摘要目的 研究2,4,6-三硝基苯磺酸( TNBS)/乙醇溃疡性结肠炎( UC)大鼠中,UC相关miRNA的调控作用,即miRNA与mRNA互作关系. 方法 用TNBS/乙醇灌肠,诱导UC大鼠模型.经过造模与14 d生理盐水灌胃后,解剖处死. 留取正常组与模型组大鼠相应的结肠组织. 通过大体及镜下观察,验证造模效果. 同时对相应结肠组织进行miRNA芯片检测,根据芯片结果及相关文献报道,筛选出与UC发病密切相关的miRNA并对其进行RT-PCR验证. 另外,利用miRWalk数据库对差异miRNA的靶基因进行预测. 为了验证预测结果与实际情况的相符度,又同步进行了mRNA水平的表达谱芯片实验,筛选出与差异miRNA确有互作作用的mRNA. 为了更好地说明miRNA与mRNA的互作关系,又利用David数据库对靶基因进行注释,最终形成miRNA与mRNA共表达网络. 结果模型组大鼠结肠大体及镜下表现均符合UC 的发病特征. 通过 miRNA芯片筛选出了68 个差异miRNA[倍数变化(fold change)>2, P<0.05,表达值(expression mean)>7],结合相关文献报道,筛选出6个与UC发病密切相关的miRNA. 同时对这6个miRNA进行了RT-PCR实验,结果提示,与正常组相比,4个差异miRNA( miR-146a-5p、miR-146b-5p、miR-126a-3p和miR-21-5p)表达明显上调( P<0.01,P<0.05),2个差异miRNA(miR-200b-3p、miR-145-5p)表达明显下调(P<0.01). 结合miRWalk数据库对这6个差异miRNA的靶基因预测结果和表达谱芯片结果,本研究发现IL-6、Ccl5、Mapk3、Smad7这4个mRNA与上述6个miRNA有互作关系. 最终通过David数据库对这种互作关系进行注释,以阐明其在UC发病中的重要作用. 结论 一个miRNA可以调控多条信号通路,而一条信号通路又受到多个miRNA的调控. 如果仅仅单纯抑制某条信号通路,可能并不能从根本上抑制炎症的发生. 而从其上游出发,研究miRNA作用,阐明相互关系,并调控mRNA的作用,可能可以从根本上逆转疾病的进程.

abstractsObjective To investigate the correlations between miRNA and mRNA ( the regulatory effects of miRNA) in a rat model of trinitro-benzene-sulfonic acid (TNBS)/ethanol induced ulcerative colitis ( UC) .Methods TNBS and ethanol were used to induce the development of UC in rats .After the modeling procedure and oral administration of normal saline ( NS) for 14 days, rats from the control and model groups were dissected to collect the samples of colonic mucosa .General and histological evaluations were performed to validate the modeling of UC .The expression of miRNA was profiled using miRNA microarray .The target miRNAs that were closely related to the pathogenesis of UC were selected out according to the results of mi -croarray and related literatures .RT-PCR was performed to verify the differentially expressed miRNAs .The mirWalk database was used to predict the target genes of miRNAs .In order to verify whether the predicted results were in accordance with the actual results , the microarray technology was used for mRNA expression profiling .The genes that showed interactions with those miRNAs were screened out .The David database was used for gene annotation .An interaction net between miRNA and mRNA was formed .Results General and histological manifestation of colon tissue samples from the model group were in accordance with the features of UC.Sixty-eight miRNAs were identified to be differentially expressed in rats from the model group and the control group (fold change>2, P<0.05, expression mean>7).Six candidate miRNAs were selected as hav-ing close relations to the pathogenesis of UC referring to reported literatures , the expression of which was checked and verified by real-time polymerase chain reaction (PCR).Compared with the control group, 4 miRNAs (miR-146a-5p, miR-146b-5p, miR-126a-3p and miR-21-5p) were up-regulated (P<0.01, P<0.05) and 2 miRNAs (miR-200b-3p and miR-145-5p) were down-regulated (P<0.01) in rats with TNBS/ethanol induced UC.Four mRNAs (IL-6, Ccl5, Mapk3 and Smad7) that interacted with the 6 miRNAs were identified based on the results of target gene prediction of the above 6 miRNAs and gene expression pro-filing.The David database was used to annotate the interactions for elucidating their significance in the path -ogenesis of UC .Conclusion A miRNA can regulate many signaling pathways and a signaling pathway can also be regulated by many miRNAs .Therefore , simply inhibiting certain pathways may not radically stop the process of inflammation .Studying the functions of miRNAs and elucidating the correlations between miRNA and mRNA might fundamentally inhibit the development of UC .