Differentially expressed microRNAs during solid endosperm development in coconut (Cocos nucifera L.)

MicroRNAs (miRNAs) are a class of 20–24 nt, endogenously expressed, non-coding RNAs that play important regulatory roles in plants and animals. To identify miRNAs potentially involved in tissue development and compound anabolism, we studied miRNA expression profiles in endosperm of coconut at different developmental stages. Based on the annotation in miRBase (release 10.1), we measured a total of 179 miRNAs in immature (95 expressed miRNAs) and mature tissues (176 expressed miRNAs) using microarrays, respectively. The comparative analyses on miRNA expression profiles between these two groups of tissues showed that 23 miRNAs were up-regulated and nine miRNAs were down-regulated in matured endosperm. We further confirmed the increased expression of four miRNAs and decreased expression of a miRNA in immature endosperm using real-time PCR. Moreover, we computationally predicted the target genes of 32 miRNAs with differential expression (p < 0.01), and identified the lowest-score targets of six miRNAs. Finally, we discussed the potential functional relevance of several differentially expressed miRNAs.     

RNA分子在植物韧皮部长距离运输的研究进展

 RNA分子存在于植物韧皮部筛分子中,且能进行长距离运输,这是植物界一个突破性的发现。现已发现有3类RNA存在于韧皮液内,包括外源类病毒或RNA病毒,植物内源mRNA和非编码小RNA。韧皮液内RNA分子的长距离移动可作为系统信号分子介导植物生长发育的调控。本文就韧皮液的取样方法,韧皮液内存在的RNA分子及其介导的信号调控和运输机制等进行了综述。     

Microarray-based miRNAs profiling in lower limb arteriosclerosis of diabetic rats

AIM: To establish the profiling of microRNAs (miRNAs) in the lower extremity arterial tissue between diabetic rats with lower limb arteriosclerosis (DAS) and diabetic rats with normal lower limb (DN), and to explore the possible molecular mechanisms involved in aberrant miRNA expression in DAS. METHODS: The rat models of DAS and DN were successfully established. The respective lower extremity arterial tissue was isolated. The total miRNAs were purified for a hybridization detection by miRNA microarray. The results of chip scanning and data were analyzed and verified by RT-qPCR. RESULTS: Ten miRNAs related to DAS, including rno-miR-206-3p, rno-miR-133a-5p, rno-miR-133b-3p, rno-miR-133a-3p, rno-miR-325-5p, rno-miR-675-3p, rno-miR-411-5p, rno-miR-329-3p, rno-miR-335 and rno-miR-126a-3p, were determined. All 10 abnormally expressed miRNAs were up-regulated. The validating results of RT-qPCR confirmed 9 of the miRNAs in line with chip expression. Just rno-miR-335 showed the opposite between PCR detection and microarray result. CONCLUSION: A group of miRNAs in diabetic rats suffering from lower limb arteriosclerosis plays an important role in the vascular atherosclerosis process. The abnormal expression of miRNAs is likely to affect the vascular atherosclerosis process.     

Effect of miRNAs on apoptotic leukemia cells induced by demethylcantharidin

AIM: To explore the role of miRNA expression in the process of antitumor drug demethylcantharidin (DMC)-induced leukemia cell apoptosis. METHODS: K562 cells were treated with DMC to induce apoptosis. Microarray assessment was performed to detect the changes of miRNAs. The expression of miRNAs was further detected by RT-PCR and real-time PCR. The miRNA-relative genes were analyzed by gene information softwares,and their expression was determined by ELISA analysis. RESULTS: The results of microarray showed that more than 290 miRNAs were differentially expressed after DMC treatment. The expression of miR-16, miR-34 and miR-125 increased at more than 1.5 folds in DMC treatment group determined by RT-PCR and real-time PCR analysis, while both miR-106 and miR-150 were down-expressed over 60％. Using microRNA TargetScan and miRanda analysis software, we found that the expression of oncogenes ( bcl-2, E2F1, E2F3 ) and tumor suppressor genes ( RB1, p53 ) may be regulated by the above miRNAs. The expression of RB1 and P53 proteins significantly increased, while Bcl-2, E2F1 and E2F3 proteins were obviously down-regulated after DMC treatment.CONCLUSION: DMC induces K562 cell apoptosis by regulating the expression of miRNAs and their relative genes.     

草莓不同器官中microRNA表达差异研究

MicroRNA(miRNA)在植物的生长发育过程中起着重要作用。以CTAB法提取的总RNA为模板,通过设计茎环反转录引物,利用实时RT-PCR技术对草莓根系、花托、茎尖和叶片4种器官中的6种miRNA进行了表达差异研究。以草莓叶片中6种miRNA的表达量为1,差异表达结果显示miR164在草莓花托中高度表达,达到6.615,明显高于根系(0.485)、茎尖(0.371)和叶片(1.000);miR172在茎尖和花托中的表达量都较低,分别为0.029和0.020,在根系中的表达量更低,只有0.0008454;miR167在根系中表达也很低,只有0.053;miR156,miR159和miR165在4种草莓器官中的表达差异不明显。这些结果为进一步验证miRNA基因在草莓植株生长发育过程中的作用奠定了基础。     

Differential microRNA expression profile in laryngeal cancer and effect of miR-125a-5p on proliferation of laryngeal cancer cell line

AIM：To investigate the differential microRNA expression profiles between laryngeal cancer and adjacent normal laryngeal mucosa. METHODS：Forty two pairs of laryngeal cancer tissue and adjacent normal laryngeal mucosa tissue were collected. Ten pairs of samples were used for determining microRNA expression by the method of miRNA microarray chip. Data analysis was performed to find out the significant differential microRNA expression profile in laryngeal cancer, and the difference was verified by quantitative real-time PCR (qRT-PCR) analysis on another 32 pairs of samples. Methyl thiazolyl tetrazolium (MTT) assay and colony-forming assay were used to analyze the proliferation of Hep2 cells induced by miR-125a-5p. RESULTS：Both miRNA microarray and qRT-PCR showed that the expression of let-7f-5p, miR-10a-5p, miR-125a-5p, miR-144-3p, miR-195-5p and miR-203 was down-regulated in laryngeal cancer tissues. miR-125a-5p suppressed the proliferation of Hep2 cells. CONCLUSION：The results of microarray are accordant with those of qRT-PCR. Significant difference of miRNA expression profiles between laryngeal cancer and adjacent normal laryngeal mucosa indicates that miRNAs may play a role in carcinogenesis and progression of laryngeal cancer. miR-125a-5p inhibits the proliferation of Hep2 cell, indicating a novel therapeutic target against laryngeal cancer.     

库尔勒香梨9个新miRNA克隆鉴定

利用在小RNA高通量测序试验中筛选出的脱萼组与宿萼组差异表达新miRNA基因,采用 Stem-loop法对总体表达量居前20位、在脱萼组和宿萼组中具有显著性差异表达、在子房和萼片组织中具有显著性差异表达的新miRNA进行成熟体的克隆鉴定、前体序列二级结构分析、qRT-PCR试验以及靶基因预测。结果显示,在不同的样本中有9个新miRNA（novel_miRNA）的成熟体序列以及4个novel_miRNA的表达量与高通量测序结果完全一致,并且预测得到大量具有生物学功能的靶基因。新发现的miRNA可能与‘库尔勒香梨’萼片脱落和宿存有密切关系。     

Up-regulation of microRNA-187* inhibits proliferation of human colon cancer cell line HCT116

AIM:To investigate the expression of microRNA-187* (miR-187*) in human colon cancer cell lines and normal colon tissues, and to determine the effects of miR-187* up-regulation on the proliferation and cell cycle of human colon cancer cell line HCT116. METHODS:The expression profiling of miRNAs in 3 colorectal adenocarcinoma samples and their matched normal tissue samples was performed using miRNA microarray chip. Total RNA was isolated from 8 colon cancer cell lines and 10 normal colon tissues. The miR-187* level was detected by Taqman real-time RT-PCR. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1), the possible target of miR-187*, was also detected. Synthetic miR-187* mimics were transfected into HCT116 cell line by LipofectamineTM 2000. The mRNA expression of miR-187* and BMI-1 in HCT116 cell line was measured by real-time RT-PCR. Cell growth and cell cycle were assayed by MTS method and flow cytometry. RESULTS:miR-187* was found to be differentially expressed between colorectal adenocarcinoma and normal tissues. The expression of miR-187* in 8 colon cancer cell lines was down-regulated, while BMI-1 mRNA was up-regulated. Compared with blank control group, miR-187* expression was remarkably increased after transfection with miR-187* mimics, and ectopic expression of miR-187* significantly inhibited the mRNA expression of BMI-1. The cell growth was inhibited in miR-187* mimics group, and proliferating cell nuclear antigen (PCNA) mRNA expression was decreased. The cells at G2/M phase in miR-187* mimics group were significantly increased. CONCLUSION: miR-187* is down-regulated in human colon cancer cell lines. Up-regulation of miR-187* not only inhibits the proliferation but also influence the cell cycle of HCT116 cells, which might act as a tumor suppressor in colorectal cancer by inhibiting the expression of BMI-1.     

马铃薯S病毒PVSO株系RT-LAMP检测方法的建立及应用

为建立一种马铃薯S病毒普通株系PVSO快速、灵敏的RT-LAMP可视化检测方法。根据马铃薯S病毒普通株系的CP核苷酸序列设计4条引物,以感染PVS的马铃薯叶片总RNA为模板,采用一步法RT-LAMP,在62 ℃下反应60 min,扩增产物利用琼脂糖电泳分析和钙黄绿素染料显色判断结果,同时对其特异性和灵敏性进行测定。结果表明,建立的RT-LAMP方法可特异地对PVSO进行检测,检测灵敏度为RT-PCR的100倍。对33份马铃薯样品检测表明,RT-LAMP与RT-PCR方法检测结果基本一致。因此建立的PVSO的 RT-LAMP可视化检测方法简便,特异性强,灵敏度高,适合PVSO的快速检测。     

MicroRNA profile analysis of ischemic myocardial tissues from rats with acute myocardial infarction

AIM：To identify differentially expressed microRNAs (miRNAs) in ischemic myocardial tissues from the rats with acute myocardial infarction (AMI) by miRNA array technique, and to predict their targets and analyze their functions using bioinformatics. METHODS：The rat models of AMI (n=3) were prepared by ligaturing the left anterior descending coronary artery (LAD) of Wistar rats. Electrocardiogram and blood pressure were detected during the operation, and the myocardial infarct size was measured by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Ischemic myocardial tissues were isolated from the infarct area 4 h after ischemia. The same procedure in sham group (n=3) was performed except for ligaturing LAD. Total RNA was extracted from ischemic and normal myocardial tissues. miRNA was isolated from total RNA, labeled with Cy3 and hybridized on miRNA array. Real-time PCR was applied to verify the reliability of miRNA array results. The targets of differentially expressed miRNAs were predicted and their functions were analyzed by bioinformatics. RESULTS：Rat model of AMI was successfully prepared and verified by electrocardiogram detection, blood pressure measurement and pathological observation. Compared with sham group, microarray screening showed that total 11 AMI-related miRNAs were selected, including 6 up-regulated and 5 down-regulated. Three of them (rno-miR-181c, rno-miR-146b and rno-miR-208) were related to the cardiovascular functions, while the functions of the others (rno-miR-672*, rno-miR-743b, rno-miR-128, rno-miR-138-1*, rno-miR-336, rno-miR-138-2*, rno-miR-325-3p and rno-miR-3572) were unknown and might be novel AMI-related biomarkers. Parts of the miRNA targets were also related to the cardiovascular functions. CONCLUSION：Differentially expressed miRNAs in AMI rats may serve as novel biomarkers for diagnosis of AMI and potential targets for treatment of AMI.     

Inhibition of 293T cell proliferation by blocking miR-20 and miR-106 expression with antisense RNA

AIM: To inhibit 293T cell proliferation by reducing miR-20 and miR-106 expression with antisense RNA. METHODS: Antisense RNA (or antisense oligonucleotides, ASO) specific to miR-20 and miR-106 was synthesized, and the 293T cells were treated with these antisense RNA. Then, the cell proliferation derived from suppression of antisense RNA was studied by microscopy and fluorescence-activated cell sorter. The expression of miRNA and its target gene Rb were analyzed by real-time PCR and ELISA, respectively. RESULTS: Our results showed that ASO could inhibit the expression of miR-20 and miR-106 effectively and inhibit 293T cell proliferation. The results also demonstrated that ASO specific to miR-106 could up-regulate anti-oncogene Rb expression. CONCLUSION: ASO could inhibit 293T cell proliferation by inhibiting miRNA expression and up-regulating Rb expression.     

Quantitative analysis of RNA levels from single hepatocytes in vivo: combined use of real-time RT-PCR and laser microdissection

AIM: The manner in which a cell responds to and influences its environment is ultimately determined by the genes that are expressed.To better understand cellular functions,the isolation of single cells and subsequent quantification of the expressed genes is essential.METHODS: Normal liver tissue was obtained from operation,snap-frozen in liquid nitrogen and sectioned in crystat.Individual hepatocytes were microdissected.RNA was extracted,then reverse transcribed and amplified using real-time quantitative polymerase chain reaction (PCR).RESULTS: Single hepatocytes were dissected by laser beam and catapulted to the microcentrifuge cap which was put above the slide.In this way,cells were collected,RNA was extracted,reverse transcribed to cDNA and used for analysis of RNA expression by real-time quantitative PCR.The amplification results showed that quantitation of the RNA inside the cell was compatible with the number of cells.CONCLUSION: The expression of RNA in single cells can be quantitated successfully by using laser microdissection and real-time PCR.These techniques provide an opportunity to monitor in vivo gene expression levels in single hepatocytes.     

3种苹果潜隐病毒多重RT-PCR检测体系的建立

 研究建立了能同时检测苹果茎沟病毒(Apple stem grooving virus, ASGV) 、苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus, ACLSV) 和苹果茎痘病毒(Apple stem pitting virus, ASPV) 的多重RT-PCR方法。以复合感染3种病毒的苹果‘望山红’组培苗为试材, 对影响多重PCR的反应条件进行了一系列的调整和优化。多重RT-PCR体系灵敏性测验显示, 最低能从RNA总量187.5 ng的样品中检测3种病毒的存在。多重RT-PCR产物的序列与报道的病毒序列有较高的同源性, 12个田间苹果样品的多重RT-PCR检测结果与单一PCR的结果一致, 初步证明了多重RT-PCR检测的准确性。     

间接原位RT-PCR法检测辣椒叶组织中辣椒轻斑驳病毒的分布

为了了解辣椒轻斑驳病毒（Pepper mild mottle virus,PMMoV）在辣椒组织中的分布特点,探索利用间接原位RT-PCR技术对病毒在辣椒组织中进行定位。利用改进的CTAB法从辣椒叶片中提取总RNA,利用RT-PCR技术扩增出PMMoV的特异片段,并经克隆测序,证明其为PMMoV的特异片段。利用PCR技术,制备出了地高辛标记的特异cDNA探针。对原位RT-PCR反应体系进行优化,经过切片制备（切片厚度7 μm、应用Superfrost plus正电荷防脱载玻片）、预处理（1 mg ? L-1蛋白酶K消化5 min）、RT-PCR（37 ℃反转录2.5 h、PCR退火温度为58 ℃）、杂交检测,建立了应用间接原位RT-PCR技术检测PMMoV在辣椒组织中分布的方法。原位RT-PCR结果显示,叶片组织中PMMoV阳性信号主要分布于栅栏组织,其次是海绵组织,表皮组织中也有少量阳性信号。此外,叶柄中可见少量阳性信号,主要位于表皮组织。     

枣树阶段转变相关microRNA家族的鉴定及其表达分析

与其他果树相比,枣树具有童期短、成花快的特征.已有研究表明,多个microRNA(miRNA)家族参与植物阶段转变和开花时间调控等过程.研究枣树阶段转变相关的miRNA家族对果树童期调控具有重要意义.以枣实生后代植株不同发育阶段(节位)的当年生枝(枣吊)为材料,通过Small RNA测序,在童期、过渡期和成年期等3个时...     

两步多重RT-PCR快速检测苹果潜隐性病毒

【目的】为建立检测3种苹果潜隐性病毒更为快速准确的方法,【方法】以携带苹果茎沟病毒(ASGV)、苹果褪绿叶斑病毒(ACLSV)和苹果茎痘病毒(ASPV)的成龄苹果树韧皮部为试材,根据基因库中苹果茎痘病毒(Apple stempitting virus,ASPV)的外壳蛋白基因序列,设计合成了2对特异性引物,分别与合成的苹果茎沟病毒(ASGV)引物和苹果褪绿叶斑病毒(ACLSV)的引物组合配伍,筛选出最佳的引物对组合。对cDNA的合成所用引物和总RNA模板进行遴选和量化,对PCR过程中cDNA的用量、引物对的终浓度、退火温度等主要影响因素进行优化。【结果】结果表明,反转录引物为Oligo(dT)18、总RNA 0.1～3.0μL时,可以得到较好的cDNA;ASPV-FR与ASGV-Pch、ACLSV-Pch引物组合优于ASPV-Pch,并且筛选出4组最佳的终浓度处理组合;cDNA用量为2.0~4.0μL、退火温度为50.0~52.9℃、循环数为35时,扩增效果较好。采用优化的多重RT-PCR体系对采自陕西和山东两省的样品进行检测,并用3种病毒的单重RT-PCR体系进行验证。【结论】结果呈现高度一致性,充分印证了该多重RT-PCR体系的准确性,适用于大量样品的快速检测。     

应用实时荧光RT-PCR检测柑橘黄化脉明病毒

为更快速准确检测柑橘黄化脉明病毒（Citrus yellow vein clearing virus,CYVCV）,根据病毒外壳蛋白基因的保守序列设计特异性引物,通过体系优化得到最佳反应条件,建立基于SYBR GreenⅠ的CYVCV实时荧光RT-PCR检测方法。该方法可特异性检测CYVCV,而测试的柑橘衰退病毒、碎叶病毒等5种柑橘病毒均不能检测出。灵敏度较普通RT-PCR提高了100倍,标准曲线循环阈值与模板浓度呈良好的线性关系,扩增效率和相关性系数分别为102%和0.999。对田间样品的检测结果表明,建立的实时荧光RT-PCR适用于检测不同柑橘品种中CYVCV的含量。     

DAS-ELISA、RT-PCR和IC-RT-PCR检测葡萄卷叶病毒Ⅲ的比较研究

在生长季节分3次对部分葡萄品种枝条上部、中部和下部叶片及韧皮部,进行双抗体夹心法(DAS-ELISA)、反转录聚合酶链式反应(RT-PCR)和免疫捕捉反转录聚合酶链式反应(IC-RT-PCR)3种方法检测卷叶病毒Ⅲ(GLRaV-3)的比较。结果表明:DAS-ELISA的检出率明显低于RT-PCR和IC-RT-PCR方法。RT-PCR可以检测出RNA提取液为1:10-4的GLRaV-3病毒,但它受到了提取RNA难以及其它物质(如蛋白质、DNA等)干扰的影响,检测难度增加。从检测的灵敏度来看,RT-PCR和IC-RT-PCR比DAS-ELISA灵敏;并且,IC-RT-PCR比其它两种方法较快,整个过程仅需8h。