Knockdown Of miR-21 Research Articles

Exosomes derived from endothelial progenitor cells ameliorate acute lung injury by transferring miR-126

Endothelial progenitor cell (EPC) has potential to attenuate pulmonary inflammation and injury. As a pivotal paracrine entity of stem cells, whether EPC-derived exosomes (EPC-Exos) contribute to acute lung injury (ALI) remains unknown. Exosomes were purified from conditional medium of EPCs, and then characterized by electron micrograph and immunoblotting. A model of ALI was induced by lipopolysaccharide (LPS) and then rats were transplanted with EPC-Exos. The underlying mechanisms of action of EPC-Exos were examined in vitro endothelial functional assays including the TEER, proliferation (CKK-8), angiogenesis and migration. A possible underlying mechanism was examined by western blotting and further animal studies. Administration of EPC-Exos ameliorated LPS-induced ALI and restored the in vivo pulmonary integrity. EPC-Exos enhanced the proliferation, migration and tube formation of the endothelial cells (ECs). Furthermore, we found that miR-126 was enriched in EPC-Exos and can be delivered onto ECs. Modification of EPCs through miR-126 knockdown can diminish their exosomes function in vitro, indicative of the abilities of EPC-Exos to protect against LPS were inherited by the horizontal shuttled miR-126. Luciferase reporter assays confirmed that miR-126 could target SPRED1. Additionally, the miR-126 transferred to target endothelial cells resulted in subsequent downregulation of SPRED1 and promoted RAF/ERK signaling pathways and subsequent improvement in endothelial cell function. Our study revealed a novel role of exosomal miRNAs in EPC-mediated therapy, suggesting that the clinical application of EPC-Exos might represent a strategy in ALI/ARDS.

A dual-targeting reconstituted high density lipoprotein leveraging the synergy of sorafenib and antimiRNA21 for enhanced hepatocellular carcinoma therapy

Sorafenib (So) is a multi-target kinase inhibitor extensively used in clinic for hepatocellular carcinoma therapy. It demonstrated strong inhibition both in tumor proliferation and tumor angiogenesis, while hampered by associated cutaneous side-effect and drug resistance. The knockdown of miR-21 with antisense oligonucleotides (antimiRNA21) was regarded as an efficient strategy for increasing tumor sensibility to chemotherapy, which could be employed to appreciate the efficacy of So. Herein, we successfully formulated a dual-targeting delivery system for enhanced hepatocellular carcinoma therapy by encapsulating So and antimiRNA21 in RGD pentapeptide-modified reconstituted high-density lipoprotein (RGD-rHDL/So/antimiRNA21). The RGD and apolipoprotein A-I (ApoA-I) on nanoparticles (NPs) could drive the system simultaneously to tumor neovascular and parenchyma by binding to the overexpressed ανβ3-integrin and SR-B1 receptors, achieving precise delivery of therapeutics to maximize the efficacy. A series in vitro and in vivo experiments revealed that co-delivery of So and antimiRNA21 by RGD-rHDL significantly strengthened the anti-tumor and anti-angiogenic effect of So with negligible toxicity towards major organs, reversed drug-resistance and was capable of remodeling tumor environments. The constructed RGD-rHDL/So/antimiRNA21 with improved efficacy and excellent tumor targeting ability provided new idea for chemo-gene combined therapy in hepatocellular carcinoma. Statement of SignificanceSorafenib (So) is a multi-target kinase inhibitor which was approved by FDA as first-line drug for hepatocellular carcinoma (HCC) therapy. However, long term application of So in clinic was hampered by serious dermal toxicity and drug resistance. Although numerous researchers were devoted to finding alternatives or therapies as combination treatments with So to reach more desired therapeutic efficacy, the therapeutic options were still limited. The present study prepares RGD pentapeptide decorated biomimic reconstituted high-density lipoprotein (rHDL) loaded with So and antimiRNA21 (RGD-rHDL/So/antimiRNA21) for enhanced HCC therapy. The RGD-rHDL/So/antimiRNA21 NPs offer an effective platform for anti-tumor and anti-angiogenesis therapy in HCC and provide new approach to reverse drug-resistance of So for feasible clinical application.

Total glucosides of paeony improves the immunomodulatory capacity of MSCs partially via the miR-124/STAT3 pathway in oral lichen planus.

Mesenchymal stem cells (MSCs) have been used clinically and experimentally to relieve severe immune-related diseases due to their immunomodulatory properties, but these are impaired by inflammation. Oral lichen planus (OLP) is a T cell-mediated chronic inflammatory mucosal disease. In the present study, we found MSCs from OLP with higher expression of interleukin (IL)-6, tumour necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β) and IL-10 compared with control. Total glucosides of paeony (TGP) significantly improves the immunomodulatory function of MSCs by inhibiting IL-6 and TNF-α expression and increasing TGF-β and IL-10 expression. Moreover, TGP can downregulate p-STAT3 expression through upregulation of miR-124. The changes of IL-6, TGF-β and p-STAT3 were further confirmed by overexpression and knockdown of miR-124 in MSCs. Taken together, the immune-regulating function of MSCs can be improved by TGP via the miR-124/STAT3 pathway.

MiR-221 alleviates the inflammatory response and cell apoptosis of neuronal cell through targeting TNFAIP2 in spinal cord ischemia–reperfusion

This study aimed to examine the role of miR-221 in inflammatory response and apoptosis of neuronal cells after spinal cord ischemia/reperfusion (I/R) injury. Blood samples were obtained from 20 I/R patients and that of 20 healthy individuals were used as a control. AGE1.HN and SY-SH-5Y neuronal cell lines subjected to oxygen-glucose deprivation (OGD) stress were used in cell experiments. Real-time PCR and western blot were used to evaluate the expression of miR-221, tumor necrosis factor-α, and TNFAIP2. TUNEL assay analyzed cell apoptosis. I/R patients had lower serum levels of miR-221 than healthy controls. In OGD-AGE1.HN and SY-SH-5Y cells, miR-221 was significantly downregulated and TNFAIP2 mRNA and protein were upregulated; meanwhile, both proinflammatory cytokine tumor necrosis factor-α and anti-inflammation cytokine interleukin-6 were elevated and the percentage of apoptotic cells was increased. This inflammatory response and cell apoptosis induced by OGD stress were attenuated by miR-221 overexpression and enhanced by miR-221 knockdown. TNFAIP2 is a target gene for miR-221 and could be regulated negatively by the miR-221 mimic or the miR-221 inhibitor with or without OGD stress. Accordingly, TNFAIP2 overexpression reversed the inflammatory response and cell apoptosis induced by miR-221 under OGD stress. Downregulation of miR-221 occurs in spinal cord I/R injury and in cell lines subjected to oxygen-glucose deprivation. miR-221 regulates the inflammatory response and apoptosis of neuronal cells through its impact on TNFAIP2.

MiR-155 inhibits the formation of hypertrophic scar fibroblasts by targeting HIF-1α via PI3K/AKT pathway

Hypertrophic scar (HS) is a serious skin fibrotic disease characterized by the excessive proliferation of fibroblasts and often considered as a kind of benign skin tumor. microRNA-155 (miR-155) is usually served as a promising marker in antitumor therapy. In view of the similarities of hypertrophic scar and tumor, it is predicted that miR-155 may be a novel therapeutic target in clinical trials. Here we found the expression levels of miR-155 was gradually down regulated and HIF-1α was upregulated in HS tissue and HS derived fibroblasts (HFs). And cell proliferation was inhibited when miR-155 was overexpressed or HIF-1α was silenced. Moreover, overexpression of miR-155 in HFs could reduce the expression of collagens in vitro and inhibit the collagen fibers arrangement in vivo, whereas miR-155 knockdown gave opposite results. Furthermore, we found that miR-155 directly targeted the HIF-1α, which could also independently inhibit the expression of collagens in vitro and obviously improved the appearance and architecture of the rabbit ear scar in vivo when it was silencing. Finally, we found that PI3K/AKT pathway was enrolled in these processes. Together, our results indicated that miR-155 was a critical regulator in the formation and development of hypertrophic scar and might be a potential molecular target for hypertrophic scar therapy.

MicroRNA-21 up-regulates metalloprotease by down-regulating TIMP3 during cumulus cell-oocyte complex in vitro maturation.

Cumulus cells and the remodeling of their extracellular matrix around oocytes are essential for oocyte maturation and ovulation in the ovary. An important extracellular metalloprotease, ADAMTS1, and its partner VERSICAN, mediate essential cumulus-oocyte-complex (COC) structural remodeling. However, how the expression of these proteinases is regulated during oocyte maturation is unclear. Here we report that both ADAMTS1 and VERSICAN significantly increased in porcine cumulus cells during COC in vitro maturation (IVM). Interestingly, one of the tissue inhibitors of the metalloproteinase family member, TIMP3, was found to be significantly decreased in cumulus cells during this process. Down-regulation of TIMP3 using specific small interfering RNA decreased TIMP3 expression, while increased the levels of ADAMTS1 and VERSICAN, suggesting an inverse relationship between TIMP3, the metalloprotease, and the breakdown product of its substrate. MiR-21 significantly increased in cumulus cells during COC maturation. Knockdown of miR-21 in cumulus cells during COC maturation resulted in increased TIMP3 and decreased ADAMTS1 and VERSICAN expression, which is accompanied by a decrease in cumulus cell expansion and the ratio of oocytes that reached MII stage. In contrast, over-expression of miR-21 decreased TIMP3 and increased ADAMTS1, and enhanced cumulus cell expansion and oocyte maturation. Moreover, in silico prediction revealed that a miR-21 binding site is present at the 3-untranslated region (3-UTR) of the TIMP3 mRNA, which was further confirmed to be the target site of miR-21 by luciferase gene reporter assays. Our findings revealed that miR-21 promotes cumulus expansion and oocyte maturation via down-regulating TIMP3, and subsequent increase of ADAMTS1 and VERSICAN during in vitro COC maturation.

A Novel Murine Chronic Obstructive Pulmonary Disease Model and the Pathogenic Role of MicroRNA-21

Chronic obstructive pulmonary disease (COPD) is a multi-pathogenesis chronic lung disease. The mechanisms underlying COPD have not been adequately illustrated. Many reseachers argue that microRNAs (miRs) could play a crucial role in COPD. The classic animal model of COPD is both time consuming and costly. This study proposes a novel mice COPD model and explores the role of miR-21 in COPD. A total of 50 wide-type (WT) C57BL/6 mice were separated into five euqlly-sized groups—(1) control group (CG), (2) the novel combined method group (NCM, cigarette smoke (CS) exposure for 28 days combined with cigarette smoke extract (CSE) intraperitoneal injection), (3) the short-term CS exposure group (SCSE, CS exposure for 28 days), (4) the CSE intraperitoneal injection group (CSEII, 28 days CSE intraperitoneal injection), and (5) the long-term CS exposure group (LCSE, CS exposure).The body weight gain of mice were recorded and lung function tested once the modeling was done. The pathological changes and the inflammation level by hematoxylin eosin (H&E) staining and immunohistochemical staining (IHS) on the lung tissue sections were also evaluated. The level of miR-21 in the mice lungs of the mice across all groups was detected by RT-qPCR and the effects of miR-21 knock-down in modeled mice were observed. The mice in LCSE and NCM exhibited the most severe inflammation levels and pathological and pathophysiological changes; while the changes for the mice in SCSE and CSEII were less, they remained more severe than the mice in the CG. The level of miR-21 was found to be negatively correlated with lung functions. Moreover, knocking miR-21 down from the modeled mice, ameliorated all those tested COPD-related changes. Our novel modeling method detected virtually the same changes as those detected in the classic method in WT mice, but in less time and cost. Further, it was determined that the level of miR-21 in the lungs could be an indicator of COPD severity and blocking functions of miR-21 could be a potential treatment for early stage COPD.

MiR-31 Mediates Inflammatory Signaling to Promote Re-Epithelialization during Skin Wound Healing.

Wound healing is essential for skin repair after injury, and it consists of hemostasis, inflammation, re-epithelialization, and remodeling phases. Successful re-epithelialization, which relies on proliferation and migration of epidermal keratinocytes, requires a reduction in tissue inflammation. Therefore, understanding the molecular mechanism underlying the transition from inflammation to re-epithelialization will help to better understand the principles of wound healing. Currently, the invivo functions of specific microRNAs in wound healing are not fully understood. We observed that miR-31 expression is strongly induced in wound edge keratinocytes, and is directly regulated by the activity of NF-κB and signal transducer and activator of transcription 3 signaling pathways during the inflammation phase. We used miR-31 loss-of-function mouse models to demonstrate that miR-31 promotes keratinocyte proliferation and migration. Mechanistically, miR-31 activates the Ras/mitogen-activated protein kinase signaling by directly targeting Rasa1, Spred1, Spred2, and Spry4, which are negative regulators of the Ras/mitogen-activated protein kinase pathway. Knockdown of these miR-31 targets at least partially rescues the delayed scratch wound re-epithelialization phenotype observed invitro in miR-31 knockdown keratinocytes. Taken together, these findings identify miR-31 as an important cell-autonomous mediator during the transition from inflammation to re-epithelialization phases of wound healing, suggesting a therapeutic potential for miR-31 in skin injury repair.

MiR-206 is required for changes in cell adhesion that drive muscle cell morphogenesis in Xenopus laevis

MicroRNAs (miRNAs) are highly conserved small non-coding RNA molecules that post-transcriptionally regulate gene expression in multicellular organisms. Within the set of muscle-specific miRNAs, miR-206 expression is largely restricted to skeletal muscle and is found exclusively within the bony fish lineage. Although many studies have implicated miR-206 in muscle maintenance and disease, its role in skeletal muscle development remains largely unknown. Here, we examine the role of miR-206 during Xenopus laevis somitogenesis. In Xenopus laevis, miR-206 expression coincides with the onset of somitogenesis. We show that both knockdown and over-expression of miR-206 result in abnormal somite formation affecting muscle cell rotation, attachment, and elongation. In particular, our data suggests that miR-206 regulates changes in cell adhesion that affect the ability of newly formed somites to adhere to the notochord as well as to the intersomitic boundaries. Additionally, we show that β-dystroglycan and F-actin expression levels are significantly reduced, suggesting that knockdown of miR-206 levels affects cellular mechanics necessary for cell shape changes and attachments that are required for proper muscle formation.

Downregulation of microRNA-21 inhibited radiation-resistance of esophageal squamous cell carcinoma

BackgroundMicroRNA-21 (miR-21) was previously reported being dysregulated in many kinds of cancer including esophageal squamous cell carcinoma (ESCC). In the present study, we aimed to investigate the role of miR-21 in ESCC, especially in its effects on radiation-sensitivity of ESCC.MethodsExpression of miR-21 was detected in 63 pairs ESCC tumor and adjacent non-tumoral tissues using qRT-PCR, correlation between miR-21 and clinicopathological feature of ESCC was analyzed. The role of miR-21 in the proliferation, cell cycle and apoptosis of ESCC cells during irradiation were studied.ResultsMicroRNA-21 expression was significantly increased in ESCC tumor tissues. Expression of miR-21 was positively associated with advanced clinical stage. Under irradiation, overexpression of miR-21 increased cell proliferation and cells in S phase, and inhibited cell apoptosis of ESCC cells. In contrast, knockdown of miR-21 had an opposite effect.ConclusionsDownregulation of miR-21 inhibited the radiation-resistance of ESCC, whereas overexpression of miR-21 increased the radiation-resistance. MiR-21 is a potential novel target for developing specific treatment interventions in ESCC in future.

Mir-126 is a conserved modulator of lymphatic development

Organ homeostasis relies upon cellular and molecular processes that restore tissue structure and function in a timely fashion. Lymphatic vessels help maintain fluid equilibrium by returning interstitial fluid that evades venous uptake back to the circulation. Despite its important role in tissue homeostasis, cancer metastasis, and close developmental origins with the blood vasculature, the number of molecular players known to control lymphatic system development is relatively low. Here we show, using genetic approaches in zebrafish and mice, that the endothelial specific microRNA mir-126, previously implicated in vascular integrity, regulates lymphatic development. In zebrafish, in contrast to mir-126 morphants, double mutants (mir-126a-/-; mir-126b-/-, hereafter mir-126-/-) do not exhibit defects in vascular integrity but develop lymphatic hypoplasia; mir-126-/- animals fail to develop complete trunk and facial lymphatics, display severe edema and die as larvae. Notably, following MIR-126 inhibition, human Lymphatic Endothelial Cells (hLECs) respond poorly to VEGFA and VEGFC. In this context, we identify a concomitant reduction in Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) and Vascular Endothelial Growth Factor Receptor-3 (VEGFR3, also known as FLT4) expression upon MIR-126 inhibition. In vivo, we further show that flt4+/- zebrafish embryos exhibit lymphatic defects after mild miR-126 knockdown. Similarly, loss of Mir-126 in Flt4+/- mice results in embryonic edema and lethality. Thus, our results indicate that miR-126 modulation of Vegfr signaling is essential for lymphatic system development in fish and mammals.

Long non-coding RNA Gas5 regulates proliferation and apoptosis in HCS-2/8 cells and growth plate chondrocytes by controlling FGF1 expression via miR-21 regulation

BackgroundLncRNA Gas5 is known to be a key control element during growth, differentiation and development in mammalian species. However, the role and function of Gas5 in growth plate chondrocytes has not been determined.MethodsThe overexpression and knockdown models of Gas5 and miR-21 in cells and animals were constructed. Cell survival was determined by MTT assay and flow cytometry. Animal biochemical indices were measured by enzyme-linked immunosorbent assay, hematoxylin/eosin staining, immunohistochemistry or in situ hybridisation. Dual luciferase reporter gene assay was carried out to study targeting.ResultsFirst, we found the expression levels of fibroblast growth factor 1(FGF1) were up-regulated and miR-21 were down-regulated in Gas5 overexpressing model cells. Meanwhile, the expression levels of FGF1 and Gas5 were up-regulated in miR-21 knockdown model cells. Furthermore, cell proliferation was significantly promoted after Gas5 knockdown or miR-21 overexpression. Subsequently, Gas5 promoted apoptosis, while miR-21 suppressed apoptosis. Animal assays demonstrated that both Gas5 and dexamethasone suppressed proliferation and promoted apoptosis of growth plate chondrocytes, up-regulated FGF1 expression but reduced miR-21 expression. Finally, there was a binding relationship between Gas5, miR-21 and FGF1.ConclusionWe concluded that Gas5 regulated proliferation and apoptosis in growth plate by controlling FGF1 expression via miR-21 regulation.

CircHIPK3 regulates cell proliferation and migration by sponging miR-124 and regulating AQP3 expression in hepatocellular carcinoma

Noncoding RNAs plays an important role in hepatocellular carcinoma (HCC). Here, we show that miR-124 was downregulated in HCC tissues and that the ectopic expression of miR-124 inhibited the proliferation and migration of HCC cells. We proposed that aquaporin 3 (AQP3) is a direct target of miR-124. AQP3 was upregulated in HCC tissues and inversely correlated with miR-124 expression. The overexpression of miR-124 decreased AQP3 expression. Indeed, AQP3 overexpression promoted cell proliferation and migration, whereas miR-124 knockdown suppressed cell proliferation and migration. Furthermore, we found that circular RNA HIPK3 (circHIPK3) acted as a miR-124 sponge and regulated the expression of the miR-124 target gene AQP3. circHIPK3 was upregulated in HCC tissues and positively correlated with AQP3 expression. Thus, silencing circHIPK3 inhibited cell proliferation and migration by downregulating AQP3 expression. Moreover, miR-124 inhibition rescued circHIPK3 knockdown induced reduction in cell proliferation and migration, as well as AQP3 expression. In vivo experiments also confirmed that circHIPK3 regulated xenograft tumor growth via the miR-124-AQP3 axis. These observations indicate a possible novel therapeutic strategy involving circular RNAs in HCC.

Preliminary study of microRNA-126 as a novel therapeutic target for primary hypertension.

The present study aimed to explore microRNA-126 (miR-126) as a novel therapeutic target for primary hypertension. The lentiviral vector containing human immunodeficiency virus 1 (HIV-1), the miR-126 gene knockdown viral vector (lenti-miR-126-KD), and control lentiviral vector (lenti-scramble-miR) were constructed. Spontaneously hypertensive rats were randomly divided into 4 groups, which received a high dose of lenti-miR-126-KD (1×108, n=5), low dose of lenti-miR-126-KD (1×107, n=6), scramble-miR (5×107, n=6), and PBS (n=6). Lentiviral vectors were injected into the tail vein. Data on the systolic blood pressure, diastolic pressure, mean arterial pressure, and heart rate were collected weekly. After 8 weeks of virus administration, the distribution of lentiviral vectors in different tissues was observed by fluorescence microscopy. Picric acid Sirius red and H&E staining were used to observe the target organ damage, and the ELISA kit was used to determine the serum nitric oxide (NO) content. The lentiviral vector was found to be constructed successfully. Eight weeks after the lentiviral vector injection, green fluorescent protein was observed in different tissues in each group. The blood pressure and heart rate were not significantly altered after lentiviral vector injection (P>0.05). No significant differences in the heart-to-body weight ratio among the four groups were observed (P=0.23). Picric acid Sirius red and H&E staining revealed that there was no significant difference in morphology among the four groups. No significant difference in the serum NO level among the four groups was noted (P=0.23). The miR-126 gene knockdown lentiviral vector was constructed successfully. No significant antihypertensive effect was observed by the knockdown of miR-126 for the treatment of primary hypertension. The target organs were not protected significantly after the treatment. The increased level of miR-126 expression in hypertensive patients may be due to a compensatory mechanism.

MicroRNA-223 Suppresses Osteoblast Differentiation by Inhibiting DHRS3

Background/Aims: In this study, we aimed to use bioinformatics tools to identify the specific miRNAs and mRNAs involved in osteogenic differentiation and to further explore the way in which miRNA regulates osteogenic differentiation. Methods: The microarray GSE80614, which includes data from 3 human mesenchymal stromal cells (hMSCs) and 3 hMSCs after 72 hours (hr) of osteogenic differentiation, was used to screen for differentially expressed mRNAs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of these mRNAs were conducted using Gene Set Enrichment Analysis (GSEA). Then, the miRanda website was employed to detect the binding sites of DHRS3. In vitro experiments, including RT-PCR and western blotting, were used to detect miR-233 and DHRS3 expression levels 7 and 14 days (d) after the induction of osteogenic differentiation using human bone marrow-derived mesenchymal stem cells (hBMSCs). The target relationship between miR-223 and DHRS3 was confirmed by a dual luciferase assay. ALP (alkaline phosphatase) staining, ARS (Alizarin Red S) staining and western blotting (Runx2, OPN, OCN) were used to detect the level of osteogenic differentiation after transfection with miR-223 mimics and DHRS3 cDNA. Results: In this study, 127 mRNAs differentially expressed during osteogenic differentiation were identified in GSE80614. GO term and KEGG pathway enrichment analyses found that the retinol metabolism pathway was activated during osteogenic differentiation and that DHRS3, which is involved in the pathway, was upregulated. During osteogenic differentiation in hBMSCs, miR-223 was gradually downregulated, while DHRS3 was upregulated. After 14 days of osteogenic differentiation, ALP and ARS staining assay results showed strong ALP activity and extracellular matrix calcification with the inhibition of miR-223 or the overexpression of DHRS3. Furthermore, the expression levels of Runx2, OPN, and OCN were upregulated with the knockdown of miR-223 or the overexpression of DHRS3, while the simultaneous transfection of a miR-223 agomir and DHRS3 cDNA resulted in no significant difference from the negative control (NC) group. Conclusion: The inhibition of miR-223 promotes the osteogenic differentiation of hBMSCs via the upregulation of DHRS3.

Resveratrol Inhibits ROS-Promoted Activation and Glycolysis of Pancreatic Stellate Cells via Suppression of miR-21.

Activation of pancreatic stellate cells (PSCs) initiates pancreatic fibrosis in chronic pancreatitis and furnishes a niche that enhances the malignancy of pancreatic cancer cells (PCCs) in pancreatic ductal adenocarcinoma (PDAC). Resveratrol (RSV), a natural polyphenol, exhibits potent antioxidant and anticancer effects. However, whether and how RSV influences the biological properties of activated PSCs and the effects of these changes on tumor remain unknown. In the present study, we found that RSV impeded hydrogen peroxide-driven reactive oxygen species- (ROS-) induced activation, invasion, migration, and glycolysis of PSCs. In addition, miR-21 expression in activated PSCs was downregulated after RSV treatment, whereas the PTEN protein level increased. miR-21 silencing attenuated ROS-induced activation, invasion, migration, and glycolysis of PSCs, whereas the overexpression of miR-21 rescued the responses of PSCs treated with RSV. Moreover, RSV or N-acetyl-L-cysteine (NAC) administration or miR-21 knockdown in PSCs reduced the invasion and migration of PCCs in coculture, and the effects of RSV were partly reversed by miR-21 upregulation. Collectively, RSV inhibits PCC invasion and migration through suppression of ROS/miR-21-mediated activation and glycolysis in PSCs. Therefore, targeting miR-21-mediated glycolysis by RSV in tumor stroma may serve as a new strategy for clinical PDAC prevention or treatment.

Melatonin prevents endothelial cell pyroptosis via regulation of long noncoding RNA MEG3/miR-223/NLRP3 axis.

Atherosclerosis (AS) is an inflammatory disease linked to endothelial dysfunction. Melatonin is reported to possess substantial anti-inflammatory properties, which has proven to be effective in AS. Emerging literature suggests that pyroptosis plays a critical role during AS progression. However, whether pyroptosis contributes to endothelial dysfunction and the underlying molecular mechanisms remained unexploited. This study was designed to investigate the antipyroptotic effects of melatonin in atherosclerotic endothelium and to elucidate the potential mechanisms.In this study, high-fat diet (HFD)-treated ApoE-/- mice were used as an atherosclerotic animal model. We found intragastric administration of melatonin for 12weeks markedly reduced the atherosclerotic plaque in aorta. Meanwhile, melatonin also attenuated the expression of pyroptosis-related genes, including NLRP3, ASC, cleaved caspase1, NF-κB/GSDMD, GSDMD N-termini, IL-1β, and IL-18 in aortic endothelium of melatonin-treated animals. Consistent antipyroptotic effects were also observed in ox-LDL-treated human aortic endothelial cells (HAECs). We found that lncRNA MEG3 enhanced pyroptosis in HAECs. Moreover, MEG3 acted as an endogenous sponge by sequence complementarity to suppress the function of miR-223 and to increase NLRP3 expression and enhance endothelial cell pyroptosis. Furthermore, knockdown of miR-223 blocked the antipyroptotic actions of melatonin in ox-LDL-treated HAECs. Together, our results suggest that melatonin prevents endothelial cell pyroptosis via MEG3/miR-223/NLRP3 axis in atherosclerosis, and therefore, melatonin replacement might be considered a new strategy for protecting endothelium against pyroptosis, thereby for the treatment of atherosclerosis associated with pyroptosis.

Chlorogenic Acid Inhibits Liver Fibrosis by Blocking the miR-21-Regulated TGF-β1/Smad7 Signaling Pathway in Vitro and in Vivo.

Aims: Chlorogenic acid (CGA) is a phenolic acid that has a wide range of pharmacological effects. However, the protective effects and mechanisms of CGA on liver fibrosis are not clear. This study explored the effects of CGA on miR-21-regulated TGF-β1/Smad7 liver fibrosis in the hepatic stellate LX2 cell line and in CCl4-induced liver fibrosis in Sprague-Dawley rats.Methods: The mRNA expression of miR-21, Smad7, connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase 1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), and transforming growth factor-β1 (TGF-β1) and the protein levels of Smad2, p-Smad2, Smad3, p-Smad3, Smad2/3, p-Smad2/3, Smad7, CTGF, α-SMA, TIMP-1, MMP-9 and TGF-β1 were assayed in LX2 cells and liver tissue. The effects of CGA after miR-21 knockdown or overexpression were analyzed in LX2 cells. The liver tissue and serum were collected for histopathological examination, immunohistochemistry (IHC) and ELISA.Results: The mRNA expression of miR-21, CTGF, α-SMA, TIMP-1, and TGF-β1 and the protein expression of p-Smad2, p-Smad3, p-Smad2/3, CTGF, α-SMA, TIMP-1, and TGF-β1 were inhibited by CGA both in vitro and in vivo. Meanwhile, CGA elevated the mRNA and protein expression of Smad7 and MMP-9. After miR-21 knockdown and overexpression, the downstream molecules also changed accordingly. CGA also lessened the degree of liver fibrosis in the pathological manifestation and reduced α-SMA and collagen I expression in liver tissue and TGF-β1 in serum. Conclusion: CGA might relieve liver fibrosis through the miR-21-regulated TGF-β1/Smad7 signaling pathway, which suggests that CGA might be a new anti-fibrosis agent that improves liver fibrosis.

Ischemic Postconditioning Protects Against Intestinal Ischemia/Reperfusion Injury via the HIF-1\u03b1/miR-21 Axis

Intestinal ischemia/reperfusion (I/R) can lead to tissue damage associated with inflammation and mucosal apoptosis. Ischemic postconditioning (IPostC), a series of repeated, brief, intermittent periods of ischemia and reperfusion, has beneficial effects against I/R-induced injury in the heart and intestine, although the underlying mechanisms for these effects remain unclear. We evaluated the involvement of microRNA-21 (miR-21) in the protective effects of IPostC in a rat model of I/R induced by superior mesenteric artery occlusion and reopening. IPostC decreased I/R injury and suppressed apoptosis in the intestinal tissues concomitant with the induction of hypoxia inducible factor 1 alpha (HIF-1α) and the upregulation of miR-21. In vitro experiments in the intestinal epithelial cell line IEC-6 showed that hypoxia induced miR-21 and this effect was abolished by silencing HIF1-α, confirming the induction of miR-21 by HIF1-α, HIF1-α or miR-21 inhibition exacerbated I/R induced apoptosis, and programmed cell death 4 (PDCD4) and Fas-L was involved in miR-21 mediated anti-apoptotic effects on intestinal epithelial cells. Knockdown of miR-21 or inhibition of HIF1-α abolished the IPostC-mediated attenuation of intestinal injury and apoptosis and the downregulation of PDCD4 and Fas-L. A potential mechanism underlying the protective effect of IPostC may therefore involve the induction of miR-21 by HIF1-α and the attenuation of apoptosis via the downregulation of PDCD4 and Fas-L.

Screening the expression characteristics of several miRNAs in G93A-SOD1 transgenic mouse: altered expression of miRNA-124 is associated with astrocyte differentiation by targeting Sox2 and Sox9.

MicroRNAs (miRNAs) are suspected to be a contributing factor in amyotrophic lateral sclerosis (ALS). Here, we assess the altered expression of miRNAs and the effects of miR-124 in astrocytic differentiation in neural stem cells of ALS transgenic mice. Differentially expressed miRNA-positive cells (including miR-124, miR-181a, miR-22, miR-26b, miR-34a, miR-146a, miR-219, miR-21, miR-200a, and miR-320) were detected by insitu hybridization and qRT-PCR in the spinal cord and the brainstem. Our results demonstrated that miR-124 was down-regulated in the spinal cord and brainstem. In vitro, miR-124 was down-regulated in neural stem cells and up-regulated in differentiated neural stem cells in G93A-superoxide dismutase 1 (SOD1) mice compared with WT mice by qRT-PCR. Meanwhile, Sox2 and Sox9 protein levels showed converse change with miR-124 invivo and vitro. After over-expression or knockdown of miR-124 in motor neuron-like hybrid (NSC34) cells of mouse, Sox2 and Sox9 proteins were noticeably down-regulated or up-regulated, whereas Sox2 and Sox9 mRNAs remained virtually unchanged. Moreover, immunofluorescence results indicated that the number of double-positive cells of Sox2/glial fibrillary acidic protein (GFAP) and Sox9/glial fibrillary acidic protein (GFAP) was higher in G93A-SOD1 mice compared with WT mice. We also found that many Sox2- and Sox9-positive cells were nestin positive in G93A-SOD1 mice, but not in WT mice. Furthermore, differentiated neural stem cells from G93A-SOD1 mice generated a greater proportion of astrocytes and lower proportion of neurons than those from WT mice. MiR-124 may play an important role in astrocytic differentiation by targeting Sox2 and Sox9 in ALS transgenic mice. Cover Image for this issue: doi: 10.1111/jnc.14171.