Up-regulation of miR-34b/c by JNK and FOXO3 protects from liver fibrosis

α1-Antitrypsin (AAT) deficiency is a common genetic disease presenting with lung and liver diseases. AAT deficiency results from pathogenic variants in the SERPINA1 gene encoding AAT and the common mutant Z allele of SERPINA1 encodes for Z α1-antitrypsin (ATZ), a protein forming hepatotoxic polymers retained in the endoplasmic reticulum of hepatocytes. PiZ mice express the human ATZ and are a valuable model to investigate the human liver disease of AAT deficiency. In this study, we investigated differential expression of microRNAs (miRNAs) between PiZ and control mice and found that miR-34b/c was up-regulated and its levels correlated with intrahepatic ATZ. Furthermore, in PiZ mouse livers, we found that Forkhead Box O3 (FOXO3) driving microRNA-34b/c (miR‐34b/c) expression was activated and miR-34b/c expression was dependent upon c-Jun N-terminal kinase (JNK) phosphorylation on Ser⁵⁷⁴. Deletion of miR-34b/c in PiZ mice resulted in early development of liver fibrosis and increased signaling of platelet-derived growth factor (PDGF), a target of miR-34b/c. Activation of FOXO3 and increased miR-34c were confirmed in livers of humans with AAT deficiency. In addition, JNK-activated FOXO3 and miR-34b/c up-regulation were detected in several mouse models of liver fibrosis. This study reveals a pathway involved in liver fibrosis and potentially implicated in both genetic and acquired causes of hepatic fibrosis.

α1-Antitrypsin (AAT) deficiency is an inherited disorder that affects ∼1 in 3,000 individuals and is an important genetic cause of lung and liver disease (1). The most common defect is the Z variant of the SERPINA1 gene, which results in the production of misfolded and polymerogenic Z α1-antitrypsin (ATZ). ATZ-dependent liver disease has a wide spectrum of clinical manifestations ranging from liver insufficiency in newborns to chronic liver disease and hepatocellular carcinoma in adults (2, 3). Because of its misfolding and polymerization, ATZ is unable to efficiently traverse the secretory pathway. Accumulation of ATZ in the endoplasmic reticulum (ER) of hepatocytes has a proteotoxic effect.Expression of microRNAs (miRNAs) is affected in several liver diseases with distinct profiles across diseases with different etiologies (4). Here, we investigated differentially expressed miRNAs in the liver of PiZ mice, a transgenic animal model expressing the human ATZ (5). We then confirmed the most relevant findings in liver samples from patients. Following the identification of an important miRNA cluster involved in liver fibrosis, the upstream molecules affecting its expression, and its effector, we showed this pathway is involved in various murine models of liver fibrosis.     

A novel alpha1-antitrypsin null variant （PiQ0Milano）nt (PiQ0Milano)

Alpha1-antitrypsin deficiency is an autosomal recessive disease characterized by reduced serum levels of alpha1-antitrypsin(AAT)due to mutations in the SERPINA1 gene causing early onset pulmonary emphysema and,occasionally,chronic liver disease.We report an incidental finding of a novel null AAT allele,Q0Milano,consisting of a 17 nucleotides deletion in exon 3 of SERPINA1 gene,in an Italian child with persistently increased liver enzymes,a mild decrease in circulating AAT levels and without any pulmonary disease.Q0Milano variant results in an unfunctional protein lacking of AAT active site,as the resultant protein is truncated near PiS locus involved in AAT protein stability.     

MiR-223-3p affects myocardial inflammation and apoptosis following myocardial infarction via targeting FBXW7

BackgroundMyocardial infarction (MI) is one of the main causes of disability and death in the world, leading to myocarditis and cardiomyocyte apoptosis. Studies have shown that microRNA (miRNA) is involved in myocarditis and apoptosis. The main purpose of this study was to explore the regulatory mechanism of miR-223-3p on myocarditis and apoptosis after MI.MethodsWe cultured H9c2 cells and detected the expression of miR-223-3p in cells treated with different concentrations of H₂O₂. Sprague Dawley (SD) rats were fed with normal diet, constructed an MI model and detect the expression of miR-223-3p in heart tissue. Overexpression or inhibition of miR-223-3p was conducted in MI model cells in vitro, and the contents of the inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) in cell supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Cell apoptosis was detected by flow cytometry. Luciferase activity assay was used to detect the targeting relationship between miR-223-3p and FBXW7, and the expression of FBXW7 in cells was also detected. Overexpression of miR-223-3p was performed in MI rats to detect the expression of inflammatory factors, FBXW7, and apoptosis in rat cardiac tissue.ResultsThe expression of miR-223-3p was down regulated in MI models established in vitro and in vivo. Overexpression of miR-223-3p can inhibit inflammatory response and apoptosis in H9c2 cells and cardiac tissues. It was revealed that miR-223-3p can inhibit the expression of FBXW7, reduce myocarditis and apoptosis after MI, and improve cardiac function.ConclusionsIt is possible that miR-223-3p reduces myocarditis and apoptosis after MI and improves cardiac function by targeted inhibition of FBXW7 expression.     

The role of targeted regulation of COX11 by miR-10a-3p in the development and progression of paediatric mycoplasma pneumoniae pneumonia

BackgroundMiR-10a-3p is associated with the pathogenesis of many immune inflammatory diseases including Mycoplasma pneumoniae pneumonia (MPP), and cytochrome coxidase assembly homologue 11 (COX11) is one of its direct target proteins. This study investigates the function and mechanism of miR-10a-3p targeting with COX11 in the development and progression of paediatric MPP.MethodsNinty-seven paediatric MPP patients and 100 age- and sex-matched healthy children were enrolled. Clinical and laboratory indicators of paediatric MPP patients were collected. The mRNA levels of the COX11 gene and miR-10a-3p were detected by qRT-PCR. THP-1 mononuclear macrophages were stimulated using MPP lipid-associated membrane proteins (Mp-LAMPs). The relative expression level of miR-10a-3p was detected after 12, 24, and 48 h. THP-1 cells were transfected to overexpress or inhibit the expression of miR-10a-3p, miR-10a-3p, COX11 mRNA, NF-κB signalling pathway-related proteins, and C-reactive protein (CRP) were detected after 48 h by Western blot.ResultsThe relative expression level of miR-10a-3p in the MPP group was 2.38±0.52, compared with 1.76±0.38 in control group (t=4.584, P<0.001) whileCOX11 in MPP group was 3.70±1.12, compared to 5.78±1.84 in control group (t=4.876, P<0.001). Pearson correlation analysis showed that miR-10a-3p and COX11 in MPP group presented a negative correlation (r=-0.679, P<0.001). By searching in the prediction website of TargetScan database, it was found that miR-10a-3p and Cox11 genes had targeted regulatory binding sites, and the targeting relationship between miR-10a-3p and Cox11 genes was confirmed by dual luciferase reporting assay in 293T cells. Among paediatric MPP patients, miR-10a-3p expression had a positive correlation with the white blood cells count, erythrocyte sedimentation rate (ESR), and CRP expression, while COX11 mRNA expression had a positive correlation with ESR and CRP. After LAMP stimulation, the miR-10a-3p expression level in THP-1 cells significantly increased (P<0.05). After THP-1 cells were transfected with the miR-10a-3p mimic or inhibitor, the relative expression level of miR-10a-3p significantly increased or decreased, respectively. COX11 expression in the mimic group significantly decreased, whereas COX11 in the inhibitor group significantly increased (both P<0.05). In addition, after transfection, IκBα expression significantly decreased and that of p-IKKα/β, p-p65, and CRP significantly increased in the mimic group, and the opposite was true in the inhibitor group.ConclusionsIn paediatric MPP, increased miR-10a-3p downregulated COX11, activating NF-κB signalling pathway to promote disease development and progression.     

Silencing LncRNA-DANCR attenuates inflammation and DSS-induced endothelial injury through miR-125b-5p

BackgroundLncRNA-DANCR is involved in inflammation and acts as a major contributor to colon cancer. The effects and mechanism of LncRNA-DANCR were first investigated in a DSS-induced colitis model in vivo and vitro.Material and methodsSprague-Dawley rats were given DSS to induce the colitis model. TNF-α, IL-1β, IL-6 levels and expression of intestinal adhesion proteins ZO-1 and MUC2 in colon tissues and DSS-induced NCM460 cells were measured using corresponding kits. A hematoxylin and eosin (H&E) staining assay was performed to evaluate colon tissue pathology conditions. Protein expression levels in DSS-induced NCM460 cells were evaluated by Western blotting, and cell apoptosis was detected using a TUNEL assay. Gene levels in DSS-induced NCM460 cells were evaluated by PCR. The StarBase online tool was used to predict the LncRNA-DANCR target. The LncRNA-DANCR target was verified using a luciferase reporter assay.ResultsLncRNA-DANCR was up-regulated in DSS-induced groups of rats. TNF-α, IL-1β and IL-6 expression was significantly increased in DSS-induced groups of rats and cells. Zo-1 and MUC2 expression levels were decreased in DSS-induced groups of rats. Silencing LncRNA-DANCR reduced inflammation, cell apoptosis and up-regulated ZO-1, MUC2 and Claudin-1 in DSS-induced cells. MiR-125b-5p was the downstream LncRNA-DANCR target. All LncRNA-DANCR effects in the colitis model were reversed by the miR-125b-5p inhibitor.ConclusionLncRNA-DANCR/miR-125b-5p, which may act as a regulatory axis in inflammation, apoptosis and barrier function dysregulation, can provide an essential reference for the development of new drugs in colitis treatment.     

miR-129 Blocks Secondary Hyperparathyroidism-Inducing Fgf23/αKlotho Signaling in Mice with Chronic Kidney Disease

BackgroundSecondary hyperparathyroidism, a condition of excess parathyroid hormone (PTH, Pth) production, is often seen in chronic kidney disease (CKD) patients with elevated fibroblast growth factor 23 (FGF23, Fgf23). Elevated FGF23 levels stimulate secondary hyperparathyroidism-associated parathyroid αKlotho signaling. As overexpression of rationally selected microRNAs can suppress target gene activation, we hypothesized that microRNA-based suppression of parathyroid FGF23/αKlotho axis activity may be a potential strategy to combat secondary hyperparathyroidism.MethodsIn vitro luciferase assays and human parathyroid adenoma cell experiments were used to determine miR-129-1-3p's effects on αKlotho expression in vitro. We also studied the effects of parathyroid-specific miR-129-1 overexpression (miR-129Ox) in CKD and non-CKD mice and parathyroid tissue cultures derived therefrom.ResultsmiR-129-1-3p directly targets the αKlotho mRNA strand in human parathyroid cells. miR-129Ox CKD mice and control CKD mice displayed comparable serum levels of calcium, phosphate, Fgf23, and 1,25-dihydroxyvitamin D (1,25(OH)₂D). However, miR-129Ox CKD mice displayed reduced parathyroid αKlotho expression and lower circulating Pth levels. In vitro culture of miR-129Ox CKD murine parathyroid tissue showed suppressed responses to Fgf23, with decreased Pth secretion and diminished cell proliferation after four days.ConclusionsmiR-129 negatively regulates pro-proliferative, Pth-inducing Fgf23/α?Klotho signaling in the parathyroid glands of CKD mice.     

B-cell malignancies in microRNA Eμ-miR-17～92 transgenic mice

miR-17∼92 is a polycistronic microRNA (miR) cluster (consisting of miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a) which frequently is overexpressed in several solid and lymphoid malignancies. Loss- and gain-of-function studies have revealed the role of miR-17∼92 in heart, lung, and B-cell development and in Myc-induced B-cell lymphomas, respectively. Recent studies indicate that overexpression of this locus leads to lymphoproliferation, but no experimental proof that dysregulation of this cluster causes B-cell lymphomas or leukemias is available. To determine whether miR-17∼92- overexpression induces lymphomagenesis/leukemogenesis, we generated a B-cell–specific transgenic mouse model with targeted overexpression of this cluster in B cells. The miR-17∼92 overexpression was driven by the Eµ-enhancer and Ig heavy-chain promoter, and a 3′ GFP tag was added to the transgene to track the miR expression. Expression analysis using Northern Blot and quantitative RT-PCR confirmed 2.5- to 25-fold overexpression of all six miRs in the transgenic mice spleens as compared with spleens from wild-type mice. Eµ-miR-17∼92 mice developed B-cell malignancy by the age of 12–18 mo with a penetrance of ∼80% (49% splenic B-cell lymphoproliferative disease, 28% lymphoma). At this stage mice exhibited severe splenomegaly with abnormal B-cell–derived white pulp expansion and enlarged lymph nodes. Interestingly, we found three classes of B-cell lymphomas/leukemias at varying grades of differentiation. These included expansion of CD19⁺ and CD5⁺ double-positive B cells similar to the aggressive form of human B-cell chronic lymphocytic leukemia, B220⁺ CD43⁺ B1-cell proliferation, and a CD19⁺ aggressive diffuse large B-cell lymphoma–like disease, as assessed by flow cytometry and histopathological analysis.MicroRNAs (miRs) are 21- to 22-nucleotide-long noncoding RNA molecules that regulate the expression of multiple cellular genes, and their dysregulation is involved in many human diseases including cancer. The MiR-17∼92 cluster frequently is up-regulated in several different malignancies including diffuse large B-cell lymphoma (DLBCL) (1) and lung cancer (2, 3). The MiR-17∼92 cluster is encoded by the chromosome 13q31 locus in humans and the 14qE4 locus in mice. This genomic region is amplified in DLBCLs and several other tumors (reviewed in ref. 4). The cluster consists of six miRs (miR-17, 18a, 19a, 20a, 19b-1, and 92a-1) and has two paralogs in the genome, miR-106a∼363 and miR-106b∼25, proposed to have arisen through series of duplication and deletion events during vertebrate evolution (5).The first suggestion for its possible role in oncogenesis came from the study in Eμ-Myc mice in which enforced expression of miR-17∼92 was shown to accelerate B-cell tumor development (6). An additional study that investigated the role of miR-17∼92 in mice by driving its overexpression under the human CD2 promoter in both B and T cells found that mice overexpressing the miR-17∼92 cluster develop autoimmunity, a lymphoproliferative disease, and die prematurely (7). No lymphomas were observed in these animals, however. Targeted deletion of this cluster, but not of its paralogs, has shown that miR-17∼92 plays an important role in B-cell development, and the KO mice die shortly after birth from lung hypoplasia and ventricular septal defects (8). Further examination of the role of individual miRs in B-cell lymphomas showed that miR-19a and miR19b are required and sufficient for the proliferative activities of the cluster (9).To understand better the role of the miR-17∼92 cluster in B-cell neoplastic progression, we generated miR-17∼92 B-cell–specific transgenic mice. These mice overexpress miR-17∼92 under the control of Ig heavy-chain promoter and Eμ enhancer and express a 3′ GFP tag to track the miRs’ expression. After screening multiple founder mice, we obtained three transgenic lines that concurrently overexpress the six miRs by about 2.5- to 25-fold in their spleens as assessed by quantitative real-time PCR of purified B cells. These mice also express GFP in their B cells which serves as a proxy for miR-17∼92 expression.These mice develop a B-cell malignancy by age of 12–18 mo with a penetrance of ∼80% (49% splenic B-cell lymphoproliferative disease, 28% lymphoma). At this stage these mice exhibit severe splenomegaly with abnormal B-cell–derived white pulp expansion and enlarged lymph nodes. Interestingly, we found three classes of B-cell lymphomas/leukemias at varying grades of differentiation. These included expansion of CD19⁺ and CD5⁺ double-positive B cells similar to the aggressive forms of human B-cell chronic lymphocytic leukemia (B-CLL), B220⁺ CD43⁺ B1-cell proliferation, and a CD19⁺ aggressive DLBCL-like disease, as assessed by flow cytometry and histopathological analysis.     

Adipocytes-derived exosomal miR-122 promotes non-alcoholic fat liver disease progression via targeting Sirt1

AimsNonalcoholic fatty liver disease (NAFLD) is a chronic liver disease that affects adipose function. This study aimed to explore the function of adipocytes-derived exosomal (ADEs) miR-122 in NAFLD.MethodsA high-fat and high-fructose diet-induced rat model and a palmitic acid (PA)-induced in vitro model were established. The RNA level of miR-122 and Sirt1 was measured using qRT-PCR. The protein levels of exosome biomarkers, and lipogenesis, inflammation and fibrosis biomarkers were determined by western blotting. Cell viability and apoptosis were assessed using cell counting kit-8 and flow cytometry, respectively. Serum alanine aminotransferase, aspartate aminotransferase, total cholesterol, triglyceride levels were measured. Liver tissue damage was assessed using haematoxylin and eosin staining. The interaction between miR-122 and Sirt1 3′UTR was assessed using a luciferase reporter gene assay.ResultsADEs exhibited abundant level of miR-122 and promoted lipogenesis, impaired hepatocyte survival, enhanced liver damage and increased serum lipid levels in vivo and in vitro. Inhibition of miR-122 in ADEs alleviated NAFLD progression, lipid and glucose metabolism, liver inflammation and fibrosis both in vivo and in vitro. miR-122 binds directly to the 3′UTR of Sirt1 to suppress its expression. Moreover, Sirt1 overexpression reversed the increase in cell apoptosis, glucose and lipid metabolism, liver inflammation and fibrosis induced by ADEs in vivo and in vitro.ConclusionsThe ADEs miR-122 promotes the progression of NAFLD via modulating Sirt1 signalling in vivo and in vitro. The ADEs miR-122 may be a promising diagnostic biomarker and therapeutic target for NAFLD.     

circZFR promotes cell proliferation and migration by regulating miR-511/AKT1 axis in hepatocellular carcinoma

BackgroundEmerging data suggest the crucial regulatory roles of circular RNAs (circRNAs) in hepatocellular carcinoma (HCC). However, the pathophysiology role of circZFR in HCC remains largely unknown.AimsThis study aims to disclose the functions of circZFR in HCC progression and its potential molecular mechanism.MethodscircZFR and miR-511 were identified by qRT-PCR. Colony formation assay, wound-healing assay, transwell assay, and flow cytometry assay were performed to determine the cell proliferation, migration, invasion and apoptosis. Western blotting and immunohistochemistry (IHC) were utilized to evaluate the expression level of AKT1, GSK3β, β-catenin and cascades of proliferation-related proteins both in vitro and in vivo. Dual luciferase reporter assay was conducted to evaluate the interactions among circZFR, miR-511 and AKT1.ResultsThe expression of circZFR was enhanced and the expression of miR-511 was down-regulated in HCC tissues and cells. Functionally, circZFR silencing or miR-511 overexpression suppressed cell proliferation, migration and invasion, and induced apoptosis of HCC cells. Mechanistically, circZFR acted as a miR-511 sponge to up-regulate its target gene AKT1, which activated cascades of proliferation-related proteins (c-Myc, cyclin D1, Survivin and Bcl-2). Furthermore, depletion of circZFR inhibited tumorigenesis and decreased the expression level of AKT1 in xenograft models.ConclusioncircZFR promotes HCC progression by directly down-regulating miR-511 to activate AKT1 signaling, suggesting that circZFR is a potential target in HCC treatment. Targeting circZFR may provide therapeutic benefits for HCC.     

Long non-coding RNA TP73-AS1 promotes pancreatic cancer growth and metastasis through miRNA-128-3p/GOLM1 axis

BACKGROUNDPrevious studies have suggested that long non-coding RNAs (lncRNA) TP73-AS1 is significantly upregulated in several cancers. However, the biological role and clinical significance of TP73-AS1 in pancreatic cancer (PC) remain unclear. AIMTo investigate the role of TP73-AS1 in the growth and metastasis of PC.METHODSThe expression of lncRNA TP73-AS1, miR-128-3p, and GOLM1 in PC tissues and cells was detected by quantitative real-time polymerase chain reaction. The bioinformatics prediction software ENCORI was used to predict the putative binding sites of miR-128-3p. The regulatory roles of TP73-AS1 and miR-128-3p in cell proliferation, migration, and invasion abilities were verified by Cell Counting Kit-8, wound-healing, and transwell assays, as well as flow cytometry and Western blot analysis. The interactions among TP73-AS1, miR-128-3p, and GOLM1 were explored by bioinformatics prediction, luciferase assay, and Western blot. RESULTSThe expression of TP73-AS1 and miRNA-128-3p was dysregulated in PC tissues and cells. High TP73-AS1 expression was correlated with a poor prognosis. TP73-AS1 silencing inhibited PC cell proliferation, migration, and invasion in vitro as well as suppressed tumor growth in vivo. Mechanistically, TP73-AS1 was validated to promote PC progression through GOLM1 upregulation by competitively binding to miR-128-3p. CONCLUSIONOur results demonstrated that TP73-AS1 promotes PC progression by regulating the miR-128-3p/GOLM1 axis, which might provide a potential treatment strategy for patients with PC.     

外周血miR-320b表达水平对颈动脉粥样硬化斑块性质的评价研究

目的探讨外周血miR-320b表达水平对颈动脉粥样硬化斑块性质的评价价值。 方法选取亳州市人民医院神经内科自2020年5月至2021年9月收治的108例颈动脉粥样硬化患者设为研究组,另选取同时期的105例健康志愿者设为对照组。测定并比较2组受试者外周血miR-320b表达水平。根据颈动脉超声检查结果将研究组患者分为无斑块组和斑块组,对比miR-320b表达水平。另根据高分辨率磁共振成像检查将斑块组患者分为不稳定斑块组与稳定斑块组,对比miR-320b表达水平,分析颈动脉粥样硬化斑块性质的影响因素及miR-320b水平对颈动脉粥样硬化不稳定斑块的评价作用。 结果研究组外周血miR-320b表达水平较对照组降低,差异有统计学意义（P<0.05）。与无斑块组相比,斑块组患者外周血miR-320b表达水平显著降低,差异有统计学意义（P<0.05）。外周血miR-320b水平评估颈动脉粥样硬化斑块的受试者工作特征（ROC）曲线结果显示,曲线下面积（AUC）为0.948,敏感度、特异度分别为88.24%、97.30%。美国心脏病协会（AHA）颈动脉斑块Ⅳ~Ⅴ型、Ⅵ型患者外周血miR-320b表达水平均低于Ⅰ~Ⅱ型、Ⅲ型、Ⅶ型、Ⅷ型患者,且Ⅵ型患者miR-320b表达水平低于Ⅳ~Ⅴ型患者,差异有统计学意义（P<0.05）。和稳定组斑块相比,不稳定斑块组外周血中的miR-320b表达水平显著降低（P<0.05）。多因素Logistic回归性分析显示,总胆固醇、低密度脂蛋白胆固醇水平均是颈动脉粥样硬化不稳定斑块的危险因素（OR=1.979、2.217,P<0.05）,而高密度脂蛋白胆固醇、外周血miR-320b表达水平是其保护因素（OR=0.641、0.548,P<0.05）。外周血miR-320b表达水平评估颈动脉粥样硬化不稳定斑块的ROC结果显示,AUC为0.915,敏感度76.47%,特异度92.50%。 结论外周血miR-320b表达水平可能和颈动脉粥样硬化斑块性质存在一定的关系,可较好地评估斑块不稳定性。     

脑梗死患者血清miR-181c、miR-128b表达水平及其与90天预后的相关性

目的探讨脑梗死患者血清微小RNA(miRNA)miR-181c、miR-128b表达水平及其与90天预后的相关性,为miRNA在脑梗死诊断和治疗上提供参考意义。方法选取128例脑梗死患者以及同期108例健康体检者作为研究对象,分别作为研究组和对照组。比较两组研究对象的miR-181c和miR-128b表达水平,并分析miR-181c、miR-128b表达水平与疾病严重程度的相关性以及脑梗死相关危险因素;根据90天随访的预后情况分为预后良好组和预后不良组,并对影响90天预后的相关因素进行单因素和Logistic回归分析,探讨影响疾病发生及预后的独立影响因素。结果研究组吸烟、高血脂、高血压、糖尿病以及心房颤动比例明显高于对照组(P0.05),研究组患者miR-181c、miR-128b表达水平显著高于对照组,差异具有统计学意义(P0.05),Logistic回归分析结果显示糖尿病、miR-181c、miR-128b表达水平是脑梗死的独立危险因素(P0.05);Spearman相关性分析显示急性脑梗死患者病情严重程度与血清miR-181c和miR-128b水平呈正相关(r=0.867,P=0.005;r=0.885,P=0.002);预后不良组患者发生高血压、糖尿病、心房颤动的比例和入院时NIHSS评分以及血清miR-181c、miR-128b水平均显著高于预后良好组(P0.05),Logistic回归分析结果显示入院时NIHSS评分及血清miR-181c、miR-128b水平均与脑梗死患者90天预后存在密切相关性(P0.05)。结论脑梗死患者血清miR-181c、miR-128b表达水平显著高于健康人群,糖尿病、miR-181c、miR-128b表达水平是脑梗死的危险因素(P0.05),miR-181c、miR-128b表达水平与患者脑梗死严重程度存在显著正相关,且两者表达水平以及NIHSS评分均与急性脑梗死患者90天预后存在密切联系。     

Potential roles of EZH2, Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma cell line Hep3B

AIM: To investigate the potential roles of enhancer of zeste homolog2(EZH2), Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma(HCC) cell line Hep3 B.METHODS: A total of 73 patients who underwent surgical resection at Fuzong Clinical Medical College of Fujian Medical University were enrolled in this study. Hep3 B cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37?℃. Vectors that containing c DNA of the EZH2 gene or mi R-203 targeted sh RNA plasmid were constructed, and then transfected into Hep3 B cells. The m RNA expression of mi R-203, EZH2, and Bmi-1 was analyzed using quantitative real-time polymerase chain reaction analysis, and the protein levels of EZH2 and Bmi-1 were detected by Western blot analysis. Effect of EZH2 or mi R-203 on cell proliferation was observed by methyl thiazolyl tetrazolium assay, and cell apoptosis was assessed using flow cytometry. Besides, effect of EZH2 or mi R-203 on tumor cell invasion was detected using Transwell assay.RESULTS: The m RNA levels of EZH2 and Bmi-1 in HCC tissues and in Hep3 B cells were significantly higher compared with those in normal samples(P 0.01), while mi R-203 level was significantly lower in HCC tissues(P 0.01). Hep3 B cells transfected with EZH2-sh RNA or mi R-203-sh RNA showed lower expression levels of EZH2 and Bmi-1(P 0.05). Compared with controls, Hep3 B cells transfected with EZH2-sh RNA had relative slow cell proliferation, indicating that low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could inhibit Hep3 B cell proliferation(P 0.05). The average apoptosis rate of Hep3 B cells transfected with EZH2-sh RNA vector was about 18.631%, while that of Hep3 B cells transfected with sh RNA vector was about 5.33%, suggesting that EZH2 was down-regulated by transfecting with EZH2-sh RNA, and the down-regulated EZH2 contributed to the cell apoptosis. Low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could reduce Hep3 B cell invasion(P 0.05).CONCLUSION: Our study suggests that EZH2 and Bmi-1 are up-regulated while mi R-203 is downregulated in Hep3 B cells. Mi R-203 may contribute to the metastasis and enhance apoptosis of HCC cells by regulating EZH2 and Bmi-1. Our study may provide a theoretical basis for metastasis of HCC and targeted therapy of HCC.     

Elevated miR-33a and miR-224 in steatotic chronic hepatitis C liver biopsies

AIM: To assess the expression of selected microRNAs (miRNA) in hepatitis C, steatotic hepatitis C, noninfected steatotic and normal liver tissues.METHODS: The relative expression levels of miR-21, miR-33a, miR-96, miR-122, miR-125b, miR-221 and miR-224 were determined in 76 RNA samples isolated from 18 non-steatotic and 28 steatotic chronic hepatitis C (CHC and CHC-Steatosis, respectively) cases, 18 non-infected, steatotic liver biopsies of metabolic origin (Steatosis) and 12 normal formalin-fixed paraffin-embedded liver tissues using TaqMan MicroRNA Assays. All CHC biopsy samples were obtained prior to initiating therapy. Patients’ serum biochemical values, which included glucose, triglyceride, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl-transferase (GGT), alkaline phosphatase (AP), were obtained and correlated with relative miRNA expression.RESULTS: When compared with control non-infected liver samples, miR-122 and miR-221 levels were reduced in CHC-Steatosis (P < 0.03) and in CHC, CHC-Steatosis and Steatosis (P < 0.01). Alternatively, the expression of miR-33a and miR-224 were elevated in CHC-Steatosis and Steatosis in comparison to control tissue (P < 0.01). The levels of miR-33a and miR-224 in CHC-Steatosis (P < 0.02) and miR-224 in Steatosis (P < 0.001) were increased in comparison to CHC samples. By contrast, the expression of miR-21 did not differ statistically between diseased and normal liver samples. Levels of miR-33a correlated negatively with serum AST and AP levels in Steatosis as well as with necroinflammatory grade in CHC, whereas miR-21 correlated positively with AST in Steatosis and displayed negative correlation with triglyceride level in CHC-Steatosis. In contrast, miRNA levels were not correlated with ALT, GGT, cholesterol levels or fibrosis stage.CONCLUSION: Differences in miRNA expression were observed between CHC and steatotic CHC, CHC and steatotic liver, but not between steatotic CHC and steatotic liver of metabolic origin.     

Differentially expressed miR-152, a potential biomarker for in-stent restenosis (ISR) in peripheral blood mononuclear cells (PBMCs) of coronary artery disease (CAD) patients

Background and aimsIn-stent restenosis (ISR) remains the most daunting challenge of current treatments of coronary artery disease (CAD). MicroRNAs (miRNAs) are prominent regulators of key pathological processes leading to restenosis and used as diagnostic tools in different studies. miR-152 and miR-148a are implicated to contribute in the putative intracellular mechanisms of ISR. The aim of present study is to investigate the potential early-stage diagnostic role of miR-152 and miR-148a expression levels for ISR in peripheral blood mononuclear cells (PBMCs) of patients who underwent stent implantation.Methods and resultsThe miRNAs that are supposed to be involved in the ISR were nominated by bioinformatics approach mainly using miRWalk3. Then by quantitative real-time PCR, we determined the relative expression of miR-152 and miR-148a of PBMCs from ISR patients with their age/sex-matched controls.ResultsThe presence of ISR significantly coincided with a decrease in the relative expression of miR-152. The area under the curve (AUC) for miR-152 receiver operating characteristic (ROC) curve was 0.717 (95% CI; 0.60–0.83) with a sensitivity of 70% and a specificity of 67%, suggesting that the miRNA expression level might be employed to identify patients at risk of ISR.ConclusionsTo the best of our knowledge, this is the first work to show that the miR-152 expression level can possibly be applied to predict CAD patients at risk of ISR. The results suggest that the expression levels of miR-152 in PBMCs may serve as a biomarker for ISR. Our finding suggests the importance of miRNA levels in PBMCs as a novel biological tool to detect diseases in their early clinical stages.