as well as a xenograft model and western blotting. The predicted binding site between the 3'-untranslated region of CDK14 and miR-26b, miR-26b promoter and TCF4 was verified by luciferase or chromatin immunoprecipitation assays. RESULTS: CDK14 overexpression inhibited p-GSK3ß, whereas it promoted p-LRP6, the nuclear translocation of ß-catenin and the transactivation of TCF4 transcription factor, thus promoting pancreatic cancer cell aggressiveness. miR-26b directly targeted CDK14 and inhibited CDK14 expression. In vitro and in vivo, miR-26b overexpression inhibited, and CDK14 overexpression promoted, cancer cell aggressiveness; CDK14 overexpression partially attenuated the miR-26b overexpression effects on cancer cells. The effects of miR-26b overexpression on tumor growth and the Wnt/ß-catenin/TCF4 signaling were partially reversed by CDK14 overexpression. TCF4 inhibited the expression of miR-26b by targeting its promoter region. CONCLUSIONS: CDK14, ß-catenin, TCF4 and miR-26b form a positive feedback regulation for modulating pancreatic cancer cell phenotypes in vitro and tumor growth in vivo.

Asporin regulated by miR-26b-5p mediates chondrocyte senescence and exacerbates osteoarthritis progression via TGF-ß1/Smad2 pathway.

OBJECTIVES: This study aimed to investigate the role and mechanism of asporin in modulating chondrocyte senescence in OA pathology. METHODS: Asporin and senescence-related hallmark expression were examined in human and experimental OA mouse cartilage samples. Twelve-week-old male C57 mice were administered with recombinant protein (rm-asporin)- or asporin-siRNA-expressing lentiviruses via intra-articular injection once a week after destabilization of the medial meniscus (DMM) surgery to induce OA. Cartilage damage was measured using the Osteoarthritis Research Society International score. Senescence-associated ß-galactosidase (SA-ß-Gal) staining, γH2AX, p21 and p16INK4a were analysed by immunofluorescence staining and western blot to assess the specific role of asporin in chondrocyte senescence. The TGF-ß1-Smad2 signalling pathway and miR-26b-5p were further evaluated to explore the mechanism of asporin in OA. RESULTS: Asporin was upregulated in articular chondrocytes of OA patients and DMM mice and accompanied by accumulation of senescent cells. Asporin overexpression exaggerated OA progression, whereas silencing asporin restored chondrocyte homeostasis and deferred chondrocyte senescence, leading to markedly attenuated DMM-induced OA. Cellular and molecular analyses showed that asporin can be inhibited by miR-26b-5p, which was significantly downregulated in OA cartilage, leading to exacerbation of experimental OA partially through inhibition of TGF-ß1-Smad2 signalling in chondrocytes. CONCLUSIONS: Our findings indicate that asporin plays an essential role in chondrocyte senescence and OA pathogenesis. Upregulated by miR-26b-5p, asporin inhibits the TGF-ß1-Smad2 pathway to accelerate chondrocyte senescence and exacerbate cartilage degeneration. Targeting the miR-26b-5p-asporin-Smad2 axis may serve as a practical therapeutic strategy to delay chondrocyte senescence and OA development.

CircPVT1 promotes the tumorigenesis and metastasis of osteosarcoma via mediation of miR-26b-5p/CCNB1 axis.

INTRODUCTION: Osteosarcoma (OS) is the most aggressive malignancy among the bone tumors in the world. Circular RNAs (circRNAs) have been reported to be participated in multiple cancers, including OS. Meanwhile, circPVT1 has been proved to be upregulated in OS. However, the mechanism by which circPVT1 mediates the tumorigenesis of OS remains to be further explored. MATERIALS AND METHODS: Protein and gene expressions in OS cells were measured by western blot and RT-qPCR, respectively. Cell growth was assessed by flow cytometry and colony formation, respectively. In addition, cell migration was assessed by wound healing, and invasion was evaluated by Transwell assay. Meanwhile, the correlation among circPVT1, miR-26b-5p and CCNB1 was explored by RNA pull-down and dual luciferase assay. Finally, in vivo model was established to explore the role of circPVT1 in OS in vivo. RESULTS: CircPVT1 and CCNB1 were significantly upregulated in OS cells, while miR-26b-5p was downregulated. Knockdown of circPVT1 notably inhibited proliferation and induced apoptosis of OS cells. CircPVT1 shRNA significantly suppressed the OS cell invasion and migration. Meanwhile, circPVT1 sponged miR-26b-5p and CCNB1 was found to be the direct target of miR-26b-5p. Furthermore, silencing of circPVT1 inhibited the growth and metastasis of OS in vivo. CONCLUSION: Silencing of circPVT1 notably suppressed the tumorigenesis and metastasis of OS via miR-26b-5p/CCNB1 axis. Therefore, circPVT1 might be used as a target for OS treatment.

MicroRNA-26b-5p Targets DAPK1 to Reduce Intestinal Ischemia/Reperfusion Injury via Inhibition of Intestinal Mucosal Cell Apoptosis.

BACKGROUND: Emerging evidence has suggested that miRNAs are important regulators of intestinal I/R injury, but their function in this context remains elusive. AIMS: To evaluate the role of miR-26b-5p in intestinal I/R injury. METHODS: We utilized in vivo murine models of intestinal I/R and in vitro Mode-K cell-based models of oxygen and glucose deprivation/reperfusion (OGD/R) to examine the function of miR-26b-5p in intestinal I/R injury. The expression of miR-26b-5p in intestinal mucosa and Mode-K cell was detected by RT-PCR. HE staining and Chiu's score were used to evaluate intestinal mucosa injury severity. Apoptosis was detected by TUNEL stain, flow cytometry, and western blot. TargetScan and StarBase prediction algorithms were applied to predict putative target genes of miR-26b-5p and validated by luciferase reporter analyses. RESULTS: We found that the expression of miR-26b-5p in intestinal mucosa was markedly decreased during I/R injury. We additionally found miR-26b-5p overexpression to markedly disrupt intestinal I/R- or OGD/R-induced injury in vivo and in vitro, whereas inhibiting this miRNA had an adverse impact and resulted in increased intestinal tissue injury and Mode-K cell damage. From a mechanistic perspective, miR-26b-5p was predicted to target DAPK1, which was related to cellular apoptosis. Luciferase reporter assay results confirmed that miR-26b-5p directly targets DAPK1 in Mode-K cells, thereby suppressing OGD/R-induced cell apoptosis. CONCLUSION: Our findings show that miR-26b-5p may prevent intestinal I/R injury via targeting DAPK1 and inhibiting intestinal mucosal cell apoptosis, suggesting that this miRNA may be a viable target for the treatment of intestinal I/R injury.

Combinatorial targeting of microRNA-26b and microRNA-101 exerts a synergistic inhibition on cyclooxygenase-2 in brain metastatic triple-negative breast cancer cells.

PURPOSE: Extravasation of triple-negative (TN) metastatic breast cancer (BC) cells through the brain endothelium (BE) is a critical step in brain metastasis (BM). During extravasation, metastatic cells induce alteration in the inter-endothelial junctions and transmigrate through the endothelial barrier. Transmigration of metastatic cells is mediated by the upregulation of cyclooxygenase-2 (COX-2) that induces matrix metalloproteinase-1 (MMP-1) capable of degrading inter-endothelial junctional proteins. Despite their important role in BM, the molecular mechanisms upregulating COX-2 and MMP-1 in TNBC cells remain poorly understood. In this study, we unraveled a synergistic effect of a pair of micro-RNAs (miR-26b-5p and miR-101-3p) on COX-2 expression and the brain transmigration ability of BC cells. METHODS: Using a gain-and-loss of function approach, we modulated levels of miR-26b-5p and miR-101-3p in two TNBC cell lines (the parental MDA-MB-231 and its brain metastatic variant MDA-MB-231-BrM2), and examined the resultant effect on COX-2/MMP-1 expression and the transmigration of cancer cells through the BE. RESULTS: We observed that the dual inhibition of miR-26b-5p and miR-101-3p in BC cells results in higher increase of COX-2/MMP-1 expression and a higher trans-endothelial migration compared to either micro-RNA alone. The dual restoration of both micro-RNAs exerted a synergistic inhibition on COX-2/MMP-1 by targeting COX-2 and potentiated the suppression of trans-endothelial migration compared to single micro-RNA. CONCLUSION: These findings provide new insights on a synergism between miR-26-5p and miR-101-3p in regulating COX-2 in metastatic TNBC cells and shed light on miR-26-5p and miR-101-3p as prognostic and therapeutic targets that can be exploited to predict or prevent BM.

The m6A methyltransferase METTL3 controls epithelial-mesenchymal transition, migration and invasion of breast cancer through the MALAT1/miR-26b/HMGA2 axis.

BACKGROUND: Previous studies have revealed the key functions of N6-methyladenosine (m6A) modification in breast cancer (BC). MALAT1 as a highly m6A modified lncRNA associated with cancer development and metastasis, but the functional relevance of m6A methyltransferase and MALAT1 in BC is still unknown. Here, our study investigated the effects of the novel m6A methyltransferase METTL3 on epithelial-mesenchymal transition (EMT) in BC via the MALAT1/miR-26b/HMGA2 axis. METHODS: Firstly, we collected clinical BC samples and cultured BC cells, and detected mRNA and protein levels in the human samples and human cell lines by RT-qPCR and Western blot, respectively. Then, the binding of MALAT1 and miR-26b and the targeting relationship between miR-26b and HMGA2 were examined by dual-luciferase assay. Moreover, the binding of MALAT1 and miR-26b was tested by RNA pull down and RNA immunoprecipitation (RIP) assays. Methylated-RNA immunoprecipitation (Me-RIP) was used to detect the m6A modification level of MALAT1. The interaction of METTL3 and MALAT1 was detected by photoactivatable ribonucleoside-crosslinking immunoprecipitation (PAR-CLIP). Finally, effects on invasion and migration were detected by Transwell. RESULTS: In BC, the level of miR-26b was consistently low, while the levels of METTL3, MALAT1 and HMGA2 were high. Further experiments showed that METTL3 up-regulated MALAT1 expression by modulating the m6A modification of MALAT1, and that MALAT1 could promote the expression of HMGA2 by sponging miR-26b. In BC cells, we found that silencing METTL3 could inhibit EMT and tumor cell invasion by suppressing MALAT1. Furthermore, MALAT1 mediated miR-26b to target HMGA2 and promote EMT, migration, and invasion. In summary, METTL3 promoted tumorigenesis of BC via the MALAT1/miR-26b/HMGA2 axis. CONCLUSIONS: Silencing METTL3 down-regulate MALAT1 and HMGA2 by sponging miR-26b, and finally inhibit EMT, migration and invasion in BC, providing a theoretical basis for clinical treatment of BC.

MiR-26b-5p inhibits cell proliferation and EMT by targeting MYCBP in triple-negative breast cancer.

BACKGROUND: The study was designed to elucidate the association and functional roles of miR-26b-5p and c-MYC binding protein (MYCBP) in triple-negative breast cancer (TNBC). METHOD: Luciferase reporter assay was used to confirm the relationship between miR-26b-5p and MYCBP in TNBC cells. The expression levels of miR-26b-5p and MYCBP in tissue specimens and cell lines were determined using reverse transcription-quantitative PCR. Cell proliferation, migration and invasion were assessed using CCK-8 assay, colony formation and transwell assay. RESULTS: We first observed that miR-26b-5p directly targets the 3'-UTR of MYCBP to inhibit MYCBP expression in MDA-MB-468 and BT-549 cells. The expression of miR-26b-5p was inversely correlated with MYCBP expression in TNBC tissues. We further demonstrated that MYCBP knockdown suppressed the proliferation, migration and invasion of TNBC cells. Furthermore, MYCBP overexpression counteracted the suppressive effect of miR-26b-5p on TNBC cell behaviors. Western blot analysis demonstrated that the E-cadherin protein level was increased, while protein levels of N-cadherin and vimentin were decreased in cells transfected with miR-26b-5p, which were all reversed by ectopic expression of MYCBP. CONCLUSIONS: In summary, our findings revealed the tumor suppressive role of miR-26b-5p in regulating TNBC cell proliferation and mobility, possibly by targeting MYCBP.