Mechanism of miRNA-31 Regulating Wnt/ß-catenin Signaling Pathway by Targeting Satb2 in the Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells.

OBJECTIVE: To explore the expression of miR-31 and Satb2 gene in the serum of postmenopausal women with osteoporosis (OP). METHODS: 97 postmenopausal women with OP and 100 healthy women were selected as research subjects. MSCs were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. Bone marrow-derived mesenchymal stem cells (BMSCs) were isolated, identified and transfected, and then quantified by alkaline phosphatase (ALP) levels. The expression levels of miR-31 and Satb2 gene mRNA were determined by qRT-PCR. The proteins of RUNX2, OCN and BMP and Wnt/ß-catenin pathway-related proteins (GSK-3, Frizzled 1, Lrp5, Lrp6 and ß-catenin) were tested by Western blotting. RESULTS: In the OP group, the relative expression of miR-31 was 3.61±0.54, significantly higher than that (1.75±0.27) in the healthy control group (t=9.422, P<0.001). The relative expression of mRNA of Satb2 gene was 0.86±0.12, significantly lower than that (1.35±0.21) in the healthy control group (t=5.897, P<0.001). CONCLUSIONS: The increase in miR-31 expression can down-regulate the Wnt/ß-catenin pathway by targeting the expression of Satb2 gene, thereby inhibiting the osteogenic differentiation of BMSCs. This provides an important reference for further understanding the mechanism of OP and identifying targets for early diagnosis and treatment.

MicroRNA-31 mediated by interferon regulatory factor 7 signaling facilitates control of Mycobacterium tuberculosis infection.

Tuberculosis (TB) induced by Mycobacterium tuberculosis (M. tuberculosis) infection remains a global most deadly infectious disease. While development of more effective TB vaccines and therapeutics relies on identifications of true biomarkers designating an immune protection against M. tuberculosis infection, exact protective immune components against M. tuberculosis infection remain largely unidentified. We previously found that severe TB induced remarkable up-regulation of interferon regulatory factor 7 (IRF7) and IRF7-related gene signatures, implicating that some unknown downstream molecules in IRF7 signaling cascades may determine the M. tuberculosis infection outcomes and serve as a protective immune component against M. tuberculosis infection. Indeed, here, we observe that genetic ablation of IRF7 leads to more severe lung pathology, increased M. tuberculosis burdens, impaired differentiation of effector/memory T subsets, and extensively elevated expression of pro-inflammatory cytokines in lungs. Importantly, IRF7 is vital for sustaining expression of PD-1/PD-L1 and PD-1/PD-L1-modulated miRNA-31. Moreover, interventions of miRNA-31 expressions via administration of miRNA-31 agomir reduces lung pathology and bacilli burdens via inducing up-regulation of gene sets involved in biological processes of defense response or cellular and chemical homeostasis in lungs. Thus, this study uncovers previously unrecognized importance and mechanisms of IRF7-mediated miRNA-31 as a protective immune component against M. tuberculosis infection.

Fabrication and Evaluation of Celecoxib Oral Oleogel to Reduce the Inflammation of Ulcerative Colitis.

Oleogel consists of hydrophobic solvent and an oleogelator. In this study, attempts were made to study the influence of Celecoxib solubility, concentration and dispersability on its release, absorption, and biological performance. Oleogels were prepared to study the formulation variables on its stability and release. Castor oil was selected as the oil and the oleogelator concentration was 4.5% w/w. F3 revealed the highest release and stability compared to other formulae. The percent permeated across the rat intestine showed a 7.5-fold increase over free Celecoxib, and its lifetime was found to be greater than 18 months. The efficacy of free Celecoxib and oleogel formulae to treat rats with ulcerative colitis was done via the induction of ulcerative colitis (UC) through administration of 5% dextran sodium sulphate (DSS). Celecoxib besides its formulae significantly reduced the release of Leucine rich 2 glycoprotein (LRG), Myeloperoxidase (MPO), Tumor necrosis factor-α (TNF-α), proinflammatory cytokine expression, High mobility group box 1 (HMGB1), Nuclear factor kappa B (NF-ΚB), Trefoil Factor 3 (TFF3), Metalloproteinase-3 (MMP3), and miRNA31. Moreover, F3 significantly increased the colonic cAMP in DSS treated rats and reduced the intestinal inflammation beside healing of mucosa and restitution of the epithelium of the gastrointestinal tract.

NF-κB-responsive miRNA-31-5p elicits endothelial dysfunction associated with preeclampsia via down-regulation of endothelial nitric-oxide synthase.

Inflammatory cytokines, including tumor necrosis factor-α (TNFα), were elevated in patients with cardiovascular diseases and are also considered as crucial factors in the pathogenesis of preeclampsia; however, the underlying pathogenic mechanism has not been clearly elucidated. This study provides novel evidence that TNFα leads to endothelial dysfunction associated with hypertension and vascular remodeling in preeclampsia through down-regulation of endothelial nitric-oxide synthase (eNOS) by NF-κB-dependent biogenesis of microRNA (miR)-31-5p, which targets eNOS mRNA. In this study, we found that miR-31-5p was up-regulated in sera from patients with preeclampsia and in human endothelial cells treated with TNFα. TNFα-mediated induction of miR-31-5p was blocked by an NF-κB inhibitor and NF-κB p65 knockdown but not by mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase inhibitors, indicating that NF-κB is essential for biogenesis of miR-31-5p. The treatment of human endothelial cells with TNFα or miR-31-5p mimics decreased endothelial nitric-oxide synthase (eNOS) mRNA stability without affecting eNOS promoter activity, resulting in inhibition of eNOS expression and NO/cGMP production through blocking of the functional activity of the eNOS mRNA 3'-UTR. Moreover, TNFα and miR-31-5p mimic evoked endothelial dysfunction associated with defects in angiogenesis, trophoblastic invasion, and vasorelaxation in an ex vivo cultured model of human placental arterial vessels, which are typical features of preeclampsia. These results suggest that NF-κB-responsive miR-31-5p elicits endothelial dysfunction, hypertension, and vascular remodeling via post-transcriptional down-regulation of eNOS and is a molecular risk factor in the pathogenesis and development of preeclampsia.

miRNA-31 over-expression improve synovial cells apoptosis induced by RA.

OBJECTIVE: The aim of this study was to evaluate the effects and mechanism of miRNA-31 in synovial cells apoptosis induced by RA. METHODS: The miRNA-31 gene expressions were extracted from synovial tissues of normal and RA patients by RT-PCR and H et E staining. The synovial cells of RA patients were isolated and randomly divided into Control, Blank and miRNA groups. The cell apoptosis of difference groups were measured by flow cytometry; the TNF-α and IL-1ß concentrations of difference groups were measured by Elisa assay; TLR4 and NF-κB proteins expressions were measured by WB assay and the correlation between TLR4 and miRNA-31 were evaluated by double luciferase target experiment. RESULTS: The miRNA-31 gene expression was significantly suppressed in RA tissues (p<0.001); Compared with control group, the cell apoptosis rate of miRNA group was significantly suppressed (p<0.001); TNF-α and IL-1ß concentrations were significantly down-regulation in culture fluid (p<0.001, respectively) and TLR4 and NF-κB proteins expressions were significantly depressed (p<0.001, respectively) in miRNA group. By double luciferase target experiment, the TLR4 was a target gene of miRNA-31. CONCLUSION: miRNA-31 is a key role in synovial cells apoptosis induced by RA (Fig. 7, Ref. 23).

miRNA-31 increases radiosensitivity through targeting STK40 in colorectal cancer cells.

OBJECTIVE: To propose and verify that miRNA-31 increases the radiosensitivity of colorectal cancer and explore its potential mechanism. METHOD: A bioinformatics analysis was performed to confirm that the expression of miRNA-31 was higher in colorectal cancer than in normal colorectal tissue. The expression of miRNA-31 was detected to verify the change in its expression in a radiotherapy-resistant cell line. Methylation was detected to explore the cause of the decrease in miRNA-31 expression. Overexpression or inhibition of miRNA-31 further confirmed the change in its expression in colorectal cancer cell lines. Bioinformatics methods were used to screen the downstream target genes and for experimental verification. A luciferase assay was performed to determine the miRNA-31 binding site in STK40. Overexpression or inhibition of STK40 in colorectal cancer cell lines further confirmed the change in STK40 expression in vitro. RESULTS: The bioinformatics results showed higher expression of miRNA-31 in tumors than in normal tissue, and miRNA-31 mainly participated in the pathway related to cell replication. Next, we observed the same phenomenon: miRNA-31 was expressed at higher levels in colorectal tumors than in normal colorectal tissue and its expression decreased in radiation-resistant cell lines after radiation, implying that miRNA-31 increased the radiosensitivity of colorectal cancer cell lines. No significant change in upstream methylation was observed. miRNA-31 regulated the radiosensitivity of colorectal cancer cell lines by inhibiting STK40. Notably, miRNA-31 played a role by binding to the 3' untranslated region of SK40. STK40 negatively regulated the radiosensitivity of colorectal cancer cells. CONCLUSIONS: miRNA-31 increases the radiosensitivity of colorectal cancer cells by targeting STK40; miRNA-31 and STK40 are expected to become potential biomarkers for increasing the sensitivity of tumor radiotherapy in clinical treatment.

Role of MicroRNA-31 (miR-31) in Breast Carcinoma Diagnosis and Prognosis.

BACKGROUND/AIM: Breast cancer (BC) is among the most widespread malignant tumors in women. In the current study, we evaluated the role of miR-31 in BC patients and its relation to the different prognostic, clinical, and pathological features. PATIENTS AND METHODS: MiR-31 levels were determined by RT-PCR in BC and adjacent normal breast tissues from 100 BC patients. BC diagnosis was established through histopathological examinations. The expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) receptor in all tumors was determined using immunohistochemistry. RESULTS: MiR-31 expression was reduced in BC tissues relative to adjacent healthy breast tissue (mean levels were 0.93 and 7.2, respectively). Also, the low expression of miR-31 in BC patients was significantly correlated with adverse clinical and pathological features such as: young patient's age, premenopausal status, infiltrative lobular carcinoma, ER and PR negative tumors, HER2 positive tumors, and advanced clinical stage. CONCLUSION: MiR-31 was expressed at low levels in BC tissues and correlated with adverse clinical and pathological features, and poor survival.

LncRNA GAS5 promotes spermidine­induced autophagy through the miRNA­31­5p/NAT8L axis in pulmonary artery endothelial cells of patients with CTEPH.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a leading cause of pulmonary hypertension. The present study investigated the mechanisms of long non­coding RNA growth arrest­specific transcript 5 (GAS5) on spermidine (SP)­induced autophagy. Pulmonary artery endothelial cells (PAECs) were collected from patients with CTEPH and the rat model. Immunofluorescence, Western blots, reverse transcription­quantitative polymerase chain reaction, bioinformatics, rapid amplification of cDNA ends assays, luciferase reporter assays, RNA­binding protein immunoprecipitation assays, GFP­LC3 adenoviruses, tfLC3 assays and transmission electron microscopy were performed. The results revealed that SP­induced autophagy increased GAS5 in PAECs. The upregulation of GAS5 enhanced and the downregulation of GAS5 reversed the roles of SP in PAECs. Furthermore, GAS5 promoted SP­induced autophagy in PAECs by targeting miRNA­31­5p. The miRNA­31­5p mimic suppressed and the inhibitor promoted SP­induced autophagy. Furthermore, N­Acetyltransferase 8 Like (NAT8L) was a target gene of miRNA­31­5p and knockdown of NAT8L inhibited the autophagic levels of PAECs. In vivo, SP treatment decreased miRNA­31­5p and increased NAT8L levels, which was reversed by the knockdown of GAS5. The downregulation of GAS5 abolished the stimulatory role of SP in PAECs of CTEPH rats. In conclusion, GAS5 promoted SP­induced autophagy through miRNA­31­5p/NAT8L signaling pathways in vitro and in vivo and GAS5 may be a promising molecular marker for therapies of CTEPH.1.

Clinical significance of the expression of miRNA-21, miRNA-31 and miRNA-let7 in patients with lung cancer.

OBJECTIVE: To explore the expression differences of miRNA-21, miRNA-31 and miRNA-let7 between lung cancer patient and healthy people, thereby providing reference for early diagnosis of lung cancer. METHOD: Real-time PCR was employed to determine the expression difference between lung cancer patients (50 cases) and healthy people (24 cases). The clinical data of lung cancer patients were analyzed to explore the correlation between clinicopathological characteristics and expression level of miRNA-21, miRNA-31, miRNA-let7. RESULTS: The relative expression levels of miRNA-21 and miRNA-31 in lung cancer group were obviously higher than those in healthy control group, and the relative expression level of miRNA-let7 in lung cancer group was slightly higher than that in healthy control group. Lung cancer patients with lymph node metastasis had higher expression level than those without lymph node metastasis. The ROC curve showed that the three miRNAs had clinical diagnosis efficiency for lung cancer, and the combined detection of the three miRNAs were more efficient in diagnosing lung cancer. Survival curve analysis suggested that the median survival times of patients in the miRNA-21 and miRNA-31 high expression groups were shorter than those in the low expression groups, and the median survival time of patients in miRNA-let7 high expression group was longer than that in the low expression group. CONCLUSION: Plasma miRNA-21, miRNA-31 and miRNA-let7 may be diagnostic marker for lung cancer.

miRNA-31-5p Mediates the Proliferation and Apoptosis of Human Spermatogonial Stem Cells via Targeting JAZF1 and Cyclin A2.

Several lines of evidence highlight the important application of human spermatogonial stem cells (SSCs) in translational medicine. The fate decisions of SSCs are mainly mediated by genetic and epigenetic factors. We have recently demonstrated that PAK1 regulates the proliferation, DNA synthesis, and early apoptosis of human SSCs through the PDK1/KDR/ZNF367 and ERK1/2 and AKT pathway. However, the underlying epigenetic mechanism of PAK1 in human SSCs remains unknown. In this study, we found that the level of miRNA-31-5p was elevated by PAK1 knockdown. CCK-8, PCNA, and 5-ethynyl-2'-deoxyuridine (EDU) assays revealed that miRNA-31-5p mimics inhibited cell proliferation and DNA synthesis of human SSCs. Annexin V/propidium iodide (PI) staining and flow cytometry showed that miRNA-31-5p increased the early and late apoptosis of human SSCs. Furthermore, JAZF1 was predicted and verified as a target of miRNA-31-5p, and the three-dimensional (3D) structure model of JAZF1 protein was illustrated. JAZF1 silencing led to a reduction of cell proliferation and DNA synthesis as well as an enhancement of the early and late apoptosis of human SSCs. Finally, miRNA-31-5p mimics decreased the level of cyclin A2 rather than cyclin D1 or cyclin E1, and JAZF1 knockdown led to the reduction of cyclin A2 in human SSCs. Collectively, miRNA-31-5p regulates the proliferation, DNA synthesis, and apoptosis of human SSCs by the PAK1-JAZF1-cyclin A2 pathway. This study thus offers a novel insight into the molecular mechanisms underlying the fate determinations of human SSCs and might provide novel targets for molecular therapy of male infertility.

MicroRNA-31 contributes to colorectal cancer development by targeting factor inhibiting HIF-1α (FIH-1).

The molecular mechanisms underlying colorectal cancer (CRC) tumorigenesis remain incompletely understood, partially contributing to the mortality of CRC. Advances in identification of novel mechanisms are therefore in an urgent need to fill the gap of our knowledge in CRC development. Here, we performed both in vitro and in vivo experiments along with in silico analysis to identify a new regulatory circuit that stimulated CRC tumorigenesis. In this report, we, for the first time, analyzed the correlation of FIH-1 level with clinicopathological features of CRC. The finding that FIH-1 was not only significantly decreased in tumor tissue as compared with the adjacent normal tissue but also was significantly correlated with tumor T stage status, indicated the role of FIH-1 as a tumor suppressor in CRC development. Moreover, we found the expression of miR-31, a short non-coding RNA which played a critical role in CRC development, was negatively correlated with FIH-1 expression in CRC samples and cell lines. Together with the result from luciferase report assay, it was demonstrated that miR-31 could directly regulate FIH-1 expression in CRC. This miR-31/FIH-1 nexus was further shown to control cell proliferation, migration and invasion in vitro and to control tumor growth in vivo. Additionally, correlation of the miR-31 expression with clinicopathologic features in CRC samples was examined in support of the driving role of newly identified miR-31/FIH-1 nexus in CRC tumorigenesis. These findings highlight the critical role of miR-31/FIH-1 nexus in CRC and reveal the contribution of miR-31 to CRC development by targeting FIH-1.

Expression of serum miR-31 in colorectal cancer patients and its effect on cell proliferation and ;apoptosis / 中国癌症杂志

Background and purpose:miRNA plays important roles in tumorigenesis. It has been reported that many kinds of serum miRNA serve as markers for tumor diagnosis and screening. This study aimed to detect the expression of serum miRNA-31 (miR-31) in colorectal cancer patients and to explore the effect of miR-31 on cell proliferation, apoptosis and cell cycle distribution. Methods: The expressions of miR-31 in 40 cases of colorectal cancer serum and 35 cases of the healthy control were examined by real-time lfuorescent quantitative polymerase chain reaction (RTFQ-PCR). The correlation between miR-31 expression and clinicopathological features of colorectal cancer (including age, gender, depth of inifltration, lymph node metastasis, clinical stage) were further analyzed. The miR-31 mimics, inhibitor and miR-control (negative control) were transfected into HCT116 cells. The effect of miR-31 on cell proliferation was evaluated by CCK-8 method. Flow cytometry was used to examine the change of cell apoptosis and cell cycle. Results:Relative expression of serum miR-31 was signiifcantly increased in cancer patients compared with healthy controls (P<0.01). Expression of serum miR-31 was higher in poorly differentiated carcinoma than that in well or moderately differentiated carcinoma (P<0.05). No correlation was found between serum miR-31 expression and other clinicopathological variables. CCK-8 assay showed that after transfection with miR-31 mimics, the cell proliferation was increased, compared with miR-31 inhibitor and negative control group. Meantime, the apoptotic cell number was signiifcantly decreased, particularly in late apoptosis. The cell number of G1 stage was remarkably increased in miR-31 inhibitor group, compared with miR-31mimics and negative control group. Conclusion:The expression of serum miR-31 is higher in colorectal cancer. miR-31 can promote cell proliferation and inhibit the apoptosis of HCT116 cells. It might be a potential biomarker for colorectal cancer.

Expression of miRNA-31 in serum of children with airway allergic disease / 国际儿科学杂志

Objective To examine miRNA-31 expression in serum of children with airway allergic disease (asthma and allergic rhinitis) and determine the correlation with CD44.Methods The expression of miR-NA-31 and CD44 in serum of 26 children with acute asthma and allergic rhinitis and non-acute phase and 36 normal control children were determined by real-time reverse transcriptase polymerase chain reaction.Results The miRNA-31 was significantly overexpressed in serum of children with acute asthma and allergic rhinitis compared with non-acute phase and normal control children and significantly correlated with CD44.Conclusion The correlation between miRNA-31 and CD44, the aberrant expression of miRNA-31 may account for the occurrence and development of airway allergic diseases.