miR-124和Otx2在牙鲆变态发育期间的表达调控及其靶向关系验证

Expression of miR-124 and Otx2 during metamorphosis and verification of their targeting relationship in Paralichthys olivaceus

牙鲆(Paralichthys olivaceus)是研究鱼类变态发育的理想模型,其右眼移位及生活习性的改变受甲状腺激素(TH)调控。同时microRNA (miRNAs)在变态期间发挥关键性作用。为研究TH、pol-miR-124及Otx2在牙鲆变态中的调控机制,本实验通过生物信息学方法预测pol-miR-124潜在靶基因Otx2,首先利用qRT-PCR检测在牙鲆各组织、正常变态及外源TH处理仔鱼后pol-miR-124和Otx2的表达模式。然后克隆400 bp含有“种子序列”的Otx2 3′UTR区序列,构建野生型重组载体pmirGLO-Otx2并转染293T细胞检测双荧光素酶活性确定靶向关系。qRT-PCR结果表明: pol-miR-124和Otx2均在脑和眼睛组织中特异性高表达;在仔鱼变态28 dph表达量最高与变态进程相一致;TH作用下,在20 dph、24 dph时期, pol-miR-124的表达量低于正常组,而Otx2的表达量高于正常组;在28 dph、32 dph、36 dph时期, pol-miR-124的表达量高于正常组,但Otx2的表达量低于正常组,两者呈现出相反的表达趋势;双荧光素酶结果显示pol-miR-124靶向负调控Otx2。本实验旨为揭示牙鲆视觉感光系统的发育机制奠定研究基础,同时为探讨牙鲆变态发育机制提供了新的理论依据。

The Japanese flounder (Paralichthys olivaceus) is an ideal model for studying the metamorphosis of fish. Thyroid hormone (TH) can regulate the right eye shift and lifestyle of P. olivaceus as it transforms from a bilateral pelagic larva into an asymmetrical benthic juvenile. At the same time, microRNAs (miRNAs) play a key role during metamorphosis. To research the regulation mechanism of TH, pol-miR-124, and Otx2 in P. olivaceus metamorphosis, we found the potential target, Otx2, based on its binding site through bioinformatics analysis. First, we detected the expression levels of pol-miR-124 and Otx2 in adult tissues in the normal group and TH treatment group larvae using real-time quantitative PCR. Then, the 400 bp alignment region of Otx2 3 UTR containing the "seed sequence" was cloned, and the target relationships between miR-124 and Otx2 were verified at the cellular level by constructing a dual luciferase reporter. The qPCR results showed that both pol-miR-124 and Otx2 were highly expressed in the brain and eye tissues;the highest expression at 28 dph was observed in the larvae. Compared with the NC group, the pol-miR-124 expression level was inhibited at 20 dph and 24 dph in the TH group, and the Otx2 expression level was higher than that in the NC group. However, the expression level of pol-miR-124 increased at 28 dph, 32 dph, and 36 dph, but the Otx2 level was lower than that in the NC group. The expression of pol-miR-124 was negatively correlated with the expression of the predicted target Otx2 expression. Luciferase results showed that the luciferase activity of the pol-miR-124 group was significantly decreased and Otx2 was the target gene of pol-miR-124. The results of this study form an important research foundation for further revealing the developmental mechanism of the photoreceptor system during P. olivaceus metamorphosis.