Expression Levels Of miR-125b Research Articles

Inactivation of lncRNA HOTAIRM1 caused by histone methyltransferase RIZ1 accelerated the proliferation and invasion of liver cancer.

Liver cancer is the second most common cause of cancer death, causing more than 700,000 deaths every year. It has been demonstrated that Long non-coding RNA (LncRNA) plays an important regulatory role in a series of diseases. However, the regulatory mechanism of LncRNAs in liver cancer has not been fully elucidated. The purpose of this study was to explore the interaction of lncRNA HOTAIRM1 and aberrant histone modification in liver cancer. qRT-PCR was used to detect the expression levels of RIZ1 and miR-125b in liver cancer cells. Cell proliferation was measured using the CCK8 assay. ChIP-Real-time PCR confirmed the binding site of the promoter of HOTAIRM1 by H3K9me1. The direct target of HOTAIRM1 and miR-125b in liver cancer cells was measured by a luciferase reporter assay. Cell proliferation was detected by Cell Counting Kit-8 (CCK8). Cell invasion was measured by transwell assays and cell migration was detected by wound healing assay. The expression level of RIZ1 and miR-125b was upregulated, and HOTAIRM1 was downregulated in liver cancer cells. Transwell and CCK-8 assay showed that RIZ1 expression is associated with the proliferation, invasion and migration of liver cancer cells, silencing of RIZ1 inhibited cell proliferation, migration, and invasion in HEPG2 and HCC-LM3 cells. RIZ1 interference could significantly inhibit H3K9me1 expression. H3K9me1 protein can bind to HOTAIRM1 promoter directly. Furthermore, the bioinformatics prediction and luciferase assay demonstrated that miR-125b can interact with HOTAIRM1 by direct binding. HOTAIRM1 down-expression promoted HEPG2 cell growth and metastasis, which was further strengthened following the co-transfection of miR-125b. Furthermore, overexpressed HOTAIRM1 inhibited HCC-LM3 cell growth and metastasis and a complete reversal of the results seen when transfected with miR-125b. For the first time, we found that RIZ1 was upregulated in liver cancer cells and RIZ1-mediated H3K9me1 enrichment on the HOTAIRM1 promoter regulated the growth and metastasis of liver cancer cells by targeting miR-125b, which could further accelerate tumor proliferation, migration and invasion. It may serve as a therapeutic marker for liver cancer treatment.

AntagomiR-29b inhibits vascular and valvular calcification and improves heart function in rats.

We aimed to investigate the role of the miR‐29b and its effect on TGF‐β3 pathway in vascular and valvular calcification in a rat model of calcific aortic valve diseases (CAVD). A rat model of CAVD was established by administration of warfarin plus vitamin K. The expression levels of miR‐29b, osteogenic markers and other genes were determined by qRT‐PCR, Western blot and/or immunofluorescence and immunohistochemistry. The calcium content and alkaline phosphatase (ALP) activity were measured. The calcium content, ALP activity and osteogenic markers levels in calcified aorta and aortic valve were augmented compared to controls. The expression of miR‐29b, p‐Smad3, and Wnt3 and β‐catenin was significantly up‐regulated, whereas TGF‐β3 was markedly down‐regulated. However, compared with the CAVD model group, the calcium content and ALP activity in rats treated with antagomiR‐29b were significantly decreased, and antagomiR‐29b administration reversed the effects of CAVD model on the expression of miR‐29b and osteogenic markers. Inhibition of miR‐29b in CAVD rats prevented from vascular and valvular calcification and induced TGF‐β3 expression, suggesting that the miR‐29b/TGF‐β3 axis may play a regulatory role in the pathogenesis of vascular and valvular calcification and could play a significant role in the treatment of CAVD and other cardiovascular diseases.

Evaluation of miR-181b and miR-126-5p expression levels in T2DM patients compared to healthy individuals: Relationship with NF-κB gene expression

BackgroundType 2 diabetes mellitus (T2DM) is a progressive metabolic disorder whose prevalence is rising very fast across the world. Diagnosis of this disease in early stages (pre-diabetic stage) plays an important role in reducing mortality associated with this disorder. miRNAs, as key players in the pathogenesis of T2DM, have been investigated in several studies. Furthermore, their expression profile changes in the early stages of diabetes mellitus in body fluids such as serum, peripheral blood, and peripheral blood mononuclear cell (PBMC) have been studied. Due to their high stability and the presence of non-invasive sensitive methods for their measurement, such as real-time PCR, they can be used for early diagnosis of T2DM as a biomarker. In this experimental study, the expression levels of miR-181b, miR-126-5p, and NF-κB were measured in patients with T2DM, pre-diabetic subjects, and healthy controls in a Yazd population. Material and methodNinety asymptomatic subjects including 30 T2DM, 30 pre-diabetic, and 30 healthy subjects (diagnosis based on WHO criteria) were included in this study. Real-time PCR was used to measure the expression levels of miR-181b and miR-126-5p. Moreover, the NF-κB expression level was also measured to determine its relationship with these two microRNAs. ResultIn this study, the expression level of miR-181b and miR-126-p decreased gradually in pre-diabetic as well as T2DM subjects compared to healthy controls. Furthermore, our study showed a significant negative correlation between these two miRNAs and NF-κB for the first time. ConclusionThese results introduce these anti-inflammatory miRNAs as powerful tools for early diagnosis of T2DM.

Fluorescent Mitoxantrone Hydrochloride Nanoparticles Inhibit the Malignant Behavior of Giant Cell Tumor of Bone via miR-125b/PTH1R Axis

Objective. To explore the therapeutic effects and mechanism of fluorescent mitoxantrone hydrochloride nanoparticles on giant cell tumor of bone. Methods. The adsorption capacity of nanoparticles to hydroxyapatite (HA), cell adsorption capacity, encapsulation rate, particle size, and potential of the nanoparticles were determined by HPLC and Zetasizer Nano ZS nanomicelle potentiometer. MTT assay was used to determine the toxicity of nanoparticles to cells. The fluorescent intensity of the nanoparticles and their location in the cells were observed under a fluorescence microscope. RT-qPCR and Western blotting were then used to measure the expression levels of miRNA, mRNA, and proteins in cells. Transwell and Annexin V-FITC/PI staining tests were used to study the cell invasion and apoptotic rate, respectively. The dual-luciferase reporter gene experiment was then carried out to verify the binding relationship between miR-125b and its predicted target. Results. ALN-FOL-MTO-NLC nanoparticles showed a stronger adsorption capacity for HA and stronger toxicity to GCTB28 cells. Compared to normal tissues, the expression level of miR-125b in giant bone tumor tissue and cells was significantly downregulated, and the expression level of miR-125b was upregulated to some extent after treatment. Overexpression of miR-125b or treatment of ALN-FOL-MTO-NLC nanoparticles can inhibit the malignant behavior of GCTB28 cells, whereas the inhibition of the expression of miR-125b can promote the malignant behavior of GCTB28 cells. The result showed that parathyroid hormone receptor 1 (PTH1R) was a downstream target gene for miR-125b. Rescue experiment showed that the treatment of GCTB28 with ALN-FOL-MTO-NLC nanoparticles while inhibiting miR-125b expression can reduce the inhibitory effect of miR-125b on the malignant behavior of GCTB28 cells, whereas upregulating the expression levels of miR-125b and PTH1R in GCTB28 cells had no significant effect on the malignant behavior of GCTB28 cells. Conclusion. ALN-FOL-MTO-NLC nanoparticles have a certain inhibitory effect on the malignant behavior of giant cell tumor of bone through the miR-125b/PTH1R molecular axis.

MiR-29b enhances the proliferation and migration of bone marrow mesenchymal stem cells in rats with castration-induced osteoporosis through the PI3K/AKT and TGF-β/Smad signaling pathways.

The aim of the present study was to investigate the role of microRNA (miR)-29b in the proliferation and migration of bone marrow mesenchymal stem cells (BMSCs) in rats with castration-induced osteoporosis and the relevant mechanisms. The gene expression profiling microarray technique was utilized to sequence the BMSCs with overexpressed miR-29b. The intersection of the potential targets and the genes downregulated in the sequencing were utilized for GO enrichment analysis. Gene set enrichment analysis (GSEA) was employed to analyze the effect of miR-29b on signaling pathways. Additionally, the effects of miR-29b overexpression on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and transforming growth factor-β (TGF-β)/Drosophila mothers against decapentaplegic protein (Smad) signaling pathways were detected via RT-qPCR assay and western blotting. The expression level of miR-29b was found to be significantly reduced in bone marrow tissues of postmenopausal osteoporosis patients and BMSCs of rats with castration-induced osteoporosis established via ovariectomy. Based on transcriptome sequencing and bioinformatics software prediction, 76 potential targets of miR-29b were obtained, which were distinctly enriched in such biological processes as cell proliferation, cell cycle, cell migration and cell adhesion. The results of CCK-8 and EdU assays showed that overexpression of miR-29b overtly promoted the proliferation of BMSCs in rats with castration-induced osteoporosis. Moreover, the Transwell assay results revealed that the overexpression of miR-29b significantly facilitated the migration of BMSCs in rats with castration-induced osteoporosis. According to RT-qPCR assay and western blotting, miR-29b activated the PI3K/AKT and TGF-β/Smad signaling pathways. miR-29b exhibited a clearly lower expression level in the bone marrow tissues of the postmenopausal osteoporosis patients and BMSCs of rats with castration-induced osteoporosis established via ovariectomy. Overexpression of miR-29b was able to enhance the proliferation and migration ability of BMSCs in rats with castration-induced osteoporosis, and such an enhancement may be correlated with the activation of the PI3K/AKT and TGF-β/Smad signaling pathways.

Expressions of miRNAs in Papillary Thyroid Carcinoma and Their Associations with the BRAFV600E Mutation and Clinicopathological Features

Objectives The microRNAs (miRNAs) are known to be commonly expressed in papillary thyroid carcinoma. The BRAFV600E mutation is the most common genetic mutation in thyroid cancer. The main aim of this study was to determine the possible association between expression of the three miRNAs and that of BRAFV600E mutation and the clinicopathological features in papillary thyroid carcinoma. Methods This study was conducted on 51 paraffin-embedded tissues (42 thyroid cancer, 9 benign tumor) obtained from patients undergoing thyroidectomy at the Endocrine Center of OOO University Hospital. Results miRNAs expression was significantly high in patients with cervical lymph node metastasis and advanced TNM stage. In addition, miR-146b expression levels were significantly higher in papillary thyroid carcinoma patients with BRAFV600E mutation. The relative quantification (2-ΔΔCt) of miR-146b was also high among the miRNAs. Individually, the AUCs for miRNA-146b was 0.923 (cutoff value −1.97, sensitivity 88.9%, specificity 85.7%). Conclusions Especially, expression of miR-146b increased higher in PTC patients with BRAFV600E mutation. These findings showed a role of miR-146b as potential biomarkers in differentiating PTC from benign tumor and as a prognostic indicator of PTCs. Further investigation will need for the roles of miRNAs in the pathogenesis of papillary thyroid carcinomas.

Expression Level and Target Gene Prediction of miR-181b in Patients with Chronic Lymphocytic Leukemia

To investigate the expression level of miR-181b in CD19+ B lymphocytes of patients with chronic lymphocytic leukemia (CLL), to analyze the relationship between its expression and the prognosis of CLL patients, and to predict the potential target gene of miR-181b in CLL by using bioinformatics. Eight-four patients with CLL treated in People's Hospital of Xinjiang Uygur Autonomous Region from June 2013 to June 2018 were selected. and 20 healthy people were selected as control group. RNA was extracted from CD19+B lymphocytes of peripheral blood by magnetic bead sorting, the expression level of miR-181b was detected, and it's expression differences in different IPI groups were analyzed. The correlation between the expression level of miR-181b and PFS of CLL patients also was analyzed. miR-181b target genes were predicted by online database and literatures, and gene annotation analysis and relevant signal pathway analysis were performed for candidate target genes. The expression level of miR-181b in CLL patients was significantly lower than that in control group (P＜0.01); The expression level of miR-181b in the low-risk group was higher than that in high-risk group and extremely high-risk group (P＜0.05), but there was no statistical difference between low-risk group and medium-risk group (P=1.00). The expression level of miR-181b in medium-risk group was higher than that in high-risk group and extremely high-risk group (P＜0.05), but there was no difference between high-risk group and extremely high-risk group (P=1.00). ROC curve results showed that the area under the curve (AUC) was 0.792 (P＜0.01).When the expression level of miR-181b was at the threshold value of 0.279, it showed a better sensitivity (62.9%) and specificity (91.8%). Survival analysis results suggested that compared with the high expression group, the miR-181b low expression group had poor PFS (log rank: P=0.047). Prediction of miR-181b by using the starBase, targetscan and picTar database and its combination with literature reports indicated that CARD11, ZFP36L1, RUNX1, NR4A3, ATP1B1, PUM1 and PLAG1 related with blood diseases, and up-regulated CARD11 and ZFP36L1 participated in lymphoid tumor formation by promoting cell proliferation and inhibiting cell aging. The expression level of miR-181b in CLL group are significantly lower than that in the controls group, and the low expression of miR-181b relates with poor prognosis of CLL patients. Through bioinformatics prediction and combined with literature reports, it is speculated that CARD11 and ZFP36L1 as target genes of miR-181b may be participated in the occurrence and development of CLL. Further experiments are needed to verify this result.

Circular RNA COL1A2 promotes angiogenesis via regulating miR-29b/VEGF axis in diabetic retinopathy

AimsThe dysregulation of circular RNAs (circRNAs) has been implicated in the progression of diabetic retinopathy (DR). This study aims to explore the role and underlying mechanism of hsa_circ_0081108 (circCOL1A2) in DR. Materials and methodscircCOL1A2, vascular endothelial growth factor (VEGF) and miR-29b expression levels in human retinal microvascular endothelial cells (hRMECs) were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting. The biological functions of hRMECs were evaluated by MTT, transwell, tube formation, and vascular permeability assays, respectively. The interaction between miR-29b and circCOL1A2/VEGF was determined by dual luciferase assay. The release of VEGF was examined by ELISA. The in vivo role of circCOL1A2 was further verified in streptozotocin (STZ)-induced DR in mice. The pathological changes and VEGF expression in retinal tissues were detected by hematoxylin and eosin (HE) and immunohistochemical staining. Key findingsHigh glucose (HG) challenge led to increased circCOL1A2, VEGF, MMP-2, MMP-9 levels, but decreased miR-29b level in hRMECs. In addition, circCOL1A2 sponged miR-29b to promote VEGF expression. Silencing of circCOL1A2 inhibited HG-induced proliferation, migration, angiogenesis and vascular permeability of hRMECs via enhancing miR-29b expression. Moreover, circCOL1A2/miR-29b axis participated in HG-induced increase in angiogenesis-related protein expression. Finally, circCOL1A2 knockdown suppressed angiogenesis via regulating miR-29b/VEGF axis in DR mice. SignificancecircCOL1A2 facilities angiogenesis during the pathological progression of DR via regulating miR-29b/VEGF axis, suggesting that targeting circCOL1A2 may be a potential treatment for DR.

MiRNAs as Biomarkers of Metabolic Disorders Associated with Visceral Adiposity, a Study Protocol

Abstract Objectives The expression of some micro RNAs is associated independently with visceral fat and metabolic disorders. However, the mechanisms of regulation by which these miRNAs could increase visceral fat and its possible association with metabolic disorders have not been fully elucidated. Therefore, the objective was to evaluate and compare the expression levels of miR-146b, miR-130, miR-181a5p, and miR-33a in subjects according to their visceral fat area and their metabolic state. Methods Men 25–45 years old (n = 118) with normal weight, overweight or obese (18.5–34.9 Kg/m2) were recruited for a cross-sectional study. After consent, anthropometric measures and body composition were evaluated by bioelectrical impedance analysis (Inbody S10). A blood sample was taken for analysis of glucose and lipid profile. Subjects were classified into six groups according to their body mass index (BMI), VAT area and metabolic alterations, (1: normal weight, <100 cm2 and no metabolic alterations, 2: overweight, <100 cm2 and no metabolic alterations, 3: overweight, ≥100 cm2 and no metabolic alterations, 4: overweight, <100 cm2 and metabolic alterations, 5: overweight, ≥100 cm2 and metabolic alterations, 6: obese, ≥140 cm2 and metabolic alterations). The expression levels of miR-33a, miR-155 y miR-181a-5p, were quantified by RT digital PCR technique, using serum samples. Reverse transcription was used to obtain complementary DNA (cDNA) (TaqMan® Reverse Transcription Kit) from RNA samples. Then cDNA was mixed with the specific miRNA primers and a fluorescent probe for DNA, and then the sample was transferred to a droplet generator (QX200 Droplet Generator, Bio-Rad). The generated droplets were amplified by conventional PCR. Finally, the PCR products will be collocated in the droplet lector QX200, and to analyze the number of droplets, the software QuantaSoft will be used. Differences between groups were tested with ANOVA and Tukey posthoc. Results A total of 300 subjects were evaluated, and 118 meet the inclusion criteria. According to their BMI, VAT area, clinical, and metabolic variables (blood pressure, glucose, and lipid profile), subjects were assigned to one of the six groups. Conclusions At the end of the study, we will have found if the proposed miRNAs could be used as biomarkers of metabolic disorders associated with visceral adiposity. Funding Sources DAIP 2019.

MicroRNAs involved in immune response as prognostic markers in early and metastatic breast cancer.

e15528 Background: MicroRNAs (miRNAs) are involved in the regulation of immune response and have an important role in immune escape. We analyzed the expression levels of plasma miR-10b, miR-126, miR-19α, miR-20α and miR-155, which regulate tumor-immune interactions and investigated their prognostic implications in patients with early (eBC) and metastatic (mBC) breast cancer. Methods: Blood samples were obtained before treatment from 140 and 64 patients with eBC and mBC, respectively. Plasma miRNA expression levels were assessed by qRT-PCR and expression was classified as high or low according to the median values. Results: A panel of four miRNAs (miR-19a, miR-20a, miR-126 and miR-155) could discriminate eBC from mBC (AUC 0.802, p < 0.001). In early disease, miR-10b (p = 0.022) and miR-155 (p = 0.005) expression was lower in relapsed (n = 46) compared to non-relapsed (n = 94) patients; miR-155 expression along with lymph node infiltration and tumor grade had increased ability to predict relapse (AUC = 0.775; p = 0.003). In addition, miR-10b (p = 0.015), miR-19a (p = 0.003), miR-20a (p = 0.012), miR-126 (p = 0.001) and miR-155 (p < 0.001) expression levels were lower in patients with early relapse (relapse at ≤2 years). MiR-155 and miR-19a had the highest performance in discriminating early relapse (AUC 0.855; p < 0.001 and AUC = 0.729; p = 0.003, respectively), whereas, combined mir-155 and miR-19a expression further increased the accuracy of prediction (AUC = 0.867; p < 0.001). In eBC, the number of infiltrated lymph nodes and low miR-10b independently predicted for shorter DFS (p = 0.001 and p = 0.03, respectively) and axillary lymph nodes for shorter OS (p = 0.003). In the triple negative subgroup, low miR-155 strongly predicted for shorter DFS (p = 0.037). In mBC, recurrent disease and low miR-10b expression independently predicted for shorter PFS (p = 0.001 and p = 0.017, respectively), whereas performance status of 2 independently predicted for shorter OS (p = 0.03). Conclusions: Deregulated expression of circulating miRNAs involved in tumor-immune interactions can discriminate disease status in BC and independently predicts for patients’ outcome in early and mBC. Our results further support the notion that circulating miRNAs represent a useful prognostic tool in patients with BC.

Association of MicroRNA Expression and BRAFV600E Mutation with Recurrence of Thyroid Cancer.

Many miRNAs and cancer-related mutations have been proposed as promising molecular markers of papillary thyroid carcinoma (PTC). However, there are limited data on the correlation between miRNA expression, BRAFV600E mutation, and PTC recurrence. Therefore, to evaluate the potential of BRAFV600E mutation and five selected miRNAs (-146b, -222, -21, -221, -181b) in predicting PTC recurrence, these molecular markers were analyzed in 400 formalin-fixed, paraffin-embedded PTC tissue specimens. The expression levels of miRNAs were measured using qRT-PCR. It was demonstrated that expression levels of all analyzed miRNAs are significantly higher in recurrent PTC than in non-recurrent PTC (p < 0.05). Moreover, higher expression levels of miR-146b, miR-222, miR-21, and miR-221 were associated with other clinicopathologic features of PTC, such as tumor size and lymph node metastases at initial surgery (p < 0.05). No significant differences in the frequency of BRAFV600E mutation in recurrent PTC and non-recurrent PTC were determined. Our results suggest that miRNA expression profile differs in PTC that is prone to recurrence when compared to PTC that does not reoccur after the initial surgery while BRAFV600E mutation frequency does not reflect the PTC recurrence status. However, the prognostic value of the analyzed miRNAs is rather limited in individual cases as the pattern of miRNA expression is highly overlapping between recurrent and non-recurrent PTC.

MiR-10b regulated by Twist maintains myofibroblasts activities in oral submucous fibrosis

Oral submucous fibrosis (OSF) is an oral precancerous disorder associated with the habit of areca nut chewing. MiR-10b has been shown to be upregulated in the oral cancer cells and induced by Twist. Our previous work has revealed that Twist participated in the pathogenesis of OSF and therefore we aimed to investigate whether Twist/miR-10b axis was involved in the activation of myofibroblast in the oral cavity. The expression levels of miR-10b in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) were examined. Besides, the expression of miR-10b was determined in fBMFs following knockdown of Twist or in BMFs after arecoline stimulation. Myofibroblast activities, including collagen gel contraction, migration and wound healing abilities, as well as the expression of α-SMA were measured in fBMFs treated with miR-10b inhibitor. Last, we investigated whether the effect of Twist overexpression could be reversed by suppression of miR-10b. MiR-10b expression was overexpressed in both OSF tissues and fBMFs. The silence of Twist resulted in the downregulation of miR-10b in fBMFs and arecoline treatment led to an increase of miR-10b in a dose-dependent manner. Inhibition of miR-10b ameliorated the activation of myofibroblasts and the expression of α-SMA. Moreover, we demonstrated that suppression of miR-10b hindered the increased collagen gel contraction caused by Twist overexpression. MiR-10b upregulation in OSF may be due to the stimulation of areca nut, leading to elevated myofibroblast activation. Our findings showed that the areca nut-induced expression of miR-10b was under the regulation of Twist and inhibition of miR-10b may provide a direction for treatment of OSF.

Exosomal miR-29b from cancer-associated fibroblasts inhibits the migration and invasion of hepatocellular carcinoma cells.

BackgroundHepatocellular carcinoma (HCC) is often characterized by poor prognosis, high invasiveness and chemotherapeutic resistance, and its migration is strongly dependent on the specific tumor microenvironment. Fibroblasts, such as cancer-associated stromal fibroblasts (CAFs), are the main supporting cells in the tumor microenvironment. Thus, an understanding of how these cells communicate is required for HCC treatment.MethodsCAFs and paracancerous fibroblasts (PAFs) were isolated from patients’ surgical specimens, followed by exosome isolation and miRNA sequencing. The expression levels of miR-29b in different cell groups were detected by qPCR assay. Cell transfection with exogenous miRNAs was used to study whether the stromal cells could transfer miRNAs to HCC cells. Based on the preliminary results, a miR-29b mimic, inhibitor or miR-nonspecific mimic (miR-NSM) was further transfected into HepG2 and Huh7 cells prior to scratch wound healing and cell invasion experiments. Finally, the transfected cells were stained with Hoechst 33348.ResultsThe direct transfer of miR-29b from CAFs to HCC cells through an exosome was observed in this study. DNA methyltransferase 3b (DNMT3b) expression was directly inhibited by miR-29b, while metastasis suppressor 1 (MTSS1) expression was upregulated in HCC cells. Such changes further induced growth arrest and inhibited HCC cell invasion.ConclusionsExosomal miR-29b from CAFs can play a crucial role in the development, progression and metastasis of HCC. By functioning as a tumor suppressor that targets DNMT3b, miR-29b may serve as a potential therapeutic agent.

The miRNA-29b Is Downregulated in Placenta During Gestational Diabetes Mellitus and May Alter Placenta Development by Regulating Trophoblast Migration and Invasion Through a HIF3A-Dependent Mechanism.

Gestational diabetes mellitus (GDM) is a disease that changes the function of microvascular of placenta. MicroRNA (miRNA) expression in placenta may contribute to the pathogenesis of GDM. Here, we evaluate the role and function of miR-29b in the development of GDM. This study discovered that miR-29b expression was lower in placentas derived from patients with GDM than that in control placentas. MiR-29b over-expression inhibited cell growth and migration, and miR-29b knockdown promoted cell migration. Then we predicted and confirmed that HIF3A was a direct target of miR-29b with two specific binding sites at the recognition sequences of miR-29b in 3′-UTR of HIF3A mRNA, which was negatively correlated with miR-29b expression level. The up-regulation of HIF3A partially antagonized the inhibitory effect of miR-29b over-expression on cell growth and migration. The enhancement of cell migration induced by miR-29b knockdown was attenuated by down-regulating HIF3A. These results imply that down-regulation of miR-29b may be related with the development of GDM partially via increasing the expression of HIF3A, which may provide a new insight for the mechanism of GDM.

Evaluation of miR-34b/c polymorphisms to the risk of ischemic stroke.

Polymorphisms in microRNA genes are related to the risk of ischemic stroke, but the association between miR-34b/c polymorphisms and the risk of ischemic stroke has not been reported. MiR-34b/c rs2187473 and rs4938723 polymorphisms were genotyped by Snapshot assay among 495 controls and 492 ischemic stroke patients. Expression levels of miR-34b and miR-34c were quantified by real-time PCR. Transcriptional activity of miR-34b/c promoter was measured by luciferase reporter assay. Rs4938723 was associated with an increased risk of ischemic stroke in our study (CC versus TT: OR = 2.34, 95% CI = 1.47-3.72, P = 0.001; C versus T: OR = 1.37, 95% CI = 1.12-1.68, P = 0.002; CC versus TT + TC: OR = 2.12, 95% CI = 1.37-3.29, P = 0.001). The expression levels of miR-34b and miR-34c were significantly downregulated in cases by contrast with controls (P < 0.05). Further analysis demonstrated that the expression levels of miR-34b and miR-34c were also downregulated in the individuals carrying rs4938723 CC genotype by contrast with that carrying TT + TC genotypes (P < 0.05). The result of luciferase reporter assay showed that rs4938723C allele decreased the transcriptional activity of miR-34b/c promoter compared with rs4938723 T allele. Our study showed a positive relation between the miR-34b/c rs4938723 polymorphism and the risk of ischemic stroke, which indicated that rs4938723 may be used for ischemic stroke prediction or therapy in the future.

Tissue microRNAs in non-small cell lung cancer detected with a new kind of liquid bead array detection system

BackgroundCommonly used miRNA detection methods cannot be applied for high-throughput analyses. However, this study was aimed to performed a liquid bead array detection system (LBAS) to detect tissue 6 miRNAs in non-small cell lung cancer (NSCLC).MethodsIn this study, evaluation of LBAS was performed to observe the precision, specificity, limitation and stability. Then, a total of 52 primary NSCLC patients who received resection operation without preoperative radiotherapy and chemotherapy between June 2013 and March 2014 were selected, and then the total RNA of the tissues were extracted. We prepared six NSCLC-related miRNAs for LBAS. After optimization and evaluation, LBAS was verified by detecting the relative expression levels of 6 microRNAs in the pathological tissues and corresponding normal tissues of 52 NSCLC patients.ResultsThe results of evaluation of LBAS showed that the Mean Fluorescence Intensity (MFI) of the reaction only added with chimeric probes and beads showed no significant change after 180 days (P > 0.05). And the intra-assay Coefficient of Variation (CV) was between 1.57 and 3.5%, while the inter-assay CV was between 4.24 and 11.27%, indicating this system was ideal for diagnostic reagents. In addition, only the beads corresponding to the additional miRNAs showed high MFIs from 8426 to 18,769, whereas the fluorescence values of the other beads were under background levels (MFIs = 20 to 55) in each reaction, indicating no cross reactivity among the miRNAs. The limit of detection of miR-21, miR-210, miR-125b, miR-155, miR-375, and miR-31 were 5.27, 1.39, 1.85, 2.01, 1.34, and 2.73 amol/μL, respectively, showing that the lowest detection limit of miRNA by this system was under pM level. Then, the relative expression levels of miR-21, miR-210, miR-125b, miR-155, miR-375, and miR-31 by using this system were significantly correlated with NSCLC (P < 0.05). And the results of AUC method indicated that specific of the LBAS system was 94.2%.ConclusionsOur findings suggest that LBAS was simple, high-throughput, and freely combined with absolute quantification. Thus, this system could be applied for tumor miRNAs detection.

MicroRNA-29b promotes cell sensitivity to Temozolomide by targeting STAT3 in glioma.

This study aimed to explore the effects of microRNA-29b (miR-29b) on chemoresistance of glioma and to examine the underlying mechanisms. MiR-29b expression in glioma tissues and cell lines was analyzed by quantitative real time-polymerase chain reaction (qRT-PCR). The cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was analyzed by Annexin V-Fluorescein isothiocyanate (FITC) assay. The relationship between miR-29b and signal transducer and activator of transcription 3 (STAT3) was examined by the Dual-Luciferase reporter gene assay. The levels of cleaved caspase-3, Bax, Bcl-2, and STAT3 were detected by Western blotting assay. The expression of miR-29b was downregulated in glioma tissues compared to normal brain tissue. In addition, the expression level of miR-29b was lower in glioma tissues from patients at late stages (III and IV) compared with early stages (I and II). Besides, miR-29b expression was significantly lower in LN229, U87MG, and U251 cells compared to normal human astrocytes (NHA) cells. Moreover, our results showed that miR-29b expression in Temozolomide (TMZ)-resistance cell lines U251/TMZ and U87MG/TMZ was markedly lower than that of TMZ-sensitivity cell lines U251 and U87MG. The protein levels of STAT3 and the phosphorylation of STAT3 were increased in U251/TMZ and U87MG/TMZ compared to U251 and U87MG. When the expression of miR-29b was repressed, cell viability was increased. Meanwhile, cell apoptosis was reduced, the protein levels of cleaved caspase-3 and (Bcl-2 Associated X Protein) Bax were decreased, whereas the protein level of B-cell lymphoma 2 (Bcl-2) was increased. Moreover, the effects of miR-29b knockdown on the cell growth and apoptosis in U251 and U87MG cells were markedly attenuated by knockdown of STAT3. In TMZ-resistant U251/TMZ and U87MG/TMZ cells, transfection with miR-29b decreased cell growth, promoted apoptotic cell death, elevated the protein levels of cleaved caspase-3, and Bax protein, while downregulated Bcl-2 protein. As expected, the effect of miR-29b upregulation on cell growth and apoptosis of TMZ-resistant glioma cells was reversed by STAT3 overexpression. The results from the Luciferase assay demonstrated miR-29b modulated STAT3 expression by directly bound with 3'-Untranslated Region (3'-UTR). MiR-29b enhances the cell sensitivity to TMZ by inhibiting STAT3 in glioma. Our study might provide a novel target for treating TMZ-resistant glioma.

Hypomethylation of nerve growth factor (NGF) promotes binding of C/EBP\u03b1 and contributes to inflammatory hyperalgesia in rats

BackgroundChronic pain usually accompanied by tissue damage and inflammation. However, the pathogenesis of chronic pain remains unclear.MethodsWe investigated the role of nerve growth factor (NGF) in chronic inflammatory pain induced by complete Freund’s adjuvant (CFA), explored the methylation status of CpG islands in the promoter region of the NGF gene, and clarified the function and mechanism of C/EBPα-NGF signaling pathway from epigenetic perspective in the chronic inflammatory pain model.ResultsCFA induced significant hyperalgesia and continuous upregulation of NGF mRNA and protein levels in the L4–6 dorsal root ganglions (DRGs) in rats. Hypomethylation of CpG islands occurred in the NGF gene promoter region after CFA treatment. At the same time, the miR-29b expression level was significantly increased, while the DNA methyltransferase 3b (DNMT3b) level reduced significantly. Moreover, CFA treatment promoted binding of C/EBPα to the NGF gene promoter region and C/EBPα siRNA treatment obviously decreased expression of NGF levels and also alleviate inflammatory hyperalgesia significantly in rats.ConclusionCollectively, the results indicated that CFA leads to the upregulation of miR-29b level, which represses the expression of DNMT3b, enhances the demethylation of the NGF gene promoter region, and promotes the binding of C/EBPα with the NGF gene promoter, thus results in the upregulation of NGF gene expression and maintenance of chronic inflammatory pain.

MiR-26b inhibits isoproterenol-induced cardiac fibrosis via the Keap1/Nrf2 signaling pathway.

A critical event in cardiac fibrosis is the transformation of cardiac fibroblasts (CFs) into myofibroblasts. MicroRNAs (miRNAs) have been reported to be critical regulators in the development of cardiac fibrosis. However, the underlying molecular mechanisms of action of miRNA (miR)-26b in cardiac fibrosis have not yet been extensively studied. In the present study, the expression levels of miR-26b were downregulated in isoproterenol (ISO)-treated cardiac tissues and CFs. Moreover, miR-26b overexpression inhibited the cell viability of ISO-treated CFs and decreased the protein levels of collagen I and α-smooth muscle actin (α-SMA). Furthermore, bioinformatics analysis and dual luciferase reporter assays indicated that Kelch-like ECH-associated protein 1 (Keap1) was the target of miR-26b, and that its expression levels were decreased in miR-26b-treated cells. In addition, Keap1 overexpression reversed the inhibitory effects of miR-26b on ISO-induced cardiac fibrosis, as demonstrated by cell viability, and the upregulation of collagen I and α-SMA expression levels. Furthermore, inhibition of Keap1 expression led to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), which induced the transcriptional activation of antioxidant/detoxifying proteins in order to protect against cardiac fibrosis. Taken together, the data demonstrated that miR-26b attenuated ISO-induced cardiac fibrosis via the Keap-mediated activation of Nrf2.

Evaluation of miR-181b and miR-126-5p expression levels in T2DM patients compared to healthy individuals: Relationship with NF-κB gene expression

BackgroundType 2 diabetes mellitus (T2DM) is a progressive metabolic disorder whose prevalence is rising very fast across the world. Diagnosis of this disease in early stages (pre-diabetic stage) plays an important role in reducing mortality associated with this disorder. miRNAs, as key players in the pathogenesis of T2DM, have been investigated in several studies. Furthermore, their expression profile changes in the early stages of diabetes mellitus in body fluids such as serum, peripheral blood, and peripheral blood mononuclear cell (PBMC) have been studied. Due to their high stability and the presence of non-invasive sensitive methods for their measurement, such as real-time PCR, they can be used for early diagnosis of T2DM as a biomarker. In this experimental study, the expression levels of miR-181b, miR-126-5p, and NF-κB were measured in patients with T2DM, pre-diabetic subjects, and healthy controls in a Yazd population. Material and methodNinety asymptomatic subjects including 30 T2DM, 30 pre-diabetic, and 30 healthy subjects (diagnosis based on WHO criteria) were included in this study. Real-time PCR was used to measure the expression levels of miR-181b and miR-126-5p. Moreover, the NF-κB expression level was also measured to determine its relationship with these two microRNAs. ResultIn this study, the expression level of miR-181b and miR-126-p decreased gradually in pre-diabetic as well as T2DM subjects compared to healthy controls. Furthermore, our study showed a significant negative correlation between these two miRNAs and NF-κB for the first time. ConclusionThese results introduce these anti-inflammatory miRNAs as powerful tools for early diagnosis of T2DM.