microRNAs与糖尿病肾病

microRNAs(miRNAs)是一类内源性非编码调控单链小RNA,通过与靶mRNA的3'端非翻译区(3'UTRs)序列相互识别而引起靶mRNA降解或蛋白翻译抑制,从而参与机体器官发育分化、细胞增殖凋亡、肿瘤发生等多种生理病理过程。目前研究表明,miRNAs在糖尿病肾病(DN)的发病进展中发挥重要作用,一些miRNAs的表达异常(如miR-192、miR-21、miR-29、miR-377、miR-93、miR-200家族、miR-451和miR-251等的上调或下调)与DN的发生、发展密切相关。近期发现miRNAs在血清、血浆和尿液等多种体液中稳定存在,并且在DN发病状态下,一些miRNAs会发生特异性改变,提示miRNAs可作为DN诊断性标志物。而且,随着对miRNAs调控机制的深入研究,特定的miRNAs有可能成为DN的治疗靶点,为疾病的预防和进展提供新的治疗策略。     

CCL4在病毒性心肌炎中的作用及其机制研究

目的:观察巨噬细胞炎性蛋白-1β(MIP-1β/CCL4)在病毒性心肌炎小鼠血清以及心肌组织中的变化,初步探讨CCL4在病毒性心肌炎中发挥的作用。方法:雄性Balb/c小鼠随机分成对照组15只和病毒性心肌炎组25只,病毒性心肌炎组腹腔注射病毒液,对照组腹腔注射Eagle's培养液。15 d处死小鼠,通过HE染色观察小鼠心肌病理的变化;RT-PCR观察CCL4以及促炎因子的m RNA水平,Western blotting观察CCL4蛋白水平的变化。结果:与正常对照组比,病毒性心肌炎组小鼠生存质量降低,生存率也显著降低;病毒性心肌炎组小鼠心肌炎性浸润严重,心肌组织中的CCL4、IL-1β、TNF-α mRNA水平显著增高,CCL4蛋白水平也明显增高,并且CCL4的浓度和蛋白表达水平与心肌病变积分呈正相关。结论:病毒性心肌炎CCL4的表达显著增加,并且与心肌病变程度相关。     

心肌肌钙蛋白T在病毒性心肌炎心肌组织中的表达及意义

目的:了解病毒性心肌炎心肌组织中肌钙蛋白T的表达情况,探讨病毒性心肌炎时心肌结构蛋白损伤的机制及意义。方法:运用免疫组化和计算机图像分析技术,观察13例明确性病毒性心肌炎和17例界限性病毒性心肌炎尸检心脏标本中心肌肌钙蛋白T的表达与分布。结果:在正常对照的心肌组织中,蛋白成强阳性表达,分布均匀,未见缺染。在明确性心肌炎及14例界限性心肌炎心肌组织中都存在着不同程度的蛋白表达缺染或脱失。缺染的范围及分布与病毒性心肌炎病变特点基本一致,但其范围往往小于炎症细胞浸润范围。计算机图像分析和数据统计结果显示缺染区域的心肌肌钙蛋白T表达量要明显小于其周边区域和正常心肌细胞(P＜0．01)。结论:病毒性心肌炎患者的心肌损害要早于炎症细胞的浸润,病毒的作用可能是心肌肌钙蛋白T脱失的主要因素。心肌肌钙蛋白T的免疫组化检查可以作为一种有效的手段,来辅助病毒性心肌炎的病理学诊断。     

三七总黄酮通过miR-223-3p/FOXO1分子轴缓解病毒性心肌炎炎症反应和细胞损伤

病毒性心肌炎严重影响患者身体健康,中药材中黄酮类物质被证实对病毒性心肌炎有治疗作用,但其中三七总黄酮对柯萨奇B3病毒导致的心肌炎发挥治疗作用的分子机制尚不明确.以探讨三七总黄酮缓解病毒性心肌炎炎症反应及细胞损伤的作用机制.采用RT-qPCR检测心肌细胞中miR-223-3p的表达水平;Western blotting检测心肌细胞中转录因子叉头框蛋白O1(Forkhead box O1,FOXO1)蛋白表达水平;MTT实验检测心肌细胞存活率;流式细胞术检测心肌细胞凋亡率;ELISA检测炎症因子肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)和白细胞介素1β(Interleukin-1β,IL-1β)、心肌酶谱磷酸肌酸激酶(Creative kinase,CK)和乳酸脱氢酶(Lactic dehydrogenase,LDH)、心肌损伤标志物心肌肌钙蛋白T(Cardiac troponin T,cTnT)和B型尿钠肽(Brain natriuretic peptide,BNP)的水平;双荧光素酶报告基因检验miR-223-3p和FOXO1之间的靶向关系.实验结果显示,三七总黄酮能够缓解病毒性心肌炎模型细胞的炎症反应及细胞损伤,并显著上调模型细胞中miR-223-3p的水平.敲除模型细胞中的miR-223-3p能够逆转三七总黄酮对病毒性心肌炎的治疗作用.通过双荧光素酶报告基因实验验证miR-223-3p靶向负调控FOXO1蛋白的表达.进一步研究发现,过表达FOXO1可抑制三七总黄酮对病毒性心肌炎的治疗作用;但同时过表达miR-223-3p后,过表达FOXO1对三七总黄酮疗效的抑制作用被逆转.由此得出结论,三七总黄酮可缓解病毒性心肌炎模型细胞的炎症反应及细胞损伤,其作用机制是通过调控miR-223-3p/FOXO1分子轴实现的.     

病毒性心肌炎所致小鼠心力衰竭心肌组织内质网应激相关的凋亡关系的研究

目的：探讨病毒性心肌炎心力衰竭小鼠心肌组织内质网应激介导的凋亡途径。方法：40只雄性Balb／c小鼠分为病毒性心肌炎组和正常对照组（n=20）,病毒性心肌炎组应用柯萨奇B3病毒制作BALB／c小鼠病毒性心肌炎模型,观察小鼠的一般情况,7d行血流动力学检查后处死取心脏标本,用TUNEL法检测心肌细胞凋亡,RT-PCR检测心肌细胞内质网伴侣蛋白葡萄糖调节蛋白（GAP）78和GRP04的mRNA表达水平。结果：①与正常对照组相比,病毒性心肌炎组小鼠血流动力学指标明显降低（P〈0．01）;②TUNEL染色显示病毒性心肌炎心力衰竭小鼠心肌组织凋亡明显增多（P〈0．01）;③病毒性心肌炎组小鼠内质网伴侣蛋白GRP78和GRP94的mRNA表达水平均明显高于对照组（P〈0．01）。结论：病毒性心肌炎心力衰竭小鼠内质网应激可能介导了心肌细胞凋亡。     

黄芪对BALB/C小鼠感染Coxsackie B_3病毒后T细胞免疫的影响

本文用抗原特异性细胞毒技术检测小鼠感染coxsackie B_3病毒后并腹腔注射黄芪一周在5—28天脾脏、外周血及心肌中T淋巴细胞亚群分布的变化情况。结果显示:黄芪治疗组小鼠外周血、脾脏中Thy1+细胞百分率在感染后第7—14天明显增高(P<0.05),在心肌中则低于同期生理盐水组。L3T4+淋巴细胞百分率黄芪治疗组在病毒感染后5—14天的脾脏,第9天的外周血和第7、14天的心肌中显著低于生理盐水组,而Lyt2+淋巴细胞百分率两实验组间均无统计学差异,提示中药黄芪对急性病毒性心肌炎小鼠T细胞免疫具有明显的调节作用,且主要是通过调节Thy1+和L3T4+淋巴细胞亚群而起到治疗目的的。     

猪伪狂犬病毒Fa株侵染对PK-15细胞microRNAs表达谱的影响

【目的】分析猪伪狂犬病毒Fa株(PRV-Fa)侵染对猪肾传代细胞PK-15 microRNAs(miRNAs)表达谱的影响。【方法】利用Illumina高通量测序技术,鉴定感染和非感染PRV-Fa的PK-15细胞的miRNAs;筛选并利用实时荧光定量RT-PCR(RT-q PCR)验证差异表达miRNAs;对差异miRNAs进行靶基因预测和Gene ontology(GO)分析。【结果】在感染和未感染PK-15细胞中分别检测到384个和405个miRNAs,其中感染PRV-Fa后差异表达的miRNAs共127个(60个上调,67个下调)。荧光定量结果显示差异miRNAs的表达趋势与高通量测序结果一致。GO分析显示,miRNAs广泛参与信号传导、细胞代谢、免疫反应、基因表达等生物学进程,其中miR-10b、miR-16、miR-18a、miR-19b、miR-20a、miR-145-5p、miR-146a、miR-181a、miR-499-5p等miRNAs与免疫相关。在靶基因调控网络图中,ssc-miR-30a-5p与ssc-miR-30d处于关键位置。研究鉴定出5个新的病毒编码miRNAs,其中PRV-miR-LLT2与PRV-miR-LLT4靶向PRV早期蛋白基因EPO。【结论】伪狂犬病毒Fa株感染对PK-15细胞编码miRNAs有显著影响。     

B3型柯萨奇病毒感染的胰岛β细胞中microRNAs表达的变化及生物学意义

B3型柯萨奇病毒（Coxsackie virus B3,CVB3）感染与1型糖尿病发病及胰岛β细胞损伤有关,但机制并不清楚。病毒性心肌炎的研究证实CVB3通过调控微小RNA(microRNAs,miRNAs)的表达引起心肌细胞损伤。本研究的目的是观察CVB3感染的胰岛β细胞中miRNAs表达的变化及生物学意义,进而初步探究CVB3感染引起胰岛β细胞损伤的分子机制。本研究培养了人胰岛β细胞株,感染CVB3后采用miRNAs芯片及荧光定量PCR检测miRNAs表达的变化;转染miR-阴性对照（NC）或miR-146a-5p后感染CVB3,检测细胞活力、肿瘤坏死因子-α（Tumor necrosis factor-α,TNF-α）及白介素-1β（IL-1β）的含量、IRAK-1及TRAF-6的表达水平;采用双荧光素酶报告基因实验验证miR-146a-5p靶向IRAK-1及TRAF-6。结果显示：CVB3组中miR-101a-3p、miR-140-5p、miR-146a-5p、miR-146b-5p、miR-340-5p的表达水平均低于对照组且miR-146a-5p表达降低最显著;miR-NC...     

黄芪总黄酮对病毒性心肌炎小鼠心律失常与内质网应激因子的影响

目的：研究黄芪总黄酮（TFA）对病毒性心肌炎小鼠心律失常与内质网应激及缝隙连接蛋白作用,明确TFA抗病毒性心肌炎合并心律失常作用机制。方法：36只雄性Balb/c小鼠分为正常对照组、病毒性心肌炎组和TFA组（n=12）,病毒性心肌炎组腹腔内无菌注射含0.1 ml/d 10-950 TCID柯萨奇B3病毒（CVB3）,注射3 d制备Balb/c小鼠病毒性心肌炎模型,TFA组给予CVB3同时尾静脉注射0.1 ml TFA （20 mg/L）,共7 d。实验结束后心电图检测心律失常发生率后处死小鼠,取心脏行HE染色,观察心肌病理改变,Western blot检测各组小鼠心肌细胞葡萄糖调节蛋白78（GRP78）、内质网应激信号通路因子激活转灵因子4（ATF4）及缝隙连接蛋白（Cx43）表达。结果：与正常组比较,病毒性心肌炎组GRP78与ATF4的表达显著升高（P<0.01）,Cx43表达明显下降（P<0.01）;与病毒性心肌炎组比较,TFA组小鼠心肌细胞GRP78与内质网应激信号通路因子ATF4表达明显减少（P<0.01）,Cx43表达明显增多（P<0.01）。结论：TFA抗心律失常作用可能与缓解内质网应激及增加Cx43表达有关。     

miR-124-3p靶向TLR4抑制流感病毒性肺炎小鼠的炎症反应

目的:探讨miR-124-3p靶向Toll样受体4(TLR4)对流感病毒性肺炎小鼠炎症反应的影响。方法:建立流感病毒亚洲甲型鼠肺适应株(FM1)感染所致病毒性肺炎小鼠模型,采用RT-qPCR测定肺组织miRNA-124-3p表达水平。病毒性肺炎小鼠尾部注射miR-124-3p agomir或agomir NC后,第4 d摘眼球取血并取出肺组织,ELISA检测血浆中白细胞介素(IL)-1β、IL-6、肿瘤坏死因子α(TNF-α)水平,HE染色观察肺组织病理变化,Western印迹测定TLR4和核转录因子κB(NF-κB)p65蛋白水平。结果:与正常肺组织比较,病毒性肺炎小鼠肺组织miR-124-3p表达量降低(P0.001);miR-124-3p agomir注射组小鼠血浆中IL-1β、IL-6、TNF-α明显低于注射agomir NC组和模型组(P0.001),而高于正常组;HE染色结果表明miR-124-3p agomir注射组小鼠的肺组织炎细胞渗出和浸润较agomir NC组和模型组减轻;与agomir NC组比较,miR-124-3p agomir注射组的TLR4、NF-κB p65表达减少(P0.01)。结论:miR-124-3p抑制TLR4/NF-κB信号通路减轻流感病毒性肺炎小鼠的炎症反应。     

Elevated Muscle-Specific miRNAs in Serum of Myotonic Dystrophy Patients Relate to Muscle Disease Progress

The discovery of reliable and sensitive blood biomarkers is useful for the diagnosis, monitoring and potential future therapy of diseases. Recently, microRNAs (miRNAs) have been identified in blood circulation and might have the potential to be used as biomarkers for several diseases and clinical conditions. Myotonic Dystrophy type 1 (DM1) is the most common form of adult-onset muscular dystrophy primarily characterized by muscle myotonia, weakness and atrophy. Previous studies have shown an association between miRNAs and DM1 in muscle tissue and, recently, in plasma. The aim of this study was to detect and assess muscle-specific miRNAs as potential biomarkers of DM1 muscle wasting, an important parameter in the disease’s natural history. Disease stable or progressive DM1 patients with muscle weakness and wasting were recruited and enrolled in the study. RNA isolated from participants’ serum was used to assess miRNA levels. Results suggest that the levels of muscle-specific miRNAs are correlated with the progression of muscle wasting and weakness observed in the DM1 patients. Specifically, miR-1, miR-133a, miR133b and miR-206 serum levels were found elevated in DM1 patients with progressive muscle wasting compared to disease stable DM1 patients. Based on these results, we propose that muscle-specific miRNAs might be useful molecular biomarkers for monitoring the progress of muscle atrophy in DM1 patients.     

Human circulating microRNA-1 and microRNA-126 as potential novel indicators for acute myocardial infarction

Circulating miRNAs have been shown as promising biomarkers for various pathologic conditions. The aim of this study was to clarify that circulating miR-1 and miR-126 in human plasma might be useful as biomarkers in acute myocardial infarction (AMI). In our study, after pre-test, two candidate miRNAs were detected by using real-time RT-PCR. Cardiac troponin I (cTnI) concentrations were measured by ELISA assay in plasma from patients with AMI (n=17) and healthy subjects (n=25), simultaneously. Increased miR-1 and decreased miR-126 in plasma from patients with AMI after the onset of symptoms compared with healthy subjects were found. A remarkable finding in this study is that miR-1, miR-126 and cTnI expression levels exhibited the same trend. Our results suggest that the plasma concentrations of miR-1 and miR-126 may be useful indicators for AMI.     

Circulating muscle-specific microRNA, miR-206, as a potential diagnostic marker for rhabdomyosarcoma

Presently there is no serum biomarker of rhabdomyosarcoma (RMS). Several studies have shown that profiles of microRNA (miRNA) expression differ among tumor types. Here we evaluated the feasibility of using muscle-specific miRNAs (miR-1, -133a, -133b and -206) as biomarkers of RMS. Expression of muscle-specific miRNAs, especially miR-206, was significantly higher in RMS cell lines than in other tumor cell lines, as well as in RMS tumor specimens. Further, serum levels of muscle-specific miRNAs were significantly higher in patients with RMS tumors than in patients with non-RMS tumors. Normalized serum miR-206 expression level could be used to differentiate between RMS and non-RMS tumors, with sensitivity of 1.0 and specificity of 0.913. These results raise the possibility of using circulating muscle-specific miRNAs, especially miR-206, as landmark biomarkers for RMS.     

MicroRNA-206 is highly expressed in newly formed muscle fibers: implications regarding potential for muscle regeneration and maturation in muscular dystrophy

miR-1, miR-133a, and miR-206 are muscle-specific microRNAs expressed in skeletal muscles and have been shown to contribute to muscle development. To gain insight into the pathophysiological roles of these three microRNAs in dystrophin-deficient muscular dystrophy, their expression in the tibialis anterior (TA) muscles of mdx mice and CXMD(J) dogs were evaluated by semiquantitative RT-PCR and in situ hybridization. Their temporal and spatial expression patterns were also analyzed in C2C12 cells during muscle differentiation and in cardiotoxin (CTX)-injured TA muscles to examine how muscle degeneration and regeneration affect their expression. In dystrophic TA muscles of mdx mice, miR-206 expression was significantly elevated as compared to that in control TA muscles of age-matched B10 mice, whereas there were no differences in miR-1 or miR-133a expression between B10 and mdx TA muscles. On in situ hybridization analysis, intense signals for miR-206 probes were localized in newly formed myotubes with centralized nuclei, or regenerating muscle fibers, but not in intact pre-degenerated fibers or numerous small mononucleated cells, possibly proliferating myoblasts and inflammatory infiltrates. Similar increased expression of miR-206 was also found in C2C12 differentiation and CTX-induced regeneration, in which differentiated myotubes or regenerating fibers showed abundant expression of miR-206. However, CXMD(J) TA muscles contained smaller amounts of miR-206, miR-1, and miR-133a than controls. They exhibited more severe and more progressive degenerative alterations than mdx TA muscles. Taken together, these observations indicated that newly formed myotubes showed markedly increased expression of miR-206, which might reflect active regeneration and efficient maturation of skeletal muscle fibers.     

Plasma Levels of MicroRNA-499 Provide an Early Indication of Perioperative Myocardial Infarction in Coronary Artery Bypass Graft Patients

Background

Recent studies indicated that microRNAs (miRNAs, miRs) were important for many biological and pathological processes, and they might be potential biomarkers for cardiovascular diseases. The present study aims to determine the release patterns of miRNAs in cardiac surgery and to analyze the ability of miRs to provide early prediction of perioperative myocardial infarction (PMI) in patients undergoing coronary artery bypass graft (CABG) surgery.

Methodology/Principal Findings

Thirty on-pump CABG patients were recruited in this study; and miR-499, miR-133a and miR-133b, cardiac troponin I (cTnI) were selected for measurement. Serial plasma samples were collected at seven perioperative time points (preoperatively, and 1, 3, 6, 12, 24, and 48 hours after declamping) and were tested for cTnI and miRs levels. Importantly, miR levels peaked as early as 1–3 hours, whereas cTnI levels peaked at 6 hours after declamping. Peak plasma concentrations of miRs correlated significantly with cTnI (miR-499, r = 0.583, P = 0.001; miR-133a, r = 0.514, P = 0.006; miR-133b, r = 0.437, P = 0.05), indicating the degree of myocardial damage. In addition, 30 off-pump CABG patients were recruited; miR-499 and miR-133a levels were tested, which were significantly lower in off-pump group than in on-pump group. A prospective cohort of CABG patients (n = 120) was recruited to study the predictive power of miRs for PMI. The diagnosis of PMI strictly adhered to the principles of universal definition of myocardial infarction. The data analysis revealed that miR-499 had higher sensitivity and specificity than cTnI, and indicated that miR-499 could be an independent risk factor for PMI.

Conclusion

Our results demonstrate that circulating miR-499 is a novel, early biomarker for identifying perioperative myocardial infarction in cardiac surgery.