MicroRNA-26b-5p alleviates murine collagen-induced arthritis by modulating Th17 cell plasticity.

Rheumatoid arthritis (RA) is a chronic autoimmune disease, and the abnormal differentiation of IL-17-producing T helper (Th17) cells is an important factor in the pathogenesis. Previous studies have shown that microRNAs (miRNAs, miR) act as key regulators of Th17 cells. However, the effects of miRNAs on Th17 cell differentiation and plasticity in RA are not clear. In this study, not only low miR-26b-5p expression and high IL-17A level were observed in the peripheral blood of RA patients, but also the negative correlation between miR-26b-5p and IL-17A was explored. The changes in collagen-induced arthritis (CIA) mice were consistent with those in RA patients. The results of in vitro experiments showed that miR-26b-5p mainly inhibited the initial differentiation of Th17 cells but did not impact the differentiation of induced-Treg into Th17-like cells. Meanwhile, miR-26b-5p mimics treatment alleviated inflammatory responses and reduced Th17 proportion in CIA mice. These results indicated that miR-26b-5p could alleviate the development of mice CIA by inhibiting the excessive Th17 cells, and that miR-26b-5p could modulate the plasticity of Th17 cell differentiation in RA, mainly block the initial differentiation. This may provide a novel strategy for the clinical treatment of RA.

Exosomal microRNA-26b-5p down-regulates ATF2 to enhance radiosensitivity of lung adenocarcinoma cells.

Lung adenocarcinoma (LUAD), as the most common subtype of non-small cell lung cancer, is responsible for more than 500 000 deaths worldwide annually. In this study, we identify a novel microRNA-26b-5p (miR-26b-5p) and elucidated its function on LUAD. The survival rate of parent LUAD cells and radiation-resistant LUAD cells were determined using clonogenic survival assay. We overexpressed or inhibited miR-26b-5p in LUAD, and the correlation between activating transcription factor 2 (ATF2) and miR-26b-5p was determined using integrated bioinformatics analysis and dual-luciferase reporter gene assay. Exosomes derived from A549 cell lines were then detected using Western blot assay, followed by co-transfection with radiation-resistant A549R cells. LUAD tissues and serum were collected, followed by miR-26b-5p relative expression quantification using RT-qPCR. miR-26b-5p was identified as the most differentially expressed miRNA and was down-regulated in LUAD. Radiation-resistant cells were more resistant to X-radiation compared with parent cells. miR-26b-5p overexpression and X-irradiation led to enhanced radiosensitivity of LUAD cells. ATF2 was negatively targeted by miR-26b-5p. Exosomal miR-26b-5p derived from A549 cells could be transported to irradiation-resistant LUAD cells and inhibit ATF2 expression to promote DNA damage, apoptosis and radiosensitivity of LUAD cells, which was verified using serum-based miR-26b-5p. Our results show a regulatory network of miR-26b-5p on radiosensitivity of LUAD cells, which may serve as a non-invasive biomarker for LUAD.

MicroRNA-26b inhibits oligodendrocyte precursor cell differentiation by targeting adrenomedullin in spinal cord injury.

Oligodendrocyte precursor cells (OPCs) serve as a reservoir of newborn oligodendrocytes (OLs) in pathological and homeostatic conditions. After spinal cord injury (SCI), OPCs are activated to generate myelinating OLs, contributing to remyelination and functional recovery; however, the underlying molecular mechanisms remain unclear. Here, microRNA-26b (miR-26b) expression in the spinal cord tissues of SCI rats was examined by real-time polymerase chain reaction analysis. The influences of miR-26b on locomotor recovery following SCI were assessed utilizing Basso, Beattie, and Bresnahan (BBB) scores. The effects of miR-26b on OPC differentiation were explored using immunofluorescence and western blot analyses in vitro and in vivo. The potential targets that are modulated by miR-26b were identified by bioinformatics, luciferase reporter assays, and western blot analyses. The effects of adrenomedullin (ADM) on OPC differentiation were explored in vitro using immunofluorescence and western blot analyses. We demonstrated that miR-26b was significantly downregulated after SCI. BBB scores showed that miR-26b exacerbated the locomotor function deficits induced by SCI. In vitro, miR-26b inhibited the differentiation of primary rat OPCs. In vivo, miR-26b suppressed OPC differentiation in SCI rats. Bioinformatics analyses and experimental detection revealed that miR-26b directly targeted ADM in OPCs. In addition, knockdown of ADM suppressed the differentiation of primary rat OPCs. Our study provides evidence that ADM may mediate miR-26b-inhibited OPC differentiation in SCI.

MiR-26a/miR-26b represses tongue squamous cell carcinoma progression by targeting PAK1.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is the most common oral malignancy. Previous studies found that microRNA (miR)-26a and miR-26b were downregulated in TSCC tissues. The current study was designed to explore the effects of miR-26a/miR-26b on TSCC progression and the potential mechanism. METHODS: Expression of miR-26a, miR-26b and p21 Activated Kinase 1 (PAK1) in TSCC tissues and cell lines was detected by reverse transcription- quantitative polymerase chain reaction (RT-qPCR). Flow cytometry analysis was performed to examine cell cycle and apoptosis. Transwell assay was conducted to evaluate the migrated and invasive abilities of SCC4 and Cal27 cells. In addition, western blot assay was employed to analyze the protein level. Glucose assay kit and lactate assay kit were utilized to analyze glycolysis. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were applied to explore the relationship between miR-26a/miR-26b and PAK1. Xenograft tumor model was constructed to explore the role of miR-26a/miR-26b in vivo. RESULTS: Both miR-26a and miR-26b were underexpressed, while PAK1 was highly enriched in TSCC. Overexpression of miR-26a and miR-26b inhibited TSCC cell cycle, migration invasion and glycolysis, while promoted cell apoptosis. Both miR-26a and miR-26b directly targeted and negatively regulated PAK1 expression. Introduction of PAK1 partially reversed miR-26a/miR-26b upregulation-mediated cellular behaviors in TSCC cells. Gain of miR-26a/miR-26b blocked TSCC tumor growth in vivo. CONCLUSION: MiR-26a/miR-26b repressed TSCC progression via targeting PAK1 in vitro and in vivo, which enriched our understanding about TSCC development and provided new insights into the its treatment.

MicroRNA-26b inhibits hepatitis B virus transcription and replication by targeting the host factor CHORDC1 protein.

Hepatitis B virus (HBV) causes acute and chronic hepatitis in humans, and HBV infection is a major threat to global health. HBV replication is regulated by a series of host factors, including microRNAs (miRNAs), which are highly conserved small noncoding RNAs that participate in a variety of physiological and pathological processes. Here, we report that a chemically synthesized mimic of miR-26b inhibited HBV antigen expression, transcription, and replication, whereas antisense knockdown of endogenous miR-26b enhanced HBV replication in HepG2 cells. Overexpression and knockdown experiments showed that miR-26b significantly decreased HBV enhancer/promoter activities. We identified the cysteine- and histidine-rich domain containing 1 (CHORDC1) as a novel host factor target of miR-26b. CHORDC1 protein but not mRNA was markedly decreased by miR-26b overexpression via base-pairing with complementary sequences in the 3'UTR of its mRNA. Overexpression and knockdown studies showed that CHORDC1 increased viral antigen expression, transcription, and replication by elevating HBV enhancer/promoter activities. Conversely, HBV infection suppressed miR-26b expression and increased CHORDC1 protein levels in human liver cells. Another mature miRNA of the hsa-miR-26 family, miR-26a, had a similar function as miR-26b in targeting CHORDC1 and affecting HBV production. These results suggest that suppression of miR-26b expression up-regulates its target gene CHORDC1, which increases HBV enhancer/promoter activities and promotes viral transcription, gene expression, and replication. Our study could provide new insights into miRNA expression and the persistence of HBV infection.

MiR-26b is down-regulated in carcinoma-associated fibroblasts from ER-positive breast cancers leading to enhanced cell migration and invasion.

Carcinoma-associated fibroblasts (CAFs) influence the behaviour of cancer cells but the roles of microRNAs in this interaction are unknown. We report microRNAs that are differentially expressed between breast normal fibroblasts and CAFs of oestrogen receptor-positive cancers, and explore the influences of one of these, miR-26b, on breast cancer biology. We identified differentially expressed microRNAs by expression profiling of clinical samples and a tissue culture model: miR-26b was the most highly deregulated microRNA. Using qPCR, miR-26b was confirmed as down-regulated in fibroblasts from 15 of 18 further breast cancers. Next, we examined whether manipulation of miR-26b expression changed breast fibroblast behaviour. Reduced miR-26b expression caused fibroblast migration and invasion to increase by up to three-fold in scratch-closure and trans-well assays. Furthermore, in co-culture with MCF7 breast cancer epithelial cells, fibroblasts with reduced miR-26b expression enhanced both MCF7 migration in trans-well assays and MCF7 invasion from three-dimensional spheroids by up to five-fold. Mass spectrometry was used to identify expression changes associated with the reduction of miR-26b expression in fibroblasts. Pathway analyses of differentially expressed proteins revealed that glycolysis/TCA cycle and cytoskeletal regulation by Rho GTPases are downstream of miR-26b. In addition, three novel miR-26b targets were identified (TNKS1BP1, CPSF7, COL12A1) and the expression of each in cancer stroma was shown to be significantly associated with breast cancer recurrence. MiR-26b in breast CAFs is a potent regulator of cancer behaviour in oestrogen receptor-positive cancers, and we have identified key genes and molecular pathways that act downstream of miR-26b in CAFs.

Downregulation of miR-26b attenuates early brain injury induced by subarachnoid hemorrhage via mediating the KLF4/STAT3/HMGB1 axis.

BACKGROUND: Early brain injury (EBI) refers to early-onset secondary complications that occur after subarachnoid hemorrhage (SAH), and associated with high rate of disability and mortality. Recent investigations have indicated microRNA-26b (miR-26b) as a biomarker in the progression of SAH. Accordingly, the present study was designed to elucidate the role of miR-26b in influencing EBI following SAH and the downstream mechanisms. METHODS: Firstly, SAH rat models and neuron injury models were developed to assess the effect of miR-26b on EBI-like symptoms and subsequent inflammation. Dual-luciferase reporter gene assay was further implemented to evaluate the binding of miR-26b to its putative target gene STAT3. Loss-of-function and rescue experiments were performed to assess the functionality of miR-26b-mediated STAT3 in both models. RESULTS: miR-26b was found to target KLF4 and negative-modulate its expression, whereby aggravating EBI and inflammatory response in SAH rat models and stimulating hemoglobin-induced apoptosis in astrocytes. On the other hand, silencing of miR-26b reversed these changes in SAH rat models and hemoglobin (Hb)-induced astrocytes. miR-26b could further activate STAT3 via down-regulation of KLF4. Furthermore, KLF4 knockdown up-regulated HMGB1 to aggravate EBI following SAH. CONCLUSIONS: Collectively, our findings highlighted the ameliorative effect of miR-26b inhibition on EBI in SAH and the possible mechanism associated with the KLF4/STAT3/HMGB1 axis.

HDAC3 Impairs Cardiac Function in Chronic Heart Failure Rats via Mediating MicroRNA-26b-3p to Target High Mobility Group AT-Hook 2.

BACKGROUND: Histone deacetylase 3 (HDAC3) has been studied in chronic heart failure (CHF), while the regulatory mechanism of HDAC3 on the development of CHF in regulating microRNA (miR)-26b-3p/high mobility group AT-hook 2 (HMGA2) axis has not been extensively investigated. This study aimed to probe the effects of HDAC3, miR-26b-3p and HMGA2 on CHF. METHODS: CHF rat models were established using aortic coarctation. HDAC3, miR-26b-3p and HMGA2 levels in CHF rats were examined. Thereafter, the CHF rats were injected with relative oligonucleotides and plasmids of HDAC3, miR-26b-3p and HMGA2 to detect the cardiac function, inflammatory reaction, myocardial tissue pathological changes, and cardiomyocyte apoptosis. The binding relationship between miR-26b-3p and HMGA2 and the interaction between HDAC3 and miR-26b-3p were validated. RESULTS: HDAC3 and HMGA2 were elevated, while miR-26b-3p was decreased in CHF rats. The reduced HDAC3 or HMGA2 or enriched miR-26b-3p attenuated cardiac dysfunction, inflammatory reaction, myocardial tissue pathological changes and cardiomyocyte apoptosis in CHF rats, while the reduction of miR-26b-3p exerted the opposite effects. Furthermore, the inhibition of the miR-26b-3p or elevation of HMGA2 reversed the effect of reduced HDAC3 on mitigating CHF progression. Mechanically, miR-26b-3p targeted HMGA2 and HDAC3 bound to miR-26-3p. CONCLUSION: Downregulation of HDAC3 relieves cardiac function in CHF rats via mediating miR-26b-3p/HMGA2 axis. This study provides novel theory references and a distinct direction for the therapy strategies of CHF.

1,4,5,6,7,8­Hexahydropyrido[4,3­d]pyrimidine inhibits HepG2 cell proliferation, migration and invasion, and induces apoptosis through the upregulation of miR­26b­5p by targeting CDK8.

1,4,5,6,7,8-Hexahydropyrido[4,3-d]pyrimidine (PPM) promotes apoptosis of HepG2 cells and serves a role in tumor suppression. However, the role of microRNA (miRNA) regulation in initiating apoptosis remains unclear. Therefore, the present study performed reverse transcription-quantitative PCR to investigate the association between PPM and miRNA, which demonstrated that PPM upregulated the expression of miR-26b-5p. Wound healing and Transwell assays showed that PPM inhibited the migration and invasion of HepG2 cells, and EdU staining experiments showed that PPM inhibited the proliferation of HepG2 cells. Transfection with miR-26b-5p inhibitor reversed the effects of PPM on HepG2 cells. Flow cytometry results showed that PPM promoted apoptosis of HepG2 cells by upregulating miRNA (miR)-26b-5p, and Western blotting results showed that PPM promoted the expression of apoptosis-associated protein Bax and inhibited the expression of Bcl-2 by upregulating miR-26b-5p. Using a proteomic approach combined with bioinformatics analysis, CDK8 was identified as a potential target of miR-26b-5p and was downregulated by miR-26b-5p overexpression. However, PPM induced HepG2 cell cycle arrest without the involvement of miR-26b-5p. Western blotting results showed that PPM upregulation of miR-26b-5p suppresses NF-κB/p65 signaling pathway in HepG2 cells by targeting of CDK8. The present results suggested that miR-26b-5p may function as a target gene of PPM and may serve a role in hepatocellular carcinoma treatment.

MicroRNA-26b Attenuates Platelet Adhesion and Aggregation in Mice.

Platelets are key regulators of haemostasis, making platelet dysfunction a major driver of thrombosis. Numerous processes that determine platelet function are influenced by microRNAs (miRs). MiR-26b is one of the highest-expressed miRs in healthy platelets, and its expression in platelets is changed in a diseased state. However, the exact effect of this miR on platelet function has not been studied yet. In this study, we made use of a whole-body knockout of miR-26b in ApoE-deficient mice in order to determine its impact on platelet function, thrombus formation and platelet signalling both ex vivo and in vivo. We show that a whole-body deficiency of miR-26b exacerbated platelet adhesion and aggregation ex vivo. Additionally, in vivo, platelets adhered faster, and larger thrombi were formed in mice lacking miR-26b. Moreover, isolated platelets from miR-26b-deficient mice showed a hyperactivated Src and EGFR signalling. Taken together, we show here for the first time that miR-26b attenuates platelet adhesion and aggregation, possibly through Src and EGFR signalling.

microRNA-26b inhibits growth and cellular invasion of ovarian cancer cells by targeting estrogen receptor α.

The current study set out to elucidate the mechanism of miR-26b in OC cell proliferation and EMT via suppression of ERα. Initial findings illustrated that miR-26b was poorly expressed in OC tissues and cells. On the other hand, over-expression of miR-26b exerted a diminishing effect on SKOV3 cell proliferation, migration, invasion and EMT, whereas silencing of miR-26b conferred an enhancing effect on CAOV3 cell proliferation, migration, invasion and EMT. Subsequently, with help from the TargetScan database, a dual-luciferase reporter gene assay was carried out to verify the targeting relation between miR-26b and ERα, which revealed that miR-26b could negatively modulate ERα. Furthermore, the in vivo experimentation illustrated that over-expression of miR-26b led to down-regulation of ERα and suppression OC tumor growth and EMT. Meanwhile, silencing of ERα inhibited OC cell proliferation, migration, invasion and EMT. In conclusion, our findings indicated that miR-26b inhibited OC cell proliferation and EMT via negative-modulation of ERα. This investigation may offer potential strategy for OC treatment. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03222-2.

MicroRNA-26b regulates BMSC osteogenic differentiation of TMJ subchondral bone through ß-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 ß-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 ß-catenin and related osteogenic and osteoclastic factors after using miR-26b mimics and inhibitors in vivo. Moreover, BMSCs were treated with the ß-catenin blocker Wnt-C59 and then transfected with miR-26b mimics or inhibitors. Then, we examined the expression of ß-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 ß-catenin in the context of TMJ-OA progression.

Circular RNA_ANKIB1 accelerates chemo-resistance of osteosarcoma via binding microRNA-26b-5p and modulating enhancer of zeste homolog 2.

Osteosarcoma is a common bone malignancy in children and adolescents. Chemotherapeutic drug resistance is the major factor impacting the surgical outcome and prognosis of patients with osteosarcoma. This investigation assessed the role and mechanism of circular RNA_ANKIB1 in the development of osteosarcoma. The circular RNA (circ) _ANKIB1, microRNA (miR)-26b-5p, enhancer of zeste homolog 2 (EZH2) expression in OS samples was investigated through RT-qPCR. The EZH2, multidrug resistance protein 1 (MRP1), P-gp, and lipoprotein receptor-related protein (LRP) protein expressions were analyzed through western blot. The association between circ_ANKIB1 and the occurrence of clinic-pathological features in OS patients was assessed; the circular features of circ_ANKIB1 were analyzed. The hFOB1.19, KHOS, U2-OS OS cells were used to study the semi-inhibitory concentration IC50 of Doxorubicin (DXR)-resistant cells, clone formation, invasion, and apoptosis. The luciferase assay was used to study the binding of circ-ANKIB1 with miR-26b-5p and the targeting of miR-26b-5p with EZH2. In vivo experiments were performed via subcutaneous tumorigenic experiments. MiR-26b-5p in OS tissues and cells and DXR-resistant OS tissues and cells was silenced while circ_ANKIB1 and EZH2 were elevated. Circ_ANKIB1 silencing elevated miR-26b-5p, repressed EZH2, MRP1, P-gp, LRP, IC50, and elevated OS advancement. Circ_ANKIB1 bind miR-26b-5p. Reduced miR-26b-5p revered the influence of silencing circ_ANKIB1 on DXR resistant OS cells. MiR-26b-5p targeted EZH2, and EZH2 elevation reversed the impact of increasing miR-26b-5p on DXR resistant cells. Circ_ANKIB1 silencing suppressed DXR-resistant OS cells in vivo. In conclusion, Circ_ANKIB1 binds miR-26b-5p and modulates EZH2 to accelerate the chemo-resistance of osteosarcoma.

miR-26b enhances the sensitivity of hepatocellular carcinoma to Doxorubicin via USP9X-dependent degradation of p53 and regulation of autophagy.

Multi-drug resistance is a major challenge to hepatocellular carcinoma (HCC) treatment, and the over-expression or deletion of microRNA (miRNA) expression is closely related to the drug-resistant properties of various cell lines. However, the underlying molecular mechanisms remain unclear. CCK-8, EdU, flow cytometry, and transmission electron microscopy were performed to determine cell viability, proliferation, apoptosis, autophagic flow, and nanoparticle characterization, respectively. In this study, the results showed that the expression of miR-26b was downregulated following doxorubicin treatment in human HCC tissues. An miR-26b mimic enhanced HCC cell doxorubicin sensitivity, except in the absence of p53 in Hep3B cells. Delivery of the proteasome inhibitor, MG132, reversed the inhibitory effect of miR-26b on the level of p53 following doxorubicin treatment. Tenovin-1 (an MDM2 inhibitor) protected p53 from ubiquitination-mediated degradation only in HepG2 cells with wild type p53. Tenovin-1 pretreatment enhanced HCC cell resistance to doxorubicin when transfected with an miR-26b mimic. Moreover, the miR-26b mimic inhibited doxorubicin-induced autophagy and the autophagy inducer, rapamycin, eliminated the differences in the drug sensitivity effect of miR-26b. In vivo, treatment with sp94dr/miR-26b mimic nanoparticles plus doxorubicin inhibited tumor growth. Our current data indicate that miR-26b enhances HCC cell sensitivity to doxorubicin through diminishing USP9X-mediated p53 de-ubiquitination caused by DNA damaging drugs and autophagy regulation. This miRNA-mediated pathway that modulates HCC will help develop novel therapeutic strategies.

Transcriptome signature of miRNA-26b KO mouse model suggests novel targets.

BACKGROUND: MicroRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. One of the miRNAs that has been shown to play a role in various pathologies like cancer, neurological disorders and cardiovascular diseases is miRNA-26b. However, these studies only demonstrated rather ambiguous associations without revealing a causal relationship. Therefore, the aim of this study is to establish and validate a mouse model which enables the elucidation of the exact role of miRNA-26b in various pathologies. RESULTS: A miRNA-26b-deficient mouse model was established using homologous recombination and validated using PCR. miRNA-26b-deficient mice did not show any physiological abnormalities and no effects on systemic lipid levels, blood parameters or tissue leukocytes. Using next generation sequencing, the gene expression patterns in miRNA-26b-deficient mice were analyzed and compared to wild type controls. This supported the already suggested role of miRNA-26b in cancer and neurological processes, but also revealed novel associations of miRNA-26b with thermogenesis and allergic reactions. In addition, detailed analysis identified several genes that seem to be highly regulated by miRNA-26b, which are linked to the same pathological conditions, further confirming the role of miRNA-26b in these pathologies and providing a strong validation of our mouse model. CONCLUSIONS: miRNA-26b plays an important role in various pathologies, although causal relationships still have to be established. The described mouse model of miRNA-26b deficiency is a crucial first step towards the identification of the exact role of miRNA-26b in various diseases that could identify miRNA-26b as a promising novel diagnostic or even therapeutic target in a broad range of pathologies.

Interference of long non-coding RNA HAGLROS inhibits the proliferation and promotes the apoptosis of ovarian cancer cells by targeting miR-26b-5p.

Ovarian cancer (OV) is the fifth most common type of cancer affecting women worldwide. Long non-coding RNAs (lncRNAs) serve essential roles in the progression of OV. As such, the present study aimed to investigate the specific role of HAGLR opposite strand lncRNA (HAGLROS) in OV and the underlying mechanism of action through which HAGLROS exerts its effects on OV cells. In the present study, the expression of HAGLROS in several OV cell lines was first detected using reverse transcription-quantitative PCR. HAGLROS was then silenced to evaluate cell viability, proliferation and apoptosis, which were determined using Cell Counting Kit-8, colony formation and TUNEL assays, respectively. Additionally, immunofluorescence staining and western blotting were used to confirm the expression profile of proliferation- and apoptosis-related proteins. Moreover, a dual luciferase reporter assay was used to verify the potential interactions between HAGLROS and microRNA (miR)-26b-5p. Subsequently, rescue assays were performed to investigate the effects of HAGLROS and miR-26b-5p on OV progression. The results indicated that HAGLROS was highly expressed in OV cells. Interference of HAGLROS led to a decrease in the proliferation, but an increase in the apoptosis of OV cells, accompanied by downregulated expression levels of Ki67 and Bcl-2, and upregulated expression levels of Bax and cleaved caspase-3. Further study revealed that HAGLROS acted as a sponge for miR-26b-5p and positively regulated its expression. miR-26b-5p inhibitor transfection partially reversed the effects of HAGLROS knockdown on the proliferation and apoptosis of OV cells. In conclusion, the results of the present study suggested that interference of HAGLROS suppressed the proliferation and promoted the apoptosis of OV cells through regulating miR-26b-5p, indicating that HAGLROS may be a promising biomarker in OV diagnosis and treatment.

Ectodermal Organ Development Is Regulated by a microRNA-26b-Lef-1-Wnt Signaling Axis.

The developmental role of Lef-1 in ectodermal organs has been characterized using Lef-1 murine knockout models. We generated a Lef-1 conditional over-expression (COEL) mouse to determine the role of Lef-1 expression in epithelial structures at later stages of development after endogenous expression switches to the mesenchyme. Lef-1 over expression (OE) in the oral epithelium creates a new dental epithelial stem cell niche that significantly increases incisor growth. These data indicate that Lef-1 expression is switched off in the dental epithelial at early stages to maintain the stem cell niche and regulate incisor growth. Bioinformatics analyses indicated that miR-26b expression increased coinciding with decreased Lef-1 expression in the dental epithelium. We generated a murine model over-expressing miR-26b that targets endogenous Lef-1 expression and Lef-1-related developmental mechanisms. miR-26b OE mice have ectodermal organ defects including a lack of incisors, molars, and hair similar to the Lef-1 null mice. miR-26b OE rescues the Lef-1 OE phenotype demonstrating a critical genetic and developmental role for miR-26b in the temporal and spatial expression of Lef-1 in epithelial tissues. Lef-1 expression regulates Wnt signaling and Wnt target genes as well as cell proliferation mechanisms, while miR-26b OE reduced the levels of Wnt target gene expression. The extra stem cell compartment in the COEL mice expressed Lef-1 suggesting that Lef-1 is a stem cell factor, which was absent in the miR-26b OE/COEL rescue mice. This is the first demonstration of a microRNA OE mouse model that has ectodermal organ defects. These findings demonstrate that the levels of Lef-1 are critical for development and establish a role for miR-26b in the regulation of ectodermal organ development through the control of Lef-1 expression and an endogenous stem cell niche.

miR-26b inhibits isoproterenol-induced cardiac fibrosis via the Keap1/Nrf2 signaling pathway.

A critical event in cardiac fibrosis is the transformation of cardiac fibroblasts (CFs) into myofibroblasts. MicroRNAs (miRNAs) have been reported to be critical regulators in the development of cardiac fibrosis. However, the underlying molecular mechanisms of action of miRNA (miR)-26b in cardiac fibrosis have not yet been extensively studied. In the present study, the expression levels of miR-26b were downregulated in isoproterenol (ISO)-treated cardiac tissues and CFs. Moreover, miR-26b overexpression inhibited the cell viability of ISO-treated CFs and decreased the protein levels of collagen I and α-smooth muscle actin (α-SMA). Furthermore, bioinformatics analysis and dual luciferase reporter assays indicated that Kelch-like ECH-associated protein 1 (Keap1) was the target of miR-26b, and that its expression levels were decreased in miR-26b-treated cells. In addition, Keap1 overexpression reversed the inhibitory effects of miR-26b on ISO-induced cardiac fibrosis, as demonstrated by cell viability, and the upregulation of collagen I and α-SMA expression levels. Furthermore, inhibition of Keap1 expression led to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), which induced the transcriptional activation of antioxidant/detoxifying proteins in order to protect against cardiac fibrosis. Taken together, the data demonstrated that miR-26b attenuated ISO-induced cardiac fibrosis via the Keap-mediated activation of Nrf2.

Effect of miR-26b-5p on cis-diamine dichloroplatinum-induced ovarian granulosa cell injury by targeting MAP3K9.

The proliferation and differentiation of granulosa cells are very important for follicular development. The dysfunction of granulosa cells leading to follicular development is an important cause of ovarian endocrine abnormalities. More and more evidence shows that microRNAs are involved in the regulation of ovarian granulosa cell function. It has been found that MiR-26b may be involved in CDDP resistance. Studies have shown that miR-26b can promote apoptosis of ovarian granulosa cells, but there are few studies on its mechanism, and no studies have been found on the damage of miR-26b-5p to rat ovarian granulosa cells induced by CDDP. Identification of ovarian granulosa cells was conducted by immunochemical staining. Cell counting kit 8 (CCK-8) was used to detect cell viability, flow cytometry was used to detect cell apoptosis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot (WB) were used to analyze the expression of miR-26b-5p, MAP3K9, cleaved Caspase-3, Bax, and Bcl-2; dual-luciferase reporter assay results further verify the targeting relation between miR-26b-5p and MAP3K9. CDDP remarkably inhibited ovarian granulosa cell viability and induced ovarian granulosa cell apoptosis; miR-26b-5p inhibitor enhanced viability and inhibited apoptosis of ovarian granulosa cells, which treated with CDDP, but had little effect on normal cells. MAP3K9 partially reversed the effect of miR-26b-5p on ovarian granulosa cells induced by CDDP. miR-26b-5p has a protective effect on CDDP-induced ovarian granulosa cells via targeting MAP3K9.

Circular RNA circRNA_000203 aggravates cardiac hypertrophy via suppressing miR-26b-5p and miR-140-3p binding to Gata4.

AIMS: Circular RNAs (circRNAs) are involved in gene regulation in a variety of physiological and pathological processes. The present study aimed to investigate the effect of circRNA_000203 on cardiac hypertrophy and the potential mechanisms involved. METHODS AND RESULTS: CircRNA_000203 was found to be up-regulated in the myocardium of Ang-II-infused mice and in the cytoplasma of Ang-II-treated neonatal mouse ventricular cardiomyocytes (NMVCs). Enforced expression of circRNA_000203 enhances cell size and expression of atrial natriuretic peptide and ß-myosin heavy chain in NMVCs. In vivo, heart function was impaired and cardiac hypertrophy was aggravated in Ang-II-infused myocardium-specific circRNA_000203 transgenic mice (Tg-circ203). Mechanistically, we found that circRNA_000203 could specifically sponge miR-26b-5p, -140-3p in NMVCs. Further, dual-luciferase reporter assay showed that miR-26b-5p, -140-3p could interact with 3'-UTRs of Gata4 gene, and circRNA_000203 could block the above interactions. In addition, Gata4 expression is transcriptionally inhibited by miR-26b-5p, -140-3p mimic in NMVCs but enhanced by over-expression of circRNA_000203 in vitro and in vivo. Functionally, miR-26b-5p, -140-3p, and Gata4 siRNA, could reverse the hypertrophic growth in Ang-II-induced NMVCs, as well as eliminate the pro-hypertrophic effect of circRNA_000203 in NMVCs. Furthermore, we demonstrated that NF-κB signalling mediates the up-regulation of circRNA_000203 in NMVCs exposed to Ang-II treatment. CONCLUSIONS: Our data demonstrated that circRNA_000203 exacerbates cardiac hypertrophy via suppressing miR-26b-5p and miR-140-3p leading to enhanced Gata4 levels.