猪长链非编码RNA（XLOC_359028）调控脂质沉积的分子机制

Pigs are important model for research on lipid metabolism and associated diseases. It has been proved that long non-coding RNA (lncRNA) participated in the regulation of lipid metabolism. Our results of high-throughput sequencing indicated that a novel lncRNA (XLOC_359028) was associated with fat deposition in pig. And bioinformatics analysis predicted that XLOC_359028 had binding sites of CCAAT enhancer binding protein α (C/EBPα) and miR-9, and its target gene was cell death-inducing DFFA-like effector c (CIDEC). They are important regulation factors in lipid metabolism, but their interaction with XLOC_359028 remain unclear. So in this project, we firstly validate the effect of XLOC_359028 on lipid deposition by using qRT-PCR and RNA interference, then we confirm their interaction by using RNA-binding protein immunoprecipitation (RIP), Dual fluorescence reporter system, and Chromatin confirmation capture (3C) technology, respectively. Hence, this project firstly research the molecular regulation mechanism of XLOC_359028 from chromatin remodeling, transcriptional and post-transcriptional level, which has vast importance for understanding the molecular regulation mechanism of lipid deposition in animal.

猪是研究脂肪代谢及相关疾病的重要动物模型，已有研究证明长链非编码RNA（lncRNA）参与脂肪代谢的调控。本课题组通过高通量测序发现lncRNA（XLOC_359028）与猪脂肪沉积相关，生物信息学分析发现在其序列附近有C/EBPα和miR-9的结合位点，靶基因为CIDEC。这三者均为脂质代谢的重要调控因子，但XLOC_359028与其具体的互作方式仍是未知的。本项目拟通过qRT-PCR和RNA干扰技术明确XLOC_359028对脂质沉积的影响，然后使用RNA结合蛋白免疫沉淀反应（RIP）、双荧光报告系统和染色质构象捕获技术（3C）技术分别验证XLOC_359028与C/EBPα、miR-9和CIDEC的具体作用机制。本研究首次从染色质重构、转录和转录后三个水平研究XLOC_359028的分子调控机制，对于理解动物脂质沉积的分子调控机制具有重要意义。

脂肪沉积对于肉质性状的研究有重要意义，已有研究表明，lncRNA也参与脂质代谢的调控。本团队前期通过高通量测序发现lncRNA（XLOC_359028），其预测靶基因为CIDEC，其序列有C/EBPα和miR-9的结合位点。这三者都是脂质代谢的重要调控因子，所以我们提出假设，XLOC_359028可能通过与这三者的互作参与猪脂质代谢的调控。首先表达谱分析发现XLOC_359028具有广谱表达性，在成脂分化过程中表达量降低。通过RNA干扰和腺病毒介导的过表达技术，验证了XLOC_359028具有抑制脂质生成的作用。FISH杂交结果提示XLOC_359028主要位于细胞质中，提示其可能通过ceRNA机制发挥作用。定量PCR结果发现XLOC_359028与miR-9的靶基因SIRT1表达显著相关，经双荧光素酶活性试验，证实XLOC_359028是miR-9的靶基因，XLOC_359028通过竞争性结合miR-9调控SIRT1的表达，参与脂质生成的调控。而通过ChIRP试验并没有发现XLOC_359028与CIDEC和C/EBPα的直接作用。本研究首次验证了XLOC_359028参与调控猪脂肪细胞脂质沉积的ceRNA机制，进一步完善了lncRNA调控猪脂肪细胞脂质沉积的调控网络，为改善猪肉肉质提供选育的靶标。