心肌细胞过表达miR-27b导致小鼠发生心肌纤维化和线粒体损伤

以往的miRNA芯片研究结果显示, miR-27b在人类心脏疾病标本和压力负荷引起的小鼠心肌肥厚模型中表达水平明显升高, 提示其在心脏疾病发生过程中发挥了重要功能。为研究miR-27b在心脏组织中的功能, 文章建立了在心肌细胞特异性 a-肌球蛋白重链(a-MHC)启动子(5.5 kb)控制下过表达miR-27b的转基因小鼠。通过Real-time PCR检测, 发现miR-27b前体和成熟体表达水平在转基因小鼠心脏组织中明显升高。miR-27b转基因小鼠不仅出现心肌肥厚, 还表现出明显的心肌纤维化。进一步研究表明心肌纤维化的关键调节分子金属基质蛋白酶13(MMP13)是miR-27b的靶分子, 在miR-27b转基因小鼠中MMP13显著下调, 胶原分子I和 III则显著上调。此外, 还发现miR-27b转基因小鼠会出现心脏超微结构的损伤。以上研究结果表明, miR-27b可能通过抑制MMP13促进心肌纤维化。     

miR-106b转基因小鼠的建立

目的:建立miR-106b转基因小鼠模型,探讨其在阿尔茨海默病(Alzheimer’s disease, AD)发病中的作用。方法:构建miR-106b表达载体,显微注射法建立miR-106b转基因小鼠。PCR鉴定转基因小鼠的基因型,real time RT-PCR检测miR-106b转基因小鼠脑组织中miR-106b的表达情况,Western blot检测miR-106b转基因小鼠脑组织中TGFBR2蛋白的表达。结果:构建了高表达miR-106b转基因小鼠;与对照相比,miR-106b转基因小鼠脑组织中TGFBR2蛋白的表达升高。结论:miR-106b转基因小鼠的建立为研究该microRNA在AD发病中的作用提供了工具。     

miR-122过表达转基因小鼠质粒构建及其功能验证

目的:比较两种miR-122转基因小鼠过表达载体构建方法,为建立miR-122过表达转基因小鼠奠定基础。方法:PCR扩增长约291bp的pre-miR-122的序列,分别定向克隆到pBROAD3-GFP载体GFP基因上游内含子或下游3'UTR区域,两种质粒分别转染293T细胞,Q-PCR检测miR-122和GFP的表达水平,并观察GFP绿色荧光。miR-122 sensor reporter是将3个miR-122成熟序列的反义序列串联克隆至psiCHECK2载体luciferase 3'UTR中,然后分别与2种miR-122过表达质粒载体共转染293T细胞,最后检测荧光素酶活性来鉴定miR-122调控功能。结果:2种构建方法的miR-122表达水平都明显增高,而只有插入到GFP基因3'UTR的质粒表达GFP功能正常。结论:构建microRNA过表达载体时,microRNA位于报告基因3'UTR区域不会影响microRNA和报告基因的功能;构建的两种miR-122过表达质粒载体都可应用到转基因小鼠研究中,而将miR-122插入到GFP下游的方法则更利于miR-122的表达。     

LncRNA RMST靶向miR-27a-3p影响过氧化氢诱导心肌细胞损伤

为了探讨LncRNA RMST对过氧化氢(H2O2)诱导的心肌细胞损伤的影响及其可能的作用机制,该研究采用H2O2诱导大鼠心肌细胞H9C2建立细胞氧化损伤模型,随机分组:si-NC+200 μmol/L H2O2组(即培养基中加入200 μmol/L H2O2干预2 h)、si-LncRNA RMST+200 μmol...     

心肌细胞特异性miR-30b转基因小鼠的建立及其功能研究

MicroRNAs(miRNAs)基因芯片结果显示,携带有MYH7基因突变的家族性肥厚性心肌病病人的心脏组织以及小鼠心力衰竭模型中miR-30b表达下调,提示miR-30b可能在心脏疾病发生发展过程中发挥了重要功能.为研究miR-30b在心脏组织中的功能,本实验室首先建立了在心肌细胞特异性启动子琢肌球蛋白重链(琢-MHC,5.5 kb)控制下过表达miR-30b的转基因小鼠.通过qRT-PCR方法,证实miR-30b表达水平在转基因小鼠心脏组织中明显升高(P0.05).miR-30b转基因小鼠心重/体重比和左心室/体重比无明显变化,心肌组织结构未见异常.目前,关于miR-30b在心肌梗死中的功能及相关机制未见报道.本文通过冠状动脉左前降支结扎法建立心肌缺血再灌注(ischemia-reperfusion,I/R)模型,以假手术组作为对照组.生化检测结果及TTC-Evans blue双染结果显示,I/R损伤后,与野生型小鼠相比,转基因小鼠LDH、CK-MB和cTn玉浓度显著减小(P0.05),并且心肌梗死面积明显减少(P0.05).超声心动图检测结果显示,转基因小鼠心功能显著改善.由此得出结论:miR-30b对缺血再灌注损伤的心肌具有保护作用,该研究成果可能为预防和治疗心肌梗死提供新策略.     

小鼠心肌纤维化模型的制备及其胶原沉积的机制

目的 建立缺血性心肌纤维化小鼠模型并探讨其胶原沉积机制.方法 将BALB/c小鼠随机分为实验组和对照组,每组10只.实验组予以腹部皮下注射异丙肾上腺素50 ms/kg,每天2次,连续10 d.对照组同法注射生理盐水.对比体表心电图,45 d后处死小鼠,天狼猩红染色观察心脏Ⅰ、Ⅲ型胶原纤维含量,荧光定量PCR检测心脏基质金属蛋白酶9(MMP-9)、基质金属蛋白酶组织抑制剂一1(TIMP-1)基因表达,免疫组化染色分析心、肝、肾组织层粘连蛋白(LN)的表达.结果 实验组小鼠室性心律失常增多,心率加快(P<0.05);心脏胶原沉积较多,MMP-9、TIMP-1、LN表达上调(P<0.05),肝、肾组织LN无明显改变(P>0.05).结论 异丙肾上腺素能制备缺血性心肌纤维化小鼠模型,其机制与MMP-TIMP失衡有关.     

miR-124和miR-520b下调Eps8表达并抑制HeLa细胞增殖

Eps8是一个多功能的信号分子,参与肌动蛋白重排、受体内吞和肿瘤的发生发展. 为了寻找靶向Eps8的microRNAs (miRNAs)并研究其在宫颈癌中的调控作用,本文采用软件预测获得4个可能调控Eps8表达的miRNAs. 利用双荧光素酶报告系统和Western印迹研究发现,miR-124 和miR-520b结合到人EPS8 mRNA的3′非翻译区(untranslated region, UTR)并有效抑制Eps8蛋白的表达. 进一步细胞存活检测、MTT法和克隆形成实验分析显示,miR-124和miR-520b过表达显著抑制HeLa细胞的生长和增殖.而且,miRNA调节的Eps8下调能提高HeLa细胞对化疗药物顺铂的敏感性. 同时证明,miR-124和miR-520b激活肿瘤抑制基因p53与下游基因p21报告基因转录活性,也相应地上调了p53与p21的蛋白表达. 这些结果提示,miR-124和miR-520b下调癌基因EPS8表达,从而抑制HeLa细胞增殖,负调控宫颈癌细胞生长.     

心肌特异性高表达热休克蛋白27转基因鼠建立

目的 建立人热休克蛋白27（heat shock protein 27, Hsp27）基因在小鼠心肌特异性表达的转基因鼠模型。 方法 将人心肌Hsp27cDNA插入含有心肌特异性表达启动子αMHC的pBSⅡ-SK+载体中,限制性内切酶EcoRI酶切、纯化后,获得含有αMHC启动子－Hsp27cDNA－HGH polyA的线性DNA片段,以显微注射法将目的基因导入受精卵, PCR筛选基因型,Western Blot鉴定转移基因的表达和表达的组织特异性。结果和结论 共获得两个转基因系小鼠,均呈心肌组织特异性高表达。     

过表达微小RNA miR-125b对人胚胎干细胞增殖的影响

目的:通过在人胚胎干细胞(hESC)中有效转染微小RNA miR-125b的真核表达载体,研究过表达miR-125b对hESC增殖的影响。方法:将在无饲养层上培养至第3 d,克隆融合达70%的hESC用Accutase酶消化为单细胞,然后用LipofectAMINE2000对hESC单细胞转染pHRS-1cla-miR125b-CMV-EGFP载体及其对照pHRS-1cla-CMV-EGFP载体,通过实时定量PCR对转染后细胞中成熟miR-125b的表达进行检测;进一步进行细胞计数和克隆计数,对miR-125b表达上调的hESC的增殖情况进行分析。结果:实时定量PCR检测结果表明,细胞转染后72 h,miR-125b的表达上调1.45倍,说明hESC转染成功;克隆计数及细胞计数结果显示过表达miR-125b的hESC增殖受到明显抑制(P<001)。结论:转染miR-125b真核表达载体的hESC能够上调成熟miR-125b的表达,hESC中miR-125b的表达上调能明显抑制hESC的增殖。     

幽门螺杆菌相关性胃疾病中miR-551b和miR-124a的表达及意义CSCD

目的 检测miR-551b、miR-124a在慢性浅表性胃炎(CSG)、慢性萎缩性胃炎(CAG)及胃癌(GC)患者胃黏膜组织中表达水平,探讨miR-551b、miR-124a与幽门螺杆菌(H.pylori)感染的关系及在胃癌发生、发展中的作用。方法 选择2018年7月-2019年8月在本院肿瘤科与消化科住院的患者120例,其中CSG患者40例,CAG患者40例,GC患者40例。采用qRT-PCR法检测各研究对象胃黏膜组织中miR-551b、miR-124a表达水平,分析miR-551b、miR-124a水平与H.pylori感染及GC患者临床病理特征的关系。结果 GC组患者胃黏膜组织中miR-551b、miR-124a表达水平低于CSG组、CAG组(P<0.05),CSG、CAG组之间比较,差异无统计学意义(P>0.05);H.pylori阳性感染GC患者胃黏膜组织中miR-551b、miR-124a表达水平低于H.pylori阴性感染患者,差异有统计学意义(P<0.05);中低分化、TNM分期为Ⅲ~Ⅳ、浸润深度为T3-T4、有淋巴结转移的GC患者胃黏膜组织中miR-551b、miR-124a表达量低于高分化、TNM分期为Ⅰ~Ⅱ、浸润深度为T1-T2、无淋巴结转移的GC患者(P<0.05)。miR-551b、miR-124a表达与性别、年龄、肿瘤大小无关(P>0.05)。结论 miR-551b、miR-124a在GC患者胃黏膜组织中低表达,且在H.pylori阳性感染患者中低表达,检测miR-551b、miR-124a表达水平可能在H.pylori感染所致胃黏膜组织癌变过程中具有一定预测作用。     

Cardiomyocyte overexpression of miR-27b induces cardiac hypertrophy and dysfunction in mice

Recent studies have begun to reveal critical roles of microRNAs (miRNAs) in the pathogenesis of cardiac hypertrophy and dysfunction. In this study, we tested whether a transforming growth factor-β (TGF-β)-regulated miRNA played a pivotal role in the development of cardiac hypertrophy and heart failure (HF). We observed that miR-27b was upregulated in hearts of cardiomyocyte-specific Smad4 knockout mice, which developed cardiac hypertrophy. In vitro experiments showed that the miR-27b expression could be inhibited by TGF-β1 and that its overexpression promoted hypertrophic cell growth, while the miR-27b suppression led to inhibition of the hypertrophic cell growth caused by phenylephrine (PE) treatment. Furthermore, the analysis of transgenic mice with cardiomyocyte-specific overexpression of miR-27b revealed that miR-27b overexpression was sufficient to induce cardiac hypertrophy and dysfunction. We validated the peroxisome proliferator-activated receptor-γ (PPAR-γ) as a direct target of miR-27b in cardiomyocyte. Consistently, the miR-27b transgenic mice displayed significantly lower levels of PPAR-γ than the control mice. Furthermore, in vivo silencing of miR-27b using a specific antagomir in a pressure-overload-induced mouse model of HF increased cardiac PPAR-γ expression, attenuated cardiac hypertrophy and dysfunction. The results of our study demonstrate that TGF-β1-regulated miR-27b is involved in the regulation of cardiac hypertrophy, and validate miR-27b as an efficient therapeutic target for cardiac diseases.     

Transgenic overexpression of cardiac actin in the mouse heart suggests coregulation of cardiac,skeletal and vascular actin expression

Previous studies have shown that depletion of cardiac actin by targeted disruption is associated with increased expression of alternative actins in the mouse heart. Here we have studied the effects of transgenic overexpression of cardiac actin using the -myosin heavy chain promoter. Lines carrying 7 or 8 copies of the transgene showed a 2-fold increase in cardiac actin mRNA and also displayed decreased expression of skeletal and vascular actin in their hearts. In contrast, a line with more than 250 copies of the transgene did not show a similar decrease in the expression of skeletal and vascular actin despite a 3-fold increase in cardiac actin mRNA. While the low copy number transgenic mice displayed hearts that were similar to non-transgenic controls, the high copy number transgenic line showed larger hearts with distinct atrial enlargement and cardiomyocyte hypertrophy. Further, while the low copy number transgenic mouse hearts were mildly hypocontractile when compared with non-transgenic mouse hearts, the high copy number transgenic mouse hearts were significantly so. We conclude that in the presence of a small number of copies of the cardiac actin transgene, homeostatic mechanisms involved in maintaining actin levels are active and negatively regulate skeletal and vascular actin levels in the heart in response to increased expression of cardiac actin. However, these putative mechanisms are either inoperative in the high copy number transgenic line or are countered by the enhanced expression of skeletal and vascular actin during cardiomyocyte hypertrophy.     

Biochanin-A alleviates fibrosis and inflammation in cardiac injury in mice

Biochanin-A (BCA), is an isoflavonoid, exhibits protective effects against various diseases. This study was conducted to observe the effect of BCA on isoprenaline (ISP)-induced cardiac fibrosis and explore the underlying mechanism. The curative effect of BCA was investigated with oral administration for 14 days in ISP-induced cardiac fibrosis in mice. The fibrotic biomarkers, like collagen I and III, were estimated by ELISA. Commercial kits were used to estimate cholesterol, triglycerides, and creatine kinase-myocardial band (CK-MB) levels. The messenger ribonucleic acid (mRNA) expression studies were performed by quantitative real-time polymerase chain reaction. Gelatin zymography was used to study the expression of matrix metalloproteinases-2 (MMP-2). BCA co-administration significantly improved the morphometric parameters; including heart weight, heart weight to body weight, heart weight to tibial length, and lipid profile. BCA treatment showed a reduction in inflammatory cells and collagen deposition as depicted in the histopathology of heart tissues. The enhanced levels of collagen-I, III, and hydroxyproline were significantly decreased by BCA co-treatment, whereas CK-MB level was reduced slightly. BCA co-administration increased the activity of reduced glutathione enzyme, showing the antioxidative effects of BCA. BCA treatment significantly reduced interleukin-6 (Il6) inflammatory cytokine along with partially decreased mRNA expression of fibrotic signaling markers such as natriuretic peptide type B (Nppb), α-smooth muscle actin (Acta2), connective tissue growth factor (Ctgf), transforming growth factor β (Tgfb), small mothers against decapentaplegic homolog-3 (Smad-3). However, BCA did not modify Mmp-2 expression, which was significantly increased by ISP. In conclusion, BCA exerts an antifibrotic effect by modulating lipid profile, enhancing antioxidant enzyme, and reducing collagen content and inflammation.     

miR-146b overexpression ameliorates lipopolysaccharide-induced acute lung injury in vivo and in vitro

Acute respiratory distress syndrome (ARDS) is a type of acute lung injury (ALI), which causes high morbidity and mortality. So far, effective clinical treatment of ARDS is still limited. Recently, miR-146b has been reported to play a key role in inflammation. In the present study, we evaluated the functional role of miR-146b in ARDS using the murine model of lipopolysaccharide (LPS)-induced ALI. The miR-146b expression could be induced by LPS stimulation, and miR-146b overexpression was required in the maintenance of body weight and survival of ALI mice; after miR-146b overexpression, LPS-induced lung injury, pulmonary inflammation, total cell and neutrophil counts, proinflammatory cytokines, and chemokines in bronchial alveolar lavage (BAL) fluid were significantly reduced. The promotive effect of LPS on lung permeability through increasing total protein, albumin and IgM in BAL fluid could be partially reversed by miR-146b overexpression. Moreover, in murine alveolar macrophages, miR-146b overexpression reduced LPS-induced TNF-α and interleukin (IL)-1β releasing. Taken together, we demonstrated that miR-146b overexpression could effectively improve the LPS-induced ALI; miR-146b is a promising target in ARDS treatment.     

miR-27b synergizes with anticancer drugs via p53 activation and CYP1B1 suppression

Liver and kidney cancers are notorious for drug resistance. Due to the complexity, redundancy and interpatient heterogeneity of resistance mechanisms, most efforts targeting a single pathway were unsuccessful. Novel personalized therapies targeting multiple essential drug resistance pathways in parallel hold a promise for future cancer treatment. Exploiting the multitarget characteristic of microRNAs (miRNAs), we developed a new therapeutic strategy by the combinational use of miRNA and anticancer drugs to increase drug response. By a systems approach, we identified that miR-27b, a miRNA deleted in liver and kidney cancers, sensitizes cancer cells to a broad spectrum of anticancer drugs in vitro and in vivo. Functionally, miR-27b enhances drug response by activating p53-dependent apoptosis and reducing CYP1B1-mediated drug detoxification. Notably, miR-27b promotes drug response specifically in patients carrying p53-wild-type or CYP1B1-high signature. Together, we propose that miR-27b synergizes with anticancer drugs in a defined subgroup of liver and kidney cancer patients.     

RASSF1 promotes cardiomyocyte apoptosis after acute myocardial infarction and is regulated by miR-125b

Cardiomyocyte apoptosis is a common pathological injury in association with acute myocardial infarction (AMI). In the current study, the relationship between Ras-association domain family 1 (RASSF1) and cardiomyocyte apoptosis was investigated. RASSF1 was significantly over expressed in infarcted myocardial tissues as well as in cardiomyocytes induced by hypoxia. Inhibition of RASSF1 expression alleviated cardiomyocytes apoptosis induced by hypoxia in vitro and reduced cardiomyocytes apoptosis after AMI in vivo. RASSF1 expression was directly modulated by miR-125b, which was further confirmed by luciferase reporter assay. The current study verified that the miR-125b/RASSF1 axis was involved in cardiomyocytes apoptosis. To sum up, these results suggest that RASSF1 downregulation alleviated infarction-induced cardiomyocytes apoptosis and was regulated by miR-125b.