miR-483-5p通过下调CDK15促进肾上腺皮质癌增殖和侵袭

目的探讨miR-483-5p在肾上腺皮质癌中的作用及其可能的作用机制。方法荧光定量PCR法检测miR-483-5p和CDK15在肾上腺皮质癌组织和细胞系中的表达,CCK-8增殖试验测定miR-483-5p对细胞增殖的影响,Transwell法检测ACC细胞侵袭性的变化。荧光素酶试验和挽救实验验证miR-483-5p与CDK15相关的分子机制。结果miR-483-5p在肾上腺皮质癌组织中高表达(2.36±1.02 vs 1.09±0.43),CDK15在肾上腺皮质癌组织中低表达(0.57±0.26 vs 1.06±0.32)。荧光素酶检测证实CDK15是miR-483-5p的直接靶点。过表达miR-483-5p可通过下调CDK15的表达促进ACC细胞的增殖(24 h:0.26±0.03 vs 0.23±0.04,48 h:0.56±0.05 vs 0.41±0.03,72 h:0.73±0.04 vs 0.59±0.03)和侵袭能力(95.78±4.66 vs 23.89±2.52)。结论miR-483-5p可通过下调CDK15的表达促进肾上腺皮质癌的发生发展,其可作为肾上腺皮质癌的潜在生物标志物及治疗肾上腺皮质癌的新的作用靶点。     

MiR-30b-3p and miR-126-3p of urinary extracellular vesicles could be new biomarkers for prostate cancer

BackgroundExtracellular vesicles (EVs) including exosomes are present in blood, urine, and saliva and contain proteins, microRNAs, and messenger RNAs. We investigated microRNAs in urinary EVs to discover new biomarkers of prostate cancer (PCa).MethodsWe isolated EVs from urine obtained following digital rectal examination (DRE) of 14 men with elevated levels of serum prostate-specific antigen (PSA) [negative biopsy (n=4) and PCa with Gleason scores of 6 (n=3), 7 (n=3), and 8–9 (n=4)]. MicroRNAs extracted from EVs were analyzed by microRNA microarray.ResultsMicroRNAs miR-30b-3p and miR-126-3p were identified as being overexpressed in urinary EVs of the PCa patients versus the biopsy-negative men, but no microRNAs were associated with the Gleason score. In the independent cohort as well, these two microRNAs were overexpressed in urinary EVs from the PCa patients versus the negative-biopsy men. Logistic regression analysis adjusted by age and PSA showed that these two microRNAs were significantly associated with the prediction of PCa in biopsy specimens. Sensitivity and specificity of miR-30b-3p and miR-126-3p for the prediction of PCa were 46.4% and 88.0% and 60.7% and 80.0%, respectively, which were better than those of serum PSA (53.5% and 64.0%, respectively).ConclusionsMiR-30b-3p and miR-126-3p in urinary EVs could be potential biomarkers of PCa.     

miR-490-5p和miR-363作为结直肠癌诊断标记的研究

目的筛查可作为结直肠癌诊断标记的微小RNA（miRNA）。方法采用实时荧光定量PCR分析miRNA在结直肠癌患者肿瘤和瘤旁组织之间的表达谱．运用非配对t检验的方法,筛查出表达水平具有统计学差异的miRNA。并通过受试者特征曲线（ROC）分析miR-363和miR-490．5p作为诊断标记筛查结直肠癌患者的特异性和敏感性。结果在男性和女性样本中分别发现73和42个表达具有显著性差异的miRNA。而33个miRNA同时在男、女样本中都具有显著性的异常表达,其中10个miRNA的异常表达水平超过5倍,这10个miRNA在男女混合样本中同样具有显著性的异常表达。结论男、女结直肠癌患者中的部分miRNA的表达水平具有显著差异：miR-363和miR-490-5p具有作为结直肠癌的临床筛查指标的潜能。     

血清miR-196a-5p和miR-339-5p表达水平在乳腺癌诊断中的应用价值

背景与目的:研究显示,miR-196a-5p在乳腺癌细胞中呈显高表达,而miR-339-5p呈低表达,两者可能是乳腺癌潜在治疗靶点和诊断标志物.因此,本研究探讨血清miR-196a-5p和miR-339-5p表达水平在乳腺癌诊断中的应用价值.方法:选择2016年1月-2017年6月收治的乳腺癌患者107例(乳腺癌组),...     

miR-339-3p和miR-339-5p在体外胃癌细胞中的表达和意义

目的观察人胃癌细胞株及正常人胃黏膜上皮细胞株中miR-339-3p和miR-339-5p的表达水平,通过功能获得型（gain of function）实验验证miR-339-3p和miR-339-5p与胃癌的相关性,并阐明二者在肿瘤发生中的意义。方法采用SYBR GreenI嵌合荧光法实时定量PCR技术分别检测人正常胃黏膜上皮细胞GES-1,胃癌MKN．45、SGC．7901及BGC-823细胞株中miR-339-3p和miR-339-5p的表达水平;将外源性miR-339-3pmimics和miR-339-5p mimics转染胃癌MKN．45细胞株,采用RT-PCR法检测其转染率,并应用流式细胞术和CCK-8法分别检测转染72h后MKN．45细胞的凋亡及增殖能力的变化。结果miR-339-3p和miR-339-5p在胃癌MKN-45、SGC一7901及BGC-823细胞株中的表达均下调;与对照组相比,转染miR-339-3pmimics及miR-339-5pmimics后,MKN-45细胞株的凋亡率明显增高P〈0．05）,而增殖能力明显减弱（P〈0．01）。结论miR-339-3p和miR-339-5p在3种胃癌细胞株中均呈低表达。miR-339-3p和miR-339-5p可能参与了胃癌细胞的增殖与凋亡机理。     

SP1被鉴定为miR-122-5p的一个转录因子

目的:miR-122-5p跟精子发生相关,本研究主要是探究miR-122-5p在睾丸内的转录因子. 方法:采用生物信息学方法预测到miR-122-5p启动子的可能转录因子为SP1和GATA4,然后分别构建双荧光素酶pGL3-miR-122-5p promoter载体、pcDNA3.1(+)-SP1表达载体和pcDNA3...     

Clinical significance of serum miR-129-5p in patients with diabetes mellitus presenting macrovascular complications

BACKGROUNDDiabetic macrovascular complications (DMCs) are the most common complications encountered during the course of diabetes mellitus (DM) with extremely high mortality rates. Therefore, there is an urgent need to identify specific and sensitive biomarkers for the early diagnosis of DMCs.AIMTo investigate the expression and significance of serum miR-129-5p in patients with DM and macrovascular complications.METHODSSerum samples were collected from 36 healthy controls, 58 patients with DM presenting no macrovascular complications, and 62 patients with DMCs. The expression of miR-129-5p was detected using quantitative real-time polymerase chain reaction. Pearson’s correlation assay was performed to analyze the correlation between serum miR-129-5p levels and clinical indicators. Receiver operator characteristic (ROC) analysis was conducted to analyze the diagnostic value of serum miR-129-5p in patients with DM or DMCs.RESULTSThere was a 4.378-fold and 7.369-fold increase in serum miR-129-5p expression in the DM (5.346 ± 0.405) and DMCs (8.998 ± 0.631) groups, respectively (P < 0.001), compared with the control group (1.221±0.090). In addition, the expression of serum miR-129-5p in patients with DMCs was higher than that in patients with DM, revealing a 1.683-fold increase (P < 0.001). Additionally, serum miR-129-5p expression significantly correlated with smoking history, disease duration, and glycated hemoglobin (HbA1c) in patients with DMCs (P < 0.001). The area under the ROC curve (AUC) of miR-129-5p as a serum marker was 0.964 (95% confidence interval [CI]: 0.930-0.997, P < 0.001) in distinguishing between patients with DM and healthy controls, whereas the AUC of miR-129-5p as a serum marker was 0.979 (95%CI: 0.959-0.999, P < 0.001) in distinguishing between patients with DMCs and healthy controls. CONCLUSIONElevated serum miR-129-5p expression levels correlate with the development of DMCs and can be utilized as a novel early diagnostic biomarker for DM combined with macrovascular complications.     

血浆外泌体miR-17-5p和miR-21对乳腺癌的诊断价值

目的探讨微小RNA-21（miR-21）和miR-17-5p在乳腺癌患者血浆外泌体中的表达水平及其诊断价值。 方法选取2017年6月至2018年3月于成都市第七人民医院就诊的86例乳腺癌患者为乳腺癌组,选取同期体检健康女性45例为对照组。采用实时定量聚合酶链反应（qRT-PCR）检测miR-21、miR-17-5p在两组血浆外泌体中的表达水平。根据qRT-PCR检测结果将乳腺癌患者分为miR-17-5p高表达组及低表达组,miR-21高表达组及低表达组,比较分析其与乳腺癌患者临床病理参数的关系。采用受试者工作特征曲线（ROC）分析血浆外泌体miR-21、miR-17-5p对乳腺癌的诊断价值。 结果乳腺癌组患者血浆外泌体miR-17-5p表达水平显著低于对照组（P<0.05）,而miR-21表达水平显著高于对照组（P<0.05）;miR-17-5p单独检测时曲线下面积（AUC）为0.677,敏感度为58.14%,特异度为75.56%,截断值为0.72;miR-21单独检测时AUC为0.694,敏感度为59.30%,特异度为77.78%,截断值为1.68;联合检测时敏感度为96.51%,特异度为95.56%,准确性为96.18%;联合检测诊断乳腺癌的敏感度、特异度及准确性均显著高于单项检测（P<0.05）。血浆外泌体miR-17-5p、miR-21表达水平均与TNM分期、分化程度、淋巴结转移、cerbB-2及Ki-67有关（P<0.05）。 结论血浆外泌体低表达的miR-17-5p与高表达的miR-21均可作为诊断乳腺癌的潜在生物学标志物。     

miR-34a在前列腺癌血清中的表达意义

目的 探讨血清miR-34a在前列腺癌发生过程中的作用及意义.方法 分别提取20例前列腺癌(PCa)、10例前列腺增生(BPH)及10例健康对照者(N)血清样本的miRNA,应用逆转录-实时定量聚合酶链反应(RT-qPCR)的方法检测三组样本中miR-34a的表达差异,并分析miR-34a的表达与PCa转移是否相关.结果 与健康对照组相比,miR-34a在PCa与BPH患者血清中明显低表达(P均＜0.05),平均下调分别约为7.16、8.53倍.但PCa及BPH的血清中miR-34a的表达水平差异无统计学意义(P＞0.05),并且miR-34a表达水平的高低与PCa是否转移不存在显著相关性(P＞0.05).结论 血清中低表达的miR-34a可能促进了PCa的发生发展,对PCa的诊断及治疗有潜在的临床意义.     

丹酚酸A调控miR-940与miR-576-5p促进退变终板软骨细胞修复的作用研究

目的:观察丹酚酸A (salvianolic acid A,SAA)对退变终板软骨细胞(cartilaginous endplates cells,CEPCs)的干预作用,及其调控的潜在非编码RNA(micro-RNA,miRNA)作用靶点。方法:从腰椎间盘手术标本中分离CEPCs,用不同浓度SAA(2、5、10μM处理24、48、72 h,利用CCK-8检测细胞活性确定SAA的最适剂量和干预时间。通过阿利新蓝染色和对血小板反应蛋白解整合素金属肽酶-5 (A disintegrin and metalloproteinase with thrombospondin-5,ADAMTS-5)、基质金属肽酶3(matrix metallopepridase 3,MMP-3)、Ⅱ型胶原a1 (clollagen typeⅡa1,Col2a1)的蛋白表达检测,分析SAA对IL-1β诱导的退变CEPCs的干预作用。进一步结合生信分析,以及实时荧光定量PCR(quantitative real-time,qRT-PCR)与Western blot检测,并应用miRNA模拟物(miR-mimics)与...     

miR-7-5p抑制胰腺癌细胞放疗后加速再增殖的体外实验研究

目的 研究放疗后濒死胰腺癌细胞（PANC-1）释放的微小RNA-7-5p（miR-7-5p）对存活PANC-1加速再增殖效应的相关机制。方法 通过慢病毒感染,建立荧光素酶标记的PANC-1-LUC胰腺癌细胞放疗后加速再增殖模型。通过RT-qPCR检测miRNA-7-5p的表达水平,流式细胞仪检测细胞周期及凋亡,双荧光素酶法验证miR-7-5p靶基因,Western blotting检测γH2A.X与DDIT4 蛋白的表达情况。结果 miR-7-5p在受辐照PANC-1（10 Gy）濒死细胞上清中表达下调,受辐照PANC-1细胞与存活PANC-1-LUC细胞共培养,可促进放疗后存活PANC-1-LUC细胞加速再增殖及其DNA损伤修复,而上调miR-7-5p可抑制放疗后胰腺癌细胞PANC-1-LUC再增殖。进一步研究表明,miR-7-5p在胰腺癌细胞中通过靶向下调DDIT4通路促进凋亡信号。结论 受辐照胰腺癌细胞可通过下调miR-7-5p促进DDIT4表达,从而抑制细胞凋亡及调控细胞周期再分布来加速再增殖,表明提高miR-7-5p的水平能够提高胰腺癌细胞的放疗敏感性,这将为改善胰腺癌放疗抵抗提供了一种新的思路和方法。     

膀胱尿路上皮癌细胞株E2F3基因与miR-17-5p和miR-20a的相互作用

目的 研究E2F3基因、miR-17-5p和miR-20a在膀胱尿路上皮癌细胞株中的表达及相互影响,探讨E2 F3基因与miR-17-5p和miR-20a在膀胱癌发生中的作用. 方法 用pcDNA3.1-HA-E2F3和pAAV-siRNA-E2F3质粒在膀胱癌细胞株5637中分别过表达和敲低E2F3基因,用miR-17-5p和miR-20a的模拟物和反义寡核苷分别过表达和敲低miR-17-5p和miR-20a后,实时荧光定量PCR法检测miR-17-5 p、miR-20a和E2F3基因的变化,蛋白质印迹法检测E2F3蛋白水平的变化.结果 E2F3基因过表达后,miR-17-5p和miR-20a的2-△ △Ct值分别为2.26±0.30和4.04 ±0.51,与对照组(1.00±0.00)比较差异有统计学意义(P＜0.05);敲低E2F3后,miR-17-5p和miR-20a的2-△△Ct值分别为0.49±0.02和0.65 ±0.04,与对照组(1.00±0.00)比较差异有统计学意义(P＜0.05);同时过表达miR-17-5p和miR-20a,E2F3的mRNA水平明显下调,蛋白质平均灰度值为55.31 ±7.89,对照组为103.67±13.61,与对照组比较差异有统计学意义(P＜0.05);同时敲低miR-17-5p和miR-20a后,E2 F3的mRNA比对照显著上调,蛋白质平均灰度值为295.68±19.25,与对照组平均灰度值为103.67±13.6l比较差异有统计学意义(P＜0.05). 结论 E2F3基因可上调miR-17-5p和miR-20a的表达,miR-17-5p和miR-20a可以下调E2F3基因的表达.E2F3基因、miR-17-5p和miR-20a在膀胱尿路上皮癌中形成调节环路,协调膀胱肿瘤细胞的生长.     

miR-129-5p inhibits gemcitabine resistance and promotes cell apoptosis of bladder cancer cells by targeting Wnt5a

Objectives

Gemcitabine resistance is a major obstacle for effective treatment of bladder cancer. This study was aimed to investigate the potential role of miR-129-5p in the development of gemcitabine resistance in bladder cancer cells and its underlying mechanism.

Methods

The IC50 for gemcitabine in 20 bladder cancer cells was first profiled from Genomics of Drug Sensitivity in Cancer. miR-129-5p level and gene mRNA expression were detected using quantitative real-time PCR (qRT-PCR). Cell viability, apoptosis, and gene protein level were assessed by MTT, flow cytometry, and Western blot, respectively. Regulatory relationship between Wnt5a and miR-129-5p was determined using luciferase reporter assay.

Results

We found that down-regulated miR-129-5p level contributed to gemcitabine resistance in bladder cancer cells and tissues. We also observed restoration of miR-129-5p could significantly increase cell sensitivity to gemcitabine and promote cell apoptosis. Mechanism analysis revealed that Wnt5a is a direct target gene of miR-129-5p and knock-down of Wnt5a reversed gemcitabine resistance.

Conclusions

Taken together, our findings indicate that miR-129-5p and Wnt5a may be novel therapeutic targets for overcoming gemcitabine resistance in bladder cancer treatment.

     

The efficiency of serum lncRNA GAS5/miR-21 as biomarkers in patients with diabetes and diabetic nephropathy

Objective To elucidate the efficiency lncRNA GAS5 and miR-21 as biomarkers in diabetes mellitus and diabetic nephropathy. Methods The patients were divided into three groups, diabetic nephropathy group (DN group proven by renal biopsy, n=25, 14 males and 11 females), diabetes group (DM group, with normal urine albumin creatinine ratio, n=10, 4 males and 6 females), and normal control group (NC group, n=9, 4 males and 5 females). The expressions of lncRNA GAS5 and miR-21 in serum samples were detected by real-time quantitative PCR. The correlation between serum lncRNA GAS5 and miR-21 expressions and the clinical parameters was analyzed by T-test, Pearson, Spearman test and multivariate linear regression analysis. Differences of lncRNA GAS5 and miR-21 in different groups were analyzed by one-way analysis of variance. The ROC curve was used to analyze the diagnostic efficacy of lncRNA GAS5 and miR-21 in diabetes and diabetic nephropathy. All data were analyzed by SPSS 20.0 and GraphPad software, with P＜0.05 as considered statistically significant. Results (1) The expression of serum lncRNA GAS5 was significantly down-regulated and serum miR-21 was significantly up-regulated in both diabetes mellitus and diabetic nephropathy patients compared to the NC group all (P＜0.05). (2) In DN patients, the expression of serum lncRNA GAS5 was gradually up-regulated along with the increment of 24 h urinary protein. The expression of serum miR-21 was gradually up-regulated along with renal biopsy stage IIb-Ⅲ of DN (P＜0.05). (3) FBG and HbA1c were all negatively correlated with serum lncRNA GAS5 (P＜0.05), and FBG was independently correlated with serum lncRNA GAS5 (P＜0.05). Urine microalbumin, Total cholesterol(TC), Scr, Urea and SBP were all positively correlated with serum miR-21(P＜0.05). Albumin (ALB) and estimated GFR (eGFR) were negatively correlated with serum miR-21(P＜0.05), and ALB was independently correlated with serum miR-21 (P＜0.05). (4) The diagnostic efficiency of serum lncRNA GAS5, miR-21 and lncRNA GAS5/miR-21 as "diagnostic signature" for DM were was good (P＜0.05). (5) The diagnostic efficiency of serum miR-21 and lncRNA GAS5/miR-21 as "diagnostic signature" for DN were was good (P＜0.05). Conclusions (1) Serum lncRNA GAS5 had good diagnostic efficiency in diabetes mellitus. The sensitivity of lncRNA GAS5/miR-21 for diagnosis of diabetes was 85.71%, and specificity was 88.89%. (2) The level of serum miR-21 can be used as a noninvasive diagnostic marker for diabetic nephropathy.     

Sputum and serum calprotectin are useful biomarkers during CF exacerbation

BackgroundAdequate monitoring of cystic fibrosis lung disease is difficult. CF exacerbation offers a unique setting to test the utility of biomarkers in the assessment of changing airways inflammation. We hypothesised that levels of calprotectin in sputum (and serum) would change informatively following treatment of an exacerbation.Methods27 patients with CF were recruited at onset of pulmonary exacerbation. Sputum and serum were collected at the start and end of anti-biotic therapy. Sputum calprotectin, interleukin-8 (IL8), and myeloperoxidase (MPO) were measured, as were serum calprotectin, CRP and vascular endothelial growth factor (VEGF).ResultsSputum calprotectin decreased following treatment of an exacerbation (p < 0.05), and was superior to other sputum markers. Serum calprotectin, CRP, and VEGF also decreased significantly (p = 0.002, p = 0.002, p = 0.013 respectively). Serum calprotectin level following treatment had predictive value for time to next exacerbation (p = 0.032).ConclusionsThis study demonstrates the superiority of calprotectin (in sputum and serum) as a biomarker of CF exacerbation over better-established markers.     

miR-34b-5p promotes renal cell inflammation and apoptosis by inhibiting aquaporin-2 in sepsis-induced acute kidney injury

ObjectiveThis study was designed to uncover the mechanism of miR-34b-5p-mediated aquaporin-2 (AQP2) in sepsis-induced injury using human renal tubular epithelial cells (HK-2).MethodsSerum levels of miR-34b-5p, TNF-α, IL-1β, IL-6, serum creatinine (SCr), and blood urea nitrogen (BUN) in septic patients with acute kidney injury (AKI) and healthy controls were detected. Lipopolysaccharide (LPS) was used to induce sepsis in HK-2 cells. LPS-induced HK-2 cells were transfected with miR-34b-5p inhibitor, miR-34b-5p mimic, pcDNA3.1-AQP2, si-AQP2, miR-34b-5p inhibitor + si-NC, or miR-34b-5p inhibitor + si-AQP2. The expressions of miR-34b-5p, AQP2, Bax, Bcl-2, cleaved caspase-3, TNF-α, IL-1β, and IL-6 in HK-2 cells were detected. TUNEL staining revealed the apoptosis of HK-2 cells. Dual-luciferase reporter assay verified the binding between miR-34b-5p and AQP2.ResultsThe expression of miR-34b-5p and the inflammatory responses were augmented in septic AKI patients. miR-34b-5p was up-regulated and AQP2 was down-regulated in LPS-induced HK-2 cells. miR-34b-5p inhibition or AQP2 overexpression ameliorated apoptosis and inflammation in LPS-induced HK-2 cells. In contrast, overexpressing miR-34b-5p deteriorated LPS-induced injury in HK-2 cells. AQP2 was a downstream target of miR-34b-5p. AQP2 silencing abolished the suppressive effects of miR-34b-5p inhibition on LPS-induced apoptosis and inflammatory response in HK-2 cells.ConclusionmiR-34b-5p inhibits AQP2 to promote LPS-induced injury in HK-2 cells.