Therapeutic implication of MicroRNA-320a antagonist in attenuating blood clots formed during venous thrombosis.

Venous thrombosis (VT) is a complex multi-factorial disease and a major health concern worldwide. Its clinical implications include deep vein thrombosis (DVT) and pulmonary embolism (PE). VT pathogenesis involves intricate interplay of various coagulants and anti-coagulants. Growing evidences from epidemiological studies have shown that many non-coding microRNAs play significant regulatory role in VT pathogenesis by modulating expressions of large number of gene involved in blood coagulation. Present study aimed to investigate the effect of human micro RNA (hsa-miR)-320a antagonist on thrombus formation in VT. Surgery was performed on Sprague-Dawley (SD) rats, wherein the inferior vena cava (IVC) was ligated to introduce DVT. Animals were divided into four groups (n = 5 in each group); Sham controls (Sham), IVC ligated-DVT (DVT), IVC ligated-DVT + transfection reagent (DVT-NC) and IVC ligated-DVT + miR320a antagonist (DVT-miR-320a antagonist). IVC was dissected after 6 h and 24 h of surgery to estimate thrombus weight and coagulatory parameters such as levels of D-dimer, clotting time and bleeding time. Also, ELISA based biochemical assays were formed to assess toxicity of miRNA antagonist in animals. Our experimental analysis demonstrated that there was a marked reduction in size of thrombus in hsa-miR-320a antagonist treated animals, both at 6 h and 24 h. There was a marked reduction in D-dimer levels in hsa-miR-320a antagonist treated animals. Also, blood clotting time was delayed and bleeding time was increased significantly in hsa-miR-320a antagonist treated rats compared to the non-treated and Sham rats. There was no sign of toxicity in treated group compared to control animals. Hsa-miR-320a antagonist could be promising therapeutic target for management of VT.

Emerging role of MicroRNA-Based theranostics in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), with its high mortality and short survival rate, continues to be one of the deadliest malignancies despite relentless efforts and several technological advances. The poor prognosis of HCC and the few available treatments are to blame for the low survival rate, which emphasizes the importance of creating new, effective diagnostic markers and innovative therapy strategies. In-depth research is being done on the potent biomarker miRNAs, a special class of non-coding RNA and has shown encouraging results in the early identification and treatment of HCC in order to find more viable and successful therapeutics for the disease. It is beyond dispute that miRNAs control cell differentiation, proliferation, and survival and, depending on the genes they target, can either promote tumorigenesis or suppress it. Given the vital role miRNAs play in the biological system and their potential to serve as ground-breaking treatments for HCC, more study is required to fully examine their theranostic potential.

miRNAs and Stem Cells as Promising Diagnostic and Therapeutic Targets for Alzheimer's Disease.

Alzheimer's disease (AD) is a cumulative progressive neurodegenerative disease characterized mainly by impairment in cognitive functions accompanied by memory loss, disturbance in behavior and personality, and difficulties in learning. Although the main causes of AD pathogenesis are not fully understood yet, amyloid-ß peptides and tau proteins are supposed to be responsible for AD onset and pathogenesis. Various demographic, genetic, and environmental risk factors are involved in AD onset and pathogenesis such as age, gender, several genes, lipids, malnutrition, and poor diet. Significant changes were observed in microRNA (miRNA) levels between normal and AD cases giving hope for a diagnostic procedure for AD through a simple blood test. As yet, only two classes of AD therapeutic drugs are approved by FDA. They are classified as acetylcholinesterase inhibitors and N-methyl-D-aspartate antagonists (NMDA). Unfortunately, they can only treat the symptoms but cannot cure AD or stop its progression. New therapeutic approaches were developed for AD treatment including acitretin due to its ability to cross blood-brain barrier in the brain of rats and mice and induce the expression of ADAM 10 gene, the α-secretase of human amyloid-ß protein precursor, stimulating the non-amyloidogenic pathway for amyloid-ß protein precursor processing resulting in amyloid-ß reduction. Also stem cells may have a crucial role in AD treatment as they can improve cognitive functions and memory in AD rats through regeneration of damaged neurons. This review spotlights on promising diagnostic techniques such as miRNAs and therapeutic approaches such as acitretin and/or stem cells keeping in consideration AD pathogenesis, stages, symptoms, and risk factors.

Programming inactive RNA-binding small molecules into bioactive degraders.

Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure-activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L1. Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function.

The miR-181 family: Wide-ranging pathophysiological effects on cell fate and function.

MicroRNAs (miRNAs) are epigenetic regulators that can target and inhibit translation of multiple mRNAs within a given cell type. As such, a number of different pathways and networks may be modulated as a result. In fact, miRNAs are known to regulate many cellular processes including differentiation, proliferation, inflammation, and metabolism. This review focuses on the miR-181 family and provides information from the published literature on the role of miR-181 homologs in regulating a range of activities in different cell types and tissues. Of note, we have not included details on miR-181 expression and function in the context of cancer since this is a broad topic area requiring independent review. Instead, we have focused on describing the function and mechanism of miR-181 family members on differentiation toward a number of cell lineages in various non-neoplastic conditions (e.g., immune/hematopoietic cells, osteoblasts, osteoclasts, chondrocytes, adipocytes). We have also provided information on how modulation of miR-181 homologs can have positive effects on disease states such as cardiac abnormalities, pulmonary arterial hypertension, thrombosis, osteoarthritis, and vascular inflammation. In this context, we have used some examples of FDA-approved drugs that modulate miR-181 expression. We conclude by discussing some common mechanisms by which miR-181 homologs appear to regulate a number of different cellular processes and how targeting specific miR-181 family members may lead to attractive therapeutic approaches to treat a number of human disease or repair conditions, including those associated with the aging process.

Macrophage miR-149-5p induction is a key driver and therapeutic target for BRONJ.

Bisphosphonate-related (BP-related) osteonecrosis of the jaw (BRONJ) is one of the severe side effects of administration of BPs, such as zoledronic acid (ZA), which can disrupt the patient's quality of life. Although the direct target of skeletal vasculature and bone resorption activity by BPs has been phenomenally observed, the underlying mechanism in BRONJ remains largely elusive. Thus, it is urgently necessary to discover effective therapeutic targets based on the multifaceted underlying mechanisms in the development of BRONJ. Here, we determined the inhibitory role of ZA-treated macrophages on osteoclast differentiation and type H vessel formation during tooth extraction socket (TES) healing. Mechanistically, ZA activated the NF-κB signaling pathway and then induced p65 nuclear translocation in macrophages to promote miR-149-5p transcription, resulting in impaired osteoclast differentiation via directly binding to the Traf6 3'-UTR region. Moreover, we identified that miR-149-5p-loaded extracellular vesicles derived from ZA-treated bone marrow-derived macrophages could regulate biological functions of endothelial cells via the Rap1a/Rap1b/VEGFR2 pathway. Furthermore, local administration of chemically modified antagomiR-149-5p was proven to be therapeutically effective in BRONJ mice. In conclusion, our findings illuminate the dual effects of miR-149-5p on skeletal angiogenesis and bone remolding, suggesting it as a promising preventive and therapeutic target for BRONJ.

Identification of benzamides derivatives of norfloxacin as promising microRNA-21 inhibitors via repressing its transcription.

MicroRNA-21 is a carcinogenic microRNA, whose overexpression arises in a variety of tumor tissues. Hence, microRNA-21 a prospective target for cancer treatment, and regulation of microRNA-21 by small molecule inhibitors is deemed as a promising approach for tumor therapy. In this work, to discover potent microRNA-21 inhibitor, series of 4-(N-norfloxacin-acyl)aminobenzamides were designed and synthesized, and their inhibitory effects were appraised by utilizing dual luciferase reporter assays. The results indicated that compound A7 was the most efficient microRNA-21 small molecule inhibitor. What's more, A7 suppressed the migration of Hela cells and the colony formation of Hela and HCT-116 cells as well as promoted apoptosis of Hela cells. In the mechanism study, results of RT-qPCR certified that A7 could reduce the level of mature microRNA-21 via disrupting its expression at the transcriptional level of its primary form "pri-miR-21", which was distinct from most previous inhibitors directly binding with pre-miR-21. Noticeably, Western blotting and RT-qPCR uncovered A7 could upregulate the expression PTEN, EGR1 and SLIT2, which are the downstream functional targets of microRNA-21. These findings demonstrated that A7 was a promising microRNA-21 small molecule inhibitor and 4-(N-norfloxacin-acyl) aminobenzamide can serve as a new scaffold for discovery of potent microRNA-21 inhibitor.

miR-340-5p Mediates Cardiomyocyte Oxidative Stress in Diabetes-Induced Cardiac Dysfunction by Targeting Mcl-1.

Diabetic cardiomyopathy (DCM) is initially characterized by early diastolic dysfunction, left ventricular remodeling, hypertrophy, and myocardial fibrosis, and it is eventually characterized by clinical heart failure. MicroRNAs (miRNAs), endogenous small noncoding RNAs, play significant roles in diabetes mellitus (DM). However, it is still largely unknown about the mechanism that links miRNAs and the development of DCM. Here, we aimed to elucidate the mechanism underlying the potential role of microRNA-340-5p in DCM in db/db mouse, which is a commonly used model of type 2 DM and diabetic complications that lead to heart failure. We first demonstrated that miR-340-5p expression was dramatically increased in heart tissues of mice and cardiomyocytes under diabetic conditions. Overexpression of miR-340-5p exacerbated DCM, which was reflected by extensive myocardial fibrosis and more serious dysfunction in db/db mice as represented by increased apoptotic cardiomyocytes, elevated ROS production, and impaired mitochondrial function. Inhibition of miR-340-5p by a tough decoy (TUD) vector was beneficial for preventing ROS production and apoptosis, thus rescuing diabetic cardiomyopathy. We identified myeloid cell leukemia 1 (Mcl-1) as a major target gene for miR-340-5p and showed that the inhibition of Mcl-1 was responsible for increased functional loss of mitochondria, oxidative stress, and cardiomyocyte apoptosis, thereby caused cardiac dysfunction in diabetic mice. In conclusion, our results showed that miR-340-5p plays a crucial role in the development of DCM and can be targeted for therapeutic intervention.

Exosomal miR-17-3p Alleviates Programmed Necrosis in Cardiac Ischemia/Reperfusion Injury by Regulating TIMP3 Expression.

OBJECTIVE: Myocardial ischemia/reperfusion (I/R) injury can aggravate myocardial injury. Programmed necrosis plays a crucial role in this injury. However, the role of exosomal miRNAs in myocardial I/R injury remains unclear. Therefore, this study is aimed at exploring the function and mechanism of exosomal miR-17-3p in myocardial I/R injury. METHODS: The myocardial I/R injury animal model was established in C57BL/6 mice. Exosomes were identified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. Programmed necrosis was detected by PI staining. Heart function and myocardial infarct size were evaluated using echocardiography and triphenyl tetrazolium chloride (TTC) staining, respectively. Histopathological changes were visualized by hematoxylin and eosin (H&E) and Masson staining. The regulation of TIMP3 expression by miR-17-3p was verified using a dual-luciferase reporter assay. Lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) levels were measured by enzyme-linked immunosorbent assays (ELISA). TIMP3 expression was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting. RESULTS: We demonstrated that miR-17-3p was significantly downregulated in peripheral blood exosomes after cardiac I/R injury. Further analysis indicated that exosomal miR-17-3p attenuated H2O2-induced programmed necrosis in cardiomyocytes in vitro. Moreover, TIMP3 was a target for miR-17-3p. TIMP3 affected H2O2-induced programmed necrosis in cardiomyocytes. This effect was modulated by miR-17-3p in vitro. Furthermore, exosomal miR-17-3p greatly alleviated cardiac I/R injury in vivo. CONCLUSIONS: The present study demonstrated that exosomal miR-17-3p alleviated the programmed necrosis associated with cardiac I/R injury by regulating TIMP3 expression. These findings could represent a potential treatment for I/R injury.

Inhibition of miRNA-100 facilitates bone regeneration defects of mesenchymal stem cells in osteoporotic mice through the protein kinase B pathway.

Osteoporotic patients suffer from bone microstructure damage and are prone to fracture and bone defect. Due to the damage of bone healing ability, the bone repair of osteoporotic patients is usually slow. Here we aimed to explore the function and potential molecular mechanism of miR-100 in osteogenic differentiation ability of bone marrow stem cells (BMSCs). Ovariectomy was performed on mice to induce osteoporosis. BMSCs were extracted from normal and ovariectomized (OVX) mice to examine the effect of microRNA (miR)-100 on BMSC osteogenic differentiation. Hematoxylin and eosin (H&E) staining and safranin O-fast green staining assays were performed on femur tissues to reveal pathological changes. The osteogenic differentiation of BMSCs were determined by Alkaline Phosphatase and Alizarin red staining assays. The results showed that miR-100 expression was significantly upregulated in bone tissues and BMSCs from osteoporotic mice. MiR-100 knockdown partially improved osteogenic function of OVX mice-derived BMSCs. Next, mechanistic target of rapamycin kinase (MTOR) was identified as the target downstream miR-100. MiR-100 deficiency can activate the protein kinase B (AKT)/mTOR pathway. MiR-100 controlled the osteogenic function of BMSCs by the AKT/mTOR pathway. Collectively, our findings demonstrate that inhibition of miR-100 facilitates bone regeneration defects of BMSCs in osteoporotic mice through AKT pathway, indicating that miR-100 might be an effective target for the treatment of osteoporotic mandibular injury and bone defect diseases.

Exosomal MALAT1 Derived from High Glucose-Treated Macrophages Up-Regulates Resistin Expression via miR-150-5p Downregulation.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays a crucial role in the pathophysiological process associated with diabetes-related complications. The effect of high glucose levels on macrophage-derived exosomal MALAT1 is unknown. Therefore, we investigated the molecular regulatory mechanisms controlling exosomal MALAT1 in macrophages under high glucose treatment and the therapeutic target of macrophage-derived exosomal MALAT1 using a balloon injury model of vascular disease in diabetic rats. High glucose (25 mM) significantly increased MALAT1 expression in macrophage-derived exosomes. MALAT1 suppressed miR-150-5p expression in macrophage-derived exosomes under high-glucose conditions. Silencing MALAT1 using MALAT1 siRNA significantly reversed miR-150-5p expression induced by macrophage-derived exosomes. Macrophage-derived exosomes under high-glucose treatment significantly increased resistin expression in macrophages. Silencing MALAT1 and overexpression of miR-150-5p significantly decreased resistin expression induced by macrophage-derived exosomes. Overexpression of miR-150-5p significantly decreased resistin luciferase activity induced by macrophage-derived exosomes. Macrophage-derived exosome significantly decreased glucose uptake in macrophages and silencing MALAT1, resistin or overexpression of miR-150-5p significantly reversed glucose uptake. Balloon injury to the carotid artery significantly increased MALAT1 and resistin expression and significantly decreased miR-150-5p expression in arterial tissue. Silencing MALAT1 significantly reversed miR-150-5p expression in arterial tissue after balloon injury. Silencing MALAT1 or overexpression of miR-150-5p significantly reduced resistin expression after balloon injury. In conclusion, high glucose up-regulates MALAT1 to suppress miR-150-5p expression and counteracts the inhibitory effect of miR-150-5p on resistin expression in macrophages to promote vascular disease. Macrophage-derived exosomes containing MALAT1 may serve as a novel cell-free approach for the treatment of vascular disease in diabetes mellitus.

MiR-21 participates in LPS-induced myocardial injury by targeting Bcl-2 and CDK6.

OBJECTIVE: This study aimed to investigate the relationship between miR-21 and lipopolysaccharide (LPS)-induced myocardial injury and its molecular and regulatory mechanisms. METHODS: We constructed LPS-mediated myocardial injury model using C57BL/6J mice and H9c2 cells. In-vivo, in-vitro, RIP and dual-luciferase reporter assays were used to determine the effect of miR-21 on myocardial injury. RESULTS: In-vivo and in-vitro results showed that the expression of miR-21 was increased in LPS-treated H9c2 cells and myocardial tissues of mice, and the pro-inflammatory cytokines (IL-1ß, IL-6, IL-8 and TNF-α) and NF-κB pathway were activated in LPS-treated H9c2 cells. Besides, the B-cell lymphoma-2 (Bcl-2) and cyclin-dependent kinase 6 (CDK6) expression levels decreased, while Bax and cleaved caspase 9 levels increased in LPS-treated H9c2 cells. Inhibition of miR-21 could suppress LPS-induced apoptosis, inflammatory reactions and NF-κB activation to attenuate LPS-induced myocardial injury in H9c2 cells, and effectively improve survival of mice with sepsis. Most importantly, Bcl-2 and CDK6 were found to be the direct target of miR-21 using dual-luciferase reporter and RNA immunoprecipitation assays. Further gain-of-function assay demonstrated that Bcl-2 or CDK6 over-expression promoted the protective effects of miR-21 inhibitor on LPS-mediated myocardial cells. CONCLUSION: Our findings revealed that the down-regulation or antagonism of miR-21 protects myocardial cells against LPS-induced apoptosis and inflammation through up-regulating Bcl-2 and CDK6 expression, which provided a new insight for prevention and treatment of myocardial injury.

lncRNA MALAT1 promotes osteogenic differentiation through the miR-217/AKT3 axis: A possible strategy to alleviate osteoporosis.

BACKGROUND: Accumulating evidence demonstrates that long non-coding RNAs (lncRNAs) are associated with the development of osteoporosis. The present study aimed to investigate the effect of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on osteogenic differentiation in osteoporosis. METHODS: MALAT1 levels were detected by a real-time polymerase chain reaction (RT-qPCR). Moreover, the levels of osteogenic differentiation-related factors (Bmp4, Col1a1 and Spp1) were measured by a RT-qPCR and western blotting. Alkaline phosphatase (ALP) activity was detected using an ALP staining assay. RESULTS: MALAT1 levels were downregulated in hindlimb unloading mice and simulated in microgravity (MG) treated MC3T3-E1 cells. Moreover, MG treatment induced the downregulation of the expression of ALP, BMP4, Col1a1 and Spp1, whereas overexpression of MALAT1 abolished the downregulation. MG also inhibited ALP activity, whereas MALAT1 reversed the effect. Furthermore, miR-217 was identified as a target of MALAT1, and AKT3 was verified as a target of miR-217. Overexpression of miR-217 rescued the promotion of osteogenic differentiation induced by MALAT1 in MG treated cells. Knockdown of AKT3 abolished the facilitation of osteogenic differentiation induced by downregulation of miR-217. CONCLUSIONS: MALAT1 promotes osteogenic differentiation through regulating miR-217/AKT3 axis, suggesting that MALAT1 is a potential target with respect to alleviating osteoporosis.

Circular RNA CCDC66 Improves Murine Double Minute 4 (MDM4) Expression through Targeting miR-370 in Colorectal Cancer.

INTRODUCTION: Colorectal cancer (CRC), a common digestive tract tumor that contains colon and rectal cancer, is one of the three most common cancers globally. circRNAs are involved in the occurrence and development of CRC, but the mechanism of how they participate in this process remains unclear. METHODS: We adopted PCR for expression measure, CCK-8 for cell proliferation detection, Transwell for cell migration and invasion detection, and dual-luciferase reporter assays to detect the potential downstream targets of CCDC66 in CRC. RESULTS: This study showed that circRNA CCDC66 was overexpressed in CRC tissues, and after knockdown, it inhibited the proliferation, migration, and invasion of CRC cells (RKO and HCT-116) in vitro. In addition, the dual-luciferase reporter assay showed that there was a binding site between circCCDC66 and miR-370, as well as between miR-370 and murine double minute 4 (MDM4). That is, circCCDC66 upregulated the expression of MDM4 through competitively binding to miR-370. The expression of circCCDC66 in CRC tissues was positively correlated with MDM4 and negatively correlated with miR-370. CONCLUSION: In summary, our results indicate that circCCDC66 is a key upregulation of CRC. circCCDC66 upregulates MDM4 through competitive binding to miR-370, thereby enhancing the metastatic ability of CRC cells and promoting the development of CRC.

microRNA-671-5p reduces tumorigenicity of ovarian cancer via suppressing HDAC5 and HIF-1α expression.

OBJECTIVE: microRNA (miR)-based therapeutic reference has been established and expanded in the treatment of cancers. For this reason, we explored how miR-671-5p regulated tumorigenicity of ovarian cancer (OC) through regulating histone deacetylase 5 (HDAC5) and hypoxia-inducible factor-1α (HIF-1α). METHODS: miR-671-5p, HDAC5 and HIF-1α expression levels were determined in OC clinical tissues. The OC cell line H8910 was screened and transfected with the vectors that altered miR-671-5p, HDAC5 and HIF-1α levels. Finally, the proliferation, migration, invasion and apoptosis of the transfected H8910 cells were determined and the role of miR-671-5p and HDAC5 in vivo tumor growth was further discussed. RESULTS: Low expression miR-671-5p and high expression HDAC5 and HIF-1α levels were tested in OC tissues. Up-regulating miR-671-5p or down-regulating HDAC5 or HIF-1α suppressed proliferation, migration, invasion and augmented apoptosis of H8910 cells while silenced miR-671-5p or enhanced HDAC5 caused the opposite consequences. Overexpression of HDAC5 reduced while depletion of HDAC5 enhanced the influence of up-regulated miR-671-5p on OC cell growth. In animal models, suppressing miR-671-5p or promoting HDAC5 encouraged OC tumor growth. CONCLUSION: A summary delineates that miR-671-5p reduces tumorigenicity of OC via suppressing HDAC5 and HIF-1α expression levels.

MicroRNA-122-5p promotes renal fibrosis and injury in spontaneously hypertensive rats by targeting FOXO3.

Hypertensive renal injury is accompanied by tubular interstitial fibrosis leading to increased risk for renal failure. This study aimed to explore the influences of miR-122-5p in hypertension-mediated renal fibrosis and damage. 14-week-old male SHR and WKY rats were randomly assigned to treat with rAAV-miR-122-5p or rAAV-GFP for 8 weeks. There were marked increases in miR-122-5p and Kim-1 levels and decreases in FOXO3 and SIRT6 levels in hypertensive rats. Transfection with rAAV-miR-122-5p triggered exacerbation of renal fibrosis, apoptosis and inflammatory injury in SHR, associated with downregulated levels of FOXO3, SIRT6, ATG5 and BNIP3 as well as upregulated expression of Kim-1, NOX4, CTGF, and TGF-ß1. In cultured primary mouse renal tubular interstitial fibroblasts, exposure to angiotensin II resulted in obvious downregulation of FOXO3, SIRT6, ATG5, BNIP3 and nitric oxide levels as well as augmented cellular migration, oxidative stress, and inflammation, which were exacerbated by miR-122-5p mimic while rescued by miR-122-5p inhibitor and rhFOXO3, respectively. Notably, knockdown of FOXO3 strikingly blunted cellular protective effects of miR-122-5p inhibitor. In summary, miR-122-5p augments renal fibrosis, inflammatory and oxidant injury in hypertensive rats by suppressing the expression of FOXO3. Pharmacological inhibition of miR-122-5p has potential therapeutic significance for hypertensive renal injury and fibrosis-related kidney diseases.

Inhibition of microRNA-297 alleviates THLE-2 cell injury induced by hypoxia/reoxygenation by inhibiting NLRP3 inflammasome activation via sirtuin 3.

It has been acknowledged that microRNAs (miRNAs/miRs) assume a critical role in hypoxia/reoxygenation (H/R) - induced hepatocyte injury. Therefore, cell experiments were performed in this study to investigate the mechanism of miR-297 in H/R-induced hepatocyte injury with the involvement of sirtuin 3 (SIRT3) and NOD-like receptor pyrin domain containing 3 (NLRP3). Initially, transformed human liver epithelial-2 (THLE-2) cells were utilized for H/R challenge. After miR-297 antagomir and NLRP3 adenovirus vector delivery, THLE-2 cell proliferation and apoptosis were measured by MTT, EdU, and TUNEL assays, respectively. Enzyme-linked immunosorbent assay was conducted to evaluate the levels of apoptosis-related indicators (Bax and Bcl-2) and inflammation-related indicators (interleukin 6 (IL-6) and IL-10), Western blot analysis to detect NLRP3, and cleaved caspase-1 expression. The binding relation between miR-297 and SIRT3 was examined using dual-luciferase assay. The results showed that miR-297 antagomir repressed the apoptosis and inflammation induced by H/R treatment in THLE-2 cells. Mechanistically, miR-297 antagomir diminished the extent of IκBα and nuclear factor-kappa B (NF-κB) phosphorylation and NLRP3 activation in H/R-induced THLE-2 cells by targeting SIRT3. Furthermore, NLRP3 overexpression normalized the promoting effects of miR-297 antagomir on proliferation and its inhibitory effects on apoptosis and inflammation in H/R-induced THLE-2 cells. In summary, our results elucidated that miR-297 antagomir repressed H/R-induced THLE-2 cell injury via SIRT3 promotion and NLRP3 inactivation.

Propolis Extract Regulates microRNA Expression in Glioblastoma and Brain Cancer Stem Cells.

BACKGROUND: Grade IV gliomas are classified as glioblastoma (GBM), which is the most malignant brain cancer type. Various genetic and epigenetic mechanisms play a role in the initiation and progression of GBM. MicroRNAs (miRNAs) are small, non-coding RNA molecules that belong to the main epigenetic regulatory RNA class that plays different roles in either physiological or pathological conditions, including GBM pathogenesis regulating expression levels of the target genes. Brain Cancer Stem Cells (BCSCs) are responsible for poor prognosis, including therapy resistance and relapse. Epigenetic regulation mediated by miRNAs is also a critical component of BCSC selfrenewal and differentiation properties. Propolis is a resinous substance collected by honey bees from various plant sources. The flavonoid content of propolis varies depending on the collection region and the extraction method. Although there are studies that include the effects of different originated-propolis on the miRNA expression levels of the glioblastoma cells, the impact on the BCSCs has not been studied yet. OBJECTIVE: This study aims to evaluate the effects of propolis obtained from Aydin, a city in western Turkey, on miRNA expression levels of BCSCs and GBM cells. METHODS: Aydin propolis was dissolved in 60% ethanol, and after evaporation, distilled water was added to prepare the propolis stock solution. The flavonoids content of the Aydin propolis was determined by MS Q-TOF analysis. Commercially obtained U87MG and BCSCs were used as in-vitro brain cancer models. Cytotoxic and apoptotic effects of Aydin propolis were determined via WST-1 assay and Annexin V test, respectively. The miRNA expression profile was investigated using the real-time qRT-PCR method. The fold changes were calculated by the2-ΔΔCt method. The miRNA-mRNA-pathway interactions, including significantly altered miRNAs, were determined using different bioinformatics tools and databases. RESULTS: Quercetin 3-methyl ether was the main component of the Aydin propolis. Aydin propolis did not show significant cytotoxic and apoptotic effects on both GBM and BCSCs up to 2mg/ml concentration. Aydin propolis treatment decreased the expression of nine miRNAs in the U87MG and five miRNAs in the BCSCs. Moreover, ten miRNAs have upregulated from 2.22 to 10.56 folds in propolis treated GBM cells compared to the control group significantly (p<0.05). In the study, the potential roles of two new miRNAs, whose regulations in glioma were not previously defined, were identified. One of them was miR-30d-5p, a novel potential oncomiR in GBM, which was 2.46 folds downregulated in Aydin propolis treated GBM cells. The other one is miR-335-5p, which is a potential tumor suppressor miR in GBM, that was 5.66 folds upregulated in Aydin propolis treated GBM cells. FOXO pathway, its upstream and downstream regulators, and critically neuronal developmental regulators, NOTCH and WNT pathways, were determined as the most deregulated pathways in Aydin propolis treated cells. CONCLUSION: The determination of the anti-cancer effect of Aydin propolis on the miRNA expression of GBM, especially on cancer stem cells, may contribute to the elucidation of brain cancer genetics by supporting further analyses.

MicroRNA­671­5p inhibits cell proliferation, migration and invasion in non­small cell lung cancer by targeting MFAP3L.

MicroRNA (miR)­671­5p serves as a tumor suppressor in several types of cancer, including gastric and breast cancer. However, the function of miR­671­5p in non­small cell lung cancer (NSCLC) has not been described in detail. The present study aimed to investigate the role of miR­671­5p in NSCLC. The expression levels of miR­671­5p were determined in NSCLC tissue samples and cell lines using reverse transcription­quantitative PCR. Prediction of miR­671­5p targets was performed using the TargetScan database and verified by luciferase reporter assay and western blot analysis. Functional experiments, including Cell Counting Kit­8, wound healing and Transwell assays, were performed in NSCLC cells. The results of the present study demonstrated that lower expression levels of miR­671­5p were observed in NSCLC tissues and cell lines compared with those in the corresponding controls. Low miR­671­5p levels were significantly associated with an advanced Tumor­Node­Metastasis stage and lymph node metastasis in patients with NSCLC. Microfibril­associated protein 3­like (MFAP3L) was confirmed to be a direct target of miR­671­5p. The proliferative, migratory and invasive abilities of NSCLC cells were suppressed following transfection with miR­671­5p mimics and promoted by the miR­671­5p inhibitor compared with those in the respective control groups. In addition, the effects of miR­671­5p on cell proliferation, migration and invasion, as well as the expression levels of proliferating cell nuclear antigen, E­cadherin, N­cadherin and vimentin were reversed by MFAP3L overexpression. In conclusion, targeting the miR­671­5p/MFAP3L signaling pathway may be a promising therapeutic strategy for NSCLC treatment.

Bioinformatic Searching for Optimal RNA Targets of Dimeric Compounds Informs Design of a MicroRNA-27a Inhibitor.

Various studies have shown that selective molecular recognition of RNA targets by small molecules in cells, although challenging, is indeed possible. One facile strategy to enhance selectivity and potency is binding two or more sites within an RNA simultaneously with a single molecule. To simplify the identification of targets amenable to such a strategy, we informatically mined all human microRNA (miRNA) precursors to identify those with two proximal noncanonically paired sites. We selected oncogenic microRNA-27a (miR-27a) for further study as a lead molecule binds its Drosha site and a nearby internal loop, affording a homodimer that potently and specifically inhibits miR-27a processing in both breast cancer and prostate cancer cells. This reduction of mature miR-27a ameliorates an oncogenic cellular phenotype with nanomolar activity. Collectively, these studies demonstrate that synergistic bioinformatic and experimental approaches can define targets that may be more amenable to small molecule targeting than others.