牦牛Linc24063的克隆鉴定及其与miRNAs表达水平的相关性分析

Cloning and Identification of Long-Chain Non-Coding RNA Linc24063 and Its Correlation with the Expression Level of miRNAs in Yak

【目的】通过对牦牛长链非编码RNA Linc24063进行克隆鉴定,分析其在乳腺组织中与miRNAs表达量的相关性,为Linc24063通过调控miRNA发挥功能的调控机制研究提供试验依据。【方法】选取4.5岁处于第一泌乳期的健康母牦牛12头,清晨空腹屠宰后,迅速采集大脑、乳腺、肾脏、心脏、肝脏和卵巢组织,提取组织总RNA,利用5′RACE和3′RACE方法克隆牦牛Linc24063序列;利用生物信息学对其序列保守性、染色体定位及编码潜能进行分析,然后利用原核表达试验对其编码能力进行验证;利用RT-qPCR检测其在大脑、乳腺、肾脏、心脏、肝脏和卵巢的表达水平。利用普通牛和绵羊的miRNA数据库,结合miRanda和mireap软件预测与Linc24063具有相互作用的物种间保守miRNAs,与前人研究表明在牛不同泌乳时期乳腺组织中具有差异表达的miRNA取交集,然后对其靶基因进行GO富集和KEGG信号通路分析;最后使用皮尔逊相关系数在乳腺组织中进行Linc24063与miRNAs表达量的相关性分析。【结果】Linc24063 5′RACE和3′RACE片段大小分别为476 bp和356 bp,测序分析表明Linc24063大小为758 bp,位于牦牛21号染色体的Dlk1-Dio3印记域,与普通牛的序列保守性最高。生物信息学预测其编码潜能较低,原核表达试验进一步验证Linc24063不能有效的翻译蛋白,表明Linc24063是一个真正的长链非编码lncRNA。组织表达谱分析表明,Linc24063在牦牛乳腺组织中表达量最高,在肝脏和卵巢中表达量较低。生物信息学分析后共筛选到与Linc24063具有相互作用的物种间保守miRNAs 21个,其中有研究报道在乳腺中有差异表达的13个;13个miRNAs靶基因富集到TGF-β、PI3K-Akt、胰岛素等信号通路中,表明Linc24063可能通过这些信号通路参与牦牛乳脂和乳蛋白的生物合成过程。在乳腺组织中,Linc24063表达水平与miR-200a(P=0.001)、miR-141(P=0.02)显著负相关,与miR-27a(P=0.023)显著正相关,与miR-24(P=0.601)不具备相关性。【结论】Linc24063位于Dlk1-Dio3印记域内,在乳腺组织中具有较高表达量,且可能通过与miR-200a、miR-141和miR-27a相互作用从而参与牦牛乳脂和乳蛋白的生物合成过程。为深入探讨牦牛Linc24063在乳脂和乳蛋白合成中的生物学功能提供了基础数据。

【Objective】The aim of this study was to clone and to identify the protein coding potential of the long-chain non-coding RNA Linc24063 in yak,and then to analyze its correlation with the expression of miRNAs in mammary gland,which might provide basis for studying the function of Linc24063 in yak.【Method】Twelve healthy female yaks at 4.5-year old in the first lactation period were selected as experiment animal.After fasting slaughter,the tissue samples of cerebrum,mammary gland,kidney,heart,liver and ovary were collected for total RNA extraction.Firstly,the sequence of Linc24063 was cloned by 5’RACE and 3RACE,and the sequence conservation,chromosomal location and coding potential were analyzed by bioinformatics,then the prokaryotic expression assays were used to analyze its protein coding ability.Its expression levels in cerebrum,mammary gland,kidney,heart,liver and ovary tissue were determined by using RT-qPCR.Secondly,the conserved miRNAs interacting with Linc24063 were predicted by using the miRNA database of cattle and sheep,combined with miRanda and mireap software.The same miRNAs were obtained from the published differentially expressed miRNAs during different stages of lactation in cattle and the conserved miRNAs,and then GO enrichment and KEGG pathway analysis were performed on their target genes.Finally,Pearson correlation coefficient was used for analyzing the correlation between the expression of Linc24063 and miRNAs in the mammary gland of yak.【Result】The length of Linc24063 5’RACE and 3’RACE fragments were 476 bp and 356 bp,respectively.Sequencing analysis showed that Linc24063 was 758 bp in length and located in the Dlk1-Dio3 imprinting domain of 21 chromosomes in yak.Bioinformatics predicts results showed that its coding potential was lower,and prokaryotic expression experiments further demonstrated that Linc24063had no protein coding ability,suggesting that Linc24063 was a real lncRNA.Tissue expression profiling showed that Linc24063 had highest expression in mammary gland and lowest expression in liver and ovary.After bioinformatics analysis,Twenty-one conserved miRNAs interacting with Linc24063 were screened,among which 13 miRNA were differentially expressed in the mammary gland reported previously.The 13 miRNAs targets were enriched in TGF-β,PI3K-Akt,insulin and other signaling pathways,suggesting that Linc24063 might participate in the biosynthesis of milk fat and protein via these signaling pathways.In mammary gland tissue the expression of Linc24063 was significantly negatively correlated with miR-200a(P=0.001)and miR-141(P=0.02),and significantly positively correlated with miR-27a(P=0.023),but no correlation with miR-24(P=0.601).【Conclusion】The results showed that Linc24063 was located in the Dlk1-Dio3 imprinting domain and expressed higher in mammary gland,and might play an important roles in the biosynthesis of milk fat and protein via miR-200a,miR-141 and miR-27a,which would be beneficial for revealing the regulatory mechanism of Linc24063 in milk fat and protein synthesis.