Overexpression Of miR-34a Research Articles

Upregulation of MicroRNA-34a Sensitizes Ovarian Cancer Cells to Resveratrol by Targeting Bcl-2.

PurposeResveratrol (REV), a natural compound found in red wine, exhibits antitumor activity in various cancers, including ovarian cancer (OC). However, its potential anti-tumor mechanisms in OC are not well characterized. Here, we tried to elucidate the underlying mechanisms of REV in OC cells.Materials and MethodsThe anti-proliferative effects of REV against OC cells were measured using CCK-8 assay. Apoptosis was measured using an Annexin V-FITC/PI apoptosis detection kit. The anti-metastasis effects of REV were evaluated by invasion assay and wound healing assay. The miRNA profiles in REV-treated cells were determined by microarray assay.ResultsOur results showed that REV treatment suppresses the proliferation, induces the apoptosis, and inhibits the invasion and migration of OV-90 and SKOV-3 cells. miR-34a was selected for further study due to its tumor suppressive roles in various human cancers. We found miR-34a overexpression enhanced the inhibitory effects of REV on OC cells, whereas miR-34a inhibition had the opposite effect in OC cells. In addition, we verified that BCL2, an anti-apoptotic gene, was found directly targeted by miR-34a. We also found that REV reduced the expression of Bcl-2 in OC cells. Further investigations revealed that overexpression of Bcl-2 significantly abolished the anti-tumor effects of REV on OC cells.ConclusionOverall, these results demonstrated that REV exerts anti-cancer effects on OC cells through an miR-34a/Bcl-2 axis, highlighting the therapeutic potential of REV for treatment of OC.

Long Noncoding RNA OIP5-AS1 Overexpression Promotes Viability and Inhibits High Glucose-Induced Oxidative Stress of Cardiomyocytes by Targeting MicroRNA-34a/SIRT1 Axis in Diabetic Cardiomyopathy.

Diabetic cardiomyopathy (DCM) is an important complication of diabetes. This study was attempted to discover the effects of long noncoding RNA OIP5-AS1 (OIP5-AS1) on the viability and oxidative stress of cardiomyocyte in DCM. The expression of OIP5-AS1 and microRNA-34a (miR-34a) in DCM was detected by qRT-PCR. In vitro, DCM was simulated by high glucose (HG, 30 mM) treatment in H9c2 cells. The viability of HG (30 mM)-treated H9c2 cells was examined by MTT assay. The reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) levels were used to evaluate the oxidative stress of HG (30 mM)-treated H9c2 cells. Dual-luciferase reporter assay was used to confirm the interactions among OIP5-AS1, miR-34a and SIRT1. Western blot was applied to analyze the protein expression of SIRT1. The expression of OIP5-AS1 was down-regulated in DCM, but miR-34a was up-regulated. The functional experiment stated that OIP5-AS1 overexpression increased the viability and SOD level while decreased the ROS and MDA levels in HG (30 mM)-treated H9c2 cells. The mechanical experiment confirmed that OIP5-AS1 and SIRT1 were both targeted by miR-34a with the complementary binding sites at 3'UTR. MiR-34a overexpression inhibited the protein expression of SIRT1. In the feedback experiments, miR-34a overexpression or SIRT1 inhibition weakened the promoting effect on viability and mitigated the reduction effect on oxidative stress caused by OIP5-AS1 overexpression in HG (30 mM)-treated H9c2 cells. OIP5-AS1 overexpression enhanced viability and attenuated oxidative stress of cardiomyocyte via regulating miR-34a/SIRT1 axis in DCM, providing a new therapeutic target for DCM.

Long Noncoding RNA DANCR Activates Wnt/β-Catenin Signaling through MiR-216a Inhibition in Non-Small Cell Lung Cancer.

Long noncoding RNA differentiation antagonizing nonprotein coding RNA (lncRNA-DANCR) is associated with poor prognosis in multiple cancers, and promotes cancer stemness and invasion. However, the exact mechanisms by which DANCR promotes non-small cell lung cancer (NSCLC) remain elusive. In this study, we determined that DANCR knockdown (KD) impeded cell migration and reduced stem-like characteristics in two NSCLC cell lines, A549 and H1755. Wnt signaling was shown to promote NSCLC proliferation, stemness, and invasion; therefore, we hypothesized that DANCR may regulate these activities through induction of the Wnt/β-catenin pathway. DANCR KD reduced β-catenin signaling and protein expression, and decreased the expression of β-catenin gene targets c-Myc and Axin2. One of the well-defined functions of lncRNAs is their ability to bind and inhibit microRNAs. Through in silico analysis, we identified tumor suppressor miR-216a as a potential binding partner to DANCR, and confirmed this binding through coimmunoprecipitation and luciferase-reporter assays. Furthermore, we show that DANCR-induced β-catenin protein expression may be blocked with miR-216a overexpression. Our findings illustrate a role of DANCR in NSCLC migration and stemness, and suggest a novel DANCR/miR-216a signaling axis in the Wnt/β-catenin pathway.

CASC8 lncRNA Promotes the Proliferation of Retinoblastoma Cells Through Downregulating miR34a Methylation.

BackgroundCASC8 lncRNA has been proven to be oncogenic in a variety of cancers, but its role in other types of cancer remains unclear. This study was to investigate the role of CASC8 in retinoblastoma (Rb).MethodsRT-qPCR was performed to determine the expression of CASC8 and miR34a in paired Rb and nontumor tissue. Overexpression of CASC8 and miR34a in Rb cells was achieved to evaluate the interaction between them. Methylation-specific PCR was used to analyze the effect of CASC8 overexpression on MIR34A gene methylation. CCK8 assays were used to analyze cell proliferation.ResultsThe results showed that CASC8 expression was upregulated and miR34a expression downregulated in Rb tissue. Moreover, miR34a expression was negatively correlated with the of CASC8 expression in Rb tissue. Overexpression of CASC8 decreased expression of miR34a and increased methylation of MIR34A in Rb cells. In addition, overexpression of CASC8 reduced the inhibitory effects of miR34a on Rb-cell proliferation.ConclusionCASC8 may promote Rb cell proliferation by downregulating miR34a methylation.

Berberine exerts antidepressant-like effects via regulating miR-34a-synaptotagmin1/Bcl-2 axis

ObjectiveBerberine, a cationic alkaloid first isolated in 1917, has been approved by the China Drug Administration for decades. Accumulating evidence demonstrated its antidepressant-like activities in vivo. Our previous study has shown that chronic stress leads to the upregulation of miR-34a in the hippocampus of mice. This study aims to evaluate the underlying miR-34a mediated mechanism of berberine in chronic stress-induced depression in mice. MethodsIn the present study, mice were administered with berberine during chronic stress. Levels of miR-34a, dendritic density, mitochondrial morphology, and neurogenesis were assessed in the hippocampus. Subsequently, miR-34a agomir was used as a pharmacological intervention for the investigation of berberine. ResultsThe results showed that berberine reversed the decrease in sucrose preference and the increase in latency to feed without altering total food consumption. Furthermore, chronic stress-induced overexpression of miR-34a decreased synaptotagmin-1 and Bcl-2 levels, thereby impairing spinal morphology, mitochondria and neurogenesis. Berberine inhibited miR-34a expression, in turn restored synaptotagmin-1 and Bcl-2 levels, and thus improved spinal morphology, mitochondria and neurogenesis in the hippocampus. However, the improvements induced by berberine were totally blocked by the pretreatment of miR-34a agomir, which caused the elevation of miR-34a levels in the hippocampus. ConclusionThis finding demonstrated that miR-34a downregulation was involved in the antidepressant-like effects of berberine in mice exposed to chronic stress.

Cardiac fibroblast miR-27a may function as an endogenous anti-fibrotic by negatively regulating Early Growth Response Protein 3 (EGR3).

Pathological myocardial fibrosis and hypertrophy occur due to chronic cardiac stress. The microRNA‐27a (miR‐27a) regulates collagen production across diverse cell types and organs to inhibit fibrosis and could constitute an important therapeutic avenue. However, its impact on hypertrophy and cardiac remodelling is less well‐known. We employed a transverse aortic constriction (TAC) murine model of left ventricular pressure overload to investigate the in vivo effects of genetic miR‐27a knockout, antisense inhibition of miR‐27a‐5p and fibroblast‐specific miR‐27a knockdown or overexpression. In silico Venn analysis and reporter assays were used to identify miR‐27a‐5p's targeting of Early Growth Response Protein 3 (Egr3). We evaluated the effects of miR‐27a‐5p and Egr3 upon transforming growth factor‐beta (Tgf‐β) signalling and secretome of cardiac fibroblasts in vitro. miR‐27a‐5p attenuated TAC‐induced cardiac fibrosis and myofibroblast activation in vivo, without a discernible effect on cardiac myocytes. Molecularly, miR‐27a‐5p inhibited transforming growth factor‐beta (Tgf‐β) signalling and pro‐fibrotic protein secretion in cardiac fibroblasts in vitro through suppressing the pro‐fibrotic transcription factor Early Growth Response Protein 3 (Egr3). This body of work suggests that cardiac fibroblast miR‐27a may function as an endogenous anti‐fibrotic by negatively regulating Egr3 expression.

Abstract 16827: miR-34a Expression in Cardiac Neonatal Mesenchymal Stem Cells is Essential for Heart Regeneration

Advancing chronological age negatively impacts the functional activity of stem cell-based therapies. Despite the early promise stem cell mediated differentiation into selected cellular lineages when injected in the chosen organ, stem cell’s functional unit is the secretome that is directly modulated by chronological aging. However, the precise mechanism driving the secretome potency is unknown. Here we show a single miRNA, influenced by aging, drives the potency of the stem cell secretome at multiple cellular levels by increasing exosome production and directly increasing the levels of independent stem cell paracrine factors. We show differential miRNA microarray analysis of cardiac neonatal mesenchymal stem cells (nMSCs) and adult MSCs (aMSCs) identified miR34a among the top ten differentially expressed miRNAs present in aMSCs but not nMSCs. We demonstrated that miR34a singly inhibits the aMSC’s regenerative abilities in the acute myocardial infarct model by decreasing the secretome potency when compared to nMSCs in knockdown studies of miR-34a. Moreover, overexpression of miR-34a in nMSCs reversed the strong regenerative properties of nMSCs by decreasing their secretome potency. Mechanistically, miR-34a regulates the secretome potency firstly by decreasing Heat Shock Factor 1 (HSF1) levels that directly decreases exosome production and alters the exosome cargo to a less-regenerative phenotype, and secondly by directly inhibiting transcriptional levels of critical stem cell paracrine factors. These results implicate the miR34a-HIF1 pathway as a critical pathway controlling the secretome of MSCs impacted by advancing chronological age. Our findings further advance miRNA-based therapeutic approaches to cardiac repair and heart regeneration by indicating how to improve the quality of secretome of transplanted stem cells.

MicroRNA-200a represses myocardial infarction-related cell death and inflammation by targeting the Keap1/Nrf2 and β-catenin pathways

BackgroundAcute myocardial infarction (MI) is a main cause of emergency death in the world. MicroRNAs (miRs/miRNAs) are a series of small non-coding RNA molecules, which regulate cardiovascular disorders that involve MI. In this study, we explored the function of miR-200a in MI treatment. MethodsWe observed down-regulation of miR-200a levels and up-regulation of Keap1 and β-catenin levels in H2O2-treated newborn murine ventricular cardiomyocytes (NMVCs) and the infarcted heart tissues of MI mouse models, compared to the non-treated NMVCs and normal heart tissues of healthy mice. ResultsCCK-8 and colony formation assays indicated the reduction in NMVC vitality due to H2O2 treatment and the recovery of cell vitality due to miR-200a overexpression, respectively. Flow cytometry with Annexin and PI staining indicated the inhibition of H2O2-triggered cell apoptosis through ectopically expressed miR-200a. Western blotting and ELISA analyses that detected pro-inflammatory cell factors [interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α] confirmed that miR-200a prevented H2O2-induced NMVC inflammation. Moreover, miR-200a inhibited up-regulation of Keap1 and β-catenin expression in H2O2-treated NMVCs by directly binding with the 3′-UTR regions of both Keap1 and β-catenin. Furthermore, overexpression of Keap1 and β-cateninin in H2O2-treated NMVCs with recovered miR-200a elevated inflammation and apoptosis, respectively. ConclusionThe results showed that miR-200a expression was inhibited in murine cardiomyocytes due to H2O2 stress in MI cardiac tissues and overexpressed miR-200a could protect the cells from death by regulating the Keap1/Nrf2 and β-catenin signal transduction pathways.

Sevoflurane induction alleviates the progression of gastric cancer by upregulating the miR-34a/TGIF2 axis.

This study aims to elucidate how sevoflurane affects the malignant progression of gastric cancer (GC) and its pharmacological mechanism. Dose-dependent and time-dependent regulations of sevoflurane on proliferation inhibition rate in AGS and BGC-823 cells were examined, and thus the optimal dose and treatment time of sevoflurane on GC cells were selected. Subsequently, proliferative and migratory abilities in sevoflurane-induced AGS and BGC-823 cells (3.4% sevoflurane induction for 6 h) were detected by CCK-8 and transwell assay, respectively. After sevoflurane induction, relative levels of miR-34a and TGIF2 in GC cells were determined by qRT-PCR and Western blot. Regulatory effects of miR-34a on GC cell phenotypes were also assessed. Furthermore, the in vivo function of miR-34a in GC growth was explored by generating xenografted GC in nude mice. Sevoflurane induction time-dependently and dose-dependently enhanced proliferation inhibition rate in AGS and BGC-823 cells. The proliferative and migratory abilities in GC cells induced with 3.4% sevoflurane for 6 h were markedly attenuated. sevoflurane induction upregulated miR-34a, but downregulated TGIF2 in GC cells. TGIF2 was negatively regulated by miR-34a. Notably, overexpression of miR-34a inhibited proliferative and migratory abilities in sevoflurane-induced GC cells, and knockdown of miR-34a yielded the opposite results. In nude mice with xenografted GC tissues, sevoflurane treatment markedly reduced tumorigenic ability, which was improved by knockdown of miR-34a. Sevoflurane weakens proliferative and migratory abilities in GC by upregulating miR-34a and downregulating TGIF2.

LncRNA OIP5-AS1 upregulates snail expression by sponging miR-34a to promote ovarian carcinoma cell invasion and migration

BackgroundAlthough OIP5-AS1 has been characterized as an oncogenic lncRNA in many types of cancer, its role and underlying mechanism in ovarian carcinoma (OC) remains unknown. This study aimed to investigate the role of OIP5-AS1 in OC.MethodsOC tissues and non-tumor tissues (ovary tissues within 3 cm around tumors) were collected from 58 OC patients (age range 36 to 67 years old, mean age 51.4 ± 5.9 years old). The expression of OIP5-AS1 and snail in paired tissues were determined by RT-qPCR. The interaction between OIP5-AS1 and miR-34a was predicted by IntaRNA2.0 and confirmed by dual luciferase reporter assay. The effects of overexpression of OIP5-AS1 and miR-34a on the expression of snail were analyzed by RT-qPCR and Western blotting. Cell invasion and migration were analyzed by Transwell assay.ResultsWe observed that the expression of OIP5-AS1 and snail was upregulated and positively correlated with each other in OC. RNA–RNA interaction analysis showed that OIP5-AS1 might sponge miR-34a. In OC cells, overexpression of OIP5-AS1 resulted in the upregulated expression of snail, while overexpression of miR-34a downregulated the expression of snail. In addition, overexpression of miR-34a reduced the effects of overexpression of OIP5-AS1 on the expression of snail. In cell invasion and migration assay, overexpression of OIP5-AS1 and snail resulted in increased OC cell invasion and migration, while overexpression of miR-34a decreased OC cell invasion and migration. Moreover, overexpression of miR-34a attenuated the effects of OIP5-AS1 overexpression on OC cell invasion and migration.ConclusionsTherefore, OIP5-AS1 may upregulate snail expression in OC by sponging miR-34a to promote OC cell invasion and migration.

MicroRNA-34a promotes apoptosis of retinal vascular endothelial cells by targeting SIRT1 in rats with diabetic retinopathy

ABSTRACT MiR-34a is associated with diabetic retinopathy (DR). This article aims to demystify the role of miR-34a in DR. We established a DR model by streptozocin injection. Rat retinal vascular endothelial cells (RVECs) were treated with high glucose (HG) to induce DR. The pathological changes of retinal tissues and blood-retinal vascular barrier permeability of DR rats were assessed by HE staining and Evans-Blue leak test. The expression of gene and protein was evaluated by quantitative real-time PCR or western blot. MTT assay and flow cytometry were performed to detect proliferation and apoptosis. The relationship between miR-34a and SIRT1 was evaluated using luciferase reporter assay. MiR-34a was up-regulated and SIRT1 was down-regulated in retinal tissues of DR rats and HG-induced RVECs. MiR-34a silencing improved DR by regulating apoptosis and VEGF expression in DR rats. Furthermore, miR-34a interacted with SIRT1 and suppressed SIRT1 expression. MiR-34a overexpression inhibited proliferation and promoted apoptosis of RVECs, which was effectively abolished by SIRT1 up-regulation. In summary, our data demonstrate that miR-34a promotes apoptosis of RVECs by targeting SIRT1 in DR rats. Our findings suggest that miR-34a/SIRT1 axis could be a valuable target for DR therapies.

MiR-34a regulates Schwann cell proliferation and migration by targeting CNTN2.

The proliferation and migration of Schwann cells contribute to axonal outgrowth and functional recovery after peripheral nerve injury. Previously, several microRNAs were abnormally expressed after peripheral nerve injury and they played important roles in peripheral nerve regeneration. However, the role and underlying mechanism of miR-34a in peripheral nerve injury remain largely unknown. The levels of miR-34a and contactin-2 (CNTN2) were detected by quantitative real-time PCR. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and transwell assays were used to examine cell proliferation and migration, respectively. The protein level of CNTN2 was measured by western blot. The binding sites of miR-34a and CNTN2 were predicted by the online software and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Following sciatic nerve injury, the expression of miR-34a was downregulated in the crushed nerve segment, reaching a minimum at the seventh day. Knockdown of miR-34a enhanced the axon outgrowth of dorsal root ganglion neurons. Moreover, miR-34a overexpression evidently inhibited the proliferation of Schwann cells, whereas its knockdown showed the opposite effects. In addition, CNTN2 was a direct target of miR-34a and its expression was negatively regulated by miR-34a in the crushed nerve segment. Besides, CNTN2 overexpression or knockdown could reverse the effects of miR-34a upregulation or downregulation on proliferation and migration of Schwann cells, respectively. Collectively, miR-34a inhibited the proliferation and migration of Schwann cells via targeting CNTN2, which might provide a new approach to peripheral nerve regeneration.

MicroRNA-26a inhibits wound healing through decreased keratinocytes migration by regulating ITGA5 through PI3K/AKT signaling pathway.

Background: Keratinocyte migration is essential for skin wound healing and recent studies demonstrated that microRNAs (miRNAs) are involved in the differentiation, migration and apoptosis in keratinocytes. However, the function of miR-26a in wound healing remains to be largely explored.Methods: Northern blot and quantitative reverse transcriptase PCR (qRT-PCR) were used to detect the miR-26a expression and Western blot was used to detect integrin α-5 (ITGA5), phosphatidylinositol-3-kinase (PI3K), p-PI3K, protein kinase B (AKT) and p-AKT protein expression in immortalized human keratinocyte cell line HaCaT and normal human epidermal keratinocytes (NHEK) after 2 ng/ml transforming growth factor-β1 (TGF-β1) treatment for 0, 6, 12 and 24 h. Transwell assay and Wound healing assay were introduced to measure the cell migration of HaCaT cells. TargetScan online database, luciferase reporter assay and RNA immunoprecipitation (RIP) were employed to confirm the relationship between miR-26a and ITGA5.Results: The RNA expression of miR-26a was down-regulated and ITGA5 protein expression was up-regulated by TGF-β1 treatment in HaCaT and NHEK cells in a time-dependent manner. MiR-26a overexpression inhibited the migration of HaCaT cells induced by TGF-β1 while miR-26a inhibitor enhanced the migration. ITGA5 was a downstream target mRNA and regulated by miR-26a. ITGA5 overexpression reversed the inhibitory effect of miR-26a on migration in HaCaT, while ITGA5 knockdown attenuated the stimulative effect of miR-26a inhibitor in HaCaT via PI3K/AKT signaling pathway.Conclusion: MiR-26a overexpression inhibited TGF-β1 induced HaCaT cells migration via down-regulating ITGA5 through activating the PI3K/AKT signaling pathway.

MiR-34a-5p Attenuates EMT through targeting SMAD4 in silica-induced pulmonary fibrosis.

Silicosis is an incurable occupational disease, and its pathological feature is diffuse pulmonary fibrosis. Pulmonary epithelial‐mesenchymal transition (EMT) is one of the important events in the pathogenesis of silicosis. Previous studies found that abnormal expression of various microRNAs (miRNAs) involved in the development of lung fibrosis. However, their roles in silicosis have not been elucidated. To research the biological effects of miR‐34a in EMT process in silica‐induced lung fibrosis, we established the silicosis model in mouse and miR‐34a intervention in a cell model of TGF‐β1 stimulated lung epithelial cells (A549). The results showed that miR‐34a expression was down‐regulated in the fibrotic lung tissue after silica treatment, and it was similarly expressed in A549 cells stimulated by TGF‐β1. Meanwhile, silica‐induced EMT process can increase expression of two mesenchymal markers, α‐SMA and vimentin. Furthermore, overexpression miR‐34a markedly inhibited EMT stimulated by TGF‐β1. Mechanistically, SMAD4 was identified as the target of miR‐34a. SMAD4 levels decreased in mRNA and protein levels in A549 cells upon miR‐34a overexpression. In addition, the knockdown of SMAD4 blocked the EMT process. Taken together, miR‐34a regulated EMT, which might be partially realized by targeting SMAD4. Our data might provide new insight into treatment targets for silica‐induced pulmonary fibrosis.

MiR-34a is upregulated in AIP-mutated somatotropinomas and promotes octreotide resistance.

Pituitary adenomas (PAs) are intracranial tumors associated with significant morbidity due to hormonal dysregulation, mass effects and have a heavy treatment burden. Growth hormone (GH)-secreting PAs (somatotropinomas) cause acromegaly-gigantism. Genetic forms of somatotropinomas due to germline AIP mutations (AIPmut+) have an early onset and are aggressive and resistant to treatment with somatostatin analogs (SSAs), including octreotide. The molecular underpinnings of these clinical features remain unclear. We investigated the role of miRNA dysregulation in AIPmut+ vs AIPmut- PA samples by array analysis. miR-34a and miR-145 were highly expressed in AIPmut+ vs AIPmut- somatotropinomas. Ectopic expression of AIPmut (p.R271W) in Aip-/- mouse embryonic fibroblasts (MEFs) upregulated miR-34a and miR-145, establishing a causal link between AIPmut and miRNA expression. In PA cells (GH3), miR-34a overexpression promoted proliferation, clonogenicity, migration and suppressed apoptosis, whereas miR-145 moderately affected proliferation and apoptosis. Moreover, high miR-34a expression increased intracellular cAMP, a critical mitogenic factor in PAs. Crucially, high miR-34a expression significantly blunted octreotide-mediated GH inhibition and antiproliferative effects. miR-34a directly targets Gnai2 encoding Gαi2, a G protein subunit inhibiting cAMP production. Accordingly, Gαi2 levels were significantly lower in AIPmut+ vs AIPmut- PA. Taken together, somatotropinomas with AIP mutations overexpress miR-34a, which in turn downregulates Gαi2 expression, increases cAMP concentration and ultimately promotes cell growth. Upregulation of miR-34a also impairs the hormonal and antiproliferative response of PA cells to octreotide. Thus, miR-34a is a novel downstream target of mutant AIP that promotes a cellular phenotype mirroring the aggressive clinical features of AIPmut+ acromegaly.

Inhibition of miRNA-34a Promotes M2 Macrophage Polarization and Improves LPS-Induced Lung Injury by Targeting Klf4.

Acute respiratory distress syndrome (ARDS) is an outcome of an accelerated immune response that starts initially as a defensive measure, however, due to non-canonical signaling, it later proves to be fatal not only to the affected tissue but to the whole organ system. microRNAs are known for playing a decisive role in regulating the expression of genes involved in diverse functions such as lung development, repair, and inflammation. In-silico analyses of clinical data and microRNA databases predicted a probable interaction between miRNA-34a (miR-34a), mitogen-activated protein kinase 1 (ERK), and kruppel like factor 4 (Klf4). Parallel to in silico results, here, we show that intra-tracheal instillation of lipopolysaccharides (LPS) to mice enhanced miR-34a expression in lung macrophages. Inhibition of miR-34a significantly improved lung histology, whereas over-expression of miR-34a worsened the lung injury phenotype. miR-34a over-expression in macrophages were also demonstrated to favour pro-inflammatory M1 phenotype and inhibition of M2 polarization. In a quest to confirm this likely interaction, expression profiles of Klf4 as the putative target were analyzed in different macrophage polarizing conditions. Klf4 expression was found to be prominent in the miR-34a inhibitor-treated group but down-regulated in the miR-34a mimic treated group. Immuno-histopathological analyses of lung tissue from the mice treated with miR-34a inhibitor also showed reduced inflammatory M1 markers as well as enhanced cell proliferation. The present study indicates that miR-34a intensified LPS-induced lung injury and inflammation by regulating Klf4 and macrophage polarization, which may serve as a potential therapeutic target for acute lung injury/ARDS.

Overexpression of miR-27a predicts poor prognosis and promotes the progression in cholangiocarcinoma.

The function of microRNA-27a (miR-27a) expression in cholangiocarcinoma (CCA) remains largely unclear; therefore, this study aimed to investigate the clinical significance and functional role of miR-27a in CCA. This study included 117 paired CCA tissues and adjacent normal tissues from CCA patients who received surgical resection. Reverse transcription-quantitative polymerase chain reaction was used to measure the expression levels of miR-27a in CCA tissues and cell lines. A Kaplan-Meier curve and Cox regression analysis were used to determine overall prognostic performance. The effects of miR-27a on cell proliferation, migration, and invasion were measured by CCK-8 and Transwell assays. The expression levels of miR-27a in patients with CCA and cell lines were higher than those in adjacent normal tissues and normal cells, respectively. Additionally, miR-27a levels were found to be associated with lymph node metastasis and TNM stages. The overall survival time of CCA patients with high miR-27a expression was poorer than that of those with low miR-27a expression. Furthermore, miR-27a overexpression promoted CCA cell proliferation, migration, and invasion, whereas knockdown of miR-27a suppressed cell proliferation, migration, and invasion. Taken together, these results suggest the potential usefulness of miR-27a in the prognosis and progression of CCA.

MiR-34a affects G2 arrest in prostate cancer PC3 cells via Wnt pathway and inhibits cell growth and migration.

To investigate the effect and mechanism of miRNA-34a overexpression on proliferation and migration of PC3 prostate cancer cells. Benign prostatic hyperplasia tissue (30 cases), prostate cancer tissue (30 cases), and prostate paracancerous tissue (30 cases) were collected. Levels of miRNA-34a in these fresh tissues were measured by fluorescence quantitative PCR. PC3 cells were divided into non-loaded group and overexpression group. Cells in the non-loaded group were transfected with non-loaded plasmid. Cells in the overexpression group were transfected with miRNA-34a plasmid, and the miRNA-34a level was determined by fluorescence quantitative PCR to confirm the overexpression. Cell proliferation was analyzed by CCK-8 assay. Cell migration rate was measured by cell scratch assay. Flow cytometry was used to detect apoptosis and analyze cell cycle. Western blot was used to measure the expression levels of β-catenin, E-cadherin and Vimentin. The expression level of miRNA-34a in prostate cancer tissue was significantly lower than that in prostate paracancerous tissue. Dual-Luciferase reporter gene assay was used to analyze the transcriptional activity of Wnt1 gene. The proliferation and migration of PC3 cells were significantly decreased after overexpression of miRNA-34a, and the differences were statistically significant compared with those in the non-loaded group (p<0.05). Flow cytometry analysis showed that in the overexpression group, the apoptotic rate, as well as the proportion of cells in the G2 phase, was significantly higher than that in the non-loaded group (p<0.05). The β-catenin level in the nucleus of PC3 cells was significantly reduced after overexpression of miRNA-34a. The total protein levels of β-catenin and Vimentin were significantly decreased, whereas the level of E-cadherin in the overexpression group was apparently increased, compared with that in the non-loaded group. The Dual-Luciferase reporter gene showed a decrease in the relative fluorescence intensity of Wnt1 after overexpression of miR-34a (p<0.05). Overexpression of miRNA-34a inhibits Wnt/β-catenin pathway by regulating the transcriptional activity of Wnt1, thereby regulating the proliferation and migration of PC3 cells and promoting apoptosis.

MicroRNA-34a Promotes Apoptosis of Rheumatoid Arthritis Fbroblast-Like Synoviocyte via AMPK/Akt/mTOR Signaling Pathway

Background and Objectives: Rheumatoid arthritis (RA) is an autoimmune arthropathy characterised by chronic synovitis, joint cartilage breakdown and bone erosions. The life quality of RA patients has been substantially effected by the disease and it is of great significance to search for more efficacious and novel therapeutic agents. Methods: In this study, mRNA levels of miR-34a in HFLS and RA-FLS were examined by RT-PCR. CCK8 assay was applied to detect the viability of RA-FLS transfected with miR-34a mimic or inhibitor. Furthermore, TUNEL assay and Hoechest-33342 assay was applied to detect the apoptosis of RA-FLS transfected with miR-34a mimic or inhibitor. The expressions of proteins related to AMPK/Akt/mTOR and autophagy were also detected by western blot. Results: The RT-PCR result showed that miR-34a mRNA levels was markedly downregulated in RA-FLS compared to HFLS. CCK8 assay results demonstrated that miR-34a overexpression significantly suppressed RA-FLS proliferation and miR-34a interference had the opposite effect. TUNEL assay and Hoechest-33342 assay demonstrated that miR-34a overexpression significantly promoted RA-FLS apoptosis and miR-34a interference had the opposite effect. The western blot results revealed that miR-34a can affect autophagy via AMPK/Akt/mTOR signaling pathway. Conclusion: This study suggested that miR-34a can regulate proliferation and apoptosis in RA-FLS by affecting autophagy through AMPK/Akt/mTOR signaling pathway.

LncRNA CTBP1-AS2 regulates miR-216a/ PTEN to suppress ovarian cancer cell proliferation

BackgroundWe analyzed TCGA dataset and observed the downregulation of CTBP1-AS2 in ovarian cancer (OC), while the function of CTBP1-AS2 has only been investigated in diabetes and cardiomyocyte hypertrophy, but not in cancer biology. We therefore analyzed the involvement of CTBP1-AS2 in OC.ResultWe found that CTBP1-AS2 was downregulated in OC and predicted poor survival. CTBP1-AS2 in luciferase activity assay interacted with miR-216a, while overexpression of CTBP1-AS2 and miR-216a had no significant effects on the expression of each other. However, increased expression level of PTEN, a target of miR-216a, was observed after CTBP1-AS2 overexpression. Increased proliferation rate of OC cells was observed after the overexpression of miR-216a. CTBP1-AS2 and PTEN overexpression resulted in the reduced proliferation rate of OC cells and reduced effects of miR-216a overexpression.ConclusionCTBP1-AS2 regulates miR-216a/PTEN to suppress OC cell proliferation.