Inhibition Of microRNA-21 Research Articles

Variation of Tetrahydrofurans in Thyclotides Enhances Oligonucleotide Binding and Cellular Uptake of Peptide Nucleic Acids.

Selective incorporation of conformational constraints into thyclotides can be used to modulate their binding to complementary oligonucleotides, increase polarity, and optimize uptake into HCT116 cells without assistance from moieties known to promote cell uptake. The X-ray structure and biophysical studies of a thyclotide-DNA duplex reveal that incorporation of tetrahydrofurans into an aegPNA backbone promotes a helical conformation that enhances binding to complementary DNA and RNA. Selective incorporation of tetrahydrofurans into the aegPNA backbone allows polarity to be increased incrementally so that uptake into HCT116 cells can be optimized. The enhanced binding, polarity, and cellular uptake properties of thyclotides were used to demonstrate effective inhibition of microRNA-21 in HCT116 cells.

Radiofrequency Ablation–Induced Tumor Growth Is Suppressed by MicroRNA-21 Inhibition in Murine Models of Intrahepatic Colorectal Carcinoma

PurposeTo investigate the role of microRNA-21 (miR21) in radiofrequency (RF) ablation–induced tumor growth and whether miR21 inhibition suppresses tumorigenesis. Material and MethodsStandardized liver RF ablation was applied to 35 C57/BL6 mice. miR21 and target proteins pSTAT3, PDCD4, and PTEN were assayed 3 hours, 24 hours, and 3 days after ablation. Next, 53 Balb/c and 44 C57BL/6 mice received Antago-miR21 or scrambled Antago-nc control, followed by intrasplenic injection of 10,000 CT26 or MC38 colorectal tumor cells, respectively. Hepatic RF ablation or sham ablation was performed 24 hours later. Metastases were quantified and tumor microvascular density (MVD) and cellular proliferation were assessed at 14 or 21 days after the procedures, respectively. ResultsRF ablation significantly increased miR21 levels in plasma and hepatic tissue at 3 and 24 hours as well as target proteins at 3 days after ablation (P < .05, all comparisons). RF ablation nearly doubled tumor growth (CT26, 2.0 SD ± 1.0 fold change [fc]; MC38, 1.9 SD ± 0.9 fc) and increased MVD (CT26, 1.9 SD ± 1.0 fc; MC38, 1.5 ± 0.5 fc) and cellular proliferation (CT26, 1.7 SD ± 0.7 fc; MC38, 1.4 SD ± 0.5 fc) compared with sham ablation (P < .05, all comparisons). RF ablation–induced tumor growth was suppressed when Antago-miR21 was administered (CT26, 1.0 SD ± 0.7 fc; MC38, 0.9 SD ± 0.4 fc) (P < .01, both comparisons). Likewise, Antago-miR21 decreased MVD (CT26, 1.0 SD ± 0.3 fc; MC38, 1.0 SD ± 0.2 fc) and cellular proliferation (CT26, 0.9 SD ± 0.3 fc; MC38, 0.8 SD ± 0.3 fc) compared with baseline (P < .05, all comparisons). ConclusionsRF ablation upregulates protumorigenic miR21, which subsequently influences downstream tumor-promoting protein pathways. This effect can potentially be suppressed by specific inhibition of miR21, rendering this microRNA a pivotal and targetable driver of tumorigenesis after hepatic thermal ablation.

MicroRNA-21 inhibition as a treatment against adverse cardiac remodeling and dysfunction induced by myocardial infarction

microRNA-21 inhibition as a treatment against adverse cardiac remodeling and dysfunction induced by myocardial infarction

MicroRNA21 inhibition affects porcine oocyte maturation and alters protein expression critical for metabolic pathway function.

MicroRNA21 (MIR21) abundance in porcine oocytes and cumulus cells increases during in vitro maturation. The mechanism by which MIR21 regulates oocyte maturation and the effect on the developmental competence of subsequent embryos remains unclear. The objective of this study was to assess the function of MIR21 during porcine oocyte maturation and its effect on embryonic development. Treatment with peptide nucleic acid MIR21 inhibitor (MIR21-PNA), designed to specifically bind to and prevent MIR21 activity during in vitro oocyte maturation, decreased cumulus cell expansion, and the oocyte ability to achieve metaphase II maturation stage when compared to control groups. Following parthenogenetic activation, the cleavage rate at 48 h in the MIR21-PNA group was decreased (p ≤ 0.03) relative to the control groups. Additionally, liquid chromatography-mass spectrometry (LC-MS/MS) of oocyte and cumulus cell total protein following MIR21-PNA treatment during in vitro maturation identified changes in signaling pathways with primary involvement of glucose metabolism (GM) pathways. Furthermore, there was no difference (p = 0.21) in oocyte maturation of control and MIR21-PNA treated oocytes when cultured in pyruvate lacking medium. Finally, MIR21-PNA treatment decreased (p = 0.04) glutathione and increased (p = 0.07) reactive oxygen species production in the oocyte. These data suggest that MIR21 influences porcine oocyte maturation by regulating GM pathways in the cumulus-oocyte complex.

Structure-Activity Relationships of Anti-microRNA Oligonucleotides Containing Cationic Guanidine-Modified Nucleic Acids.

Anti-microRNA oligonucleotides (AMOs) are valuable tools for the treatment of diseases caused by the dysregulation of microRNA expression. However, the correlation between chemical modifications in AMO sequences and the microRNA-inhibitory activity has not been fully elucidated. In this study, we synthesized a series of AMOs containing cationic guanidine-bridged nucleic acids (GuNA) and evaluated their activities using a dual luciferase assay. We also optimized the site of GuNA substitution and found an effective design for the inhibition of microRNA-21, which was partially different from that of conventional nucleic acid derivatives. This study showed that GuNA-substituted AMOs are effective in inhibiting the function of microRNA.

MicroRNA-21 inhibition attenuates airway inflammation and remodelling by modulating the transforming growth factor β-Smad7 pathway.

Background/AimsCurrent asthma therapies remain unsatisfactory for controlling airway remodelling in asthma. MicroRNA-21 is a key player in asthma pathogenesis, but the molecular mechanisms underlying its effects on airway remodelling are not completely understood. We investigated the effects of inhibition of microRNA-21 on allergic airway inflammation and remodelling.MethodsFemale BALB/c mice were divided into four groups: control, ovalbumin-sensitized and -challenged for 3 months, microRNA-negative control-treated ovalbumin-treated, and microRNA-21 inhibitor-treated ovalbumin-treated groups. Parameters related to airway remodelling, cytokine production, airway inflammation, and airway hyperresponsiveness were compared between groups. Human bronchial smooth muscle cells were used in a mechanism study.ResultsIn this asthma model, ovalbumin-sensitized and -challenged mice exhibited allergic airway inflammation and airway remodelling. MicroRNA-21 inhibitor-treated mice had fewer inflammatory cells, lower TH2 cytokine production, and suppressed parameters related to remodelling such as goblet cell hyperplasia, collagen deposition, hydroxyproline content, and expression of smooth muscle actin. Inhibition of microRNA-21 decreased transforming growth factor β1 expression and induced Smad7 expression in lung tissue. In human bronchial smooth muscle cells stimulated with transforming growth factor β1, microRNA-21 inhibition upregulated Smad7 expression and decreased markers of airway remodelling.ConclusionsInhibition of microRNA-21 had both anti-inflammatory and anti-remodelling effects in this model of ovalbumin-induced chronic asthma. Our data suggest that the microRNA-21–transforming growth factor β1–Smad7 axis modulates the pathogenesis of ovalbumin-induced chronic asthma and in human bronchial smooth muscle cells. MicroRNA-21 inhibitors may be a novel therapeutic target in patients with allergic asthma, especially those with airway remodelling.

MicroRNA-21 Contributes to Acute Liver Injury in LPS-Induced Sepsis Mice by Inhibiting PPARα Expression.

The severity of sepsis may be associated with excessive inflammation, thus leading to acute liver injury. MicroRNA-21 is highly expressed in the liver of a variety of inflammation-related diseases, and PPARα is also proved to participate in regulating inflammation. In the present study, the LPS-induced sepsis model was established. We found that microRNA-21 expression was upregulated in the liver of sepsis mice, and microRNA-21 inhibition significantly reduced the liver injury. The expression of liver injury markers, inflammation cytokines, and PPARα in the septic mice was higher than in antagomir-21 treated septic mice. In addition, we also found that PPARα is the target gene of microRNA-21; PPARα antagonist GW6471 could reverse the effect of antagomir-21. In conclusion, our study illustrated that microRNA-21 exacerbate acute liver injury in sepsis mice by inhibiting PPARα expression.

Therapeutic inhibition of microRNA-21 (miR-21) using locked-nucleic acid (LNA)-anti-miR and its effects on the biological behaviors of melanoma cancer cells in preclinical studies

BackgroundMelanoma is a cancer that has a high mortality rate in the absence of targeted therapy. Conventional therapies such as surgery, chemotherapy, and radiotherapy are associated with poor prognosis. The expression of miR-21 appears to be of clinical importance, and the regulation of its expression appears to be an opportunity for treatment.MethodsIn this current study, we aimed to evaluate the effects of miR-21 inhibition in- vitro and in-vivo. In-vitro studies have investigated LNA-anti-miR-21 in mouse melanoma cells (B16F10), and in-vivo studies have proposed a model of melanoma in male C57BL/6 mice. To evaluate the anticancer effects of LNA-anti-miR-21, a QRT-PCR analysis was performed using the 2−ΔΔCT method to determine the degree of inhibition of oncomiR-21. The MTT test, propidium iodide/AnnexinV in-vitro, and tumor volume measurement using the QRT-PCR test with the 2−ΔΔCT method were used to estimate the inhibition of miR-21 and the expression of downstream genes including: SNAI1, Nestin (Nes), Oct-4, and NF-kB following miR-21 inhibition. Finally, immunohistochemistry was conducted for an in-vivo animal study.ResultsMiR-21 expression was inhibited by 80% after 24 h of B16F10 cell line transfection with LNA-anti-miR-21. The MTT test showed a significant reduction in the number of transfected cells with LNA-anti-miR-21. The transfected cells showed a significant increase in apoptosis in comparison with the control and scrambled LNA groups. According to our in vivo findings, anti-miR-21 could reduce tumor growth and volume in mice receiving intraperitoneal anti-miR after 9 days. The expression of the SNAI1gene was significantly reduced compared to the controls. Immunohistochemical analysis showed no change in CD133 and NF-kB markers.ConclusionOur findings suggest LNA-anti-miR-21 can be potentially used as an anticancer agent for the treatment of melanoma.

Transcriptional and free radical responses to LVAD therapy

BackgroundMyocardial recovery with Left ventricular assistant device (LVAD) therapy is dichotomous with some patients obtaining remission from end-stage heart failure whereas most require transplantation or remain on pump support long term. Our goal was to determine transcriptional and free radical responses to LVAD treatment.MethodsTissues were collected from patients before and after LVAD placement in non-ischemic dilated cardiomyopathy patients (n = 14) along with controls (n = 3). RNA sequencing (RNASeq) analysis quantified transcriptional profiles by using a custom targeted panel of heart failure related genes on the PGM sequencer. The differential expression analysis between groups was conducted using edgeR (Empirical analysis of digital gene expression data in R) package in Bioconductor. Ingenuity Pathway Analysis (IPA) was carried out on differentially expressed genes to understand the biological pathways involved. Electron Paramagnetic Resonance (EPR) Spectroscopy was utilized to measure levels of free radicals in whole blood collected pre- and post-LVAD implantation (n = 16).ResultsThirty-five genes were differentially expressed in pre-LVAD failing hearts compared to controls. In response to LVAD therapy, only Pyruvate dehydrogenase kinase 4 (PDK4) and period circadian protein homolog 1 (PER1) were altered with 34 heart failure related genes still differentially expressed post-LVAD compared to controls. IPA showed that DNA methylation-related genes were upregulated in both pre- and post-LVAD and was persistent with a Z-score of 2.00 and 2.36 for DNA Methyltransferase 3A (DNMT3A) and DNA methyltransferase 3B (DNMT3B), respectively. Inhibition of micro RNA21 (mir21) was also significant on pathway analysis in the post-LVAD population with a Z-score of − 2.00. Levels of free radicals in blood of pre- and post-LVAD patients did not change significantly.ConclusionLVAD therapy does not reverse many of the transcriptional changes associated with heart failure. Persistent changes in gene expression may be related to ongoing oxidative stress, continued DNA methylation, or changes in metabolism. PDK4 is a key regulator of glucose metabolism and its increased expression by LVAD therapy inhibited pyruvate metabolism.

MicroRNA‐21 Ablation Attenuates Acetaminophen‐Induced Hepatoxtoxicity in Male Mice

IntroductionAcetaminophen (APAP) induced acute liver failure (ALF) is recognized as the most common cause of ALF in Western societies, occurring in approximately 40% of all cases. The disease is characterized by coagulopathy, hepatic encephalopathy, multi‐organ failure, and death. Micro‐RNAs are small, non‐coding RNAs capable of altering gene expression at the post‐transcriptional level. MicroRNA‐21 is dynamically expressed in the liver and several target genes of microRNA‐21 are highly expressed in the pericentral hepatocytes. We hypothesize that microRNA‐21 genetic ablation attenuates hepatotoxicity following acetaminophen intoxication.MethodsEight‐week old microRNA‐21 knockout (miR21KO) or wild‐type (WT) C57BL/6N male mice were injected with acetaminophen (APAP, 300 mg/kg BW) or saline. Animals were sacrificed 6 or 24 hours post‐injection, and serum and livers were harvested.ResultsWT animals showed significantly higher hepatic injury markers serum ALT (20,077±2,084 U/L vs 13,364±1,667 U/L, p&lt;0.05, n=11) and AST (29,125±3,485 U/L vs 20,282±2,445 U/L, p&lt;0.05, n=11) than their miR21KO counterparts after 6 hours of APAP treatment, while no significant difference was observed in animals treated for 24 hours. miR21KO mice presented attenuated liver necrosis compared with WT animals (59.8±5.8 vs 42.5±7.9 %, p&lt;0.05, n=10) 24 hours post‐APAP treatment. Moreover, miR21KO mice had decreased hepatic DNA fragmentation analyzed by TUNEL assay than WT animals (58.26±4.5 vs 42.60±4.9 %, p&lt;0.05, n=8) after 24 hours of APAP treatment. MicroRNA‐21 ablation did not modify hepatic GSSH/GSH or APAP‐Cysteine adducts at either 6 or 24 hours post treatment. MicroRNA‐21 ablation upregulated mRNA expression of autophagy (Beclin‐1, Map1LC3a, Pdcd4, p62) and decreased inflammation (PAI‐1) markers compared with WT mice 24 post APAP treatment. MicroRNA‐21 ablation exacerbated hepatic autophagy marker LC3A/B‐II protein expression at both 6 (2.8 vs 1.5‐fold) and 24 (2.2 vs 1.2‐fold 1.2) hours post APAP, correlating with increased Map1lc3a/b mRNA levels.ConclusionOur results suggest that miRNA‐21 mediates APAP‐induced hepatotoxicity. MicroRNA‐21 inhibition could be a novel therapeutic approach to abolish or mitigate APAP‐induced hepatotoxicity. The beneficial effect of microRNA‐21 is due to an upregulation of hepatic autophagy. Specifically, microRNA‐21 inhibition could be particularly useful when APAP intoxication is detected at its late stages and the only available therapy, NAC, is minimally effective.Support or Funding InformationSupported by NIH grants R21 DK‐113500 and P20 GM‐121334.

Low expression of microRNA-21 inhibits trophoblast cell infiltration through targeting PTEN.

We investigate whether microRNA-21 could increase the infiltration ability of trophoblast cells via regulating PTEN expression, thus participating in the occurrence and development of preeclampsia. MicroRNA-21 expression in the placenta tissues of preeclampsia women and normal pregnant women was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The effects of microRNA-21 on cell proliferation and infiltration were examined by cell counting kit-8 (CCK-8) and transwell assay, respectively. The dual-luciferase reporter gene assay was used to determine the binding relationship between microRNA-21 and PTEN. Western blot was performed to detect PTEN and microRNA-21 in trophoblasts. QRT-PCR results showed that the microRNA-21 expression was significantly lower in the placenta of the preeclampsia women than those of normal pregnant women. Overexpression of microRNA-21 in HTR-8/SVneo cells had no effect on cell proliferation, but enhanced cell infiltration ability. Inhibition of microRNA-21 in trophoblasts showed the opposite effects. The results of luciferase activity assay and Western blot showed that microRNA-21 could target PTEN and downregulate its expression. Overexpression of PTEN in HTR-8/SVneo cells partially reversed the enhanced invasive ability induced by microRNA-21 overexpression. Low expression of microRNA-21 attenuated cell infiltration of trophoblasts via direct regulating PTEN expression.

Role of microRNA-21 in uveal melanoma cell invasion and metastasis by regulating p53 and its downstream protein.

To reveal the insight mechanism of liver metastasis in uveal melanoma, we investigated cell functions of microRNA-21 in three different uveal melanoma cell lines and analyze the relationship of target gene p53 and its downstream targets. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect microRNA-21 expression in normal uveal tissue and uveal melanoma cell lines. Lenti-virus expression system was used to construct OCM-1, MuM-2B and M619 cell line with stable overexpression and inhibition of microRNA-21. In vitro cell function tests such as cell proliferation, cell apoptosis, cell circle and abilities of migration and invasion were examined by MTT, BrdU assay, flow cytometry, transwell assay and Matrigel invasion assay respectively. The target gene was predicted by bioinformatics and confirmed by using a dual luciferase reporter assay. The expression of p53 and its suspected downstream targets LIM and SH3 protein 1 (LASP1) and glutathione S transferase pi (GST-Pi) were determined by qRT-PCR in mRNA level and Western blotting analysis in protein level. Finally, the effect of microRNA-21 in a xenograft tumor model was assessed in four-week-old BALB/c nude mice. Compared to normal uveal melanoma, expressions of microRNA-21 were significantly higher in uveal melanoma cell lines. Overexpression of microRNA-21 promoted proliferation, migration, and invasion of OCM-1, M619 and MuM-2B cells, while inhibition of microRNA-21 reveal opposite effects. Wild type p53 was identified as a target gene of microRNA-21-3p, and proved by dual luciferase reporter assay. Up-regulated microRNA-21 inhibited the expression of wild type p53 gene, and the increased expression of LASP1 in mRNA level and protein level, while down-regulated microRNA-21 presented opposite way. However, GST-pi showed the potential pattern as expected, but relative mRNA level showed no statistically significant difference in OCM-1 cells. Furthermore, the mRNA expression of GST-pi was decreased in microRNA-21 overexpressing MuM-2B, and increased in M619 cells with inhibition of microRNA-21. In vivo, inhibition of microRNA-21 reduced tumor growth with statistically significant difference. These findings provide novel insight into molecular etiology of microRNA-21 in uveal melanoma cell lines, and suggest that microRNA-21 might be a potential candidate for the diagnosis and prognostic factor of human uveal melanoma.

Abstract 553: Inhibition of Microrna-21 Prevents Myocardial Remodelling and Dysfunction in a Pig Model of Ischemia/reperfusion Injury

Background: Upregulation of microRNA-21 (miR-21) has been associated with myocardial disease and its systemic inhibition was effective in reducing cardiac fibrosis and dysfunction in rodent models. However, the translation to human disease is challenging, as systemic application of miR inhibitors does not achieve adequate delivery in most organs including the heart. Here, we sought to test the applicability and therapeutic efficacy of local, catheter-based delivery of antimiR-21 in a large animal model of heart failure. Methods and results: Pigs underwent percutaneous occlusion of the left coronary artery for 60 minutes and were followed up for 33 days. A synthetic inhibitor against miR-21 (LNA-antimiR-21, 10 mg per application) was applied locally by intracoronary infusion at days 5 and 19 after the injury. AntimiR-21 effectively suppressed the I/R-induced increase of miR-21. At 33 days after I/R, LNA-21-treated hearts exhibited reduced cardiac fibrosis and hypertrophy, and improved cardiac function (ejection fraction: I/R control 22±1% vs. I/R LNA-21 33±2%). RNA sequencing of ischemic myocardium revealed a significant enrichment for miR-21 targets in anti-miR-21 treated hearts. Mechanistically, antimiR-21 interfered with a number of signalling pathways involved in myocardial remodelling including the MAPK-ERK signalling cascade. Conclusion: Taken together, our study provides the first evidence for the feasibility and therapeutic efficacy of miR-21 inhibition in a large animal model of myocardial ischemia. AntimiR-21 prevented the structural and functional consequences of ischemic myocardial damage.

Abstract 300: MicroRNA-21 Affects Platelets and Their Releasate: A Novel Mechanism for the Anti-Fibrotic Effects of MicroRNA-21 Inhibition

Introduction: Fibrosis is a major contributor to cardiac disease. MicroRNA-21 (miR-21) has been implicated as a regulator of fibrosis, with inhibitors of miR-21 currently undergoing clinical trials. We aimed to explore how miR-21 inhibition attenuates fibrosis using a proteomics approach. Methods and Results: As expected, transfection with miR-21 mimics and inhibitors altered the proliferation of murine cardiac fibroblasts. However, proteomic analysis of their conditioned media revealed limited effects on ECM secretion. Similarly, proteomic analysis of hearts from miR-21 null mice showed no major difference in extracellular matrix (ECM) composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In the community-based Bruneck study (year 2000 follow-up, n=671), we compared circulating miR-21 with a panel of 229 proteins associated with cardiovascular disease. Several platelet-derived pro-fibrotic factors correlated with plasma miR-21 levels, including the latency-associated peptide of transforming growth factor beta-1 (TGF-β1). This correlation was confirmed by ELISA measurements of active TGF-β1 in plasma (Bruneck 2015 follow-up, n=332). In agreement with this observation in men, systemic inhibition of miR-21 with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich Syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. AntagomiR-21 treatment, however, had no effect on the platelet count or their aggregation response. In contrast, miR-21 null mice had significantly lower platelet counts compared to littermate controls. Conclusions: This study reports a previously unrecognized effect of miR-21 inhibition on platelets. Both genetic deletion of miR-21 and antagomiR treatment affect platelets. The effect of miR-21 inhibition on platelet TGF-β1 release, in particular, may contribute to the anti-fibrotic effects of miR-21 inhibitors.

Glucose-sensing microRNA-21 disrupts ROS homeostasis and impairs antioxidant responses in cellular glucose variability

BackgroundAntioxidant enzymes play a fundamental role in counteracting oxidative stress induced by high glucose. Although mitochondrial superoxide dismutase (SOD2) is the principal defence against the toxicity of superoxide anions, the mechanism of its inactivation in diabetic subjects is still poorly understood. Recently, microRNA-21 has been associated with diabetes, although its function remains unclear. We sought to explore the mechanism underlying defective SOD2 antioxidant response in HUVECs during exposures to constant high glucose and oscillating glucose (as glucose variability model, GV) and the role of miR-21 in increasing the susceptibility to oxidative stress by disrupting reactive oxygen species (ROS) homeostasis.MethodsHUVECs exposed for 1 week to constant high glucose and GV were subjected to quantitative electron paramagnetic resonance for ROS measurements. Superoxide anions, SOD2 protein levels and mitochondrial membrane potential (ΔΨm) were also evaluated. Endogenous miR-21 and its putative ROS-homeostatic target genes (KRIT1, FoxO1, NFE2L2 and SOD2) were tested using mimic-miR-21 and quantified by qPCR. Luciferase assays were performed to test miR-21/3′-UTR-SOD2 binding.ResultsWe observed upregulation of microRNA-21, overproduction of superoxide anions and total ROS generation, depolarisation of the mitochondrial membrane potential (ΔΨm) and defective SOD2 antioxidant response in HUVECs subjected to constant high glucose and GV exposures. We also found that exogenous mimic-microRNA-21 targeted putative microRNA-21 ROS-homeostatic target genes, e.g., KRIT1, NRF2 and SOD2, which were significantly downregulated. All these effects were reverted by a microRNA-21 inhibitor, which improved SOD2 and KRIT1 expression, reduced the levels of ROS production and ameliorated ΔΨm.ConclusionsOur data demonstrate the association of microRNA-21 with oscillating and high glucose and early mitochondrial dysfunction. We found that microRNA-21 may promote the suppression of homeostatic signalling that normally limits ROS damage. These data provide novel clues about the inhibition of microRNA-21 as a new therapeutic approach to protect against cellular oxidative injury in glucose variability and diabetes.

MiRNA-21 ablation protects against liver injury and necroptosis in cholestasis.

Inhibition of microRNA-21 (miR-21) prevents necroptosis in the mouse pancreas. Necroptosis contributes to hepatic necro-inflammation in the common bile duct ligation (BDL) murine model. We aimed to evaluate the role of miR-21 in mediating deleterious processes associated with cholestasis. Mechanistic studies established a functional link between miR-21 and necroptosis through cyclin-dependent kinase 2-associated protein 1 (CDK2AP1). miR-21 expression increased in the liver of primary biliary cholangitis (PBC) patients and BDL wild-type (WT) mice at both 3 and 14 days. Notably, under BDL, miR-21 -/- mice displayed decreased liver injury markers in serum compared with WT mice, accompanied by reduced hepatocellular degeneration, oxidative stress and fibrosis. Hallmarks of necroptosis were decreased in the liver of BDL miR-21 -/- mice, via relieved repression of CDK2AP1. Further, miR-21 -/- mice displayed improved adaptive response of bile acid homeostasis. In conclusion, miR-21 ablation ameliorates liver damage and necroptosis in BDL mice. Inhibition of miR-21 should arise as a promising approach to treat cholestasis.

MicroRNA-21 inhibits mitochondria-mediated apoptosis in keloid.

MicroRNA-21 acts as an oncogene by promoting cell proliferation and migration, whereas inhibiting apoptosis in majority of cancers. MicroRNA-21 is upregulated in human keloid fibroblasts. We hypothesized that microRNA-21 may contribute to pathogenesis of keloid fibroblasts. First, enhanced miR-21 but reduced mitochondrial-mediated apoptosis observed in keloid tissues indicated its importance in keloids development. Second, upregulation of microRNA-21 induced a decrease in the ratio of BAX to BCL-2 and suppressed mitochondrial fission in keloid fibroblasts. Third, by attenuating the decline in cellular mitochondrial membrane potential, overexpression of miR-21 suppressed cytochrome c release to the cytoplasm, followed by a decrease in the activity of intracellular caspase-9 and caspase-3, suggesting that mitochondrial-mediated proapoptotic pathway was impaired. Simultaneously, intracellular reactive oxygen species were decreased, indicating microRNA-21 undermined oxidative stress. This phenotype was reversed by miR-21 inhibition. Therefore, our study demonstrates that inhibition of microRNA-21 induces mitochondrial-mediated apoptosis in keloid fibroblasts, proposing microRNA-21 as a potential therapeutic target in keloid fibroblasts.

Inhibition of MicroRNA-21 by an antagomir ameliorates allergic inflammation in a mouse model of asthma

ABSTRACTAim of the Study: MicroRNA-21 (miR-21) is up-regulated during allergic airway inflammation, reflecting a Th2 immune response. We investigated the effects of an miR-21 antagomir and its mechanism of action in a mouse model of acute bronchial asthma. Materials and Methods: BALB/c mice were sensitized and challenged with ovalbumin (OVA). The anti-miR-21 antagomir was administered by intranasal inhalation from the day of sensitization. Changes in cell counts, Th2 cytokine levels in bronchoalveolar (BAL) fluid, and airway hyper-responsiveness (AHR) were examined. Histopathological changes and expression levels of miR-21 in lung tissues were analyzed. The mechanism of action of the antagomir was investigated by counting CD4+/CD8− T cells in splenocytes and by measuring the expression levels of transcription factors associated with T cell polarization. Results: MiR-21 expression was selectively down-regulated in the lung tissues of mice treated with anti-miR-21. The antagomir suppressed AHR compared with that of the OVA-challenged and scrambled RNA-treated groups. It also reduced the total cell and eosinophil counts in BAL fluid and the levels of Th2 cytokines, including IL-4, IL-5, and IL-13. The direct target of miR-21, IL-12p35, was induced in the antagomir-treated group, decreasing the CD4+/CD8− T cell proportions in splenocytes. The levels of transcription factors involved in the Th2-signaling pathway were reduced in lung tissues on treatment with the antagomir. Conclusions: The miR-21 antagomir suppresses the development of allergic airway inflammation in a mouse model of acute bronchial asthma, inhibiting Th2 activation. These results suggest that this antagomir might be useful for treating bronchial asthma.

Inhibition of microRNA-21 via locked nucleic acid-anti-miR suppressed metastatic features of colorectal cancer cells through modulation of programmed cell death 4

Colorectal cancer is among the most lethal of malignancies, due to its propensity to metastatic spread and multifactorial-chemoresistance. The latter property supports the need to identify novel therapeutic approaches for the treatment of colorectal cancer. MicroRNAs are endogenous non-coding small RNA molecules that function as post-transcriptional regulators of gene expression. Recently, programmed cell death 4 has been identified as a protein that increases during apoptosis. This gene is among the potential targets of miR-21 (OncomiR). Locked nucleic acid-modified oligonucleotides have recently emerged as a potential therapeutic option for targeting microRNAs. The aim of this study was to explore the functional role of locked nucleic acid-anti-miR-21 in the LS174T cell line in vitro and in vivo models. LS174T cells were treated with locked nucleic acid-anti-miR-21 for 24, 48, and 72 h in vitro. The expression of miR-21 and PDCD4 at messenger RNA (mRNA) level was evaluated by quantitative real-time polymerase chain reaction, while the protein level of PDCD4 was determined by Western blotting. Cell migratory behavior and the cluster-forming ability of cells were assessed before and after therapy. The disseminated tumor cells were assessed in the chick chorioallantoic membrane model by Alu quantitative polymerase chain reaction. Locked nucleic acid-anti-miR-21 was transfected successfully into the LS174T cells and inhibited the expression of miR-21. Locked nucleic acid-anti-miR-21 inhibited the migration and the number of cells forming clusters. Moreover, we found that locked nucleic acid-anti-miR-21 transfection was associated with a significant reduction in metastatic properties as assessed by the in ovo model. Our findings demonstrated the novel therapeutic potential of locked nucleic acid-anti-miR-21 in colon adenocarcinoma with high miR-21 expression.

Inhibition of MicroRNA-21 induces apoptosis in dermal fibroblasts of patients with systemic sclerosis.

Prolonged activation of dermal fibroblasts is the main cause of progressive fibrosis in systemic sclerosis (SSc). It seems that inhibition of apoptosis in SSc fibroblasts deregulates fibrosis. MicroRNA-21 (miR-21) is a pro-fibrotic factor with high expression in lesional areas of SSc skin and fibroblasts. The effects of miR-21 on expression of Bcl-2 and Bax, two apoptotic genes, in dermal fibroblasts of SSc patients were evaluated using real-time polymerase chain reaction and Western blot analysis. Apoptotic cells were detected using flow cytometry and Hoechst 33258 staining assays. Overexpression of miR-21 using synthetic miR-21 RNA increased expression of Bcl-2, an inhibitor of apoptosis, and decreased the Bax : Bcl-2 expression ratio, a cell fate determinant, in SSc fibroblasts. Antisense inhibition of miR-21 induced a high rate of apoptosis in SSc fibroblasts. We propose that this may be associated with a decrease in Bcl-2 expression and a shift in the Bax : Bcl-2 ratio. Although further studies are necessary to determine the underlying apoptotic pathway, we propose that inhibition of miR-21 in dermal fibroblasts from lesional skin may be useful in harnessing progressive fibrosis in SSc.