猪miR-204组织表达与重要靶基因筛选

基于Illumina Hiseq 4000高通量测序技术筛选得到仔猪感染C型产气荚膜梭菌耐受和易感组中显著上调表达的miR-204。为研究miR-204在各组织中的表达,进一步筛选miR-204参与调控仔猪C型产气荚膜梭菌感染的关键靶基因,采用实时荧光定量PCR方法检测了miR-204在7日龄仔猪各组织中的表达,利用MEGA7.0软件对其成熟体序列保守性进行分析,运用TargetScan、miRDB、PicTar软件进行靶基因预测,并用DAVID软件对靶基因进行Gene Ontology和KEGG Pathway通路富集分析,进一步采用qRT-PCR方法对筛选得到的关键靶基因在猪肠上皮细胞(IPEC-J2)中进行靶向关系验证。结果表明,miR-204在7日龄仔猪肾脏、肝脏、胸腺、淋巴、十二指肠和回肠组织中均高度表达,其成熟体序列在脊椎动物间高度保守。3种软件预测到的共同靶基因有114个,这些靶基因显著(P<0.05)富集在15个GO功能和13个信号通路。结合前期得到的抗性基因,筛选到4个关键靶基因NR3C1、SIRT1、BCL2L2和DLG5,转染miR-204 mimics后,靶基因SIRT1、BCL2L2和DLG5极显著(P<0.01)下调表达。miR-204是参与调控仔猪抵抗C型产气荚膜梭菌感染的重要因子,SIRT1、BCL2L2和DLG5可能是miR-204的关键靶基因,其功能还需进一步验证。

Tissue expression and significant target genes analysis of swine miR-204

Based on the earlier research, miR-204 was screened out with significantly up-regulated expression in the resistance and susceptibility group for piglets infected with Clostridium perfringens type C by using Illumina Hiseq 4000 high-throughput sequencing technology. In the present study, real-time quantitative PCR was used to detect the miR-204 expression in different tissues of piglets at 7-day-old. Meanwhile the mature gene sequences was analyzed using MEGA7.0 software, and target genes were predicted using TargetScan, miRDB, and PicTar. Then, the online software DAVID was used to perform gene ontology and KEGG pathway enrichment analysis of target genes, and qRT-PCR method was used to verify the targeted relationship of the selected target genes in porcine intestinal epithelial cells (IPEC-J2). The results showed that miR-204 was highly expressed in kidney, liver, thymus, lymph, duodenum and ileum tissues of 7-day-old piglets, and its mature gene sequences was highly conserved among vertebrates. There were 114 common target genes predicted by the three softwares, and these target genes were significantly (P<0.05) enriched in 15 GO functions and 13 signaling pathways. Combined with the previously obtained resistance genes, the four key target genes nuclear receptor subfamily 3 group C member 1(NR3C1), Silence information regulator 1 (SIRT1), BCL2 like 2(BCL2L2) and Discs, large homolog 5(DLG5) were screened. After transfection of miR-204 mimics, the target genes SIRT1, BCL2L2 and DLG5 were significantly (P<0.01)down-regulated. In summary, miR-204 is an important factor involved in regulating the piglets' resistance caused by Clostridium perfringens type C infection. The genes SIRT1, BCL2L2, and DLG5 may be key target genes of miR-204, and the further study should be conducted to testify their functions.