CAJ | 학술논문

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。
채용생물신식학방법대반점차미(Ictalurus punctatus)ipu-miR-143적파기인진행예측，병대예측도적ipu-miR-143파기인진행생물학감정。생물신식학예측결과현시，반점차미미관상관단백기인RP/EB가족EB1기인적3′-UTR구구유ipu-miR-143적잠재작용위점。결합대궤충근연물충중RP/EB가족EB1기인화miR-143적진화보수성진행분석，추측ipu-miR-143재반점차미중가이통과파향EB1기인적3′-UTR구이발휘기조공공능。인차，본연구장반점차미 EB1기인적3′-UTR구(함유miR-143파위점)구건도pMIR-REPORTTM Luciferase재체적하유，통과쌍형광소매보고기인검측계통대ipu-miR-143적파기인진행감정。채용HEK293화CCK량충세포진행세포전염，량충세포중전염 miR-143 mimics 조형광상대활성여대조조상비균표현위현저성강저(P<0.05)。공전염miR-143 inhibitors 후， CCK 세포중형광소매상대활성(8.27±1.02)여대조조상비(5.44±1.55)현저상조(P<0.05)；HEK293세포중형광소매상대활성(6.30±1.19)여대조조(4.26±0.84)상비유소상승，단무현저성차이(P>0.05)。초보연구결과제시， miR-143가이통과파향EB1기인적3′-UTR구이발휘기조공공능。본연구지위후속심입연구반점차미 EB1기인적전록후조공궤제제공기출의거。
The potential target of ipu-miR-143 was predicted and identified using a luciferase reporter gene assay with HEK293 and CCK cell lines in vitro. Bioinformatic analyses indicated that the 3′untranslated region (3'-UTR) of the microtubule-associated protein RP/EB family member 1 gene (EB1) contains an ipu-miR-143 target site, which per-fectly complements the seed region (positions 2-8) of the mature ipu-miR-143 sequence. Therefore, we speculate that the EB1 gene might act as a direct regulatory target of miR-143 in channel catfish. The pMIR-EB1 report vector con-taining the ipu-miR-143 complementary sequence was constructed to investigate the target of ipu-miR-143 in channel catfish. Then, pMIR-EB1 and miR-143 mimics were co-transfected into the HEK293 and CCK cells to detect the bio-logical activity of ipu-miR-143. In comparison with the control groups, the miR-143 mimic groups showed significantly lower levels of luciferase expression in the two cell lines (P<0.05). Luciferase activity of the ipu-miR-143 inhibitor groups was significantly higher than that of the control groups in the CCK cell line (P<0.05). Contrarily, there were no significant differences between the inhibitors and control groups in the HEK293 cell line (P>0.05). The results demon-strate that the EB1 gene could be a target gene of ipu-miR-143 and provide valuable information for further research on the post-transcriptional mechanisms of ipu-miR-143 in channel catfish.