Target Of miR-200c Research Articles

Isoliquiritigenin Suppresses Breast Tumor Development by Enhancing Host Antitumor Immunity.

Isoliquiritigen (ISL), a constituent of licorice, has been shown to possess antitumorigenic effects in diverse cancer types. In this study, we observed that ISL suppressed breast tumor development significantly more effectively in immunocompetent mice than in immunocompromised ones. In exploring the cause of such a discrepancy, we detected robust tumor infiltration of CD8[Formula: see text] T lymphocytes in mice treated with ISL, not seen in tumors derived from vehicle-treated mice. Moreover, we found a dramatic reduction in PD-L1 in both experimental breast tumors and cultured breast cancer cells upon ISL treatment. In further experiments, we showed that ISL selectively elevated miR-200c in breast cancer and confirmed that PD-L1 mRNA is the target of miR-200c in both murine and human breast cancer cells. ISL suppression of PD-L1 was functionally linked to miR-200c/ZEB1/2 because (1) ISL diminished ZEB1/2; (2) knockdown of ZEB1/2 led to the disappearance of PD-L1; and (3) miR-200c antagomiR disabled ISL to reduce PD-L1. We found evidence that ISL reduced the level of PD-L1 by simultaneously intercepting the ERK and Src signaling pathways. In agreement with clinical finding that PD-L1 antibodies enhance efficacy of taxane-based therapy, we showed that ISL improved the tumoricidal effects of paclitaxel in an orthopedic murine breast tumor model. This study demonstrates that ISL-led tumor suppression acts through the augmentation of host antitumor immunity.

Exosomal miR-200c and miR-141 as cerebrospinal fluid biopsy biomarkers for the response to chemotherapy in primary central nervous system lymphoma

BackgroundTo improve early diagnosis and chemotherapy efficacy monitoring in primary central nervous system lymphoma (PCNSL), cerebrospinal fluid (CSF) exosomal microRNA (miRNA) studies were performed.MethodSmall RNA sequencing was performed to identify candidate exosomal miRNAs as CSF biopsy biomarkers from two patients with de novo PCNSL and two patients in remission after chemotherapy. miR-200c and miR-141 expression in CSF exosomes was further validated using relative quantitative real-time polymerase chain reaction in patients with PCNSL (n = 20), patients with other neurological diseases (n = 10), and normal subjects (n = 10). Receiver operating characteristic (ROC) curve analyses of miR-200c and miR-141 in the diagnosis and prediction of chemotherapy efficacy in PCNSL were performed in patients treated with methotrexate. Additionally, bioinformatics tools were utilized to predict the potential targets of miR-200c and miR-141.ResultsExosomal miR-200c and miR-141 levels in CSF from patients with PCNSL were significantly lower than those in control subjects. Importantly, miR-200c and miR-141 were upregulated in patients with PCNSL after chemotherapy (P = 0.002). There was a significant correlation between the levels of miR-141 and IL-10 in CSF (P = 0.04). The combination of miR-200c and miR-141 yielded an area under the ROC curve of 0.761 for distinguishing PCNSL with sensitivity and specificity of 60.0% and 96.7%, respectively. The potential target genes of miR-200c and miR-141 in PCNSL included ATP1B3, DYNC1H1, MATR3, NUCKS1, ZNF638, NUDT4, RCN2, GNPDA1, ZBTB38, and DOLK.ConclusionCollectively, miR-200c and miR-141 are likely to be upregulated in CSF exosomes after chemotherapy in patients with PCNSL, highlighting their potential as reliable liquid biopsy biomarkers for PCNSL diagnosis and chemotherapy efficacy monitoring.

MiR-29c alleviates hyperglycemia-induced inflammation via targeting TGF-β in cardiomyocytes.

This study aims to investigate whether miR-29c is involved in regulating transforming growth factor-β (TGF-β) mediated inflammation in diabetic cardiomyopathy (DCM). Our data showed increased inflammation and oxidative stress in diabetic myocardium together with decrease of miR-29c and elevation of TGF-β expression. In vitro experiments, we transfected miR-29c mimic and antagomir into HL-1 cells to explore the effect of miR-29c on inflammation in hyperglycemic conditions. Overexpression of miR-29c down-regulated the elevated TNF-α level, ROS production and NADPH oxidase activity which caused by high glucose. However, above changes were reversed by miR-29c antagomir. Interestingly, TGF-β protein rather than mRNA expression was changed significantly after transfection with miR-29c mimic, indicating that the modulation of TGF-β mediated by miR-29c was at the posttranslational level. Meanwhile, we found that 3'-UTR of TGF-β was the direct target of miR-29c confirmed by dual-luciferase assay. In conclusion, our study revealed that miR-29c could alleviate hyperglycemic-induced inflammation and ROS production via targeting TGF-β in cardiomyocytes, which provides a potential target for the treatment of DCM.

Long non-coding RNA MALAT1 sponges miR-30c to promote the calcification of human vascular smooth muscle cells by regulating Runx2

Objectives Recent evidence suggested that long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) play critical roles in the pathogenesis of vascular calcification (VC). In this study, we tried to explore the expression and role of a lncRNA, i.e., metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), and a miRNA, i.e., miR-30c, in VC. Methods In vitro VC model was induced in human vascular smooth muscle cells (VSMCs) after 10 days culture in calcifying medium containing 2 mM Na2HPO4. Alizarin red S staining, calcium assay and western blot analysis of runt-related transcription factor 2 (Runx2) and alpha smooth muscle actin (α-SMA) were performed to evaluate VC. Knockdown of MALAT1 and up-regulation of MALAT1, miR-30c and Runx2 was performed to determine the impact of these molecules on VSMCs calcification. Dual-luciferase report assay was performed to confirm the relationship between MALAT1 and miR-30c or miR-30c and Runx2. In addition, quantitative reverse transcription PCR and western blot were used to determine gene and protein expression. Results MALAT1 was increased, while miR-30c was decreased in calcified VSMCs. Knockdown of MALAT1 suppressed VSMCs calcification; on the contrary, up-regulation of MALAT1 promoted VSMCs calcification. The effect of MALAT1 over-expression on VSMCs calcification was reversed by upregulation of miR-30c, which was reversed again by upregulation of Runx2. Dual-luciferase report assay confirmed that there is a direct interaction between MALAT1 and miR-30c, and Runx2 is a direct target of miR-30c. Conclusion MALAT1 over-expression promoted VSMCs calcification, which was at least partially through regulating the miR-30c/Runx2 axis.

Sperm-borne microRNA-34c regulates maternal mRNA degradation and preimplantation embryonic development in mice

BackgroundStudies have shown that sperm-borne microRNAs (miRNAs) are involved in mammalian preimplantation embryonic development. In humans, spermatozoan miR-34c levels are correlated with in vitro fertilization outcomes, such as embryo quality and the clinical pregnancy and live birth rates. In rabbits and cows, miR-34c improves the developmental competence of embryos generated by somatic cell nuclear transfer. However, the mechanisms underlying the regulation of embryonic development by miR-34c remain unknown.MethodsFemale C57BL/6 mice (6–8 weeks old) were superovulated, and pronucleated zygotes were collected and microinjected with an miR-34c inhibitor or a negative-control RNA. The embryonic development of the microinjected zygotes was evaluated, and the messenger RNA (mRNA) expression profiles of the embryos at the two-cell, four-cell and blastocyst stages (five embryos per group) were determined by RNA sequencing analysis. Gene expression levels were verified by reverse transcription–quantitative polymerase chain reaction. Cluster analysis and heat map visualization were performed to detect differentially expressed mRNAs. Pathway and process enrichment analyses were performed using ontology resources. Differentially expressed mRNAs were systematically analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins database to determine their biological functions.ResultsEmbryonic developmental potential was significantly reduced in zygotes microinjected with the miR-34c inhibitor compared with those microinjected with a negative-control RNA. Two-cell stage embryos microinjected with an miR-34c inhibitor presented altered transcriptomic profiles, with upregulated expression of maternal miR-34c target mRNAs and classical maternal mRNAs. Differentially expressed transcripts were mainly of genes associated with lipid metabolism and cellular membrane function at the two-cell stage, with cell-cycle phase transition and energy metabolism at the four-cell stage; and with vesicle organization, lipid biosynthetic process and endomembrane system organization at the blastocyst stage. We also showed that genes related to preimplantation embryonic development, including Alkbh4, Sp1, Mapk14, Sin3a, Sdc1 and Laptm4b, were significantly downregulated after microinjection of an miR-34c inhibitor.ConclusionsSperm-borne miR-34c may regulate preimplantation embryonic development by affecting multiple biological processes, such as maternal mRNA degradation, cellular metabolism, cell proliferation and blastocyst implantation. Our data demonstrate the importance of sperm-derived miRNAs in the development of preimplantation embryos.

Extracellular vesicles derived from hypoxia-preconditioned bone marrow mesenchymal stem cells ameliorate lower limb ischemia by delivering miR-34c.

Hypoxic mesenchymal stem cell-derived extracellular vesicles (EVs) have been suggested as a promising therapy for various diseases. This study aims to determine the effect of EVs derived from bone marrow mesenchymal stem cells (BMMSCs) under hypoxia on lower limb ischemia and the underlying mechanism. Human BMMSCs were subjected to hypoxia or normoxia followed by the isolation of EVs. Nanoparticle trafficking analysis (NTA), transmission electron microscopy (TEM), and Western Blotting using corresponding markers were performed to confirm the EVs. The EVs from BMMSCs under hypoxia condition (Hyp-EVs) or normoxia condition (Nor-EVs) were subjected to hindlimb ischemia (HI) mice. MiR-34c expression in BMMSCs and BMMSC-EVs was detected. The role of miR-34c in regulating M2 macrophage polarization, as well as the target of miR-34c, were explored. HI mice with Hyp-EV treatment, as compared to the Nor-EV or the PBS group, had better blood flow and higher capillary density. MiR-34c expression was increased in BMMSCs, BMMSC-EVs, and the adductor muscle of HI mice. Hyp-EVs promoted the M2 macrophage polarization and anti-inflammatory cytokine production, and enhanced the blood flow and capillary density in HI mice, while the knockdown of miR-34c partly reversed these effects. PTEN is a target of miR-34c, and the PTEN silencing facilitated M2 macrophage polarization, whereas the inhibition of AKT signaling partly abolished the effect. Hyp-EVs promoted M2 macrophage polarization by delivering miR-34c via PTEN/AKT pathway, which could be a promising therapeutic strategy to ameliorate lower limb ischemia.

Glomerular Endothelial Cell-Derived miR-200c Impairs Glomerular Homeostasis by Targeting Podocyte VEGF-A.

Deciphering the pathophysiological mechanisms of primary podocytopathies that can lead to end-stage renal disease and increased mortality is an unmet need. Studying how microRNAs (miRs) interfere with various signaling pathways enables identification of pathomechanisms, novel biomarkers and potential therapeutic options. We investigated the expression of miR-200c in urine from patients with different renal diseases as a potential candidate involved in podocytopathies. The role of miR-200c for the glomerulus and its potential targets were studied in cultured human podocytes, human glomerular endothelial cells and in the zebrafish model. miR-200c was upregulated in urine from patients with minimal change disease, membranous glomerulonephritis and focal segmental glomerulosclerosis and also in transforming growth factor beta (TGF-β) stressed glomerular endothelial cells, but not in podocytes. In zebrafish, miR-200c overexpression caused proteinuria, edema, podocyte foot process effacement and glomerular endotheliosis. Although zinc finger E-Box binding homeobox 1/2 (ZEB1/2), important in epithelial to mesenchymal transition (EMT), are prominent targets of miR-200c, their downregulation did not explain our zebrafish phenotype. We detected decreased vegfaa/bb in zebrafish overexpressing miR-200c and could further prove that miR-200c decreased VEGF-A expression and secretion in cultured human podocytes. We hypothesize that miR-200c is released from glomerular endothelial cells during cell stress and acts in a paracrine, autocrine, as well as context-dependent manner in the glomerulus. MiR-200c can cause glomerular damage most likely due to the reduction of podocyte VEGF-A. In contrast, miR-200c might also influence ZEB expression and therefore EMT, which might be important in other conditions. Therefore, we propose that miR-200c-mediated effects in the glomerulus are context-sensitive.

MiR-30c facilitates natural killer cell cytotoxicity to lung cancer through targeting GALNT7.

MicroRNAs (miRNAs) have been reported to play important roles in regulating natural killer (NK) cell cytotoxicity to cancer cells. This study aimed to investigate the effects and potential mechanism of miR-30c in regulating NK cell cytotoxicity to lung cancer cells. Primary NK cells were derived from the peripheral blood of lung cancer and normal participants. Exosomes were isolated and validated via transmission electron microscopy and nanoparticle tracking analysis. The levels of miR-30c, polypeptide N-acetylgalactosaminyltransferase 7 (GALNT7) and proteins in PI3K/AKT pathway were determined using quantitative real-time polymerase chain reaction or western blot. Tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) levels and the cytotoxicity of effector NK cells to target lung cancer cells were measured via enzyme linked immunosorbent assay, cell apoptosis or xenograft experiments. The relationship between miR-30c and GALNT7 was analyzed by luciferase activity, RNA pull-down and RNA immunoprecipitation assays. And a xenograft mice model was established to verify the effect of miR-30c in regulating NK cell cytotoxicity to lung cancer cells in vivo. NK cell-derived exosomes carrying miR-30c, and miR-30c level was significantly downregulated in primary NK cells of lung cancer patients. MiR-30c overexpression promoted TNF-α and IFN-γ secretion and enhanced the cytotoxicity of interleukin 2 (IL-2)-treated NK cells to lung cancer cells, while knockdown of miR-30c played an opposite effect in regulating the cytotoxicity of NK cells to lung cancer cells. GALNT7 was a target of miR-30c and was negatively regulated by miR-30c. Besides, miR-30c targeted GALNT7 to exert its function in regulating NK cell cytotoxicity. Furthermore, GALNT7 prompted the activation of PI3K/AKT pathway in NK cells. Additionally, miR-30c overexpression enhanced NK cell cytotoxicity to lung cancer cells and inhibited tumor growth in vivo. miR-30c enhanced NK cell cytotoxicity to lung cancer cells via decreasing GALNT7 and inactivating the PI3K/AKT pathway, suggesting that regulating miR-30c expression maybe a promising approach for enhancing NK cell-based antitumor therapies.

Low levels of miR-34c in nasal washings as a candidate marker of aggressive disease in wood and leather exposed workers with sinonasal intestinal-type adenocarcinomas (ITACs)

IntroductionSinonasal intestinal-type adenocarcinomas (ITACs) are rare and aggressive tumors, closely related to professional exposure to wood dusts or leather. Here we explored the role of non-coding RNAs controlling MUC2 in liquid biopsies and tumors from ITAC patients with the aim of identifying biomarkers and molecular mechanisms to improve early diagnosis, prognosis, and therapeutic approaches for this rare cancer. MethodsMiR-34c-3p, lncRNA AF147447 and MUC2 were measured in tumors and normal mucosa, in nasal washings (NW) from the affected and non-affected nostril and in plasma from 17 ITAC patients. The Apparent Diffusion Coefficient (ADC) was also evaluated by Magnetic Resonance Imaging. ResultsMiR-34c was higher in ITACs compared to the corresponding normal mucosa (p = 0.021). Differentiated tumors exhibited higher miR-34c levels (p = 0.025) and lower ADC values (p<0.001) compared to mucinous ones and these parameters were also inversely correlated (r = 0.87; p = 0.001). High MUC2 tumor expression was associated with orbital extension (p = 0.010). Low miR-34c levels in NW were associated with orbital (p = 0.009) and intracranial (p = 0.031) extension and with advanced TNM stage (p = 0.054). Functional analysis identified Wnt, Focal adhesion, MAPK and inflammatory signalings among the pathways most enriched in mir-34c targets. DiscussionOur results suggest measuring miR-34c in NW as a biomarker for early diagnosis and monitoring of ITAC patients and for the surveillance of wood and leather exposed workers. Further research on the involvement of miR-34c regulated pathways in ITAC tumorigenesis may also allow the development of new therapeutic approaches for this rare cancer.

Overexpression of circ PTK2 suppresses the progression of nonalcoholic fatty liver disease via the miR-200c/SIK2/PI3K/Akt axis.

Excessive hepatic lipid accumulation is involved in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). A previous study showed that the circular RNA (circRNA) PTK2 was significantly downregulated in NAFLD mice. However, the detailed function of circ PTK2 in NAFLD remains unclear. A high-fat diet (HFD) was used to establish a mouse model of NAFLD, and free fatty acid (FFA) treatment was used to establish an in vitro model of NAFLD. Oil red O staining was used to evaluate lipid accumulation. The pathological changes in mice were observed by HE staining. Western blotting and RT-qPCR were applied to assess protein and mRNA levels, respectively. A dual luciferase reporter assay and RIP were used to explore the relationship among circ PTK2, miR-200c and SIK2. Circ PTK2 and SIK2 were downregulated and miR-200c was upregulated in NAFLD. Upregulation of circ PTK2 reversed lipid accumulation in FFA-treated HepG2 cells. Moreover, circ PTK2 bound to miR-200c, and SIK2 was identified as the direct target of miR-200c. Moreover, the miR-200c inhibitor-induced decrease in lipid accumulation was reversed by SIK2 knockdown. Furthermore, the impact of circ PTK2 overexpression on PI3K/Akt signaling was partially reversed by SIK2 silencing. Circ PTK2 overexpression alleviates NAFLD development via the miR-200c/SIK2/PI3K/Akt axis. Thus, our work might provide new methods for NAFLD treatment.

Melatonin Alleviates Hyperglycemia-Induced Cardiomyocyte Apoptosis via Regulation of Long Non-Coding RNA H19/miR-29c/MAPK Axis in Diabetic Cardiomyopathy.

Recent studies revealed that non-coding RNAs (ncRNAs) play a crucial role in pathophysiological processes involved in diabetic cardiomyopathy (DCM) that contribute to heart failure. The present study was designed to further investigate the anti-apoptotic effect of melatonin on cardiomyocytes in diabetic conditions, and to elucidate the potential mechanisms associated with ncRNAs. In animal models, we induced diabetes in SD rats by single intraperitoneal injection of streptozotocin (STZ) solution (55 mg/kg) at 18:00 in the evening, after a week of adaptive feeding. Our results indicate that melatonin notably alleviated cardiac dysfunction and cardiomyocyte apoptosis. In the pathological situation, lncRNA H19 level increased, along with a concomitant decrease in miR-29c level. Meanwhile, melatonin significantly downregulated lncRNA H19 and upregulated miR-29c levels. In our in vitro experiments, we treated H9c2 cells with high-concentration glucose medium (33 mM) to simulate the state of diabetes. It was verified that positive modulation of miR-29c and inhibition of lncRNA H19, as well as mitogen-activated protein kinase (MAPK) pathways, distinctly attenuated apoptosis in high-glucose-treated H9c2 cells. A luciferase activity assay was conducted to evaluate the potential target sites of miR-29c on lncRNA H19 and MAPK13. LncRNA H19 silencing significantly downregulated the expression of miR-29c target gene MAPK13 by inducing miR-29c expression. Most importantly, our results show that melatonin alleviated apoptosis by inhibiting lncRNA H19/MAPK and increasing miR-29c level. Our results elucidate a novel protective mechanism of melatonin on diabetic cardiomyocyte apoptosis, which involved the regulation of lncRNA H19/miR-29c and MAPK pathways, providing a promising strategy for preventing DCM in diabetic patients.

Hyaluronan synthase 2, a target of miR-200c, promotes carbon tetrachloride-induced acute and chronic liver inflammation via regulation of CCL3 and CCL4

Liver fibrosis occurs during wound healing after repeated liver injury and is characterized by extensive extracellular matrix deposition. We previously identified hyaluronan synthase 2 (HAS2) as a driver of liver fibrosis and hepatic stellate cell (HSC) activation. Developing strategies to suppress HSC activation is key to alleviating liver fibrosis, and HAS2 is an attractive candidate for intervention. To gain insight into the molecular function of HAS2, we investigated its posttranscriptional regulation. We found that miR-200c directly targets the 3’ untranslated regions of HAS2. Moreover, miR-200c and HAS2 were inversely expressed in fibrotic human and mouse livers. After establishing the direct interaction between miR-200c and HAS2, we investigated the functional outcome of regulating HAS2 expression in three murine models: CCl4-induced acute liver injury, CCl4-induced chronic liver fibrosis, and bile duct ligation-induced liver fibrosis. Hepatic Has2 expression was induced by acute and chronic CCl4 treatment. In contrast, miR-200c expression was decreased after CCl4 treatment. HSC-specific Has2 deletion reduced the expression of inflammatory markers and infiltration of macrophages in the models. Importantly, hyaluronidase-2 (HYAL2) but not HYAL1 was overexpressed in fibrotic human and murine livers. HYAL2 is an enzyme that can cleave the extracellular matrix component hyaluronan. We found that low-molecular-weight hyaluronan stimulated the expression of inflammatory genes. Treatment with the HA synthesis inhibitor 4-methylumbelliferone alleviated bile duct ligation-induced expression of these inflammatory markers. Collectively, our results suggest that HAS2 is negatively regulated by miR-200c and contributes to the development of acute liver injury and chronic liver inflammation via hyaluronan-mediated immune signaling.

Ozone protects cardiomyocytes from myocardial ischemia–reperfusion injury through miR-200c/FOXO3 axis

Purpose Myocardial ischemia-reperfusion injury (I/R) is a detrimental process contributing to the pathological progression of coronary artery diseases. Studies indicate that miRNAs are implicated in ischemic heart disease, and ozone therapy could protect the heart from ischemic heart disease. In this study, we investigated the effect of ozone on miR-200c expression and the potential role of miR-200c in an I/R myocardial injury model. Methods A myocardial cellular model of I/R was established to detect the expression of miR-200c. Cardiomyocytes with I/R induction were treated with ozone as a cellular model to detect miR-200 expression and investigate its functional roles. The downstream target of miR-200c was predicted with Starbase online tools and validated by dual luciferase reporter assay. The function of miR-200c/FOXO3 axis in I/R was examined by CCK-8 proliferation and apoptotic assays. Results miR-200c was upregulated in primary cardiomyocytes of the I/R model. In cardiomyocyte cells, cell proliferation in the I/R group was significantly impaired, which could be partially rescued by miR-200c inhibitor or ozone treatment. Cell death detected by LDH release and apoptosis assay in the I/R model could also be inhibited by miR-200c inhibitor or ozone treatment. FOXO3 was identified as a downstream target of miR-200c, which was induced by ozone treatment and suppressed by miR-200c. Silencing FOXO3 abrogated the protective effect of ozone treatment on the I/R cell model. Conclusion Overall, our results suggest that ozone plays a cardio-protective role in I/R through regulating miR-200/FOXO3 axis, and indicate that targeting miR-200/FOXO3 axis could potentially alleviate I/R.

MiR-200c-3p targets SESN1 and represses the IL-6/AKT loop to prevent cholangiocyte activation and cholestatic liver fibrosis

AbstractCholestasis causes ductular reaction in the liver where the reactive cholangiocytes not only proliferate but also gain a neuroendocrine-like phenotype, leading to inflammatory cell infiltration and extracellular matrix deposition and contributing to the development and progression of cholestatic liver fibrosis. This study aims to elucidate the role of miR-200c in cholestasis-induced biliary liver fibrosis and cholangiocyte activation. We found that miR-200c was extremely abundant in cholangiocytes but was reduced by cholestasis in a bile duct ligation (BDL) mouse model; miR-200c was also decreased by bile acids in vitro. Phenotypically, loss of miR-200c exacerbated cholestatic liver injury, including periductular fibrosis, intrahepatic inflammation, and biliary hyperplasia in both the BDL model and the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) model. We identified sestrin 1 (SESN1) as a target of miR-200c. Sesn1−/−-BDL mice showed mitigation of cholestatic liver injury. On a molecular level, the pro-proliferative IL-6/AKT feedback loop was activated in Mir200c−/− livers but was inhibited in Sesn1−/− livers upon cholestasis in mice. Furthermore, rescuing expression of miR-200c by the adeno-associated virus serotype 8 ameliorated BDL-induced liver injury in Mir200c−/− mice. Taken together, this study demonstrates that miR-200c restrains the proliferative and neuroendocrine-like activation of cholangiocytes by targeting SESN1 and inhibiting the IL-6/AKT feedback loop to protect against cholestatic liver fibrosis. Our findings provide mechanistic insights regarding biliary liver fibrosis, which may help to reveal novel therapeutic targets for the treatment of cholestatic liver injury and liver fibrosis.Cholangiocytes are the primary targets of cholangiopathies. This study elucidates the role of miR-200c in maintaining cholangiocyte homeostasis making use of cell culture and mouse models of cholestasis. MiR-200c restrains the proliferative and neuroendocrine-like activation of cholangiocytes by targeting sestrin 1(SESN1) and inhibiting the IL-6/AKT feedback loop to prevent cholestatic liver injury. The findings provide critical mechanistic insights into biliary liver fibrosis and suggest miR-200c may be a novel therapeutic target of cholangiopathies.

Efficacy of post‐injury anti‐miR‐181a and anti‐miR‐200c intravenous treatment in protection against experimental stroke in aged mice

IntroductionStroke remains the second leading cause of death worldwide and the primary cause of long‐term disability in the US. To date, the only pharmacological treatment for stroke remains tissue plasminogen activator. A major factor for translational failure is likely overwhelming use of young adult rodent models despite the fact that stroke occurs predominantly in aged populations. MicroRNAs (miRs) are non‐coding RNAs that regulate protein translation, and we have previously demonstrated in young adult mice neuroprotection against experimental stroke with anti‐miR‐181a and anti‐miR‐200c. In the present study, to extend translational impact, we tested efficacy of post‐injury intravenous anti‐miR‐181a and anti‐miR‐200c in clinically relevant aged mice.MethodsAll studies were conducted in accordance with National Institutes of Health guidelines for the use of experimental animals, with protocols approved by the Stanford Animal Care and Use Committee. Animal treatment groups were randomized by coin flip, and all analyses were performed by an observer blinded to conditions. Transient focal cerebral ischemia was induced by suture occlusion of the middle cerebral artery (MCAO) for 1h in 20 month‐old male C57BL/6 mice (n=8 per cohort and treatment group). Sham surgery consisted of anesthesia and internal carotid exposure without suture occlusion (n=4 per group). Intravenous infusion: animals were re‐anesthetized and 30 pmol/g anti‐miR‐181a, anti‐miR‐200c or control (MM) in sterile saline (100 μl) was infused over 1 minute into the internal jugular vein at 2h reperfusion. Neurological status was assessed by 4‐point neurologic deficit score at 48h after MCAO. Infarct volume was assessed after sacrifice by anesthesia overdose at 48h after MCAO with triphenyl tetrazolium chloride staining. Infarct volume was calculated as percentage of hemisphere corrected for edema.ResultsMCAO in all groups resulted in significant decreases in neurological score and increased infarct volume relative to sham treated animals. Both anti‐miR‐181a and anti‐miR‐200c treatment resulted in significantly (p&lt;0.05) higher neurological scores and significantly lower infarct volumes relative to MM‐control treated animals. No differences in neurological score or in infarct volume were observed between anti‐miR‐181a and anti‐miR‐200c treatments.ConclusionsBoth anti‐miR‐181a and anti‐miR‐200c are effective non‐invasive post‐stroke interventions to reduce injury in clinically‐relevant aged mice. Future investigations should be extended to testing in aged female cohorts and identifying relevant downstream targets of miR‐181a and miR‐200c.

CircRIMKLB promotes myoblast proliferation and inhibits differentiation by sponging miR-29c to release KCNJ12

ABSTRACT Muscle development is a complex process that was regulated by many factors, among which non-coding RNAs (ncRNAs) play a vital role in regulating multiple life activities of muscle cells. Circular RNA (circRNA), a type of non-coding RNA with closed-loop structure, has been reported to affect multiple life processes. However, the roles of circRNAs on muscle development have not been fully elucidated. The present study aimed to determine whether and how circRIMKLB affects muscle development. Our study revealed that circRIMKLB promoted myoblast proliferation and inhibited differentiation. Besides, miR-29c was proved as a downstream target of circRIMKLB using dual-luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay. Also, potassium inwardly rectifying channel subfamily J member 12 (KCNJ12) was identified as a novel target of miR-29c via dual-luciferase reporter assay, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and western blot. CircRIMKLB and KCNJ12 were both proved to regulate cell cycle on muscle regeneration after injury in vivo. In conclusion, we demonstrated that circRIMKLB sponged miR-29c, releasing KCNJ12 to regulate myoblast proliferation and differentiation and regulating cell cycle during muscle regeneration after injury in vivo.

RNA uridyl transferases TUT4/7 differentially regulate miRNA variants depending on the cancer cell type.

The human terminal uridyl transferases TUT4 and TUT7 (TUT4/7) catalyze the additions of uridines at the 3′ end of RNAs, including the precursors of the tumor suppressor miRNA let-7 upon recruitment by the oncoprotein LIN28A. As a consequence, let-7 family miRNAs are down-regulated. Disruption of this TUT4/7 activity inhibits tumorigenesis. Hence, targeting TUT4/7 could be a potential anticancer therapy. In this study, we investigate TUT4/7-mediated RNA regulation in two cancer cell lines by establishing catalytic knockout models. Upon TUT4/7 mutation, we observe a significant reduction in miRNA uridylation, which results in defects in cancer cell properties such as cell proliferation and migration. With the loss of TUT4/7-mediated miRNA uridylation, the uridylated miRNA variants are replaced by adenylated isomiRs. Changes in miRNA modification profiles are accompanied by deregulation of expression levels in specific cases. Unlike let-7s, most miRNAs do not depend on LIN28A for TUT4/7-mediated regulation. Additionally, we identify TUT4/7-regulated cell-type-specific miRNA clusters and deregulation in their corresponding mRNA targets. Expression levels of miR-200c-3p and miR-141-3p are regulated by TUT4/7 in a cancer cell-type-specific manner. Subsequently, BCL2, which is a well-established target of miR-200c is up-regulated. Therefore, TUT4/7 loss causes deregulation of miRNA–mRNA networks in a cell-type-specific manner. Understanding of the underlying biology of such cell-type-specific deregulation will be an important aspect of targeting TUT4/7 for potential cancer therapies.

Human Islet MicroRNA-200c Is Elevated in Type 2 Diabetes and Targets the Transcription Factor ETV5 to Reduce Insulin Secretion.

MicroRNAs (miRNAs) are part of deregulated insulin secretion in type 2 diabetes (T2D) development. Rodent models have suggested miR-200c to be involved, but the role and potential as therapeutic target of this miRNA in human islets are not clear. Here we report increased expression of miR-200c in islets from T2D as compared with nondiabetic (ND) donors and display results showing reduced glucose-stimulated insulin secretion in EndoC-βH1 cells overexpressing miR-200c. We identify transcription factor ETV5 as the top rank target of miR-200c in human islets using TargetScan in combination with Pearson correlation analysis of miR-200c and mRNA expression data from the same human donors. Among other targets were JAZF1, as earlier shown in miR-200 knockout mice. Accordingly, linear model analysis of ETV5 and JAZF1 gene expression showed reduced expression of both genes in islets from human T2D donors. Western blot analysis confirmed the reduced expression of ETV5 on the protein level in EndoC-βH1 cells overexpressing miR-200c, and luciferase assay validated ETV5 as a direct target of miR-200c. Finally, LNA knockdown of miR-200c increased glucose-stimulated insulin secretion in islets from T2D donors approximately threefold. Our data reveal a vital role of the miR-200c-ETV5 axis in β-cell dysfunction and pathophysiology of T2D.

Down-regulation of miR-29c promotes the progression of oral submucous fibrosis through targeting tropomyosin-1

Various microRNAs (miRs) have been found to be associated with the development of the precancerous condition of the oral cavity, oral submucous fibrosis (OSF). The expression of miR-29c is dysregulated in oral cancer, but its role in OSF has not been investigated. The purpose of the study is to investigate the functional role of miR-29c and its target in OSF. The expression levels of miR-29c in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) were assessed using next-generation sequencing and real-time Polymerase Chain Reaction (PCR) analysis. MiR-29c mimic and inhibitors were employed to examine its functional role of myofibroblast transdifferentiation. In addition, several myofibroblast phenotypes, such as collagen gel contraction and migration were tested, and a luciferase reporter assay was conducted to confirm the relationship between miR-29c and its predicted target, tropomyosin-1 (TPM1). We observed that miR-29c expression was downregulated in fBMFs. fBMFs transfected with miR-29c mimics exhibited reduced migration ability and collagen gel contractility, whereas inhibition of miR-29c in normal BMFs induced the myofibroblast phenotypes. Results from the luciferase reporter assay showed that TPM1 was a direct target of miR-29c and the expression of TPM1 was suppressed in the fBMFs transfected with miR-29c mimics. Besides, we confirmed that the expression of miR-29c was indeed downregulated in OSF specimens. MiR-29c seems to exert an inhibitory effect on myofibroblast activation, such as collagen gel contractility and migration ability, via suppressing TPM1. These results suggested that approaches to upregulate miR-29c may be able to ameliorate the progression of OSF.

Quaking I-5 protein inhibits invasion and migration of kidney renal clear cell carcinoma via inhibiting epithelial-mesenchymal transition suppression through the regulation of microRNA 200c.

BackgroundIt has been demonstrated that quaking I-5 protein (QKI-5) plays crucial roles in the metastasis of various kinds of cancers. However, the function and mechanism of QKI-5 in kidney renal clear cell carcinoma (KIRC) metastasis remains unclear. Therefore, this study aimed to explore the mechanism of QKI-5 in the metastasis of KIRC.MethodsThe expression of QKI-5 was detected using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot in KIRC tissues and different cell lines. Immunohistochemical staining was used to detect the quantity of QKI-5 in primary and metastases of KIRC. Cell migration and invasion were measured using wound healing and transwell assays respectively. The quantity of epithelial mesenchymal transition marker proteins was detected using western blot and immunofluorescence staining. The interaction of QKI-5 via microRNA 200c (miR-200c) was confirmed using dual luciferase reporter assay.ResultsAlthough QKI-5 was significantly more likely to be downregulated in KIRC tissues than that in normal Kidney tissues, it was dramatically elevated in metastatic KIRC tumors. Upregulation of QKI-5 promoted cell migration and invasion and elevated the expression of epithelial-mesenchymal transition (EMT) marker proteins, including vimentin, snail and slug, while it was downregulated for E-cadherin. Furthermore, a dual luciferase reporter assay demonstrated that QKI-5 was a direct target of miR-200c, and that miR-200c could reverse the effect of QKI-5 on cell migration, invasion, and expression of EMT marker proteins.ConclusionsOur results revealed that downregulation of QKI-5 by miR-200c attenuated KIRC migration and invasion via the EMT process, indicating that QKI-5 may be a potential therapeutic target and a key indicator of KIRC progression.