miR-217参与高糖诱导的内皮细胞凋亡的调控

目的:研究miR-217对高糖诱导的内皮刺激内皮细胞凋亡的作用。方法:培养人冠状动脉内皮细胞,用含D-葡萄糖(30mmol/L)的培养液刺激:(1)利用实时定量PCR检测内皮细胞相关微小RNA(miR-217、miR-137、miR-29c、miR-218、miR-451、miR-328、miR-517c和miR-216a等)的表达变化;(2)利用慢病毒感染技术干预内皮细胞miR-217水平,利用流式细胞术检测细胞凋亡水平;(3)生物信息学预测、双荧光素酶报告基因验证以及蛋白免疫印迹法(western blot)确定miR-217的靶基因。结果:(1)实时定量PCR检测发现高糖刺激内皮细胞后,miR-217、miR-137、miR-29c、miR-218等的表达上调(P0.01),miR-451、miR-328、miR-517c和miR-216a的表达下调(P0.01),其中miR-217比对照细胞升高了5.67倍;(2)慢病毒感染内皮细胞后再经高糖刺激,流式细胞术检测发现过表达miR-217的内皮细胞凋亡水平有显著提高;(3)生物信息学分析发现SIRT1基因的3'非翻译区上存在一个miR-217的结合位点,双荧光素酶报告基因和western blot结果均证明SIRT1是miR-217的靶基因。结论:miR-217可能通过抑制SIRT1的表达参与高糖诱导的内皮细胞凋亡的调控。     

氧化应激诱导内皮细胞凋亡microRNA表达改变的研究

目的:研究氧化应激诱导的内皮细胞micro RNA的表达变化。方法:ECM(Endothelial Cell Medium)培养人脐静脉内皮细胞,利用不同浓度双氧水(0μmol/L,200μmol/L,500μmol/L,800μmol/L)刺激24小时后应用流式细胞术检测其凋亡水平。提取细胞总RNA,利用实时定量PCR(Quantitive real-time PCR;q RT-PCR)检测micro RNA表达量变化,并利用生物信息学软件预测可能的靶基因。结果:加入不同浓度双氧水处理24 h后的内皮细胞总凋亡率均显著高于对照组,200μmol/L、500μmol/L和800μmol/L组的凋亡率分别为(13.31%vs 4.75%,35.9%vs 4.75%,89.75%vs 4.75%,P0.01)。200μmol/L的双氧水处理内皮细胞后,micro RNA的表达出现了明显的改变。其中mi R-92a、mi R-126的表达明显下调(P0.05),mi R-181a、mi R-217、mi R-34a和mi R-320的表达明显上调(P0.05)。靶基因预测显示mi R-320、mi R-92a可能调控多个和内皮细胞凋亡相关的基因表达。结论:在氧化应激诱导的内皮细胞凋亡中,mi RNA表达发生改变并可能参与调控内皮细胞功能。     

MiR-324-5p可能通过靶向淀粉样前体蛋白抑制棕榈酸诱导的3T3-L1脂肪细胞凋亡

miRNAs是一种非编码的小RNA,通过靶向mRNA的3′UTR调控基因的转录后翻译。为明确miR-324-5p对棕榈酸诱导的3T3-L1脂肪细胞凋亡的作用和机制,体外培养3T3-L1脂肪细胞,利用棕榈酸诱导细胞凋亡的同时过表达或抑制miR-324-5p,通过Annexin-V/FITC染色、RT-qPCR等方法检测miR-324-5p对3T3-L1脂肪细胞凋亡的作用。通过在线软件预测miR-324-5p的靶基因并进行验证。结果显示,过表达miR-324-5p能够显著抑制凋亡相关基因BCL-2相关X蛋白(BCL2-associated X protein, Bax)和胱天蛋白酶3(caspase3)的表达水平(P0.05);而抑制miR-324-5p后能够显著促进这些基因的表达(P0.05);靶基因预测及验证结果表明,miR-324-5p能够显著降低淀粉样前体蛋白(amyloid precursor protein, APP)的表达水平(P0.05)。本研究认为,miR-324-5p可能通过靶定APP抑制棕榈酸诱导的3T3-L1脂肪细胞凋亡。     

microRNA在胰腺癌中的研究进展

胰腺癌是预后很差的恶性肿瘤,其分子机制的研究是治愈胰腺癌的希望.microRNA(miRNA)是一类小分子非编码RNA,通过降解或抑制靶基因mRNA的翻译调节靶基因的功能.近年来,研究发现miRNA在胰腺癌中异常表达,有上调,也有下调.虽然很多miRNA异常表达的机制尚不清楚,但启动子CpG甲基化与在胰腺癌中某些miRNA的下调有关.研究发现miRNA表达谱可用于胰腺癌和单个miRNA表达如miR-21、miR-34、miR-10a、miR-155、miR-196a及miR-200和let-7家族成员等可作为肿瘤标志物用于胰腺癌与正常胰腺、慢性胰腺炎、胰腺内分泌肿瘤等的诊断和鉴别诊断,预测胰腺癌的预后等.研究发现miRNA在胰腺癌中上调或下调参与胰腺癌的增殖、凋亡、侵袭、转移以及对化疗药物的耐药.研究发现miR-34、miR-200c等与胰腺癌干细胞的自我更新有关.这些miRNA研究将为胰腺癌的早期诊断、分子靶向治疗打下坚实的基础.     

LPS诱导脓毒症小鼠脾组织microRNA表达的筛选研究

目的采用Small RNA测序技术检测脓毒症小鼠24 h脾组织microRNA(miRNA)的表达变化,并从非编码基因层面分析脓毒症脾功能损伤的可能机制。方法将30只SPF级健康BALB/C小鼠随机分为对照组和脓毒症组,每组15只。脓毒症组采用LPS 10 mg/kg腹腔注射进行建模,对照组仅予等量生理盐水腹腔注射。两组术毕均行肌肉注射平衡液5 ml/kg,并于术后24 h各组随机取5只小鼠的脾组织提取RNA,采用Small RNA测序技术进行miRNA检测,然后应用生物信息学软件分析脓毒症小鼠脾组织的miRNA表达差异及靶基因预测。结果小鼠建模后24 h,与对照组相比,脓毒症小鼠脾脏组织已知及未知miRNA表达上调数分别为39个、3个,下调数分别为16个、3个。其中已知miRNA表达上、下调倍数最大的前5个基因分别为Rno-miR-217-5p,rno-mir-216a-5p,rnomiR-216b-5p,rno-miR-375-3p,rno-mir-216b-3p。下调倍数前5的miRNA:Rno-miR-138-5p,rno-miR-451a-3p,rno-miR-138-1-3p,rno-miR-202-5p,rno-miR-592。结论LPS诱导脓毒症小鼠可出现miRNA表达量的改变,其上、下调倍数最显著的miRNA可能对预测LPS诱导的脓毒症脾功能损伤有一定指导意义。     

大鼠肝纤维化相关microRNA及其候选靶基因的筛选

microRNAs (miRNAs)是一类功能性非编码RNA,在多种生物过程中具有重要作用.然而,miRNA的表达模式、调控网络以及参与肝纤维化的miRNA仍有待阐明.为了探讨与肝纤维化相关的miRNA及其靶基因的功能,为临床肝纤维化治疗提供理论依据,本研究前期已采用胆管结扎法(BDL)建立大鼠胆汁淤积性肝纤维化模型.从大鼠肝脏中提取总RNA,应用基因芯片技术对胆汁淤积性肝纤维化肝组织中miRNA和mRNA表达谱进行综合分析;结合生物信息方法分析在胆汁淤积性肝纤维化中差异表达miRNA可能的靶基因;实时荧光定量PCR技术检测TGF-β1处理人肝星状细胞LX-2细胞中miR-29a-3p、miR-194-5p和miR-22-3p相对表达水平.结果 表明,与正常肝组织相比,纤维化肝组织中有48个差异表达miRNA (FC＞2,P＜0.05),其中36个上调,12个下调;筛选出18个预测靶基因参与与纤维化相关的生物过程;TGF-β1处理LX-2细胞中miR-29a-3p、miR-194-5p和miR-22-3p相对表达水平显著下调(P＜0.05).本研究筛选的差异表达miRNAs通过调节靶基因的表达在肝纤维化中可能发挥重要作用,将为miRNA在肝纤维化中的作用提供新的见解.     

miR-451a促进胰腺癌细胞对吉西他滨的敏感性

目的:探究miR-451a在胰腺癌吉西他滨耐药中的功能。方法:通过低浓度梯度递增法建立胰腺癌吉西他滨耐药细胞株,microRNA(miRNA)测序筛选耐药相关miRNA;细胞存活曲线、克隆形成实验及流式凋亡实验分析miR-451a对胰腺癌细胞耐药的影响;裸鼠成瘤实验检测在动物体内模型中miR-451a对胰腺癌吉西他滨耐药的调控作用;调取TCGA数据分析miR-451a表达水平与胰腺癌病人预后的相关性。结果:胰腺癌吉西他滨耐药细胞株中miR-451a表达水平明显下调;miR-451a过表达增加胰腺癌细胞对吉西他滨的敏感性,增强了吉西他滨抑制细胞增殖和诱导细胞凋亡的作用;miR-451a诱导了裸鼠皮下肿瘤对吉西他滨敏感;miR-451a低表达与胰腺癌患者不良预后相关。结论:miR-451a增强了胰腺癌细胞对吉西他滨的敏感性。     

microRNA-151-5p在肾血管性高血压大鼠血管内皮细胞中的表达及意义

目的:研究microRNA-151-5p(miR-151-5p)在两肾一夹(2K1C)肾血管性高血压大鼠中的表达,并为miR-151-5p参与调节血管内皮细胞功能提供理论依据。方法:建立2K1C大鼠模型,获得胸主动脉血管内皮,采用荧光定量PCR技术检测血管内皮细胞中miR-151-5p的表达,通过数据库及生物信息学软件预测miR-151-5p的靶基因,并对靶基因进行GO富集和KEGG pathway分析。结果:与假手术组大鼠相比较,实验组大鼠胸主动脉血管内皮细胞miR-151-5p的表达量显著升高(P0.05)。GO分析显示miR-151-5p的靶基因参与蛋白加工水解、Notch受体加工等多种生物学功能(P0.01);KEGG pathway分析显示miR-151-5p的靶基因参与Notch信号通路、血管平滑肌收缩、代谢途径、细菌感染和RNA转运等信号通路。结论:2K1C大鼠血管内皮细胞中miR-151-5p表达升高,可能通过对其靶基因APH1A的调控参与血管内皮细胞功能调节。     

自噬对高糖诱导的人冠状动脉内皮细胞凋亡的保护作用

目的:研究自噬对高糖诱导的人冠状动脉内皮细胞凋亡的影响。方法:将人冠状动脉内皮细胞,分别用常规培养基(正常对照组)、含30 mmol/L D-葡萄糖的高糖培养基(高糖组)、高糖培养基合并雷帕霉素(Rapamycin,RAPA;100 nmol/L)干预(RAPA组)和高糖培养基合并3-甲基腺嘌呤(3-Methyladenine,3-MA,5 mmol/L)干预(3-MA组)培养。利用CCK-8法检测细胞生长活力,使用流式细胞术检测细胞凋亡水平,western blot检测细胞自噬标记蛋白(Beclin1)的表达水平。结果:(1)高糖溶液刺激内皮细胞24 h后,细胞生长活力为正常组的55.0%(P0.01),自噬标记蛋白Beclin1的表达水平明显增加,凋亡水平为正常组的2.0倍;(2)与高糖组相比,RAPA组细胞生长活力明显增加,Beclin1的表达明显升高(P0.01),凋亡水平为高糖组的70.1%;(3)与高糖组相比,3-MA组细胞生长活力明显减少,Beclin1的表达明显降低(P0.01),凋亡水平为高糖组的1.42倍。结论:细胞自噬可能对高糖诱导的人冠状动脉内皮细胞具有凋亡保护作用。     

猪圆环病毒2型通过外泌体miR-125a-5p靶向Bcl-2诱导淋巴细胞凋亡

为研究miR-125a-5p在猪圆环病毒2型(porcine circovirus type 2,PCV2)诱导淋巴细胞凋亡中的作用及其作用机制,以PCV2感染PK-15细胞外泌体孵育的淋巴细胞为研究对象,采用流式细胞术、蛋白质免疫印迹试验(Western blotting)和实时荧光定量PCR,检测淋巴细胞凋亡率及凋亡相关miRNA表达;合成miR-125a-5p模拟物和抑制物转染PK-15细胞,检测miR-125a-5p过表达或抑制表达后细胞凋亡率;采用生物信息学方法预测miR-125a-5p的靶基因,双荧光素酶报告基因检测miR-125a-5p对靶基因的调控;Western blotting检测外泌体孵育淋巴细胞的线粒体凋亡信号通路相关蛋白Bcl-2、Bax、细胞色素C和caspase-3的表达。结果显示,感染PCV2的PK-15细胞分泌的外泌体极显著提高淋巴细胞凋亡率,在一定浓度范围内呈剂量依赖性;与PCV2诱导细胞凋亡相关的miRNA中,miR-125a-5p表达量极显著升高,miR-125a-5p模拟物转染细胞后极显著提高细胞凋亡率;利用TargetScan预测发现,miR-125a-5p与Bcl-2 3''UTR区有结合位点,miR-125a-5p模拟物极显著抑制pmir-Bcl-2 3''UTR-WT荧光素酶活性,对pmir-Bcl-2 3''UTR-MuT的荧光素酶活性无明显改变;外泌体孵育的淋巴细胞Bcl-2表达量显著降低,Bax、细胞色素C的释放和caspase-3表达量显著升高,Bcl-2/Bax的比值极显著降低。这表明,PCV2通过外泌体诱导淋巴细胞上调miR-125a-5p的表达,进而抑制Bcl-2 mRNA和蛋白表达,激活淋巴细胞线粒体凋亡通路诱导细胞凋亡。     

Deep Sequencing of RNA from Three Different Extracellular Vesicle (EV) Subtypes Released from the Human LIM1863 Colon Cancer Cell Line Uncovers Distinct Mirna-Enrichment Signatures

Secreted microRNAs (miRNAs) enclosed within extracellular vesicles (EVs) play a pivotal role in intercellular communication by regulating recipient cell gene expression and affecting target cell function. Here, we report the isolation of three distinct EV subtypes from the human colon carcinoma cell line LIM1863 – shed microvesicles (sMVs) and two exosome populations (immunoaffinity isolated A33-exosomes and EpCAM-exosomes). Deep sequencing of miRNA libraries prepared from parental LIM1863 cells/derived EV subtype RNA yielded 254 miRNA identifications, of which 63 are selectively enriched in the EVs - miR-19a/b-3p, miR-378a/c/d, and miR-577 and members of the let-7 and miR-8 families being the most prominent. Let-7a-3p*, let-7f-1-3p*, miR-451a, miR-574-5p*, miR-4454 and miR-7641 are common to all EV subtypes, and 6 miRNAs (miR-320a/b/c/d, miR-221-3p, and miR-200c-3p) discern LIM1863 exosomes from sMVs; miR-98-5p was selectively represented only in sMVs. Notably, A33-Exos contained the largest number (32) of exclusively-enriched miRNAs; 14 of these miRNAs have not been reported in the context of CRC tissue/biofluid analyses and warrant further examination as potential diagnostic markers of CRC. Surprisingly, miRNA passenger strands (star miRNAs) for miR-3613-3p*, -362-3p*, -625-3p*, -6842-3p* were the dominant strand in A33-Exos, the converse to that observed in parental cells. This finding suggests miRNA biogenesis may be interlinked with endosomal/exosomal processing.     

lncRNA PVT1沉默对高糖诱导的血管内皮细胞增殖、凋亡及氧化应激的影响*

目的: 探讨抑制lncRNA PVT1对高糖诱导的血管内皮细胞的增殖,凋亡和氧化应激的影响。方法: 体外培养人脐静脉内皮细胞(HUVECs),分为四组:对照组(5.5 mmol/L葡萄糖),高糖组(30 mmol/L葡萄糖),高糖+siNC组(30 mmol/L葡萄糖+siNC,细胞转染阴性对照组),高糖+siPVT1组(30 mmol/L葡萄糖+siPVT1,抑制lncRNA PVT1组)。采用荧光定量PCR的方法检测转染后PVT1的表达水平。MTT检测siPVT1(短片段干扰RNA PVT1)对高糖诱导的HUVECs细胞增殖能力的影响。流式细胞术检测siPVT1对高糖诱导的HUVECs细胞ROS和凋亡水平。Western blot检测HUVECs细胞中凋亡相关蛋白如Bax,Bcl-2和cleaved-caspase-3的表达水平。结果: 与对照组比较,转染siPVT1后,PVT1的表达水平显著降低(P＜0.05)。MTT结果显示,与对照组比较,培养24 h和48 h后高糖组中HUVECs细胞增殖活力均显著降低,与高糖+siNC组(阴性对照组)比较,培养24 h和48 h后,高糖+siPVT1组中的HUVECs细胞增殖活力显著增加(P＜0.05)。流式细胞术检测结果表明,与对照组比较,高糖组HUVECs细胞中ROS和凋亡率均显著增加;和高糖+siNC组比较,高糖+siPVT1组中HUVECs细胞中ROS和凋亡率均有减少(P＜0.05)。Western blot结果表明,与对照组比较,高糖组中cleaved-caspase-3和Bax表达水平均显著上调,Bcl-2的表达水平显著下调(P＜0.05,P＜0.01)。与高糖+siNC组比较,高糖+siPVT1组cleaved-caspase-3和Bax表达水平显著下调,Bcl-2的表达显著上调(P＜0.05,P＜0.01)。结论: 抑制lncRNA PVT1可以显著增加高糖诱导的HUVECs细胞增殖活力,减轻氧化应激,抑制细胞凋亡。     

MicroRNA-222 alleviates radiation-induced apoptosis by targeting BCL2L11 in cochlea hair cells

Radiation-induced hair cell injury is detrimental for human health but the underlying mechanism is not clear. MicroRNAs (miRNAs) have critical roles in various types of cellular biological processes. The present study investigated the role of miR-222 in the regulation of ionizing radiation (IR)-induced cell injury in auditory cells and its underlying mechanism. Real-time PCR was performed to identify the expression profile of miR-222 in the cochlea hair cell line HEI-OC1 after IR exposure. miRNA mimics or inhibitor-mediated up- or down-regulation of indicated miRNA was applied to characterize the biological effects of miR-222 using MTT, apoptosis and DNA damage assay. Bioinformatics analyses and luciferase reporter assays were applied to identify an miRNA target gene. Our study confirmed that IR treatment significantly suppressed miR-222 levels in a dose-dependent manner. Up-regulation of miR-222 enhances cell viability and alleviated IR-induced apoptosis and DNA damage in HEI-OC1 cells. In addition, BCL-2-like protein 11 (BCL2L11) was validated as a direct target of miR-222. Overexpression of BCL2L11 abolished the protective effects of miR-222 in IR-treated HEI-OC1 cells. Moreover, miR-222 alleviated IR-induced apoptosis and DNA damage by directly targeting BCL2L11. The present study demonstrates that miR-222 exhibits protective effects against irradiation-induced cell injury by directly targeting BCL2L11 in cochlear cells.     

MicroRNA expression changes during human leukemic HL-60 cell differentiation induced by 4-hydroxynonenal,a product of lipid peroxidation

4-Hydroxynonenal (HNE) is one of several lipid oxidation products that may have an impact on human pathophysiology. It is an important second messenger involved in the regulation of various cellular processes and exhibits antiproliferative and differentiative properties in various tumor cell lines. The mechanisms by which HNE affects cell growth and differentiation are only partially clarified. Because microRNAs (miRNAs) have the ability to regulate several cellular processes, we hypothesized that HNE, in addition to other mechanisms, could affect miRNA expression. Here, we present the results of a genome-wide miRNA expression profiling of HNE-treated HL-60 leukemic cells. Among 470 human miRNAs, 10 were found to be differentially expressed between control and HNE-treated cells (at p < 0.05). Six miRNAs were down-regulated (miR-181a*, miR-199b, miR-202, miR-378, miR-454-3p, miR-575) and 4 were up-regulated (miR-125a, miR-339, miR-663, miR-660). Three of these regulated miRNAs (miR-202, miR-339, miR-378) were further assayed and validated by quantitative real-time RT-PCR. Moreover, consistent with the down-regulation of miR-378, HNE also induced the expression of the SUFU protein, a tumor suppressor recently identified as a target of miR-378. The finding that HNE could regulate the expression of miRNAs and their targets opens new perspectives on the understanding of HNE-controlled pathways. A functional analysis of 191 putative gene targets of miRNAs modulated by HNE is discussed.     

A Novel miR-451a isomiR,Associated with Amelanotypic Phenotype,Acts as a Tumor Suppressor in Melanoma by Retarding Cell Migration and Invasion

miRNAs are key regulatory small non-coding RNAs involved in critical steps of melanoma tumorigenesis; however, the relationship between sequence specific variations at the 5′ or 3′ termini (isomiR) of a miRNA and cancer phenotype remains unclear. Deep-sequencing and qRT-PCR showed reduced expression of miR-144/451a cluster and most abundant isomiR (miR451a.1) in dysplastic nevi, in-situ and invasive melanomas compared to common nevi and normal skin (n = 101). miRNA in situ hybridization reproducibly confirmed lost miR-451a.1 in melanoma compared to nevus cells or adjacent keratinocytes. Significantly higher expression of miR-451a.1 was associated with amelanotic phenotype in melanomas (n = 47). In contrast, miR-451a was associated with melanotic phenotype, absent pagetoid scatter of intraepidermal melanocytes, superficial spreading histological subtype and tumor inflammation. Sequencing miRNAs from cultured melanocytes with cytoplasmic melanin gradient (light, medium to dark) showed absent miR-451a while revealing other melanin-associated miRNAs, e.g. miR-30b, miR-100 and miR-590 in darkly and let-7a, let-7i and let-7f in lightly to moderately pigmented cultured melanocytes. Ectopic expression of miR-144/451a in melanoma cell lines resulted in markedly higher levels of mature miR-451a.1 than miR451a or miR-144; and significantly retarded cell migration and inhibited invasion in a glucose-sensitive manner. Surprisingly, these effects were not mediated by calcium binding protein 39 (CAB39), a proven miR451a gene target. miR-144/miR-451a cluster is a novel miRNA locus with tumor suppressive activity in melanoma.     

Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types

ABSTRACT: BACKGROUND: MicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs), and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported. RESULTS: Small RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146 overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs; pathway analysis of their predicted target genes suggests a potential role in regulation of endocytosis. RT-qPCR in additional cell types and analysis of publicly available data revealed that many of these miRNAs tend to be widely preferentially exported. Whilst overexpressed miR-146a was highly enriched both in transfected cells and their EVs, the cellular:EV ratios of endogenous miRNAs were not grossly altered. MiR-451 was consistently the most highly exported miRNA in many different cells types. Intriguingly, Argonaute2 (Ago2) is required for miR-451 maturation and knock out of Ago2 has been shown to decrease expression of other preferentially exported miRNAs (eg miR-150 and miR-142-3p). CONCLUSION: The global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Observation of similar patterns in a range of cell types suggests that a common mechanism for selective miRNA export may exist.