Target Of miR-125b Research Articles

Tumor-secreted extracellular vesicles promote the activation of cancer-associated fibroblasts via the transfer of microRNA-125b

ABSTRACTTumour cells release large quantities of extracellular vesicles (EVs) to mediate their interactions with other cells in the tumour microenvironment. To identify host cells that naturally take up EVs from tumour cells, we created breast cancer cell lines secreting fluorescent EVs. These fluorescent EVs are taken up most robustly by fibroblasts within the tumour microenvironment. RNA sequencing indicated that miR-125b is one of the most abundant microRNAs secreted by mouse triple-negative breast cancer 4T1 and 4TO7 cells. Treatment with 4T1 EVs leads to an increase in fibroblast activation in isogenic 4TO7 tumours, which is reversed by blocking miR-125b in 4T1 EVs; hence, miR-125b delivery by EVs is responsible for fibroblast activation in mouse tumour models. miR-125b is also secreted by human breast cancer cells and the uptake of EVs from these cells significantly increases cellular levels of miR-125b and expression of multiple cancer-associated fibroblast markers in resident fibroblasts. Overexpression of miR-125b in both mouse and human fibroblasts leads to an activated phenotype similar to the knockdown of established miR-125b target mRNAs. These data indicate that miR-125b is transferred through EVs from breast cancer cells to normal fibroblasts within the tumour microenvironment and contributes to their development into cancer-associated fibroblasts.

Di-n-butyl phthalate epigenetically induces reproductive toxicity via the PTEN/AKT pathway

Di-n-butyl phthalate (DBP) is a kind of ubiquitous chemical linked to hormonal disruptions that affects male reproductive system. However, the mechanism of DBP-induced germ cells toxicity remains unclear. Here, we demonstrate that DBP induces reduction of proliferation, increase of apoptosis and DNA damage dependent on the PTEN/AKT pathway. Mechanistically, DBP decreases PTEN promoter methylation and increases its transcriptional activity, leading to increased PTEN expression. Notably, DNMT3b is confirmed as a target of miR-29b and miR-29b-mediated status of PTEN methylation is involved in the effects of DBP treatment. Meanwhile, DBP decreases AKT pathway expression via increasing PTEN expression. In addition, the fact that DBP decreases the sperm number and the percentage of motile and progressive sperm is associated with downregulated AKT pathway and sperm flagellum-related genes. Collectively, these findings indicate that DBP induces aberrant PTEN demethylation, leading to inhibition of the AKT pathway, which contributes to the reproductive toxicity.

MiR-196b inhibits cell migration and invasion through targeting MAP3K1 in hydatidiform mole

MicroRNAs (miRNAs) are a class of small non-coding RNAs that are closely associated with carcinogenesis. Accumulating data indicate that miR-196b participates in the development of various types of cancers. However, the role of miR-196b in the formation of hydatidiform mole (HM) is still unclear. Our previous studies have demonstrated that miR-196b levels were decreased in JAR and BeWo cells and in HM tissue samples, as demonstrated by RT-PCR analysis. Furthermore, we discovered that overexpression of miR-196b in JAR and BeWo cells inhibited cellular proliferation, migration and invasion, as shown by Cell counting kit-8 (CCK-8) and transwell assays, respectively. Subsequently, we explored the interaction of miR-196b with its target gene in human choriocarcinoma cell lines. MAP3K1 is a target gene predicted by bioinformatic analysis that was previously shown to exhibit reduced expression levels following treatment with miR-196b in JAR and BeWo cells. We demonstrated that MAP3K1 was a direct target of miR-196b using the dual-luciferase reporter assay in Hela cells. In summary, the present study demonstrated that miR-196b suppressed proliferation, migration and invasion of human choriocarcinoma cells by inhibiting its transcriptional target MAP3K1. miR-196b and MAP3K1 may be considered potential targets for the clinical treatment of HM.

MiR-29a/b cluster suppresses high glucose-induced endothelial-mesenchymal transition in human retinal microvascular endothelial cells by targeting Notch2.

Several studies have previously reported that endothelial cells contributed to pathological fibrosis in proliferative diabetic retinopathy (PDR) through endothelial-mesenchymal transition (EndMT); however, the precise mechanism of this interaction has not been completely elucidated. The present study investigated the expression of microRNA (miR)-29a/b cluster in human retinal microvascular endothelial cells (HRMECs) and examined its functional role in high glucose (HG)-induced EndMT. HRMECs were exposed to glucose at concentrations of 5, 15, 30 and 50 mM for 7 days and reverse transcription-quantitative polymerase chain reaction, western blotting and immunofluorescence were conducted to determine the expression of genes associated with miR-29a/b and EndMT. A luciferase reporter gene assay was also performed to confirm the association between miR-29a/b and neurogenic locus notch homolog protein 2 (Notch2). The expression levels of miR-29a/b, and endothelial markers vascular endothelial cadherin and cluster of differentiation 31 were decreased, whereas the expression levels of Notch2 and mesenchymal markers, including α-smooth muscle actin, fibroblast-specific protein 1 (also named S100 calcium binding protein A4, S100A4), fibronectin and SNAI1 were increased in HRMECs under HG (30 nM) conditions. In addition, Notch2 was identified as a target of miR-29a and miR-29b. Overexpression of miR-29a/b downregulated the expression of Notch2 and subsequently suppressed HG-induced EndMT. Taken together, the results of the present study revealed that the miR-29/Notch2 signaling pathway may participate in the regulation of HG-induced EndMT, and may serve as a potential molecular target during fibrosis in PDR.

MiR-125b suppresses oral oncogenicity by targeting the anti-oxidative gene PRXL2A

Oral squamous cell carcinoma (OSCC) is a globally prevalent malignancy. The molecular mechanisms of this cancer are not well understood and acquire elucidation. Peroxiredoxin like 2A (PRXL2A) has been reported to be an antioxidant protein that protects cells from oxidative stress. Our previous study identified an association between PRXL2A upregulation in OSCC and a worse patient prognosis. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in the modulation of biological/pathological properties. The miR-125 family of genes drive pluripotent regulation across a wide variety of cancers. In this study, we identify the oncogenic eligibility of PRXL2A and clarify miR-125b as its upstream regulator. Downregulation of miR-125b can be observed in OSCC tumors. Lower miR-125b expression in tumors results in a worse patient prognosis at the relatively early stage. Reporter assays are able to validate that PRXL2A is a direct target of miR-125b. Exogenous miR-125b expression in OSCC cells results in increased oxidative stress, increased drug sensitivity, and suppressor activity that is paralleled by the knockout of PRXL2A gene. The suppressor activity of miR-125b is able to be rescued by PRXL2A, which suggests the existence of a miR-125b-PRXL2A regulatory axis that is part of OSCC pathogenesis. Nuclear factor-erythroid 2-related factor 2 (NRF2) was found to be a downstream effector of the miR-125b-PRXL2A cascade. As a whole, this study has pinpointed novel clues demonstrating that downregulation of miR-125b suppressor underlies upregulation of PRXL2A in OSCC, and this then protects the affected tumor cells from oxidative stress.

MiR-125b Contributes to Ovarian Granulosa Cell Apoptosis Through Targeting BMPR1B, a Major Gene for Sheep Prolificacy.

Bone morphogenetic protein receptor 1B (BMPR1B) is a major gene for ovine ovulation rate and litter size, which plays a crucial role in follicle development. However, its role and regulation in the ovine granulosa cells (GCs) are unclear. Here, we showed that silencing of BMPR1B enhanced cell apoptosis, whereas overexpression of BMPR1B inhibited cell apoptosis in the ovine GCs. Luciferase reporter assays revealed that BMPR1B is a target of miR-125b, and miR-125b reduced BMPR1B messenger RNA and protein levels in the ovine GCs by directly binding to the 3' untranslated region of the ovine BMPR1B gene. Furthermore, miR-125b enhanced cell apoptosis by attenuating BMPR1B in the ovine GCs. Our data demonstrated that BMPR1B is an important factor in the ovine GC function in vitro and targeted by a specific regulator miR-125b.

Abstract 129: Brain-Heart Interaction After Stroke: Therapeutic Effects of Exosomes in Type 2 Diabetic Mice Subjected to Stroke

Aim: Diabetes exacerbates post-stroke cardiac injury. We tested whether treatment with exosomes harvested from human umbilical cord blood derived CD133+/KDR+ cells (CD133+Exo) improves neurological, cognitive and cardiac functional outcome in type 2 diabetes mellitus (T2DM) stroke mice. Methods: Adult (3-4m), male, db/db (T2DM) mice were subjected to distal middle cerebral artery occlusion (dMCAo) and treated with PBS or CD133+Exo (3х10 10 , i.v.) at 3 days after stroke (n=8/group). Serum exosomal microRNA (miR) expression was measured. Echocardiography, neurological and cognitive tests were performed, and mice were sacrificed at 28 days after stroke. Results: 1) Compared to T2DM-control, T2DM-stroke mice exhibit significantly decreased left ventricular ejection fraction (LVEF: db/db: 67.6±0.6%, db/db-dMCAo: 60.6±1.9%, p<0.05), significant cardiomyocyte hypertrophy, interstitial fibrosis, and decreased capillary density in heart indicating that stroke in T2DM mice induces cardiac dysfunction at 28 days after stroke. 2) Serum exosomal miR29b and miR126 expression was significantly decreased in T2DM-stroke mice compared to T2DM-control mice. CD133+Exo contain high levels of miR126 and miR29b. 3) CD133+Exo treatment in T2DM-stroke mice significantly promotes neurological functional recovery (foot-fault test), significantly improves cognitive outcome (Morris water maze (MWM) test and odor test), and significantly improves cardiac function (LVEF: db/db-dMCAo: 60.6±1.9%, db/db-dMCAo+CD133+Exo: 68.9±0.7%, p<0.05). 4) In T2DM-stroke mice with PBS or CD133+Exo treatment, cardiac function (LVEF) significantly correlates with cognitive outcome. 5) CD133+Exo treatment in T2DM-stroke mice significantly decreases myocardial fibrosis, decreases TGF-β and NOX2 expression, and increases heart tissue miR126 and miR29b expression, while decreasing expression of miR126 and miR29b target genes such as Spred-1 and DPP4. Conclusions: Stroke in T2DM mice induces significant cardiac dysfunction and CD133+Exo treatment improves neurological and cognitive outcome, and provides significant cardio-protection. The improvement in cardiac functional outcome highly correlates with cognitive functional recovery.

Decreased miR-29b expression is associated with airway inflammation in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a common chronic airway inflammatory disease. MicroRNAs are shown to be involved in the regulation of inflammation. We investigated the role of microRNA-29b (miR-29b) in the airway inflammation in COPD. The expression of miR-29b in the lung and plasma was examined. The target of miR-29b, bromodomain protein 4 (BRD4), was predicted by online algorithms and verified in human bronchial epithelial (HBE) cells. The expression of BRD4, interleukin (IL)-8, and IL-6 in the lung was also examined. The role of miR-29b in the inflammatory cytokine expression of airway epithelial cells was studied using an in vitro model system. In total, 60 subjects were recruited, including 10 nonsmokers, 24 smokers, and 26 patients with COPD. Both lung and plasma miR-29b are decreased in patients with COPD, and miR-29b expression levels are correlated with pulmonary function and inflammation. BRD4 is increased in the lung of patients with COPD and is correlated with miR-29b and IL-8 expression. miR-29b regulates cigarette smoke extract (CSE)-induced IL-8 expression by targeting BRD4 in HBE cells. The antioxidant N-acetylcysteine prevents CSE-induced miR-29b downregulation and BRD4 and IL-8 upregulation. Our findings indicate that miR-29b may participate in the airway inflammation in COPD by regulating inflammatory cytokine expression through targeting BRD4, plasma miR-29b may serve as a biomarker for disease severity in COPD, and oxidative stress may contribute to the decrease of miR-29b induced by cigarette smoke.

Elevated Expression of miR-19b Enhances CD8+ T Cell Function by Targeting PTEN in HIV Infected Long Term Non-progressors With Sustained Viral Suppression

Human immunodeficiency virus (HIV)-infected long-term non-progressors (LTNPs) are of particular importance because of their unique disease progression characteristics. Defined by the maintenance of normal CD4+T cells after more than 8 years of infection, these LTNPs are heterogeneous. Some LTNPs exhibit ongoing viral production, while others do not and are able to control viral production. The underlying basis for this heterogeneity has not been clearly elucidated. In this study, the miRNA expression profiles of LTNPs were assessed. The levels of microRNA-19b (miR-19b) were found to be significantly increased in peripheral blood mononuclear cells of LTNPs with lower rather than higher viral load. We made clear that miR-19b may regulate CD8+T cell functions in HIV infection, which has not been addressed before. Overexpression of miR-19b promoted CD8+T cell proliferation, as well as interferon-γ and granzyme B expression, while inhibiting CD8+T cells apoptosis induced by anti-CD3/CD28 stimulation. The target of miR-19b was found to be the “phosphatase and tensin homolog”, which regulates CD8+T cells function during HIV infections. Furthermore, we found that miR-19b can directly inhibit viral production in in-vitro HIV infected T cells. These results highlight the importance of miR-19b to control viral levels, which facilitate an understanding of human immunodeficiency virus pathogenesis and provide potential targets for improved immune intervention.

LncRNA-H19 Induces Retinal Müller Cell Apoptosis via MiR-29b/FOXO4 Axis in Diabetic Retinopathy

Background: The pathological development of Diabetic retinopathy (DR) is an intricate process with multiple steps, and implicates a battery of dysregulated genes and regulators. We aimed to investigate the pathological mechanism of DR involving the regulatory association among H19, miR-29b and FOXO4. Methods: Sprague-Dawley (SD) rats were used to establish the diabetes mellitus (DM) model by injection streptozotocin. The levels of miR-29b, H19 and the mRNA level of FOXO4 were detected by qRT-PCR. The expression of FOXO4 at protein level was assessed by Western blot assay. The apoptosis of rMC-1 cells were analyzed by flow cytometry. To further investigate the relationship between H19 and miR-29b, the RIP and pull down assays were performed. Results: The expression of H19 and FOXO4 were enhanced obviously, and the level of miR-29b was decreased in the retina tissues of DM rats and high glucose (HG)-treated rMC-1 cells. HG, as well as over-expression of H19, stimulated the apoptosis of rat retinal Muller cells. Knockdown of H19 reversed HG stimulation on cell apoptosis and FOXO4 up-regulation. RIP assay and RNA pull-down assay indicated that H19 was a target of miR-29b and inhibition of miR-29b reversed H19 down-regulation effect on cell apoptosis and FOXO4 expression. Down-regulation of FOXO4 reversed the effect of miR-29b knockdown on cell apoptosis. Conclusion: MiR-29b targeting lncRNA-H19 mediates the apoptosis of rat retinal Muller cells via regulating FOXO4 in diabetic retinopathy. Key words: LncRNA-H19, miR-29b, FOXO4, Retinal Muller cell apoptosis, Diabetic retinopathy

MiR29b regulates aberrant methylation in In-Vitro diabetic nephropathy model of renal proximal tubular cells

The role of DNA methylation has not been enough explored in pathophysiology of diabetic nephropathy (DN). However, according to recent reports it has been inferred that hypermethylation could be one of the principle cause associated with the enhancement of DN. An interrelationship between miR29b and DNA methylation has been studied via in-silico analysis. We have validated that miR29b prominently targets DNA methyl transferase (DNMT), specifically DNMT1, DNMT3A and DNMT3B. We have developed in vitro DN model using renal proximal tubule epithelial cells (RPTECs), contributed to a significant alleviation in RNA and protein expression levels of DNMT3A, DNMT3B and DNMT1. The developed model has also demonstrated downregulation in expression of miR29b. Our studies have also suggested that miR29b targets DNMT1 via targeting its transcription factor SP1. In addition to this, downregulation of a specific biomarker for kidney injury, tubular kidney injury molecule-1 (KIM-1) and fibrosis causing glycoprotein i.e. fibronectin, was also demonstrated. Hence, the developed model revealed that hypermethylation is a key factor incorporated in DN, and miR29b could effectively ameliorate defensive actions in DN pathogenesis.

MiR-19b Functions as a Potential Protector in Experimental Autoimmune Encephalomyelitis.

MicroRNA-19b (miR-19b) is essential in determining oligodendroglia proliferation. Phosphatase and tensin homologue on chromosome 10 (PTEN) is considered the target of miR-19b and participates in oligodendrocyte differentiation and proliferation. Murine EAE was induced by myelin oligodendrocyte glycoprotein (MOG35- 55). For EAE reversal, artificially synthesized agomiR-19b was intravenous injected after immunization. We found that the expression of miR-19b is significantly reduced in an experimental autoimmune encephalomyelitis (EAE) mouse model. This downregulation, which is associated with the neurological scores, can be dramatically ameliorated by agomiR-19b. Our results show that agomiR-19b increases the expression of myelin basic protein (MBP) and cyclic nucleotide phosphodiesterase (CNP), which are regularly utilized as molecular markers of oligodendrocytes. Furthermore, our study also revealed that miR-19b probably affects the expression of PTEN in the EAE model. These results indicate that the restoration of miR-19b probably exerts its therapeutic effect by affecting PTEN in the pathogenesis of EAE.

Downregulation of long noncoding RNA HOTAIRM1 variant 1 contributes to osteoarthritis via regulating miR-125b/BMPR2 axis and activating JNK/MAPK/ERK pathway

Objective: Osteoarthritis (OA) is a leading cause of pain and disability worldwide, which is characterized by degenerative and inflammatory processes affecting joints and surrounding tissues. This study aimed to elucidate the role and possible mechanism of long non-coding RNA HOX antisense intergenic RNA myeloid 1 variant 1 (HOTAIRM1-1) in the development of osteoarthritis (OA).Material and methods: The expressions of HOTAIRM1-1 and miR-125b in OA cartilages and normal articular cartilages were determined. Moreover, human mesenchymal stem cells (MSCs) were cultured in MSC growth medium for 8 days to induce chondrogenic differentiation. The effects of aberrant expression of HOTAIRM1-1 on MSCs viability, apoptosis and differentiation were investigated. Furthermore, the regulatory relationships between HOTAIRM1-1 and miR-125b as well as the target of miR-125b were explored. Besides, the association between HOTAIRM1-1 and JNK/MAPK/ERK pathway was detected.Results: Inverse expression of HOTAIRM1-1 (downregulated) and miR-125b (upregulated) was revealed in OA cartilages. In addition, HOTAIRM1-1 expression increased at 4 days after induction of chondrogenic differentiation and further enhanced after 8 days, while miR-125b had opposite expression changes. Suppression of HOTAIRM1-1 inhibited MSCs viability, induced apoptosis and suppressed cell differentiation. Moreover, HOTAIRM1-1 negatively regulated the expression of miR-125b, and the effects of HOTAIRM1-1 suppression on cell viability, apoptosis and differentiation were achieved by negative regulation of miR-125b. Furthermore, miR-125b regulated MSCs viability, apoptosis and differentiation via targeting bone morphogenetic protein receptor 2 (BMPR2). Besides, overexpression of HOTAIRM1-1 inhibited the activation of JNK/MAPK/ERK pathway.Conclusions: Our results indicate that HOTAIRM1-1 is downregulated in OA cartilages, and its downregulation may inhibit MSCs viability, induce apoptosis and suppress differentiation via regulating miR-125b/BMPR2 axis. JNK/MAPK/ERK pathway may be a possible downstream mechanism to mediate the role of HOTAIRM1-1 in OA development.

Abstract 16728: Cigarette Smoke Impairs the Anti-Oxidative Response by Reducing Heme Levels and Inducing Bach1

Introduction: Cigarette smoke is a major risk factor of cardiovascular diseases. Heme regulates the activity of enzymes and transcription factors including Bach1. Bach1 competes with Nrf2 transcriptional activity and inhibits the expression of anti-oxidative enzymes. However, the molecular mechanism of Bach1 activation and its impact on the anti-oxidative response after exposure to cigarette smoke is not clear. Hypothesis: We aim to understand the mechanism through which cigarette smoke impairs the anti-oxidative response. Methods: Primary human umbilical vein endothelial cells (HUVEC) were exposed to a cigarette smoke extract (CSE) (3 mg/ml nicotine) under static and unidirectional shear stress conditions. Shear stress was induced using the ibidi pump system and cone-and-plate viscometer. Llevels of reduced glutathione (GSH) were measured using the fluorescent probe o-phthalaldehyde. Heme levels were measured after oxalic acid extraction. Gene and miR expression was quantified by RT-qPCR. Immunofluorescence imaging was performed on fixed HUVEC. MiR-125b overexpression was achieved by transfecting mimics. Inhibition of Bach1 was done using siRNA transfection. Apoptosis was assessed using the caspase 3/7 assay. Human umbilical veins as an ex vivo model were exposed to CSE under static conditions. Results: Exposure to CSE induced the expression of BACH1 (1.90±0.18; p<0.05) and reduced total heme levels (0.30±0.04; p<0.001) in HUVEC. It also reduced miR-125b expression in HUVEC (0.39±0.09; p<0.001) and umbilical veins (0.72±0.12; p<0.05), whereas the expression of miR-125b target AhR repressor ( AHRR ) increased (14.93±4.08; p<0.05). Inhibiting BACH1 rescued miR-125b expression (1.02±0.45; p>0.05) and reduced AHRR expression (0.66±0.12; P<0.05). Acute exposure to CSE led to a high oxidative stress load. This observation was supported by a 2-fold reduction in glutathione levels, an increase in nuclear:cytosolic ratio of the upstream transcription factor Nrf2 (1.33±0.05; p<0.01) and an increase in expression of anti-oxidative HMOX1 (18.18±1.8; p<0.001) and NQO1 (16.12±1.99; p<0.001). CSE-induced apoptosis (1.23±0.04; p<0.001) was reversible by pre-conditioning with 1 mM N-acetyl cysteine (0.99±0.02; p>0.05) or overexpression of miR-125b (0.99±0.07; p>0.05). Conclusions: These results show that cigarette smoke-induced Bach1 impairs the anti-oxidative response through inhibiting miR-125b in the human endothelium. Reduced heme levels could be involved in the upregulation of Bach1 activity.

Retraction: MiR-125b Increases Nasopharyngeal Carcinoma Radioresistance By Targeting A20/NF-κB Signaling Pathway.

Radioresistance poses a major challenge in nasopharyngeal carcinoma (NPC) treatment, but little is known about how miRNA regulates this phenomenon. In this study, we investigated the function and mechanism of miR-125b in NPC radioresistance, one of upregulated miRNAs in the radioresistant NPC cells identified by our previous microarray analysis. We observed that miR-125b was frequently upregulated in the radioresistant NPCs, and its increment was significantly correlated with NPC radioresistance, and was an independent predictor for poor patient survival. In vitro radioresponse assays showed that miR-125b inhibitor decreased, whereas miR-125b mimic increased NPC cell radioresistance. In a mouse model, therapeutic administration of miR-125b antagomir dramatically sensitized NPC xenografts to irradiation. Mechanistically, we confirmed that A20 was a direct target of miR-125b and found that miR-125b regulated NPC cell radioresponse by targeting A20/NF-κB signaling. With a combination of loss-of-function and gain-of-function approaches, we further showed that A20 overexpression decreased while A20 knockdown increased NPC cell radioresistance both in vitro and in vivo Moreover, A20 was significantly downregulated while p-p65 (RelA) significantly upregulated in the radioresistant NPCs relative to radiosensitive NPCs, and miR-125b expression level was negatively associated with A20 expression level, whereas positively associated with p-p65 (RelA) level. Our data demonstrate that miR-125b and A20 are critical regulators of NPC radioresponse, and high miR-125b expression enhances NPC radioresistance through targeting A20 and then activating the NF-κB signaling pathway, highlighting the therapeutic potential of the miR-125b/A20/NF-κB axis in clinical NPC radiosensitization. Mol Cancer Ther; 16(10); 2094-106. ©2017 AACR.

1 - Cigarette smoke impairs the anti-oxidative response by reducing heme levels and inducing Bach1

Introduction Cigarette smoke is a major risk factor of cardiovascular diseases. Heme regulates the activity of enzymes and transcription factors including Bach1. Bach1 competes with Nrf2 transcriptional activity and inhibits the expression of anti-oxidative enzymes. However, the molecular mechanism of Bach1 activation and its impact on the anti-oxidative response after exposure to cigarette smoke is not clear. We aim to understand the mechanism through which cigarette smoke impairs the anti-oxidative response. Methods Primary human umbilical vein endothelial cells (HUVEC) were exposed to a cigarette smoke extract (CSE) under static and unidirectional shear stress conditions. Shear stress was induced using the ibidi pump system and cone-and-plate viscometer. Levels of reduced glutathione (GSH) were measured using the fluorescent probe o-phthalaldehyde. Heme levels were measured after oxalic acid extraction. Gene and miR expression was quantified by RT-qPCR. Immunofluorescence imaging was performed on fixed HUVEC. MiR-125b overexpression was achieved by transfecting mimics. Inhibition of Bach1 was done using siRNA transfection. Apoptosis was assessed using caspase 3/7 activity. Human umbilical veins as an ex vivo model were exposed to CSE under static conditions. Results Exposure to CSE induced the expression of BACH1 and reduced total heme levels in HUVEC. It also reduced miR-125b expression in HUVEC and umbilical veins, whereas the expression of miR-125b target AhR repressor (AHRR) increased. Inhibiting BACH1 increased miR-125b expression and reduced AHRR expression. Acute exposure to CSE led to a high oxidative stress load. This observation was supported by a decrease in glutathione levels, an increase in nuclear:cytosolic ratio of the upstream transcription factor Nrf2 and an increase in expression of HMOX1 and NQO1. CSE-induced apoptosis was reversible by preconditioning with N-acetyl cysteine or overexpression of miR-125b. Conclusions cigarette smoke-induced Bach1 impairs the anti-oxidative response through inhibiting miR-125b. Reduced heme levels could be involved in the upregulation of Bach1 activity.

MicroRNA-125b Regulates Apoptosis by Targeting Bone Morphogenetic Protein Receptor 1B in Yak Granulosa Cells.

The intronic microRNA, miR-125b, plays a vital role in promyelocytic and hematopoietic stem cells, and in the development and apoptosis of cancer cells. In this study, we showed that miR-125b regulates granulosa cell (GC) apoptosis in the yak ovary. Bioinformatic analyses and luciferase reporter assays demonstrated that bone morphogenetic protein receptor type 1B (BMPR1B) is an miR-125b target. miR-125b overexpression induced apoptosis in yak GC, and affected the mRNA and protein expression of BMPR1B and the ratio of Bcl2/Bax. Silencing of miR-125b decreased the rate of yak GC apoptosis and increased the ratio of Bcl2/Bax. In addition, the effects of an miR-125b inhibitor were overturned by cotransfection with siRNA-BMPR1B2 (siRNA-299) in yak GC. Together, these results demonstrated that miR-125b regulates GC apoptosis in the yak ovary by targeting BMPR1B.

Long noncoding RNA SNHG12 promotes the progression of cervical cancer via modulating miR-125b/STAT3 axis.

Increasing evidence showed that long noncoding RNAs (lncRNAs) played an important role in the occurrence and development of tumors. To date, lncRNA small nucleolar RNA host gene 12 (SNHG12) has revealed an oncogenic role in various tumors. However, the role of SNHG12 in cervical cancer is still unclear. Therefore, we focused on the biological function and molecular mechanism of SNHG12 in the tumorigenesis of cervical cancer. In this study, the expression of miR-125b was observably downregulated in cervical cancer cells. Meanwhile, the expression of SNHG12 was obviously upregulated in cervical cancer cell lines (HeLa, SiHa, Caski, C4-1, and C33A) compared with the immortalized cervical epithelial cells. The further assay showed that miR-125b was a target of SNHG12 in cervical cancer. Moreover, a negative relationship between miR-125b and SNHG12 was found in cervical cancer. In addition, SNHG12 inhibition restrained the proliferation, migration, and invasion of cervical cancer cells. Meanwhile, miR-125b mimics repressed the expression of signal transducer and activator of transcription 3 (STAT3). The further assay showed that STAT3 was a target of miR-125b in cervical cancer. In addition, sh-STAT3 repressed the migration and invasion of cervical cancer cells. Furthermore, it showed that miR-125b inhibitors reversed STAT3 expression restrained by the reduction of SNHG12 expression. In general, SNHG12 modulated STAT3 by sponging miR-125b in cervical cancer and played an important role in the development of cervical cancer.

Interaction of miR-125b-5p with Human antigen R mRNA: Mechanism of controlling HCV replication

Cellular miRNAs influence Hepatitis C virus (HCV) infection in multiple ways. In this study, we demonstrate that miR-125b-5p is upregulated in HCV infected patient serum samples as well as in HCV infected liver carcinoma cells and is involved in translational regulation of one of its predicted targets, Human antigen R (HuR). We used miRNA mimics and antagomiRs to confirm that HuR is a bonafide miR-125b target. Previously, we have shown that HuR is a positive regulator of HCV replication, whereas we noticed that miR-125b is a negative regulator of HCV infection. As a connecting link between these two observations, we showed that knockdown of miR-125b-5p increased HuR protein levels and rescued HCV replication when the availability of HuR in the cytoplasm was compromised using siRNAs against HuR or an inhibitor of HuR export to the cytoplasm. Overall, the study sheds light on the ability of host cell to use a miRNA as a tool to control virus propagation.

MiR-125b promotes tau phosphorylation by targeting the neural cell adhesion molecule in neuropathological progression.

MicroRNAs, small noncoding RNAs, not only regulate gene expression at the post-transcriptional level in a variety of physiological processes but also accompany the initiation and progression of a vast number of diseases, including dementia. While miR-125b has been shown to be aberrantly expressed in some dementia patients, its role in the pathological process remains ambiguous. Presenilin-1/2 conditional double knockout mice exhibit a range of symptoms, including impaired cognition and memory, increased tau phosphorylation, neuroinflammation, and apoptosis, and are therefore regarded as a useful dementia model. In the prefrontal cortices of double knockout mice, miR-125b was found to be abnormally increased in an age-dependent manner. We further verified the neural cell adhesion molecule (NCAM) as an miR-125b target using the dual luciferase reporter assay. The NCAM protein level was decreased when miR-125b was overexpressed (OE) in neuronal growth factor-induced differentiated PC12cells, which further inhibited the neuronal growth factor-induced phosphorylation of glycogen synthase kinase 3 beta (GSK3β) at the Ser9 site and ultimately increased the GSK3β activity and tau phosphorylation. Moreover, on serum deprivation, high GSK3β activity in differentiated miR-125b-OE PC12cells induced increased caspase-3 activation. Finally, adeno-associated virus-mediated miR-125b overexpression in the prefrontal cortexes of wild-type C57B/L6 mice resulted in decreased dendritic spine density. In addition, similar to the invitro data, elevated GSK3β activity and hyperphosphorylation of the tau protein were confirmed. Taken together, our findings reveal a direct regulation of miR-125b on NCAM, which leads to further effects on downstream GSK3β activity and tau phosphorylation and may contribute to the generation of neurofibrillary tangles in neuropathological progression.