高通量miRNA活性谱检测发现BHK21细胞中miR-206高活性

用我们建立的活细胞miRNA活性谱检测技术测定BHK21、HEK293和Vero三种肾来源细胞系中58种miRNA活性谱,发现miR-206在BHK21细胞中具有特征性高活性.鉴于miR-206是典型的横纹肌特征性miRNA,进一步以小鼠成肌细胞C2Cl2及人胚肾细胞HEK293为对照,检测了BHK21细胞中miR-206的活性及表达水平.之后,用马血清诱导培养BHK21细胞,检测诱导前后BHK21细胞中骨骼肌肌球蛋白重链(Slow skeletal myosin heavy chain,MHC)表达情况,miR-206活性和表达水平以及受miR-206负调控的Connexin43(Cx43)的表达水平.结果发现,miR-206在BHK21细胞中活性和表达水平都明显高于C2C12细胞;马血清诱导后,BHK21细胞中MHC表达水平升高,miR-206活性和表达水平都升高,而Cx43表达水平下降.结果提示BHK21细胞具有成肌细胞特性.本研究首次发现BHK21细胞中miR-206的高活性,从miRNA角度证实了BHK21细胞来源于肾间质细胞,而不是肾实质细胞.研究结果还提示BHK21细胞有可能作为一种体外模型用于miR-206的功能研究.     

利用分泌型荧光素酶体外长期监测小鼠肝脏microRNA活性

活体动物microRNA（miRNA）检测技术对阐明miRNA的生物学功能非常重要.但是,目前缺乏一种方便灵敏的实时反映活体动物miRNA活性及其变化的体外监测手段.通过在分泌型荧光素酶Gluc（Gaussia princeps luciferase）mRNA的3′非翻译区插入相应miRNA的靶序列构建一系列miRNA传感器.将miRNA传感器体外转染培养细胞和通过水动力注射方法转染小鼠肝脏,通过检测细胞培养上清和外周血液中的Gluc来反映细胞内和活体动物肝脏的miRNA活性.结果显示,CMV启动子驱动的miRNA传感器可以长期监测体外培养细胞中的miRNA和短期监测小鼠肝脏的miRNA,但由于启动子在肝脏的沉默不能用于长期监测肝脏miRNA;CAG启动子驱动的传感器则成功地实现了对肝脏内源性miR-122,miR-142和miR-34a以及对肝脏过表达的miR-34a的长期实时监测.利用以Gluc为报告基因的miRNA传感器,通过检测外周血Gluc可以方便地在体外长期连续监测小鼠肝脏的miRNA.     

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。     

鸡miR-21的组织表达谱及其功能预测分析

已知miR-21在多种生物学过程中发挥重要的调控作用,然而有关鸡miR-21功能的研究尚未见报道。为了解鸡miR 21的潜在生物学功能,采用qRT-PCR检测了固始鸡5个发育阶段、15种组织中miR-21的表达情况。同时,利用Pictar和TargetScan算法预测了miR-21的靶基因并对预测的靶基因进行了Gene Ontology分析和通路分析。结果显示,miR-21的表达具有明显的时序特征,除14胚龄鸡的小脑和腺胃外,胚胎期各组织中miR-21的表达水平均显著低于出壳后对应组织的表达水平;出壳后鸡的下丘脑、大脑、小脑、小肠、肝脏、胸肌和肾脏等组织中miR-21的表达水平随发育进程均显著上调。生物信息学分析显示,预测的miR-21的靶基因在基因表达调控、大分子代谢调控、转录调控、细胞代谢调控、细胞衰老和增殖、呼吸系统发育、骨骼发育、心脏发育和神经系统发育等广泛的生物学过程中显著富集。总之,鸡miR-21为广泛性时序表达miRNA,可能参与鸡出壳后诸多器官发育相关的生物学过程的调控。     

植物多酚通过microRNA调控脂质代谢研究进展

microRNA（简称miRNA）是长度18~25个核苷酸的非编码RNA分子,具有调控mRNA的翻译和/或稳定性的功能,从而在转录后水平调节不同基因的表达。人体内约60%编码蛋白的基因的表达受到miRNA调节,其中包括脂质代谢调控相关基因。植物多酚具有良好的生物活性,可以通过调节脂质代谢相关miRNAs,如miR-122和miR-33的表达进而发挥降血脂等活性。该文综述了miRNA调控脂质代谢相关mRNA的作用机制以及植物多酚在这一过程中的可能作用。     

电脉冲刺激法提高小鼠miR-122表达的研究

目的:电脉冲能够改变细胞膜的通透性,促进外源DNA进入细胞内。通过基因导入仪电脉冲刺激已注入质粒pcDNA3.0-miR-122的小鼠肌肉细胞,检测对小鼠miR-122表达的影响。方法:将扩增得到的pre-miR-122克隆到真核表达载体pcDNA3.0上,构建pcDNA3.0-miR-122表达质粒。利用基因导入仪分别将生理盐水、pcDNA3.0、pcDNA3.0-miR-122质粒DNA导入小鼠腿部肌肉,然后进行电脉冲刺激。qRT-PCR测定各组小鼠血清及肝脏中的miR-122水平,同时检测肝癌组织和经Cecropin XJ处理的肝癌组织中的miR-122水平。结果:基因导入仪电脉冲刺激已注入pcDNA3.0-miR-122的腿部肌肉组织后,小鼠血清和肝脏中miR-122的表达分别提高了23和11倍,经Cecropin XJ处理的小鼠肝癌组织中miR-122的表达比对照高1.6倍。结论:电脉冲刺激能够显著提高小鼠体内miR-122的表达,为探讨miR-122表达对肝癌生长影响的调控作用奠定基础。     

人肝脏特异性miR-122表达载体的构建及鉴定

人肝脏特异性miRNA-122是肝脏中表达丰度最高的miRNA。为研究该miR-122的生物学功能,从HepG2细胞基因组中用PCR的方法扩增了miR-122的前体,构建了miR-122的表达载体pLMP-miR-122。pLMP-miR-122质粒转染人正常肝细胞系L-O2和肝癌细胞系HepG2后,细胞内成熟miRNA-122的表达量显著增加。该质粒与HBV1.3共转染HepG2细胞72h后,HBV的HBs和HBe蛋白水平的表达量均下降,说明miRNA-122参与了HBV基因的复制和表达的调控,为进一步研究miRNA-122的功能和其他一些肝病如HCC的调控机制打下基础。     

含p53保守结合位点的microRNA表达载体的构建及应用

目的:构建含p53保守结合位点的microRNA(miRNA)表达载体,促进相关miRNA在具有野生型p53蛋白细胞中的高效表达。方法:改构miRNA表达载体pCMV-miR,在其多克隆位点前插入p53保守结合位点,分别将miR-138、miR-34a和miR-21前体序列pre-miR-138、pre-miR-34a和pre-miR-21插入上述改构的载体pCMV/p53-miR,将构建的pCMV/p53-miR-138、pCMV/p53-miR-34a和pCMV/p53-miR-21表达载体转染具有野生型p53的HeLa细胞和不表达p53的H1299细胞,分析p53对上述miRNA表达调控的影响。结果:转染改构的miRNA表达载体后,HeLa细胞中miR-138、miR-34a和miR-21的表达水平明显提高,它们对应的已知靶基因Cyclin D3、CDK2和PTEN的表达同时被显著下调。结论:在p53转录调控作用下,具有p53保守结合位点的miRNA表达载体能够更加有效地提高miRNA的表达水平;构建的载体不但可用于促进相关miRNA的表达,也能用于miRNA是否受p53调控的检测。     

LncRNA-GAS5抑制miR-21介导的非完全匹配靶mRNA降解

LncRNA-GAS5作为miR-21的"分子海绵",通过"吸收"miR-21从而调控miR-21对靶基因的抑制.此外,miR-21直接调控非完全匹配靶基因PTEN和TPM1的表达.我们首先在HEK293T和HeLa细胞中确认,miR-21通过碱基互补配对调控非完全匹配靶基因PTEN和TPM1的蛋白表达,但不影响PTEN和TPM1mRNA的水平.此外,过表达miR-21后,qRT-PCR检测PTEN和TPM1mRNA的半衰期,发现它们的半衰期显著缩短,miR-21加速PTEN和TPM1mRNA的降解.通过转染lncRNA-GAS5的过表达质粒,发现lncRNA-GAS5竞争性地与miR-21结合能够延长PTEN和TPM1mRNA的半衰期,而miR-21与lncRNA-GAS5碱基互补配对结合后,又对lncRNA-GAS5存在调节作用,减弱lncRNA-GAS5的稳定性并加速lncRNA-GAS5的降解.LncRNA-GAS5作为miR-21的"分子海绵"能够抑制miR-21对非完全匹配靶mRNAPTEN和TPM1的降解,同时,miR-21与lncRNA-GAS5结合后又存在对lncRNA-GAS5的反馈调节,这些相互作用机制的发现有助于了解lncRNA、miRNA、mRNA之间这个复杂又精细的调控环路.     

MiR-206在骨骼肌发育中的功能

微RNA(microRNA,miRNA)是一类在分子进化中十分保守的非编码RNA,长度约22个核苷酸,一般情况下它在转录后水平抑制基因表达。miRNA在细胞增殖、分化、凋亡等诸多生理过程中发挥着重要作用。有些miRNA具有组织特异性表达,其中miR-206是目前发现的唯一在骨骼肌中特异表达的miRNA,它在调节骨骼肌发生过程中扮演重要角色。miR-206表达异常与一些肌肉相关疾病如肌肉营养不良、肌萎缩性侧索硬化症等有关。此外,在Texel羊中,myostatin基因的一个点突变就产生了一个miR-206和miR-1的靶点,抑制了myostain基因的表达,从而产生了双肌表型。因此,miR-206有可能成为治疗肌肉相关疾病和畜禽改良育种的重要候选分子。     

Long-term <Emphasis Type="Italic">ex vivo</Emphasis> monitoring of <Emphasis Type="Italic">in vivo</Emphasis> microRNA activity in liver using a secreted luciferase sensor

Technology for monitoring in vivo microRNA (miRNA) activity is extremely important for elucidating miRNA biology. However, in vivo studies of miRNA have been hampered by the lack of a convenient approach to reliably reflect real-time functional changes in miRNAs. Sensors for miRNA were developed by adding miRNA target sequences to the 3′-untranslated region of Gaussia princeps luciferase (Gluc) mRNA. These sensors were then evaluated in vitro and in vivo by measuring Gluc activity in cell supernatants and in peripheral blood. Sensors driven by the CMV promoter were effective for monitoring miR-122 in living cells, but not for the long-term monitoring of miR-122 or miR-142 in mouse liver because of CMV-promoter silencing. Replacing the CMV promoter with a CAG promoter rendered these sensors effective for the long-term monitoring of relevant liver miRNA activities. We subsequently used the CAG-promoter-based sensor for the long-term monitoring of endogenous liver miR-122, miR142 and miR-34a activities, as well as for exogenous miR-34a activity. Our study demonstrates that real-time in vivo activities of miRNAs can be continuously and conveniently detected in mouse liver using the sensors that we have developed.     

双萤光素酶共表达载体构建及特性研究

利用来源于TaV的自剪切多肽2A的编码序列构建一种分泌型萤光素酶Gluc和非分泌型萤光素酶Fluc共表达的载体,对其体内外表达及活体成像特点进行研究。采用重叠PCR技术获得Gluc-2A-Fluc片段,克隆入表达质粒pAAV2neoCAG中,获得重组质粒pAAV2neoCAG-Gluc-2A-Fluc。将重组质粒瞬时转染BHK-21细胞,24h后在细胞上清液和细胞裂解液中均能检测到Gluc和Fluc的表达,其中Gluc98%以上分布在上清液中,而Fluc98%以上存在于细胞中,随时间延长Gluc活性在上清液中逐步增加,而细胞内Fluc活性则保持相对平稳。用水动力法经小鼠尾静脉注射pAAV2neoCAG-Gluc-2A-Fluc质粒DNA,通过尾静脉微量采血(2.5μl/次)即可实时地监测体内Gluc的表达情况。活体成像结果显示,注射Gluc的底物腔肠素时小鼠明显表现为全身显像,显像在10min内迅速衰减;而注射Fluc的底物D-Luciferin时显像主要集中在肝脏,显像在30min内都比较稳定。本研究设计和构建的pAAV2neoCAG-Gluc-2A-Fluc质粒实现了分泌型和非分泌型萤光素酶的共表达,既可以在不裂解细胞或处死动物的情况下直接在细胞培养上清或血液中动态检测Gluc的活性,又可以利用活体成像技术准确定位Fluc表达部位,比单一的萤光素酶报告载体在细胞标记和体内示踪研究方面更具优越性。     

Disease-linked microRNA-21 exhibits drastically reduced mRNA binding and silencing activity in healthy mouse liver

MicroRNAs (miRNAs) bind to mRNAs and fine-tune protein output by affecting mRNA stability and/or translation. miR-21 is a ubiquitous, highly abundant, and stress-responsive miRNA linked to several diseases, including cancer, fibrosis, and inflammation. Although the RNA silencing activity of miR-21 in diseased cells has been well documented, the roles of miR-21 under healthy cellular conditions are not well understood. Here, we show that pharmacological inhibition or genetic deletion of miR-21 in healthy mouse liver has little impact on regulation of canonical seed-matched mRNAs and only a limited number of genes enriched in stress response pathways. These surprisingly weak and selective regulatory effects on known and predicted target mRNAs contrast with those of other abundant liver miRNAs such as miR-122 and let-7. Moreover, miR-21 shows greatly reduced binding to polysome-associated target mRNAs compared to miR-122 and let-7. Bioinformatic analysis suggests that reduced thermodynamic stability of seed pairing and target binding may contribute to this deficiency of miR-21. Significantly, these trends are reversed in human cervical carcinoma (HeLa) cells, where miRNAs including miR-21 show enhanced target binding within polysomes and where miR-21 triggers strong degradative activity toward target mRNAs. Taken together, our results suggest that, under normal cellular conditions in liver, miR-21 activity is maintained below a threshold required for binding and silencing most of its targets. Consequently, enhanced association with polysome-associated mRNA is likely to explain in part the gain of miR-21 function often found in diseased or stressed cells.     

Hepatitis B core protein promotes liver cancer metastasis through miR-382-5p/DLC-1 axis

The hepatitis B virus core protein (HBc), also named core antigen, is well-known for its key role in viral capsid formation and virus replication. Recently, studies showed that HBc has the potential to control cell biology activity by regulating host gene expression. Here, we utilized miRNA microarray to identify 24 upregulated miRNAs and 21 downregulated miRNAs in HBc-expressed HCC cells, which were involved in multiple biological processes, including cell motility. Consistently, the in vitro transwell assay and the in vivo tail-vein injection model showed HBc promotion on HCC metastasis. Further, the miRNA-target gene network analysis displayed that the deleted in liver cancer (DLC-1) gene, an important negative regulator for cell motility, was potentially targeted by several differentially expressed miRNAs in HBc-introduced cells. Introduction of miRNAs mimics or inhibitors and 3′UTR luciferase activity assay proved that miR-382-5p efficiently suppressed DLC-1 expression and its 3′-UTR luciferase reporter activity. Importantly, cotransfection of miR-382-5p mimics/inhibitors and the DLC-1 expression vector almost abrogated HBc promotion on cell motility, indicating that the miR-382-5p/DLC-1 axis is important for mediating HBc-enhanced HCC motility. Clinical HCC samples also showed a negative correlation between miR-382-5p and DLC-1 expression level. Furthermore, HBc-positive HCC tissues showed high miR-382-5p level and reduced DLC-1 expression. In conclusion, our findings revealed that HBc promoted HCC motility by regulating the miR-382-5p/DLC-1 axis, which might provide a novel target for clinical diagnosis and treatment.     

胃癌与癌旁组织miRNA差异表达谱的研究

目的研究胃癌中表达失调的miRNA及其生物学功能,从而进一步阐明miRNA在胃癌发生中的分子机制。方法将总RNA加ploy（A）尾后进行反转录PCR扩增特异miRNA,使用QuantityOne软件进行定量分析,计算每对样本胃癌与癌旁组织比值（T／NRatio）,使用SAM软件进行统计分析。MTT法检测miR-21和miR-17-5p对胃癌细胞系生长的影响。结果在8对胃癌及癌旁组织样本中对237个miRNA进行了表达谱分析。对于检测到表达的161个miRNA,使用SAM软件进行统计分析,确认22个在胃癌中表达上调,2个在胃癌中表达下调（FDR=0．0963）。进一步通过生长抑制试验证实在胃癌组织中表达异常增高的miR-21和miR-17-5p对胃癌细胞的生长有明显的促进作用。结论这些在胃癌中异常表达的miRNAs具有成为新一代胃癌标记物的潜力,能够为胃癌的精确诊断分型提供依据,同时针对这些靶点可以开发新的核酸治疗技术,通过抑制或增强其功能来达到治疗胃癌的目的。