Functional Study of the Chicken microRNAs miR-143 and miR-10a

MicroRNA (miRNA) is a family of small regulatory RNA that post-transcriptionally regulates many biological functions including growth and development. Chicken miR-143 and miR-10a are expressed in both the embryonic and post-hatch chick in a spatio-temporal manner. In order to study the functions of these miRNAs, loss of function and microarray approaches were employed. Spleen cells were isolated from embryonic day 18 chickens and immediately transfected with either a synthetic miR-143 inhibitor or a synthetic miR-10a inhibitor or a negative control oligo-nucleotide (Dharmacon). Cultures were maintained for 48 hrs and microarray analysis was performed using the Arizona Gallus gallus 20.7K long oligoarray (GPL6049). Differentially expressed genes were identified for each miRNA by comparing the miRNA knock-down group to the negative control group. The differentially expressed genes were functionally categorized using the DAVID Functional Annotation Tool (http://david.abcc.ncifcrf.gov/). The 3’-UTR of the up-regulated genes (de-repressed after the miRNAs knock-down) were scanned for potential miR-143 or miR-10a binding sites using the miRanda algorithm version 3.1 (http://www.microrna.org/microrna). In addition, the Chicken (Gallus gallus) Unigene database (NCBI) was also used to predict potential targets of these miRNAs. A set of predicted targets for both miRNAs was selected and validated by dual luciferase reporter gene assay. Overall, many of the identified targets for miR-143 are associated with cell proliferation, tumerigenesis and apoptosis. Many of potential targets for miR-10a are associated with immune response. A reference designed microarray was performed in which samples (Cy3) were co-hybridized with the reference RNA pool (Cy5) on the array. The microarray data set was processed using within- and across-array loess normalization method in JMP Genomics 3.0, SAS (Cary, NC). Data quality was evaluated using the MA plots process and the correlation and grouped scatter plots procedure (JMP Genomics 3.0). Differentially expressed genes were identified using ANOVA process on JMP Genomics 3.0 with fixed effect of treatments and random effect of arrays.

MicroRNA (miRNA) 是一类小型调控RNA家族，可在转录后水平调控包括生长发育在内的诸多生物学功能。鸡源miR-143与miR-10a在胚胎期及孵化后的雏鸡体内呈时空特异性表达。为探究这两种miRNA的功能，本研究采用了功能缺失（loss of function）与基因芯片（microarray）技术手段。从孵化第18天的鸡胚中分离脾脏细胞，随即分别使用合成的miR-143抑制剂、miR-10a抑制剂，或阴性对照寡核苷酸（Dharmacon）进行转染。细胞培养维持48小时后，使用亚利桑那大学鸡（Gallus gallus）20.7K长寡核苷酸芯片（GPL6049）进行基因芯片分析。通过将miRNA敲低（knock-down）组与阴性对照组进行比较，分别鉴定出对应miRNA的差异表达基因。使用DAVID功能注释工具（DAVID Functional Annotation Tool，http://david.abcc.ncifcrf.gov/）对差异表达基因进行功能富集分类。针对miRNA敲低后表达上调（即去抑制）的基因，使用miRanda算法v3.1（http://www.microrna.org/microrna）扫描其3’非翻译区（3’-UTR）中潜在的miR-143或miR-10a结合位点。此外，本研究还借助鸡（Gallus gallus）Unigene数据库（NCBI）预测这两种miRNA的潜在靶基因。选取上述两种miRNA的部分预测靶基因，通过双荧光素酶报告基因实验进行验证。整体而言，所鉴定出的miR-143靶基因多与细胞增殖、肿瘤发生及细胞凋亡相关；而miR-10a的潜在靶基因则多参与免疫应答过程。本研究采用参考基因芯片设计方案，将样品标记为Cy3荧光基团，与标记为Cy5荧光基团的参考RNA混合池共同在芯片上进行杂交。使用JMP Genomics 3.0与SAS软件（Cary, NC）中的芯片内及芯片间局部加权回归散点平滑法（loess normalization）对基因芯片数据集进行标准化处理。借助JMP Genomics 3.0中的MA图分析、相关性分析及分组散点图流程对数据质量进行评估。使用JMP Genomics 3.0中的方差分析（ANOVA）流程鉴定差异表达基因，其中将处理方式设为固定效应，芯片批次设为随机效应。