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采用生物信息学方法对斑点叉尾(Ictalurus punctatus)ipu-miR-143的靶基因进行预测,并对预测到的ipu-miR-143靶基因进行生物学鉴定。生物信息学预测结果显示,斑点叉尾微管相关蛋白基因RP/EB家族EB1基因的3′-UTR区具有ipu-miR-143的潜在作用位点。结合对几种近缘物种中RP/EB家族EB1基因和miR-143的进化保守性进行分析,推测ipu-miR-143在斑点叉尾中可以通过靶向EB1基因的3′-UTR区而发挥其调控功能。因此,本研究将斑点叉尾EB1基因的3′-UTR区(含有miR-143靶位点)构建到pMIR-REPORTTM Luciferase载体的下游,通过双荧光素酶报告基因检测系统对ipu-miR-143的靶基因进行鉴定。采用HEK293和CCK两种细胞进行细胞转染,两种细胞中转染miR-143 mimics组荧光相对活性与对照组相比均表现为显著性降低(P<0.05)。共转染miR-143 inhibitors后,CCK细胞中荧光素酶相对活性(8.27±1.02)与对照组相比(5.44±1.55)显著上调(P<0.05);HEK293细胞中荧光素酶相对活性(6.30±1.19)与对照组(4.26±0.84)相比有所上升,但无显著性差异(P>0.05)。初步研究结果提示,miR-143可以通过靶向EB1基因的3′-UTR区而发挥其调控功能。本研究旨为后续深入研究斑点叉尾EB1基因的转录后调控机制提供基础依据。
The bioinformatics method was used to predict the target gene of Ictalurus punctatus ipu-miR-143, and the biological characteristics of the predicted target gene of ipu-miR-143 were identified. Bioinformatics prediction showed that the 3’-UTR region of the EB1 gene of Picrosurus microtubule-associated microtubule-associated protein gene had the potential role of ipu-miR-143. Combining the evolutionary conservation of EB1 gene and miR-143 in RP / EB family in several species, we speculate that ipu-miR-143 can target EBV gene in 3’-UTR region in the channel catfish Play its regulatory functions. Therefore, in this study, the 3’-UTR region of the EBV gene (containing miR-143 target site) was constructed downstream of the pMIR-REPORTTM Luciferase vector. The dual luciferase reporter assay was used to detect the expression of ipu-miR- 143 target genes were identified. The HEK293 and CCK cells were used for cell transfection. The relative fluorescence activities of miR-143 mimics transfected cells in both groups were significantly decreased compared with the control group (P <0.05). The luciferase activity in CCK cells (8.27 ± 1.02) was significantly increased compared with that in control cells (5.44 ± 1.55) (P <0.05) after transfection with miR-143 inhibitors. The relative luciferase activity in HEK293 cells ± 1.19) compared with the control group (4.26 ± 0.84), but there was no significant difference (P> 0.05). Preliminary results suggest that miR-143 can exert its regulatory function by targeting the 3’-UTR region of EB1 gene. The aim of this study is to provide a basis for further studies on the post-transcriptional regulation of EBV gene in Channel Catfish.