荧光报告系统验证miR-142-5p的靶基因Becn1

目的验证miR-142-5p的靶基因——自噬相关基因Becn1。方法生物信息学预测miR-142-5p与Becn1的结合位点,构建含有结合位点的Becn1基因3'UTR野生型和突变型质粒,将重组质粒与miR-142-5p-mimics或miR-NC共转染C2C12细胞,检测荧光素酶活性。结果 miR-142-5p与Becn1的3'UTR存在一个结合位点(123~129);成功构建野生型重组质粒p GL3-Becn1-3'UTR和靶位点含3个碱基突变的重组质粒Mut-3'UTR;与共转染p GL3-Becn1-3'UTR和miR-NC组相比,共转染p GL3-Becn1-3'UTR和miR-142-5p-mimics组荧光素酶活性显著下降,约为41.2%(P0.01);而与对照组相比,共转染Mut-3'UTR和miR-142-5p-mimics组的荧光素酶活性下降不明显(P0.05)。结论初步验证自噬相关基因Becn1是miR-142-5p的靶基因。     

miR-142-5p靶向调控TGF-β2促进人巨噬细胞凋亡

目的探讨miR-142-5p在动脉粥样硬化组织中的表达及对人巨噬细胞凋亡的作用。方法构建动脉粥样硬化大鼠模型,qRT-PCR检测动脉粥样硬化组织中miR-142-5p的表达水平。50μg/L氧化型低密度脂蛋白(ox-LDL)刺激人巨噬细胞、血管平滑肌细胞(VSMC)、内皮细胞24 h后,提取细胞RNA,qRT-PCR检测细胞中miR-142-5p的表达水平。靶基因预测软件预测miR-142-5p的靶基因,双荧光素酶报告基因鉴定靶基因的正确性。细胞转染miR-142-5p mimic、mimic control、miR-142-5p inhibitor、inhibitor control,Western blot和qRT-PCR检测miR-142-5p对靶基因的调控作用,流式细胞仪检测miR-142-5p和靶基因对巨噬细胞凋亡的作用。结果 miR-142-5p在动脉粥样硬化组织中表达上调,与正常组织相比差异显著(P0.01)。血管平滑肌细胞和内皮细胞经ox-LDL刺激后miR-142-5p的表达水平与刺激前没有明显变化,而巨噬细胞经ox-LDL刺激后miR-142-5p的表达水平较刺激前明显升高(P0.01)。预测miR-142-5p的靶基因为转化生长因子β2(TGF-β2)。miR-142-5p mimic与TGF-β2共转染后荧光素酶活性最低;miR-142-5p mimic组TGF-β2蛋白和mRNA的表达水平与mimic control组相比明显下降,miR-142-5p inhibitor组TGF-β2蛋白和mRNA的表达水平与inhibitor control组相比明显升高(P0.01);miR-142-5p mimic组细胞凋亡率明显高于mimic control组,TGF-β2 siRNA组细胞凋亡率明显高于siRNA control组(P0.01)。结论 miR-142-5p在动脉粥样硬化中过度表达,miR-142-5p通过下调靶基因TGF-β2促进人巨噬细胞凋亡。     

阿尔茨海默病患者血清miR-98-5p和miR-142-5p水平变化及其意义

目的 探讨阿尔茨海默病（AD）患者血清微小RNA-98-5p(miR-98-5p)、微小RNA-142-5p(miR-142-5p)水平变化及其临床意义。方法 选择AD患者103例（观察组）、体检健康的志愿者50例（对照组）,采集两组清晨空腹外周静脉血,离心留取血清,采用RT-qPCR法检测血清miR-98-5p、miR-142-5p,采用ELISA法检测血清Aβ1-40、Aβ1-42,同期采用简易精神状态检查量表（MMSE）评分评估认知功能。采用Pearson/Spearman相关分析法分析AD患者血清miR-98-5p、miR-142-5p水平与血清Aβ1-40、Aβ1-42水平和MMSE评分的关系。采用受试者工作特征（ROC）曲线分析血清miR-98-5p、miR-142-5p水平对AD的诊断价值。结果 观察组血清miR-98-5p、miR-142-5p水平均显著高于对照组（t/Z分别为7.557、5.827,P均<0.01）。观察组血清Aβ1-40水平显著高于对照组（Z=7.519,P<0.01）,血清Aβ1-42水平和MMSE评分均显著低于对照组（t/Z分别为5....     

结肠癌组织中runx3、突变型p53蛋白的表达变化及意义

目的观察runx3和突变型p53蛋白在结肠癌组织中的表达变化,并探讨其意义。方法采用免疫组化SP法检测50例结肠癌、30例正常结肠组织中的runx3和突变型p53蛋白。结果 runx3蛋白在结肠癌组织中的阳性表达率为46.00%,在正常结肠组织中为86.67%,两者相比,P〈0.01;突变型p53蛋白在结肠癌组织中的阳性表达率为76.00%,在正常结肠组织中为36.67%,两者相比,P〈0.01;runx3和突变型p53蛋白在结肠癌组织中的表达与肿瘤分化程度和淋巴结转移有关（P均〈0.01）。结论结肠癌组织中runx3蛋白表达减少、突变型p53蛋白表达增加,二者在结肠癌的发生发展过程中发挥作用。     

miR-485-5p对结肠癌细胞顺铂耐药的影响

目的:探讨miR-485-5p通过PI3K/Akt-PAK1信号通路对结肠癌细胞顺铂耐药的影响。方法:构建LoVo/DDP细胞株,将建LoVo/DDP细胞株分为NC组(未做转染处理)、miR-485-5p mimics组(转染miR-485-5p mimics)、miR-485-5p inhibitors组(转染miR...     

sPE患者血清miR-199a-5p、miR-142-3p表达变化及与产妇血管内皮损伤、围产儿结局的关系

目的 观察重度子痫前期（sPE）患者血清微小核糖核酸-199a-5p(miR-199a-5p)、微小核糖核酸-142-3p(miR-142-3p)表达变化,并分析其与产妇血管内皮损伤、围产儿结局的关系,为围产儿结局的改善提供参考。方法选取sPE患者200例（观察组）和产检健康孕妇200例（对照组）,采用RT-PCR法测算两组血清miR-199a-5p、miR-142-3p,酶联免疫吸附试验检测两组血清可溶性细胞内皮因子（sEng）、可溶性血管内皮生长因子受体-1(sFlt-1)、内皮素-1(ET-1)、血管内皮生长因子（VEGF）,Pearson相关分析法分析观察组血清miR-199a-5p、miR-142-3p与血管内皮功能相关指标的相关性。根据围产儿结局将观察组分为结局不良组（29例）和结局良好组（171例）,采用单因素分析法及多因素Logistic回归分析法分析患者血清miR-199a-5p、miR-142-3p表达与围产儿结局的关系。结果 与对照组比较,观察组血清miR-199a-5p表达及VEGF水平降低,血清miR-142-3p表达及sEng、sFlt-1、ET-1水平升高...     

miR-139-5p在多形性胶质母细胞瘤中的表达与靶基因验证

目的探索miR-139-5p在多形性胶质母细胞瘤(GBM)患者中的表达水平,预测和验证miR-139-5p的靶基因。方法利用实时定量PCR检测GBM患者和对照组脑组织中miR-139-5p的相对表达水平。选择star Base在线工具预测miR-139-5p的靶基因,通过Target Scan数据库分析miR-139-5p与靶基因的结合位点,并采用萤光素酶报告实验验证。检索miRPathDB数据库分析miR-139-5p靶基因显著富集的pathways,运用miRTarget Link工具构建miR-139-5p与其靶基因之间的交互网络。结果相对于对照组,miR-139-5p在GBM患者脑组织中的表达水平显著下调。star Base预测工具取交集后得到8个靶基因,其中真核翻译起始因子4γ2(EIF4G2)为miR-139-5p靶基因之一。萤光素酶报告实验表明EIF4G2基因3’UTR区中含有明确的miR-139-5p结合位点。pathway富集分析发现靶基因显著富集于胰岛素样生长因子(IGF)-1信号通路、神经营养因子通路、趋化因子通路、T细胞受体通路、Ras信号通路和转化生长因子(TGF)-β信号通路等。结论 miR-139-5p在GBM患者中低表达,可能通过抑制靶基因EIF4G2表达参与肿瘤发生相关的过程。     

结直肠癌组织中miR-338-3p、SMO的表达变化及意义

目的观察结直肠癌组织中微小RNA(miRNA)-338-3p(miR-338-3p)与Smoothened(SMO)的表达变化,并探讨其意义。方法 30例结直肠癌患者,手术时留取癌组织及癌旁组织,抽提其中的总RNA及蛋白质,采用实时荧光定量PCR检测标本中的miR-338-3p,用半定量RT-PCR检测SMO mRNA,Western blot检测SMO蛋白。结果结直肠癌组织中miR-338-3p的表达量(2-ΔΔCt)为0.153 7±0.126 4,SMO mRNA的相对表达量为0.371±0.116,SMO蛋白的灰度值为3.195±1.623,癌旁组织分别为0.901 5±0.426 3、0.366±0.117、0.733±0.305,两种组织中的miR-338-3p、SMO蛋白表达量相比,P均<0.01;miR-338-3p与SMO蛋白的表达呈负相关(r=-0.877,P<0.01)。结论结直肠癌组织中miR-338-3p表达明显下调,SMO蛋白表达明显上升;miR-338-3p可能通过转录后基因沉默机制使SMO蛋白表达下降,从而抑制结直肠癌的发生发展。     

甘木通醇提物通过miR-142-3p对ox-LDL诱导HUVECs损伤及SOD、MDA、CAT的影响

目的 探讨甘木通醇提物对氧化低密度脂蛋白(ox-LDL)诱导的人脐静脉血管内皮细胞(HUVECs)损伤和氧化应激的影响及潜在机制。方法 利用100 mg/L ox-LDL损伤HUVECs,同时给予不同浓度的甘木通醇提物。细胞计数试剂盒(CCK)-8法检测HUVECs增殖,流式细胞术评估细胞凋亡,Western印迹分析细胞核相关抗原(Ki-67)、天冬氨酸特异性半胱氨酸蛋白酶(Caspase)3蛋白表达,荧光定量聚合酶链反应(qRT-PCR)测定微小RNA(miRNA)-142-3p表达,比色法检测超氧化物歧化酶(SOD)、丙二醛(MDA)、过氧化氢酶(CAT)水平。在HUVECs中转染anti-miR-142-3p并利用100 mg/L ox-LDL诱导细胞损伤,或转染anti-miR-142-3p并给予100 ng/ml浓度的甘木通醇提物和100 mg/L ox-LDL,观察细胞的增殖、凋亡和SOD、MDA、CAT变化。结论 ox-LDL诱导HUVECs损伤后,细胞的存活率、Ki67蛋白表达量、SOD、CAT活性明显降低,凋亡率、Caspase3蛋白表达量、miR-142-3p表达量...     

miRNA-142-5p在肾细胞癌中高表达的研究

目的 用实时荧光定量PCR的方法 探讨miRNA-142-5p在肾透明细胞癌中的表达,为进一步研究其功能做准备.方法 提取30例手术患者肾癌组织和癌旁正常组织中的miRNAs,在基因芯片判断的基础上,用RT-PCR的方法 初步检测miRNA-142-5p在肾癌组织和癌旁正常组织中的表达情况,后采用Real-time PCR的方法 定量分析miRNA-142-5p在肾癌组织及癌旁正常组织中的表达情况.结果 相对于癌旁正常组织,miRNA-142-5p在肾癌组织中高表达,平均表达升高4.13倍.30例标本中有21例肾癌标本中miRNA-142-5p的表达明显高于其在癌旁正常组织中的表达,仅有9例肾癌标本中miRNA-142-5p的表达低于其在癌旁正常组织中的表达.7例G1期的肾癌标本均较对应的癌旁正常组织低表达,而较癌旁正常组织高表达的肾癌组织分布在G2和G3期,初步显示与分期有一定的关系.结论 miRNA-142-5p在肾癌组织中显著存在高表达,提示其与肾透明细胞癌的发生与演进有关,有望成为指导肾细胞癌诊断、治疗、预后的新靶点.     

PRL-3基因对结直肠癌细胞增殖能力的影响

目的:探讨PRL-3的过表达或敲低对结直肠癌细胞增殖能力的影响.方法:利用MTT法、平板克隆形成实验检测PRL-3对细胞体外增殖的影响;应用流式细胞术检测PRL-3对细胞周期的影响.结果:应用MTT法,检测PRL-3对SW480/EGFP、SW480-EGFP-PRL-3、SW480/EGFP/Mock及SW480-PRL-3-KD1细胞体外增殖能力的影响,经析因方差分析,4组差异具有显著性(F=23.463,P=0.000);不同时间点对细胞体外增殖的影响差异具有显著性(F=71.515,P=0.000);各组细胞与各时间组两因素交互效应显著(F=2.128,P=0.008);除第1天外,其他各时间点细胞组间的细胞增殖差异具有显著性.经LSD法多重比较,结果表明,与SW480/EGFP/Mock和SW480/EGFP细胞相比,SW480-EGFP-PRL-3细胞的增殖速度加快,而SW480-PRL-3-KD1细胞的增殖速度减慢.平板克隆形成实验显示SW480-EGFP-PRL-3细胞克隆形成能力明显增强,而SW480-PRL-3-KD1细胞克隆形成能力显著下降,差异具有显著的统计学意义(F=44.411,P=0.000).结论:PRL-3基因可促进结直肠癌细胞的增殖.     

Upregulated miR-328-3p and its high risk in atrial fibrillation: A systematic review and meta-analysis with meta-regression

Background:Several studies have shown miR-328-3p increased in atrial fibrillation (AF), but some researches indicated no difference or even decreased. This inconsistent result confuses researchers, and it is urgent to know the truth. This study is to assess the association between miR-328-3p levels in plasma/atrial tissue and patients with AF.Methods:PubMed, EMBASE, Scopus, Web of Science, and ProQuest were searched from inception to February 1, 2021. The standardized mean differences (SMD) with their 95% confidence interval (CI) were calculated to evaluate the association between miR-328-3p levels and AF.Results:Twelve studies met the inclusion criteria and were used for our meta-analysis. Overall, the levels of miR-328-3p were higher in patients with AF than in the control group (SMD = 0.69, 95% CI [0.10, 1.28], P = .022). After adjustment, the overall SMD was 0.82 (95% CI [0.22, 1.42], P = .007). Sensitivity analysis indicated that the results were stable, and the trim-fill analysis showed that the results were credible. Subgroup analyses showed that AF patients, n ≥ 30, various of comorbidity, articles published earlier, and Asia groups had higher levels of expression of miR-328-3p.Conclusions:High levels of miR-328-3p are significantly associated with an increased risk of AF. It implies that miR-328-3p played an important role in diagnosis and may serve as a potential momentous, and useful biomarker to identify AF.     

Patterns of microRNAs 142-3p, 106a, 17 and 20a expression in patients with systemic lupus erythematosus

IntroductionSystemic lupus erythematosus (SLE) is a connective tissue disorder which involves immune system dysregulation. Micro-ribonucleic acids (MiRNAs) up and down regulation are implicated in its development.Aim of the workTo examine the expression levels of certain miRNAs (miR-17, miR-20a, miR-106a, and miR-142-3p) in SLE patients and to investigate which miRNAs are involved in the pathogenesis of SLE, in order to be used as diagnostic or prognostic biomarkers.Patients and methods60 patients and 60 matched control were included. SLE disease activity index 2000 (SLEDAI-2 K) was assessed. Assessment of serum miRNAs was done using real-time quantitative polymerized chain reaction.ResultsThe median age of patients was 29.5 years (24–32) and they were 59 females and 1 male with a median disease duration of 24 (20–48) months and SLEDAI of 5.5 (2.3–9). miR-17a, miR-142, miR-20a and miR-106a were significantly lower in patients (22.5 ± 2.2; 22.8 ± 2.2; 23.4 ± 2.4 and 22.6 ± 2.2) compared with control (23.8 ± 2.1; 24.7 ± 2.2; 25.1 ± 2.4 and 24.4 ± 2.3) (p = 0.002, p < 0.001, p < 0.001 and p < 0.001 respectively). The 4 markers significantly correlated with the SLEDAI (p = 0.002; p = 0.002; p = 0.004 and p = 0.001 respectively). The diagnostic capability of miR-142, miR-20a and miR-106a in predicting SLE showed a specificity of 95%, 98% and 90% at cut-off values of >22.6, >22.1 and >22.8 respectively; area under the curve was 67, 72, 70 and 76% at p-values p = 0.19, p < 0.001, p < 0.001, p < 0.001 respectively.ConclusionMiR-17, miR-142-3p, miR-20a, and miR-106a have a diagnostic value in SLE and may serve as a therapeutic target for treatment. The studied markers were also related to the disease activity.     

miR-574-3p在结直肠癌中的表达和作用机制研究

目的 检测并分析结直肠癌组织和细胞系中miR-574-3p的表达情况及其对结直肠癌细胞发生、发展的影响.方法 收集武汉市第三医院11对手术切除结直肠癌患者的癌组织和对应癌旁组织,3种结直肠癌细胞系和1种正常结直肠上皮细胞,通过实时荧光定量聚合酶链反应(qRT-PCR)检测临床样本及结直肠癌细胞系中miR-574-3p的...     

miR-152-3 p靶蛋白差异表达及调控机制在肝癌复发中的作用

目的比较肝癌复发与预后良好患者癌组织的蛋白表达,分析miR-152-3p相关的靶蛋白差异及调控机制,探讨miR-152-3p在肝癌复发中的作用。方法应用TMT标记全蛋白质组学测序方法和RT-PCR分别检测肝癌切除术后2年复发(n=6)与5年预后良好(n=6)患者肝癌组织的蛋白质表达和miR-152-3p的表达水平;联合六大数据库检索分析miR-152-3p靶基因,并运用Gene Ontology、DAVID和REACTOME数据库进行靶基因筛选、富集注释和信号转导通路富集分析;将筛选的miR-152-3p靶基因进行基因突变频率和生存曲线分析,验证miR-152-3p靶基因在肝癌复发中的作用。计量资料2组间比较采用独立样本t检验。肝脏组织有关基因生存率分析采用Kaplan-Meier分析。结果肝癌切除术后预后良好患者癌组织的miR-152-3p转录表达水平显著高于复发患者癌组织的水平(P<0.05)。蛋白测序结果显示,预后良好患者与复发患者的癌组织存在365个差异表达蛋白,联合肝癌复发数据库分析显示,其中有17个蛋白受miR-152-3p调控。进一步的信号通路分析发现,17个受miR-152-3p调控的靶基因其功能集中于线粒体核糖体翻译调控;多重富集发现靶基因与线粒体呼吸链复合体密切关系的蛋白6个,分别为AKAP1、FOXRED1、MRPL28、MRPL50、SHC1、STAU1。基因突变频率及生存曲线分析发现,线粒体呼吸链相关靶蛋白的功能缺失或减弱会严重影响患者的生存率。结论miR-152-3p在HCC切除术后预后良好和复发患者的癌组织表达差异显著,miR-152-3p在肝癌复发中作用机制可能是通过调控靶基因AKAP1、FOXRED1、MRPL28、MRPL50、SHC1、STAU1作用于线粒体呼吸链,影响细胞氧化呼吸功能导致肝癌复发。     

Circular RNA AKT3 governs malignant behaviors of esophageal cancer cells by sponging miR-17-5p

BACKGROUND Recent studies have demonstrated that circular RNA AKT3(circAKT3)plays a crucial role in regulating the malignant phenotypes of tumor cells.However,the potential effects of circAKT3 on esophageal cancer have not been investigated.AIM To illuminate the role of circAKT3 in malignant behaviors of esophageal cancer cells and its underlying mechanism.METHODS Clinical samples were collected to detect the expression of circAKT3.The role of circAKT3 in proliferation,migration,invasion,and apoptosis of esophageal cancer cells was evaluated using Cell Counting Kit-8,wound healing assays,Transwell assays,and fluorescence analysis,respectively.The target of circAKT3 was screened and identified using an online database and luciferase reporter assay.A xenograft nude mouse model was established to investigate the role of circAKT3 in vivo.RESULTS In vitro assays showed that proliferative,migratory,and invasive capacities of esophageal cancer cells were significantly enhanced by circAKT3 overexpression.Furthermore,miR-17-5p was screened as the target of circAKT3,and miR-17-5p antagonized the effects of circAKT3 on esophageal cancer cells.Moreover,we identified RHOC and STAT3 as the direct target molecules of miR-17-5p,and circAKT3 facilitated expression of RHOC and STAT3 by inhibiting miR-17-5p.In vivo assays showed circAKT3 knockdown inhibited growth of esophageal cancer.CONCLUSION CircAKT3 contributed to the malignant behaviors of esophageal cancer in vitro and in vivo by sponging miR-17-5p thus providing a potential target for treatment of esophageal cancer.