Ccr-lncRNA172145靶向miR-206在锦鲤体色调控中的作用初探

Preliminary study on the role of Ccr-lncRNA172145 in targeting miR-206 in the regulation of skin color of Cyprinus carpio var. koi

  • 摘要: 为了解非编码RNA在锦鲤体色分化变异中的分子调控机制,在课题组前期对锦鲤皮肤组织转录组测序的基础上,筛选到4条在3种皮肤(黑色、红色、白色)组织中显著差异表达的lncRNA;基于RNAhybrid 和TargetScan 靶基因预测软件,发现lncRNA172145与黑色素合成通路中miR-206之间存在靶向结合位点。借助CPC、CPAT及CNIT软件,对lncRNA172145编码能力进行分析,证实该序列为lncRNA,不具备编码蛋白的能力。然后,利用qRT-PCR 技术对该序列时空表达水平进行了检测,发现在眼睛、黑色皮肤、鳍条及血液中表达量显著高于其他组织;自原肠胚时期表达量开始显著上升,高水平趋势一直持续到孵化后20 d。通过双荧光素酶报告实验,进一步证实lncRNA172145与miR-206之间存在靶向调控关系。最后,通过合成miR-206拮抗剂,对miR-206进行体内沉默,发现与注射阴性对照拮抗剂组和PBS组相比,miR-206拮抗剂组的lncRNA172145表达水平显著升高。研究表明,lncRNA172145可能通过靶向到miR-206,参与到黑色素合成通路的调控,这为后续深入挖掘二者在黑色素合成通路中具体的分子机制提供了基础资料。

     

    Abstract: Long non-coding RNA (lncRNA) is a type of RNA with a length of over 200 nucleotides that can interact with DNA, RNA, or proteins to regulate target genes at the transcriptional, post-transcriptional, and epigenetic levels. Based on the previous transcriptome sequencing data of Cyprinus carpio var. koi skin tissues, we screened four lncRNAs that were significantly differentially expressed in three skin tissues (black, red and white). In order to further explore the function of these lncRNAs in skin color differentiation and synthesis, a preliminary study of the relationship between their functions and miRNAs was also conducted. Firstly, the target binding sites between lncRNA172145 and miR-206 in the melanogenesis pathway were identified through RNAhybrid and TargetScan target gene tools. Then, the coding ability of lncRNA172145 was detected by CPC, CPAT, and CNIT software, and the results confirmed that this sequence was lncRNA with no protein coding ability. The temporal and spatial expression levels of this sequence were further analyzed by qRT-PCR, and it was demonstrated that the levels were significantly higher in the eyes, black skin, fins and blood than those in other tissues; and the expression levels started to increase significantly from the gastrulation stage, and the high-level trend continued until 20 days after hatching. Additionally, with the help of dual luciferase reporter assay, the existence of a targeted regulatory relationship between lncRNA172145 and miR-206 was further verified. Finally, miR-206 was silenced in vivo by synthesizing miR-206 antagonist, and the results showed that the expression level of lncRNA172145 in the miR-206 antagonist group, was significantly higher compared to the negative control antagonist group and the PBS group. These findings suggest that lncRNA172145 could be involved in the regulation of the melanogenesis pathway by targeting to miR-206, providing the fundamental information for further research into the specific molecular mechanisms of their roles in the melanogenesis pathway, as well as the role of long non-coding RNAs in regulating fish body pigmentation and differentiation.

     

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