Expression Levels Of miR-146a Research Articles

Circulating miR-181a as a Potential Novel Biomarker for Diagnosis of Acute Myocardial Infarction

Background: In this study, we tested the hypothesis that miR-181a levels increase during acute myocardial infarction. We investigated circulating miR-181a as a potential novel biomarker for early diagnosis of acute myocardial infarction (AMI). Methods: From June 2014 to June 2016, 120 consecutive eligible patients with AMI (n = 60) or unstable angina (UA; n = 60) and 60 control subjects were enrolled. Plasma miR-181a levels were determined by quantitative reverse transcriptase-polymerase chain reaction. Results: Circulating miR-181a expression levels detected immediately after admission were higher in the AMI group than in the UA and control groups. Relative miR-181a levels in AMI patients were positively correlated with the concentrations of the creatine kinase-MB fraction and cardiac troponin I. Correlation analysis showed that plasma miR-181a was positively correlated with coronary Gensini score (r = 0.573, P < 0.05) and negatively correlated with left ventricular ejection fraction (r = -0.489, P < 0.05). Receiver operating characteristic curve analyses showed that plasma miR-181a was of significant diagnostic value for AMI (AUC, 0.834; 95% CI, 0.756-0.912, P < 0.05). Conclusion: Circulating miR-181a levels in patients with AMI were significantly changed in a time-dependent manner, indicating the value of plasma miR-181a as a novel biomarker for diagnosing AMI.

Impaired regulatory T cell function in autoimmune diseases: are microRNAs the culprits?

Impaired regulatory T cell function in autoimmune diseases: are microRNAs the culprits?

Granulocyte Colony-Stimulating Factor Inhibits CXCR4/SDF-1 Signaling and Overcomes Stromal-Mediated Drug Resistance in Acute Myeloid Leukemia Via Mir-146a

CAG regimen was widely used in the clinical treatment of acute myeloid leukemia (AML). However, the mechanisms of G-CSF in the CAG regimen remain unknown. For the purpose of better elucidating the function of G-CSF, we evidenced that G-CSF could enhance HL60 and primary leukemia cells proliferation in vitro. Meanwhile, transwell migration experiments demonstrated that G-CSF, similar as the CXCR4 antagonist AMD3100, could remarkably inhibit the chemotaxis of HL60 cells induced by the chemokines released from marrow stromal cells (Fig 1). qRT-PCR and Western blotting results showed that the expression of miR-146a was up-regulated after G-CSF treatment, while the expression levels of CXCR4 and smad4 were down-regulated (Fig 2). CXCR4 and Smad4 expression levels in miR-146a over expression lentivirus infected HL60 cells were significantly decreased, which manifested the direct or indirect targeted relationship between miR-146a and CXCR4/Smad4. Our qRT-PCR and Western blotting results also showed an involvement of NF-KB in G-CSF induced up-regulation of miR-146a in AML cells. G-CSF activated NF-KB in the cells. Activated NF-KB induced the up-regulation of miR-146a expression. Sanguinarine, an inhibitor of NF-KB significantly inhibited miR-146a expression. We further demonstrated the involvement of NF-KB in the regulation of G-CSF in CXCR4 and Smad4 expression (Fig 3). Our study demonstrated that G-CSF not only could induce resting leukemia cells into proliferation cell cycle, but also could inhibit chemotaxis of leukemia cells. We elucidated the mechanism of G-CSF/NF-KB/miR-146a/CXCR4 signaling pathway in CAG treatment of AML. The expression levels of MiR-146a /CXCR4 /Smad4 may be selected as the clinical markers for CAG protocol choosing. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Evaluation of MicroRNA‐146a and Its Targets in Gingival Tissues of Patients With Chronic Periodontitis

MicroRNAs (miRNAs) are a group of small non-coding RNAs that play an important role in the regulation of gene expression. miRNA-146a (miR-146a), a member of the miR-146 family, is involved in the control of inflammation. Periodontitis is a set of chronic inflammatory disorders of the tissues surrounding the teeth that lead to the breakdown of alveolar bone and tooth loss. In this study, expression levels of miR-146a and its targets, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, are evaluated in human patients with chronic periodontitis (CP). The study population consisted of 10 healthy controls and 20 individuals with CP. For each participant, clinical parameters including probing depth and clinical attachment level were measured, and a gingival tissue sample was collected. Levels of miR-146a, TNF-α, IL-1β, and IL-6 were quantified using real-time polymerase chain reaction. Levels of miR-146a were significantly higher in patients with CP (P <0.001). There was a positive correlation between levels of miR-146a and clinical parameters (P <0.05). Elevated miR-146a was accompanied by a significant reduction in TNF-α and IL-6 (P <0.001). Patients with CP had higher levels of miR-146a than healthy individuals, accompanied by reduced levels of TNF-α and IL-6. A positive relationship between miR-146a levels and clinical parameters suggests a pathophysiologic role of miR-146a in CP.

Association between microRNA polymorphisms and chronic pancreatitis.

MicroRNAs play important roles in the development and progression of many human diseases. mir-146a could significantly suppress the induction of proinflammatory cytokines IL-1β, IL-6, TNF-α, NF-κB and chemokine MCP-1, which might play important roles in chronic pancreatitis. This study was conducted to evaluate the association between mir-146a rs2910164, a functional polymorphism in the pre-mir-146a, and chronic pancreatitis risk. The rs2910164 genotypes were determined in 165 patients with chronic pancreatitis and 200 healthy controls who were frequency matched for age and gender. One single nucleotide polymorphism (rs2910164) was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RLFP). The frequency of individuals who carried [G] allele was significantly higher in cases (62.7%) than in controls (53.7%, p=0.015), which resulted in a statistically significant pathogenic effect associated with this variant allele (OR: 1.448, CI: 1.076-1.950; p=0.015). The GC and GG genotypes showed strong and significant increased risk for complication of chronic pancreatitis (OR=3.668, 95%CI=1.233-10.916, p=0.019; OR=5.667, 95%CI=1.852-17.336, p=0.002). The individuals carrying G allele confer a lower expression level of mature mir-146a. These findings suggest that the mir-146a rs2910164 may contribute to genetic susceptibility to chronic pancreatitis, and that mir-146a might be involved in chronic pancreatitis development.

A pilot study on the relationship between miR-181a and RGCs in retinal ischemia-reperfusion injury model

Background Retinal ischemia-reperfusion (RIR) injury is a common pathologic change.Its mechanism has not been identified. Objective This study was to investigate the relationship of microRNA-181a (miR-181a), tumor necrosis factor-α (TNF-α) and retinal ganglial cells (RGCs) in RIR injury. Methods RIR models were induced in 68 rats, then the rats were randomly divided into control group and RIR groups, including 0 hour group, 24-hour group and 72-hour group by random number table.Predicted target gene TNF-α was chosen, according to MiRanda, Targetscan and miRBase databases.Immunofluorescent labeling, Western blot and quantitative real-time PCR were used to identify the expression levels of miR-181a, TNF-α and RGCs.Immunofluorescent labeling of RGCs in retinal flat mounts was analyzed for RGCs counts. Results Compared with the control group, RGCs densitiy was obviously decreased in 24-hour and 72-hour RIR groups (P<0.001). The expression level of mir-181a significantly decreased with reperfusion time in the RIR groups (P<0.05). Futhermore, the expression level of miR-181a was positively correlated with RGCs numbers (r=0.995, P=0.005). TNF-α and miR-181a were mainly located in inner layers of retina.As opposed to the changes in RGCs numbers and miR-181a expression, TNF-α in 24-hour group was obviously higher than that of the 0-hour group, though there was no statistical significance in overall correlation analysis. Conclusions In RIR, miR-181a may be involved in regulating RGCs apoptosis.TNF-α may be a target gene of miR-181a.Interventions within 24 hours after reperfusion might be critical.Further study of miR-181a may help to explore new molecular targets for neuroprotection treatment. Key words: Retinal ischemia-reperfusion; Retinal ganglion cells; Tumor necrosis factor-α; microRNA-181a

Polymicrobial infection alter inflammatory microRNA in rat salivary glands during periodontal disease

Periodontal disease initiated by subgingival pathogens is linked with diminished secretion of saliva, and implies pathogenic bacteria dissemination to or affects secondary sites such as the salivary glands. MicroRNAs activated in response to bacteria may modulate immune responses against pathogens. Therefore, Sprague–Dawley rats were infected by oral lavage consisting of polymicrobial inocula, namely Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, or sham-infected for 12 weeks (n = 6). We quantified inflammatory miRNA expression levels of miRNA-132, miR-146a, and miR-155 at secondary sites to the primary infection of the gingiva, including submandibular salivary glands, lacrimal glands, and pancreas. The presence of bacteria was detected in situ at secondary sites. Infected rat gingiva showed increased relative expression of miR-155. In contrast, miRNA-155 expression was decreased in submandibular salivary glands, along with positive identification of P. gingivalis in 2/6 and T. denticola in 1/6 rat salivary glands. Furthermore, miRNA-132 and miRNA-146a were significantly decreased in the pancreas of infected rats. This study is the first to show primary periodontal infections can alter miRNA profiles in secondary sites such as the salivary gland and pancreas. Whether these alterations contribute to pathologies of salivary glands in Sjögren's syndrome or of pancreas in diabetes warrants further investigation.

Abnormal expression and clinical significance of miR-146a in the peripheral blood mononuclear cells of patients with type 2 diabetic peripheral neuropathy

Objective To investigate the expression levels of miR-146a in the peripheral blood mononuclear cells(PBMCs)of patients with diabetic peripheral neuropathy(DPN)and their correlation with the disease severity. Methods The expression levels of miR-146a in the PBMCs were measured by using real time PCR in 62 patients with type 2 diabetes(32 patients without DPN, 30 with DPN)and 33 healthy individuals. The correlation of miR-146a expression with clinical parameters was analyzed. Results The expression level of miR-146a in patients with type 2 diabetes mellitus was significantly lower than that in the healthy individuals((1.92±1.99 vs 4.15±1.56, P<0.05). Furthermore, the miR-146a level was significantly lowered in diabetic patients with DPN as compared with those without DPN(1.22±1.61 vs 2.51±2.00, P<0.05). The expression levels of miR-146a in patients with DPN were positively correlated with duration of diabetes, blood glucose, and Toronto clinical scoring system scores. Conclusion The decreased expression of miR-146a in patients with DPN is correlated with the severity of DPN.(Chin J Endocrinol Metab, 2015, 31: 748-751) Key words: Diabetes mellitus, type 2; miR-146a; Diabetic peripheral neuropathy

MicroRNA-141 regulates the expression level of ICAM-1 on endothelium to decrease myocardial ischemia-reperfusion injury.

A growing number of studies have suggested microRNAs (miRNAs) are involved in the modulation of myocardial ischemia-reperfusion (MI/R) injury; however, the role of endogenous miRNAs targeting endothelial cells (ECs) and its interaction with ICAM-1 in the setting of MI/R remain poorly understood. Our microarray results showed that miR-146a, miR-146b-5p, miR-155*, miR-155, miR-497, and miR-451 were significantly upregulated, whereas, miR-141 and miR-564 were significantly downregulated in the ECs challenged with TNF-α for 6 h. Real-time PCR analyses additionally validated that the expression levels of miR-146a, miR-155*, and miR-141 were consistent with the microarray results. Then, ICAM-1 was identified as a novel target of miR-141 by Target Scan software and the reporter gene system. Further functional experiments showed that elevated levels of miR-141 inhibited ICAM-1 expression and diminished leukocytes adhesion to ECs in vitro. In an in vivo murine model of MI/R injury, pretreatment with miR-141 mimics through the tail vein downregulated the expression level of ICAM-1 in heart and attenuated MI/R injury as evidenced by decreased infarct size and decline of serum cardial troponin I (cTnI) and lactate dehydrogenase (LDH) concentration. The cardioprotective effects of miR-141 mimics may be attributed to the decreased infiltration of CD11b(+) cells and F4/80(+) macrophages into ischemic myocardium tissue. In conclusion, our results demonstrate that miR-141, as a novel repressor of ICAM-1, is involved in the attenuation of MI/R injury via antithetical regulation of ICAM-1 and inflammatory cells infiltration. Thus miR-141 may constitute a new therapeutic target in the setting of ischemic heart disease.

MicroRNA-181a enhances the chemotherapeutic sensitivity of chronic myeloid leukemia to imatinib.

MicroRNA-181 (miR-181) has been recently demonstrated to participate in the differentiation and development of immune cells, including natural killer cells and B and T lymphocytes, and myeloid linages, including erythroid and megakaryocytic cells. The aberrant expression of miR-181, particularly low expression levels, has been observed in a number of leukemia types, including B-cell chronic lymphocytic leukemia and cytogenetically abnormal acute myeloid leukemia. However, the expression and function of miR-181 in chronic myeloid leukemia (CML) remains unknown. In the present study, the aberrant expression of miR-181a was analyzed in a patient with CML and in the CML K562 cell line. In addition, the function and potential mechanisms of miR-181a in K562 cells with regard to their chemotherapeutic sensitivity to imatinib were investigated. The expression levels of miR-181a were significantly reduced in the patient with CML and in the CML K562 cell line. Furthermore, the overexpression of miR-181a in the K562 cells enhanced the chemotherapeutic sensitivity of these cells to imatinib. The potential mechanism mediating these effects may be associated with the capacity of miR-181a to inhibit cell growth and/or to induce cells apoptosis and differentiation in K562 cells. The results of the present study suggested that miR-181a may be a target for the treatment of CML and a useful indicator of the therapeutic sensitivity of CML to imatinib.

Integrative Data Analysis of Multi-Platform Cancer Data with a Multimodal Deep Learning Approach.

Identification of cancer subtypes plays an important role in revealing useful insights into disease pathogenesis and advancing personalized therapy. The recent development of high-throughput sequencing technologies has enabled the rapid collection of multi-platform genomic data (e.g., gene expression, miRNA expression, and DNA methylation) for the same set of tumor samples. Although numerous integrative clustering approaches have been developed to analyze cancer data, few of them are particularly designed to exploit both deep intrinsic statistical properties of each input modality and complex cross-modality correlations among multi-platform input data. In this paper, we propose a new machine learning model, called multimodal deep belief network (DBN), to cluster cancer patients from multi-platform observation data. In our integrative clustering framework, relationships among inherent features of each single modality are first encoded into multiple layers of hidden variables, and then a joint latent model is employed to fuse common features derived from multiple input modalities. A practical learning algorithm, called contrastive divergence (CD), is applied to infer the parameters of our multimodal DBN model in an unsupervised manner. Tests on two available cancer datasets show that our integrative data analysis approach can effectively extract a unified representation of latent features to capture both intra- and cross-modality correlations, and identify meaningful disease subtypes from multi-platform cancer data. In addition, our approach can identify key genes and miRNAs that may play distinct roles in the pathogenesis of different cancer subtypes. Among those key miRNAs, we found that the expression level of miR-29a is highly correlated with survival time in ovarian cancer patients. These results indicate that our multimodal DBN based data analysis approach may have practical applications in cancer pathogenesis studies and provide useful guidelines for personalized cancer therapy.

Regulation of connexin 43 and microRNA expression via β2-adrenoceptor signaling in 1321N1 astrocytoma cells.

Connexin 43 (Cx43) is the main gap junction protein in astrocytes and exerts the same effects on growth inhibition in astrocytoma and glioma as microRNA-146a (miR-146a) in glioma. β2-adrenergic receptor (AR) signaling modulates Cx43 expression in myocytes via components downstream of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). However, it remains to be elucidated how expression of Cx43 is modulated in astrocytes. In the present study, 1321N1 astrocytoma cells were treated with β2-AR signaling agents in order to evaluate the expression of Cx43 and miRNAs. RNA and protein were extracted from the cells for use in reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The results revealed that clenbuterol increased miR-146a level and upregulated Cx43 expression via cAMP/PKA at the mRNA and protein level. Pre-inhibition of adenyl cyclase decreased expression of Cx43 and miR-146a. PKA activation and overexpression of miR-146a in A-1321N1 cells increased the expression of Cx43. β2-AR stimulation and 6Bnz, a PKA activator, suppressed oncomiRs miR-155 and miR-27a, while 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, an Epac activator, increased their levels. The current findings demonstrated that β2-AR signaling has growth inhibitory effects via modulation of the cAMP/PKA pathway in A-1321N1 cells through increasing the expression level of Cx43 and miR-146a as well as decreasing miR-155 and miR-27a levels. Thus, stimulation of the β2-AR and PKA signaling pathway may be a useful approach for astrocytoma therapy.

Using a novel microRNA delivery system to inhibit osteoclastogenesis.

Previously, we developed a novel microRNA (miRNA) delivery system based on bacteriophage MS2 virus-like particles (MS2 VLPs). In this current study, we used this system to transport miR-146a into human peripheral blood mononuclear cells (PBMCs), and demonstrated the inhibition of osteoclastogenesis in precursors. Two cytokines, receptor activator of NF-κB ligand (RANKL), and macrophage-colony stimulating factor (M-CSF) were used to induce osteoclastogenesis. MS2 VLPs were transfected into PBMCs. qRT-PCR was applied to measure expression levels of miR-146a and osteoclast (OC)-specific genes. Western blot (WB) was conducted to evaluate miR-146a downstream target proteins: epidermal growth factor receptor (EGFR) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6). The formation and activity of OCs were assessed by cytochemical staining and bone resorption assay, respectively. In PBMCs treated with MS2-miR146a VLPs, qRT-PCR assays showed increased expression of miR-146a (p < 0.01) and decreased expression of all four OC-specific genes (p < 0.05). WB results indicated decreased expression of EGFR (p < 0.01) and TRAF6 (p < 0.05). The number of OCs decreased markedly and bone resorption assay demonstrated inhibited activity. This miR-146a delivery system could be applied to induce overexpression of miR-146a and to inhibit the differentiation and function of OCs.

Upregulation of microRNA-146a by hepatitis B virus X protein contributes to hepatitis development by downregulating complement factor H.

ABSTRACTHepatic injuries in hepatitis B virus (HBV) patients are caused by immune responses of the host. In our previous study, microRNA-146a (miR-146a), an innate immunity-related miRNA, and complement factor H (CFH), an important negative regulator of the alternative pathway of complement activation, were differentially expressed in HBV-expressing and HBV-free hepatocytes. Here, the roles of these factors in HBV-related liver inflammation were analyzed in detail. The expression levels of miR-146a and CFH in HBV-expressing hepatocytes were assessed via analyses of hepatocyte cell lines, transgenic mice, adenovirus-infected mice, and HBV-positive human liver samples. The expression level of miR-146a was upregulated in HBV-expressing Huh-7 hepatocytes, HBV-expressing mice, and patients with HBV infection. Further results demonstrated that the HBV X protein (HBx) was responsible for its effects on miR-146a expression through NF-κB-mediated enhancement of miR-146a promoter activity. HBV/HBx also downregulated the expression of CFH mRNA in hepatocyte cell lines and the livers of humans and transgenic mice. Furthermore, overexpression and inhibition of miR-146a in Huh-7 cells downregulated and upregulated CFH mRNA levels, respectively. Luciferase reporter assays demonstrated that miR-146a downregulated CFH mRNA expression in hepatocytes via 3′-untranslated-region (UTR) pairing. The overall effect of this process in vivo is to promote liver inflammation. These results demonstrate that the HBx–miR-146a–CFH–complement activation regulation pathway might play an important role in the immunopathogenesis of chronic HBV infection. These findings have important implications for understanding the immunopathogenesis of chronic hepatitis B and developing effective therapeutic interventions.

Effect of a single nucleotide polymorphism in miR-146a on COX-2 protein expression and lung function in smokers with chronic obstructive pulmonary disease.

ObjectiveTo evaluate the effect of a single nucleotide polymorphism (rs2910164) in the miR-146a precursor on the expression level of miR-146a, cyclooxygenase-2 (COX2), and production of prostaglandin E2 (PGE2) in lung tissue harvested from smokers with chronic obstructive pulmonary disease, as well as the lung function and disease stages from the same patient population.Methods and resultsOne-hundred and sixty-eight smokers with diagnosed chronic obstructive pulmonary disease were recruited. The patients were genotyped for rs2910164 polymorphism using Sanger sequencing, and their lung function/disease stages were evaluated following Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria. Meanwhile, messenger ribonucleic acid and protein expression levels of miR-146a and COX2 as well as PGE2 production were determined in 66 lung tissue samples collected in the patients who received surgical treatment. We confirmed that COX2 is a validated target of miR-146a in human fibroblast cells, and identified the differential expression patterns of miR-146a and COX2 in each rs2910164 genotype group. We observed a significant association between rs2910164 in miR-146a and the levels of either COX2 or PGE2 using real-time polymerase chain reaction and Western blot. Consistently, we were able to demonstrate that the rs2910164 single nucleotide polymorphism has a functional effect on the baseline lung function in the study population.ConclusionIn the present study, the rs2910164 CC and GC genotype was found to be associated with an improved lung function and milder disease stages, at least partially, mediated by its ability to increase in COX2 expression and PGE2 production.

Differentially Expressed miRNA in Inflammatory Mucosa of Chronic Rhinosinusitis.

Chronic sinusitis (chronic rhinosinusitis, CRS) is a chronic inflammatory disease of the nasal cavity and paranasal sinuses, pathogenesis of which is not yet completely elucidated. MicroRNA has been shown to extensively be involved in immune response. To analyze the differential expression of miRNAs in chronic sinusitis, with or without nasal polyps (nasal polyps, NP), seven miRNAs (miR- 181b, miR-26b, miR-155, miR-146a, miR-125b, miR-124 and miR-92a) that are associated with inflammation were selected to be quantifying by RT-qPCR in 40 clinical samples and 5 controls. When compared to the normal control group, results showed that, in all patients with CRS, miR- 125b, miR-155 and miR-146a were up-regulated (P < 0.05), while miR-92a, miR-26b and miR- 181b were down-regulated (P < 0.05). MiR124 expression levels were not found to have significant changes. In relation to CRS without NP, miR-125b and miR-155 were significantly up-regulated while miR-92a, miR-26b, miR-181b were down-regulated in NP patients. Furthermore, the miR-92a and miR-26b expression levels were significantly reduced while miR-146a and miR124 expression levels had no significant changes in the NP samples. The RT-qPCR results indicate that the miRNAs were differentially expressed in CRS patients and various inflammation severities could lead to this difference. The results from this study may further reveal the relationship between miRNA expressions and inflammation. These results can also provide an important mechanism (primitive data) on the occurrence of chronic sinusitis and nasal polyps.

MicroRNA Expression Analysis in Patients with Primary Myelofibrosis, Polycythemia vera and Essential Thrombocythemia.

MicroRNAs (miRNA) are small non-coding RNA molecules that play critical roles in cell differentiation, proliferation and apoptosis and thus regulate haematopoietic stem cells and committed progenitor cells. We analyzed expressions of miRNAs associated with hematopoietic transformation of myeloid, erythroid and megakaryocytic progenitor cells during haematopoiesis (mir155, mir181a, mir221, mir222, mir223, mir451), in patients with primary myelofibrosis (PMF) (n=22), polycythemia vera (PV) (n=33), essential thrombocythemia (ET) (n=49) and in healthy controls (n=40) by quantitate/real time polymerase chain reaction. RT-PCR testing was negative for BCR-ABL1 fusion gene in all the patients. Mir155 was expressed in higher levels in all 3 disorders (p<0.05). Mir221 was higher especially in ET and PMF group (p<0.05). Mir222 expression was lower in PV patients (p<0.05) and higher in ET and PMF patients compared to control group. Mir223 expression was higher in ET and PMF group than control group (p>0.05). Mir451 levels were lower in all three groups compared to control group (p<0.05). There was no difference in expression levels of mir181a between groups. JAK2V617F positivity, co-morbidities, drugs, and gender did not affect miRNA expressions. This study holds promise for the future application of these molecules for differential diagnosis and as therapeutic targets in Philadelphia chromosome negative myeloproliferative neoplasms.

The expression and functional study of miR-181a in pediatric acute lymphoblastic leukemia

目的检测microRNA-181a（miR-181a）在急性淋巴细胞白血病（ALL）患儿中的表达水平，并研究其在ALL细胞株CCRF-CEM细胞及耐药株CEM-C1细胞中的功能。方法采用实时荧光定量PCR方法检测ALL患儿骨髓样本、ALL细胞株CCRF-CEM细胞及其耐药株CEM-Cl细胞中miR-181a的表达水平。采用电穿孔转染的方法抑制耐药株CEM-C1细胞并上调非耐药株CCRF-CEM细胞中miR-181a的表达，予不同浓度梯度（终浓度分别为0.01、0.1、1、10、100、1 000 ng/ml）喜树碱处理后，采用CCK-8法观察各浓度梯度喜树碱处理后细胞的存活情况，绘制细胞增殖抑制曲线并计算半数抑制浓度（IC50）。结果初诊-复发组患儿初诊及复发骨髓样本miR-181a相对表达水平（4.84±2.71及6.53±2.20）均高于对照组（1.41±0.53）（P=0.017、0.001），初诊-完全缓解组患儿初诊骨髓样本miR-181a水平（7.58±2.50）较对照组明显升高（P=0.000），而完全缓解后miR-181a水平下降至1.35±0.35，与对照组比较差异无统计学意义（P=0.863）。CEM-C1细胞miR-181a相对表达水平（−4.39±0.08）较CCRF-CEM细胞（−2.32±0.03）明显升高（P=0.000）。转染miR-181a抑制剂CEM-C1细胞较转染阴性对照组增殖抑制率明显升高（P<0.05），IC50分别为30.61、2 255.00 ng/ml，耐药指数（RI）=73.67。miR-181a过表达CCRF-CEM细胞较阴性对照组增殖抑制率明显降低（P<0.05），IC50分别为126.60、1.34 ng/ml，RI=94.26。结论ALL患儿骨髓及CEM-C1细胞中miR-181a异常高表达，抑制CEM-C1细胞中miR-181a的表达可明显增加CEM-C1细胞的药物敏感性，在CCRF-CEM细胞中上调miR-181a的表达能明显增加CCRF-CEM细胞的耐药性。

Rs2910164 Polymorphism Confers a Decreased Risk for Pulmonary Hypertension by Compromising the Processing of microRNA-146a

Objective: To identify the association between rs2910164 polymorphism and development of pulmonary hypertension, as well as underlying molecular mechanism. Methods and Results: 281 patients diagnosed with pulmonary hypertension and 325 normal controls were recruited, and rs2910164 genotype was determined in each participant: As a result, the rs2910164 polymorphism was significantly associated with the development of pulmonary hypertension after adjusting some potential confounding factors. Additionally, lung tissue samples were obtained from 39 patients who received surgical intervention for lung cancer, and mRNA and protein expression levels of miR-146a, COX-2 and PGI₂ production were examined. Furthermore, we confirmed COX-2 is a target of miR-146a in pulmonary smooth muscle cells, and identified a differentially expressed miR-146a and COX-2 in each rs2910164 genotype group. We observed a significant association between rs2910164 polymorphism and the levels of either COX-2 or PGI₂ using real-time PCR and western blot. In conclusion, the results of this study demonstrate that the rs2910164 CC and GC genotype is associated with a decreased risk of pulmonary hypertension, which could be attributed to defective miRNA processing and compromised ability to inhibit production of COX-2 and PGI₂.

Downregulation of Dicer1 Promotes Cellular Senescence and Decreases the Differentiation and Stem Cell-Supporting Capacities of MSCs in Patients with Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is characterized by decreased blood counts caused by ineffective hematopoiesis. Although the insufficient hematopoietic support capabilities of mesenchymal stromal cells (MSCs) have been reported in patients with MDS, the underlying mechanisms remain elusive. In this study, we found that MSCs from patients with MDS displayed a significant increase in senescence, as evidenced by their decreased proliferative capacity, flattened morphology and increased expression of SA-β-gal and p21.Senescent MDS-MSCs exhibited decreased osteoblastic and adipogenic differentiation potential and decreased stem cell support capacity. Next, we found that Dicer1 expression was downregulated in MSCs from patients with lower-risk (LR)-MDS and higher-risk (HR)-MDS. Gene knockdown of Dicer1 induced senescence in the MSCs. The differentiation of MSCs into osteoblasts and adipocytes was significantly inhibited by Dicer1 knockdown (Dicer1-KD). Moreover, Dicer1-KD MSCs exhibited less vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor (G-CSF) expression than normal MSCs. Long-term culture assays showed that layers of MSCs with the Dicer1 gene knocked down are inefficient at supporting hematopoiesis. Further, we observed that the key senescence mediators, p21,but not p16, was elevated in Dicer1-KD MSCs. We also identified reduced expression in the miR-17 family (miR-17-5p, miR-20a/b, miR-106a/b and miR-93) as a potential factor responsible for increased p21 expression in Dicer1-KD MSCs. A verification experiment showed that miR-93 and miR-20a expression levels were significantly reduced in MSCs from patients with MDS. Taken together, our study shows that MSCs from MDS patients are prone to senescence and that Dicer1downregulation promotes cellular senescence and decreases the differentiation and stem cell-supporting capacities of MSCs in MDS patients. Dicer1 downregulation seems to contribute to the insufficient hematopoietic support capacities of MSCs in patients with MDS. DisclosuresNo relevant conflicts of interest to declare.