Sex-dependent phosphorylation of Argonaute 2 reduces the mitochondrial translocation of miR-181c and induces cardioprotection in females.

Obesity-induced cardiac dysfunction is growing at an alarming rate, showing a dramatic increase in global prevalence. Mitochondrial translocation of miR-181c in cardiomyocytes results in excessive reactive oxygen species (ROS) production during obesity. ROS causes Sp1, a transcription factor for MICU1, to be degraded via post-translational modification. The subsequent decrease in MICU1 expression causes mitochondrial Ca2+ accumulation, ultimately leading to a propensity for heart failure. Herein, we hypothesized that phosphorylation of Argonaute 2 (AGO2) at Ser 387 (in human) or Ser 388 (in mouse) inhibits the translocation of miR-181c into the mitochondria by increasing the cytoplasmic stability of the RNA-induced silencing complex (RISC). Initially, estrogen offers cardioprotection in pre-menopausal females against the consequences of mitochondrial miR-181c upregulation by driving the phosphorylation of AGO2. Neonatal mouse ventricular myocytes (NMVM) treated with insulin showed an increase in pAGO2 levels and a decrease in mitochondrial miR-181c expression by increasing the binding affinity of AGO2-GW182 in the RISC. Thus, insulin treatment prevented excessive ROS production and mitochondrial Ca2+ accumulation. In human cardiomyocytes, we overexpressed miR-181c to mimic pathological conditions, such as obesity/diabetes. Treatment with estradiol (E2) for 48 h significantly lowered miR-181c entry into the mitochondria through increased pAGO2 levels. E2 treatment also normalized Sp1 degradation and MICU1 transcription that normally occurs in response to miR-181c overexpression. We then investigated these findings using an in vivo model, with age-matched male, female and ovariectomized (OVX) female mice. Consistent with the E2 treatment, we show that female hearts express higher levels of pAGO2 and thus, exhibit higher association of AGO2-GW182 in cytoplasmic RISC. This results in lower expression of mitochondrial miR-181c in female hearts compared to male or OVX groups. Further, female hearts had fewer consequences of mitochondrial miR-181c expression, such as lower Sp1 degradation and significantly decreased MICU1 transcriptional regulation. Taken together, this study highlights a potential therapeutic target for conditions such as obesity and diabetes, where miR-181c is upregulated. NEW AND NOTEWORTHY: In this study, we show that the phosphorylation of Argonaute 2 (AGO2) stabilizes the RNA-induced silencing complex in the cytoplasm, preventing miR-181c entry into the mitochondria. Furthermore, we demonstrate that treatment with estradiol can inhibit the translocation of miR-181c into the mitochondria by phosphorylating AGO2. This ultimately eliminates the downstream consequences of miR-181c overexpression by mitigating excessive reactive oxygen species production and calcium entry into the mitochondria.

Neuronal small extracellular vesicles carrying miR-181c-5p contribute to the pathogenesis of epilepsy by regulating the protein kinase C-δ/glutamate transporter-1 axis in astrocytes.

Information exchange between neurons and astrocytes mediated by extracellular vesicles (EVs) is known to play a key role in the pathogenesis of central nervous system diseases. A key driver of epilepsy is the dysregulation of intersynaptic excitatory neurotransmitters mediated by astrocytes. Thus, we investigated the potential association between neuronal EV microRNAs (miRNAs) and astrocyte glutamate uptake ability in epilepsy. Here, we showed that astrocytes were able to engulf epileptogenic neuronal EVs, inducing a significant increase in the glutamate concentration in the extracellular fluid of astrocytes, which was linked to a decrease in glutamate transporter-1 (GLT-1) protein expression. Using sequencing and gene ontology (GO) functional analysis, miR-181c-5p was found to be the most significantly upregulated miRNA in epileptogenic neuronal EVs and was linked to glutamate metabolism. Moreover, we found that neuronal EV-derived miR-181c-5p interacted with protein kinase C-delta (PKCδ), downregulated PKCδ and GLT-1 protein expression and increased glutamate concentrations in astrocytes both in vitro and in vivo. Our findings demonstrated that epileptogenic neuronal EVs carrying miR-181c-5p decrease the glutamate uptake ability of astrocytes, thus promoting susceptibility to epilepsy.

Long noncoding RNA ANRIL alleviates hypoxia-induced pulmonary microvascular endothelial cell damage.

BACKGROUND: High-altitude pulmonary oedema (HAPE) is a form of noncardiogenic pulmonary oedema. Studies have found that long noncoding RNA (lncRNA) plays an important role in HAPE. ANRIL is significant in pulmonary illnesses, which implies that alterations in ANRIL expression levels may be involved in the beginning and development of HAPE. However, the specific mechanism is indistinct. The present study is meant to explore the effect and mechanism of ANRIL on hypoxic-induced injury of pulmonary microvascular endothelial cells (PMEVCs). METHODS: In the hypoxic model of PMVECs, overexpression of ANRIL or knockdown of miR-181c-5p was performed to assess cell proliferation, apoptosis, and migration. Furthermore, the levels of apoptosis-related proteins, inflammatory factors, and vascular active factors were also measured. RESULTS: The results showed that, after 24 h of hypoxia, PMVECs proliferation and migration were suppressed in comparison to the control group, along with an increase in apoptosis, a decrease in the expression of ANRIL, and an increase in the expression of miR-181c-5p (all p < .05). The damage caused by hypoxia in PMVECs can be lessened by overexpressing ANRIL, which also inhibits the production of TNF-α, iNOS, and VEGF as well as BAX and cleaved caspase-3 (all p < .05). Further experimental results showed that overexpression of ANRIL and knockdown of miR-181c-5p had the same protection against hypoxic injury in PMVECs (all p < .05). CONCLUSIONS: Our study suggests that ANRIL may prevent hypoxia injury to PMVECs in HAPE through the negative regulation of miR-181c-5p.

MDM2-p53 mediate a miR-181c-3p/LIF axis to regulate low dose-rate radiation-induced DNA damage in human B lymphocytes.

PURPOSE: Prolonged exposure to low dose-rate radiation (LDRR) is of growing concern to public health. Recent evidences indicates that LDRR causes deleterious health effects and is closely related to miRNAs. The aim of our study is to investigate the relationship between miRNAs and DNA damage caused by LDRR. MATERIALS AND METHODS: In this study, we irradiated C57BL/6J mice with 12.5µGy/h dose of γ ray emitted from uranium ore for 8 h a day for 120 days at a total dose of 12 mGy, and identified differentially expressed miRNAs from the mice long-term exposed to LDRR through isolating serum RNAs, constructing small RNA library, Illumina sequencing. To further investigate the role of differential miRNA under LDRR，we first built DNA damage model in Immortal B cells irradiated with 12.5µGy/h dose of γ ray for 28 days at a total dose of 9.4 mGy. Then, we chose the highly conserved miR-181c-3p among 12 miRNA and its mechanism in alleviating DNA damage induced by LDRR was studied by transfection, quantitative PCR, luciferase assay, and Western blot. RESULTS AND CONCLUSIONS: We have found that 12 differentially expressed miRNAs including miR-181c-3p in serum isolated from irradiated mice. Analysis of GO and KEGG indicated that target genes of theses 12 miRNA enriched in pathways related to membrane, protein binding and cancer. Long-term exposure to LDRR induced upregulation of gamma-H2A histone family member X (γ-H2AX) expression, a classical biomarker for DNA damage in B cells. miR-181c-3p inhibited Leukemia inhibitory factor (LIF) expression via combining its 3'UTR. LIF, MDM2, p53, and p-p53-s6 were upregulated after exposure to LDRR. In irradiated B cells, Transfection of miR-181c-3p reduced γ-H2AX expression and suppressed LIF and MDM2 protein levels, whereas p-p53-s6 expression was increased. As expected, the effect of LIF inhibition on irradiated B cells was similar to miR-181c-3p overexpression. Our results suggest that LDRR alters miRNA expression and induces DNA damage. Furthermore, miR-181c-3p can alleviate LDRR-induced DNA damage via the LIF/MDM2/p-p53-s6 pathway in human B lymphocytes. This could provide the basis for prevention and treatment of LDRR injury.

Saponin from Platycodi radix inactivates PI3K/AKT signaling pathway to hinder colorectal cancer cell proliferation, invasion, and migration through miR-181c/d-5p/RBM47.

Colorectal cancer (CRC) is the third frequent cancer and second leading reason of cancer-related mortality all over the globe. Saponins from Platycodi radix (SPR) and microRNAs (miRNAs) have been reported to regulate CRC cell progression. Real-time quantitative polymerase chain reaction (RT-qPCR) detected miR-181c-5p, miR-181d-5p, and RBM47 expression level. Cell counting kit-8 (CCK-8), 5-ethynyl-20-deoxyuridine (EdU), colony formation, transwell, and wound healing assays validated that miR-181c-5p and miR-181d-5p promote CRC cell proliferation, migration and invasion and SPR exerts opposite effects. Cignal Finder Reporter Array and western blot proved that the activity of PI3K/AKT pathway was decreased by RBM47 overexpression. RNA pulldown, luciferase reporter, and RNA-binding protein immunoprecipitation (RIP) assays proved the interaction between miR-181c/d-5p and RBM47, and RBM47 and PTEN. Rescue experiments were carried out to validate that RBM47 reverses the influence of miR-181c/d-5p on the progression of CRC cells. The stability of PTEN was probed by real-time quantitative polymerase chain reaction in CRC cells treated with Actinomycin D (Act D). To be concluded, SPR inactivates PI3K/AKT signaling pathway to suppress CRC cell proliferation, invasion, and migration via miR-181c/d-5p/RBM47. Elucidating the mechanisms of SPR underlying CRC may offer novel insight into CRC treatment.

Sertoli cell survival and barrier function are regulated by miR-181c/d-Pafah1b1 axis during mammalian spermatogenesis.

Sertoli cells contribute to the formation of the blood-testis barrier (BTB), which is necessary for normal spermatogenesis. Recently, microRNAs (miRNAs) have emerged as posttranscriptional regulatory elements in BTB function during spermatogenesis. Our previous study has shown that miR-181c or miR-181d (miR-181c/d) is highly expressed in testes from boars at 60 days old compared with at 180 days old. Herein, we found that overexpression of miR-181c/d via miR-181c/d mimics in murine Sertoli cells (SCs) or through injecting miR-181c/d-overexpressing lentivirus in murine testes perturbs BTB function by altering BTB-associated protein distribution at the Sertoli cell-cell interface and F-actin organization, but this in vivo perturbation disappears approximately 6 weeks after the final treatment. We also found that miR-181c/d represses Sertoli cell proliferation and promotes its apoptosis. Moreover, miR-181c/d regulates Sertoli cell survival and barrier function by targeting platelet-activating factor acetylhydrolase 1b regulatory subunit 1 (Pafah1b1) gene. Furthermore, miR-181c/d suppresses PAFAH1B1 expression, reduces the complex of PAFAH1B1 with IQ motif-containing GTPase activating protein 1, and inhibits CDC42/PAK1/LIMK1/Cofilin pathway which is required for F-actin stabilization. In total, our results reveal the regulatory axis of miR-181c/d-Pafah1b1 in cell survival and barrier function of Sertoli cells and provide additional insights into miRNA functions in mammalian spermatogenesis.

Extracellular vesicle-packaged miR-181c-5p from epithelial ovarian cancer cells promotes M2 polarization of tumor-associated macrophages via the KAT2B/HOXA10 axis.

OBJECTIVES: The molecular mechanistic actions of tumor-derived extracellular vesicles (EVs) in modulating macrophage polarization in the tumor microenvironment of epithelial ovarian cancer (EOC) is largely unknown. The study was performed to clarify the effect and downstream mechanism of microRNA-181c-5p (miR-181c-5p)-containing EVs from EOC cells in the M2 polarization of tumor-associated macrophages (TAMs). METHODS: EVs were isolated from normoxic and hypoxic human EOC cells SKOV3. Human mononuclear cell THP-1 was induced by phorbol-12-myristate-13-acetate to differentiate into TAMs. The targeting relationship between miR-181c-5p and KAT2B was verified by dual luciferase reporter gene assay. The interaction between KAT2B and HOXA10 was detected by immunofluorescence, Co-IP and ChIP assays. EdU staining, the scratch test and Transwell assay were used to assess the resultant cell proliferation, migration and invasion. The mouse xenograft model and the pulmonary metastasis model were developed through intraperitoneal injection of SKOV3 cells and tail vein injection of THP-1 cells, respectively. RESULTS: Hypoxic SKOV3 cell-derived EVs could be internalized by TAMs. SKOV3 cell-derived EVs induced by hypoxia (H-EVs) promoted M2 polarization of TAMs and facilitated the proliferation, migration and invasion of SKOV3 cells. miR-181c-5p was highly expressed in H-EVs and promoted the M2 polarization of TAMs. Further, miR-181c-5p targeted KAT2B, upregulated HOXA10 and activated the JAK1/STAT3 pathway, thereby promoting the M2 polarization of TAMs. In both mouse models, H-EV-derived miR-181c-5p promoted growth and metastasis of EOC cells. CONCLUSION: The miR-181c-5p-containing EVs from hypoxic EOC cells may upregulate HOXA10 by targeting KAT2B and activate the JAK1/STAT3 pathway to promote the M2 polarization of TAMs, ultimately promoting growth and metastasis of EOC cells in vitro and in vivo.

Trehalose Attenuates Learning and Memory Impairments in Aged Rats via Overexpression of miR-181c.

MicroRNAs have been recognized as important regulators of the aging process. Trehalose, a natural disaccharide, displays protective effects against neuronal impairment through several mechanisms. However, little is known about the interactive effects of aging and trehalose on behavioral function and underlying miRNA expression patterns in the hippocampus of young and old rats. Male Wistar rats were divided into four groups. Two groups of aged (24 months) and young (4 months) rats were administered 2% trehalose solution for 30 days. Two other groups of aged and young rats received regular tap water. At the end of treatment, rats were assessed for cognitive behavior using the Morris water maze test. The expression level of miR-181c and mir-34c was also measured by qRT-PCR. We found that trehalose treatment reduced learning and memory impairment in old rats compared to control old animals (p < 0.05). In contrast, cognitive performance was not significantly improved in trehalose-treated young rats in comparison with young controls (p > 0.05). We also showed that the expression level of miR-181c was significantly increased in trehalose-treated rats (p < 0.01). However, analysis of miR-34c expression level indicated no significant difference between trehalose-treated old rats and non-treated old animals (p > 0.05). Our results indicated that trehalose treatment improved learning and memory function in aged rats by targeting miR-181c. Therefore, trehalose administration may provide a therapeutic strategy to ameliorate age-associated cognitive impairment.

MicroRNA-181c-5p modulates phagocytosis efficiency in bone marrow-derived macrophages.

OBJECTIVE AND DESIGN: Phagocytosis and clearance of apoptotic cells are essential for inflammation resolution, efficient wound healing, and tissue homeostasis. MicroRNAs are critical modulators of macrophage polarization and function. The current study aimed to investigate the role of miR-181c-5p in macrophage phagocytosis. MATERIALS AND METHODS: miR-181c-5p was identified as a potential candidate in microRNA screening of RAW264.7 macrophages fed with apoptotic cells. To investigate the role of miR-181c-5p in phagocytosis, the expression of miR-181c-5p was assessed in phagocyting bone marrow-derived macrophages. Phagocytosis efficiency was measured by fluorescence microscopy. Gain- and loss-of-function studies were performed using miR-181c-5p-specific mimic and inhibitor. The expression of the phagocytosis-associated genes and proteins of interest was evaluated by RT2 profiler PCR array and western blotting, respectively. RESULTS: miR-181c-5p expression was significantly upregulated in the phagocyting macrophages. Furthermore, mimic-induced overexpression of miR-181c-5p resulted in the increased phagocytic ability of macrophages. Moreover, overexpression of miR-181c-5p resulted in upregulation of WAVE-2 in phagocyting macrophages, suggesting that miR-181c-5p may regulate cytoskeletal arrangement during macrophage phagocytosis. CONCLUSION: Altogether, our data provide a novel function of miR-181c-5p in macrophage biology and suggest that targeting macrophage miR-181c-5p in injured tissues might improve clearance of dead cells and lead to efficient inflammation resolution.

lncRNA SNHG1 induced by SP1 regulates bone remodeling and angiogenesis via sponging miR-181c-5p and modulating SFRP1/Wnt signaling pathway.

BACKGROUND: We aimed to investigate the functions and underlying mechanism of lncRNA SNHG1 in bone differentiation and angiogenesis in the development of osteoporosis. METHODS: The differential gene or proteins expressions were measured by qPCR or western blot assays, respectively. The targeted relationships among molecular were confirmed through luciferase reporter, RIP and ChIP assays, respectively. Alkaline phosphatase (ALP), alizarin red S (ARS) and TRAP staining were performed to measure the osteoblast/osteoclast differentiation of BMSCs. The viability, migration and angiogenesis in BM-EPCs were validated by CCK-8, clone formation, transwell and tube formation assays, respectively. Western blot and immunofluorescence detected the cytosolic/nuclear localization of ß-catenin. Ovariectomized (OVX) mice were established to confirm the findings in vitro. RESULTS: SNHG1 was enhanced and miR-181c-5p was decreased in serum and femoral tissue from OVX mice. SNHG1 directly inhibited miR-181c-5p to activate Wnt3a/ß-catenin signaling by upregulating SFRP1. In addition, knockdown of SNHG1 promoted the osteogenic differentiation of BMSCs by increasing miR-181c-5p. In contrast, SNHG1 overexpression advanced the osteoclast differentiation of BMSCs and inhibited the angiogenesis of BM-EPCs, whereas these effects were all reversed by miR-181c-5p overexpression. In vivo experiments indicated that SNHG1 silencing alleviated osteoporosis through stimulating osteoblastogenesis and inhibiting osteoclastogenesis by modulating miR-181c-5p. Importantly, SNHG1 could be induced by SP1 in BMSCs. CONCLUSIONS: Collectively, SP1-induced SNHG1 modulated SFRP1/Wnt/ß-catenin signaling pathway via sponging miR-181c-5p, thereby inhibiting osteoblast differentiation and angiogenesis while promoting osteoclast formation. Further, SNHG1 silence might provide a potential treatment for osteoporosis.

CircPTK2-miR-181c-5p-HMGB1: a new regulatory pathway for microglia activation and hippocampal neuronal apoptosis induced by sepsis.

BACKGROUND: Circular RNA hsa_circ_0008305 (circPTK2), miR-181c-5p and High mobility group box-1 (HMGB1) had a targeted regulatory relationship through bioinformatics analysis. This study explained the effects of these genes in microglia and sepsis mice. METHODS: Lipopolysaccharide (LPS) or Cecal Ligation and Puncture (CLP) was used to induce inflammation cell model or sepsis mouse model, as needed. Gene levels were measured by enzyme linked immunosorbent assay (ELISA), quantitative real-time PCR or Western blot, as required. Apoptosis was detected by TUNEL assay, and RNase R was used to test the stability of circPTK2. Targeting relationships between genes were analyzed using bioinformatics analysis and dual luciferase assay. Morris water maze test and mitochondrial membrane potential (MMP) detection were conducted to analyze the effects of genes on cognitive dysfunction of mice. RESULTS: Lipopolysaccharide induction triggered the release of pro-inflammatory cytokines, the upregulation of HMGB1 and circPTK2, and the downregulation of miR-181c-5p in microglia. Overexpression of HMGB1 enhanced the effect of LPS, while silencing HMGB1 partially counteracted the effect of LPS. Moreover, miR-181c-5p was a target of circPTK2 and bound to HMGB1. MiR-181c-5p mimic partially reversed the functions of LPS and HMGB1 overexpression, reduced the levels of TNF-α, IL-1ß, and HMGB1, and inhibited apoptosis. CircPTK2 knockdown had the same effect as miR-181c-5p up-regulation. In vivo, sicircPTK2 improved cognitive function, restored MMP level, inhibited apoptosis, reduced the levels of inflammatory factors and apoptotic factors, and increased the survival rate of CLP-induced mice. CONCLUSION: Our research reveals that circPTK2 regulates microglia activation and hippocampal neuronal apoptosis induced by sepsis via miR-181c-5p-HMGB1 signaling.

Nuclear-mitochondrial communication involving miR-181c plays an important role in cardiac dysfunction during obesity.

AIMS: In cardiomyocytes, there is microRNA (miR) in the mitochondria that originates from the nuclear genome and matures in the cytoplasm before translocating into the mitochondria. Overexpression of one such miR, miR-181c, can lead to heart failure by stimulating reactive oxygen species (ROS) production and increasing mitochondrial calcium level ([Ca2+]m). Mitochondrial calcium uptake 1 protein (MICU1), a regulatory protein in the mitochondrial calcium uniporter complex, plays an important role in regulating [Ca2+]m. Obesity results in miR-181c overexpression and a decrease in MICU1. We hypothesize that lowering miR-181c would protect against obesity-induced cardiac dysfunction. METHODS AND RESULTS: We used an in vivo mouse model of high-fat diet (HFD) for 18 weeks and induced high lipid load in H9c2 cells with oleate-conjugated bovine serum albumin in vitro. We tested the cardioprotective role of lowering miR-181c by using miR-181c/d-/- mice (in vivo) and AntagomiR against miR-181c (in vitro). HFD significantly upregulated heart levels of miR-181c and led to cardiac hypertrophy in wild-type mice, but not in miR-181c/d-/- mice. HFD also increased ROS production and pyruvate dehydrogenase activity (a surrogate for [Ca2+]m), but the increases were alleviated in miR-181c/d-/- mice. Moreover, miR-181c/d-/- mice fed a HFD had higher levels of MICU1 than did wild-type mice fed a HFD, attenuating the rise in [Ca2+]m. Overexpression of miR-181c in neonatal ventricular cardiomyocytes (NMVM) caused increased ROS production, which oxidized transcription factor Sp1 and led to a loss of Sp1, thereby slowing MICU1 transcription. Hence, miR-181c increases [Ca2+]m through Sp1 oxidation and downregulation of MICU1, suggesting that the cardioprotective effect of miR-181c/d-/- results from inhibition of Sp1 oxidation. CONCLUSION: This study has identified a unique nuclear-mitochondrial communication mechanism in the heart orchestrated by miR-181c. Obesity-induced overexpression of miR-181c increases [Ca2+]m via downregulation of MICU1 and leads to cardiac injury. A strategy to inhibit miR-181c in cardiomyocytes can preserve cardiac function during obesity by improving mitochondrial function. Altering miR-181c expression may provide a pharmacologic approach to improve cardiomyopathy in individuals with obesity/type 2 diabetes.

miR125a attenuates BMSCs apoptosis via the MAPK-ERK pathways in the setting of craniofacial defect reconstruction.

Bone mesenchymal stem cell (BMSC)-based regenerative therapy is critical for the craniofacial defect reconstruction. However, oxidative stress microenvironment after transplantation limits the therapeutic efficiency of BMSC. The miR-181c has been found to be associated with cell survival and proliferation. Herein, we investigated whether prior miR-181c treatment promoted BMSC proliferation and survival under oxidative stress injury. The results in our study demonstrated that hydrogen peroxide (H2 O2 ) treatment reduced BMSC viability and this effect could be reversed via additional supplementation of miR181-c. Mechanistically, oxidative stress increased cell apoptosis, augmented caspase-3 activity, promoted reactive oxygen species synthesis, impaired mitochondrial potential, and induced mitochondrial dynamics imbalance. However, miR-181c pretreatment reversed these effects of oxidative stress on BMSC. Moreover, miR-181c treatment improved BMSC proliferation, migration and paracrine, which are very important for craniofacial reconstruction. In addition, we identified that AMP-activated protein kinase (AMPK)-mitofusins-1 (Mfn1) axis was the direct targets of miR-181c in BMSC. Mfn1 silencing impaired the protective effects miR-181c on BMSC viability and proliferation under oxidative stress environment. Collectively, our results indicate that miR-181c participates in oxidative stress-mediated BMSC damage by modulating the AMPK-Mfn1 signaling pathway, suggesting miR-181c-AMPK-Mfn1 axis may serves as novel therapeutic targets to facilitate craniofacial defect reconstruction.

The lncRNA Malat1 functions as a ceRNA to contribute to berberine-mediated inhibition of HMGB1 by sponging miR-181c-5p in poststroke inflammation.

Long non-coding RNAs (lncRNAs) have been identified as essential mediators in neurological dysfunction. Our previous study shows that berberine (BBR) hampers the nuclear-to-cytosolic translocation of high-mobility group box 1 (HMGB1) in the process of poststroke inflammation. In this study, we explored the role of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1) in the process of BBR-induced inhibition of HMGB1 in ischemic brain. Before the 60-min MCAO surgery, the mice were pretreated with BBR (50 mg· kg-1 per day, ig) for 14 days or ICV injected with specific lentiviral vector or shRNA. We showed that MCAO caused marked increase in the expression Malat1 and HMGB1 in the ipsilateral cortex, which was significantly attenuated by pretreatment with BBR. Knockdown of Malat1 attenuated the inflammatory injury after brain ischemia, whereas overexpression of Malat1 exacerbated ischemic brain inflammation. Overexpression of Malat1 also reversed BBR-induced reduction of HMGB1 and proinflammatory cytokines. The above results suggested a potential correlation between Malat1 and stroke inflammation. Based on informatics analysis we predicted that HMGB1 was a direct downstream target of miR-181c-5p, whereas Malat1 acted as a competitive endogenous RNA (ceRNA) for miR-181c-5p targeted the 3'-UTR of HMGB1 to promote inflammation after ischemic stroke. Knockdown of Malat1 significantly decreased HMGB1 level, which could be abrogated by transfection with miR-181c-5p inhibitors. Taken together, our results demonstrate for the first time that Malat1/miR-181c-5p/HMGB1 axis may be a key pathway of BBR-induced antiinflammation effects in stroke, and they may provide a novel avenue for targeted therapy.

miR-181c-5p mediates simulated microgravity-induced impaired osteoblast proliferation by promoting cell cycle arrested in the G2 phase.

Impaired osteoblast proliferation plays fundamental roles in microgravity-induced bone loss, and cell cycle imbalance may result in abnormal osteoblast proliferation. However, whether microgravity exerts an influence on the cell cycle in osteoblasts or what mechanisms may underlie such an effect remains to be fully elucidated. Herein, we confirmed that simulated microgravity inhibits osteoblast proliferation. Then, we investigated the effect of mechanical unloading on the osteoblast cell cycle and found that simulated microgravity arrested the osteoblast cell cycle in the G2 phase. In addition, our data showed that cell cycle arrest in osteoblasts from simulated microgravity was mainly because of decreased cyclin B1 expression. Furthermore, miR-181c-5p directly inhibited cyclin B1 protein translation by binding to a target site in the 3'UTR. Lastly, we demonstrated that inhibition of miR-181c-5p partially counteracted cell cycle arrest and decreased the osteoblast proliferation induced by simulated microgravity. In conclusion, our study demonstrates that simulated microgravity inhibits cell proliferation and induces cell cycle arrest in the G2 phase in primary mouse osteoblasts partially through the miR-181c-5p/cyclin B1 pathway. This work may provide a novel mechanism of microgravity-induced detrimental effects on osteoblasts and offer a new avenue to further investigate bone loss induced by mechanical unloading.

LncRNA SNHG6 functions as a ceRNA to regulate neuronal cell apoptosis by modulating miR-181c-5p/BIM signalling in ischaemic stroke.

Long non-coding RNAs (lncRNAs) play important roles in the pathogenesis of brain and neurodegenerative disorders. As far as we know, the functions and potential mechanisms of small nucleolar RNA host gene 6 (SNHG6) in ischaemic stroke have not been explored. This study aimed to examine the functional role of SNHG6 in the ischaemic stroke. Middle cerebral artery occlusion (MCAO) in mice and the oxygen glucose deprivation (OGD)-induced injury in neuronal cells were applied to mimic ischaemic stroke. TTC staining, quantitative real-time PCR, cell apoptosis assay, caspase-3 activity assay, Western blot, RNA immunoprecipitation and luciferase reporter assay were performed to evaluate the function and possible mechanisms of SNHG6 in the pathogenesis of ischaemic stroke. The results show that SNHG6 expression was significantly increased both OGD-induced neuronal cells and MCAO model mice. In vitro results showed that inhibition of SNHG6 increased cell viability, inhibited cell apoptosis and caspase-3 activity in OGD-induced neuronal cells. Consistently, knockdown of SNHG6 reduced brain infarct size and improved neurological scores in the MCAO mice. Mechanistic study further revealed that SNHG6 functioned as a competing endogenous RNA (ceRNA) for miR-181c-5p, which in turn repressed its downstream target of Bcl-2 interacting mediator of cell death (BIM) and inhibiting cell apoptosis. This study revealed a novel function of SNHG6 in the modulating neuronal apoptosis in the ischaemic stroke model, and the role of SNHG6 in the regulating of neuronal apoptosis was at least partly via targeting miR-181c-5p/BIM signalling pathway.

Pretreatment of bone mesenchymal stem cells with miR181-c facilitates craniofacial defect reconstruction via activating AMPK-Mfn1 signaling pathways.

Context: Bone mesenchymal stem cells (BMSC)-based regenerative therapy is critical for the craniofacial defect reconstruction. However, oxidative stress micro-environment after transplantation limits the therapeutic efficiency of BMSC. The miR-181c has been found to be associated with cell survival and proliferation. Objective: Herein, we investigated whether prior miR-181c treatment promoted BMSC proliferation and survival under oxidative stress injury. Materials and methods: Cells were treated with hydrogen peroxide (H2O2) and then cell viability was determined via MTT assay, TUNEL staining and ELISA. Western blotting and immunofluorescence assay were used to detect those alterations of mitochondrial function. Results: H2O2 treatment reduced BMSC viability and this effect could be reversed via additional supplementation of miR181-c. Mechanistically, oxidative stress increased cell apoptosis, augmented caspase-3 activity, promoted reactive oxygen species (ROS) synthesis, impaired mitochondrial potential, and induced mitochondrial dynamics imbalance. However, miR-181c pretreatment reversed these effects of oxidative stress on BMSC. Moreover, miR-181c treatment improved BMSC proliferation, migration and paracrine, which are very important for craniofacial reconstruction. In addition, we identified that AMPK-Mfn1 axis was the direct targets of miR-181c in BMSC. Mfn1 silencing impaired the protective effects miR-181c on BMSC viability and proliferation under oxidative stress environment. Conclusions: Collectively, our results indicate that miR-181c participates in oxidative stress-mediated BMSC damage by modulating the AMPK-Mfn1 signaling pathway, suggesting miR-181c-AMPK-Mfn1 axis may serves as novel therapeutic targets to facilitate craniofacial defect reconstruction.

MiR-181c affects estrogen-dependent endometrial carcinoma cell growth by targeting PTEN.

MicroRNAs (miRNAs), which is a type of non-coding and single-stranded small molecule RNA, bind either completely or incompletely to 3'-UTR of the target gene mRNA to inhibit mRNA translation or degradation. In our study, we aimed to explore the roles and mechanisms of miR-181c in the apoptosis of RL95-2 human endometrial carcinoma cells. Cell activity and apoptosis were detected by cell counting Kit-8 (CCK-8) assay and flow cytometry (FCM), respectively. Related mRNAs and proteins expression was determined by quantitative real-time reverse transcription PCR (qRT-PCR) and western blot assays, respectively. The binding capacity of PTEN-3'-UTR and miR-181c was assessed by luciferase reporter assay. The obtained results suggested that E2 evidently increased the cell activity of RL95-2 cells. In addition, miR-181c inhibitor suppressed the cell viability and enhanced the apoptosis capacity of E2-induced RL95-2 cells and distinctly reduced the miR-181c expression. We also found that miR-181c could bind to PTEN-3'-UTR and miR-181c inhibitor up-regulated the expression level of PTEN in E2-induced RL95-2 cells. Besides, overexpression of PTEN markedly promoted the apoptosis of E2-induced RL95-2 cells through regulating the Bax and Bcl-2 expression, and modulated the expression of AKT pathway, p53 and Cyclin D. In conclusion, our findings revealed that miR-181c affected the estrogen-dependent endometrial carcinoma cell growth by targeting PTEN. The potential effects of miR-181c on the apoptosis of E2-induced RL95-2 cells suggest that miR-181c could be an effective target for endometrial carcinoma therapies.

MicroRNA181c inhibits prostate cancer cell growth and invasion by targeting multiple ERK signaling pathway components.

BACKGROUND: The ERK signaling pathway is frequently deregulated in tumorigenesis, mostly by classical mechanisms such as gene mutation of its components (eg, RAS and RAF). However, whether and how multiple key components of ERK pathway are regulated by microRNAs are not clear. METHODS: We firstly predicted post-transcriptional regulation of multiple key components of the ERK signaling pathway by miR181c through bioinformatics analysis, and then confirmed the post-transcriptional regulation by dual luciferase reporter gene assays and Western blot analysis. The biological effects of miR181c on prostate cancer cell proliferation, apoptosis, migration, and invasion were measured by CCK-8 assay, flow cytometry, wound scratch assay, transwell cell migration, and invasion assays. RESULTS: miR181c post-transcriptionally regulated multiple key members of the ERK signaling pathway, including extracellular signal-regulated kinase 2 (ERK2), ribosomal S6 kinase 2 (RSK2), serum response factor (SRF), and FBJ murine osteosarcoma viral oncogene homolog (c-Fos). Ectopic expression of miR181c mimics effectively suppressed prostate cancer cell proliferation, migration, and invasion, but promoted cell apoptosis. Furthermore, miR181c treatment combined with the multi-kinase inhibitor sorafenib significantly enhanced these anti-tumor effects. CONCLUSIONS: Downregulation of miR181c results in deregulated ERK signaling and promotes prostate cancer cell growth and metastasis.

MicroRNA-181c prevents apoptosis by targeting of FAS receptor in Ewing's sarcoma cells.

BACKGROUND: MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that play important roles in multiple biological processes. Here, we show that miRNAs play an important function in the down-regulation of FAS expression in Ewing's sarcoma (ES) cells. METHODS: To identify and characterize possible oncogenic factors in ES, we employed a microarray-based approach to profile the changes in the expression of miRNAs and their target mRNAs in five ES cell lines and human mesenchymal stem cells (hMSCs). RESULTS: MiRNA, miR-181c, was significantly up-regulated, whereas FAS receptor expression was significantly down-regulated in all tested ES cells compared with hMSCs. Introducing anti-miR-181c into ES cell lines resulted in an increased expression of FAS2. Additionally, anti-miR-181c prohibited cell growth and cell cycle progression in ES cells. Anti-miR-181c also promoted apoptosis in ES cells. Furthermore, the down-regulation of miR-181c in ES cells significantly suppressed tumor growth in vivo. CONCLUSIONS: These results suggest that unregulated expression of miR-181c could contribute to ES by targeting FAS. Reduction of miR181c increased expression of FAS. This proves that retardation of cell cycle progression removes apoptosis resistance, thereby repressing the growth of Ewing sarcoma. Since FAS signaling is involved in regulation of apoptosis and tumor proliferation, our findings might contribute to new therapeutic targets for ES.