2个候选lncRNA的克隆、序列分析及其在奶牛乳腺上皮细胞炎症反应中的表达

Cloning and sequence analysis of two candidate lncRNAs and their expression in bovine mammary epithelial cell with inflammation

【目的】探究2个候选lncRNA(lncRNA TCONS⁃72171和lncRNA TCONS⁃62349)在奶牛乳腺上皮细胞炎症模型中的表达与功能分析。【方法】利用基因克隆和测序技术验证候选lncRNA的真实性;并进行候选lncRNA序列同源性、亚细胞定位、靶基因预测及KEGG信号通路分析。此外,利用脂多糖(Lipopolysaccharide,LPS)诱导奶牛乳腺上皮细胞产生炎症反应;采用RT⁃qPCR技术检测LPS诱导0、3、6和12 h的奶牛乳腺上皮细胞内上述2个候选lncRNAs的表达情况。【结果】奶牛lncRNA TCONS⁃72717和ln⁃cRNA TCONS⁃62349真实存在,且在部分物种间高度保守;上述2个lncRNA在LPS诱导的奶牛乳腺上皮细胞中表达均呈极显著上调(P<0.01),且分别主要位于细胞质与细胞核。靶基因预测与KEGG信号通路分析预测结果显示,上述2个lncRNA的潜在靶基因(LCP2、ALOX12和ASGR1等)可能通过JAK⁃STAT、NF⁃κB和MAPK等炎症相关的信号通路而发挥调控细胞炎症的作用。【结论】表达上调的lncRNA TCONS⁃72171和lncRNA TCONS⁃62349可能在LPS诱导的奶牛乳腺上皮细胞炎症中发挥重要的调控作用,该结果可为深入研究奶牛乳腺炎分子调节机制奠定基础。

【Objective】The present paper aimed to investigate the expression pattern and function of two candidate lncRNAs(lncRNA TCONS⁃72171 and lncRNA TCONS⁃62349)in bovine mammary epithelial cell with inflammation.【Method】In the study,gene cloning and sequencing techniques were used to verify the authenticity of lncRNAs.Then,we performed sequence homology,subcellular localization,tar⁃get gene prediction and KEGG functional annotation analysis.In addition,an inflammatory response in bovine mammary epithelial cells was challenged with lipopolysaccharide(LPS);RT⁃qPCR was used to detect the expression of the above two candidate lncRNAs in bovine mam⁃mary epithelial cells at 0,3,6 and 12 hours of LPS induction.【Result】The bovine lncRNA TCONS⁃72717 and lncRNA TCONS⁃62349 were authentic and highly conserved among some species.The expression of the above two lncRNAs were highly significantly up⁃regulated in LPS⁃induced bovine mammary epithelial cells(P<0.01).LncRNA TCONS⁃72717 and lncRNA TCONS⁃62349 were mainly located in the cytoplasm and nucleus,respectively.The results of target gene prediction and KEGG functional annotation showed that the potential target genes(LCP2,ALOX12 and ASGR1,etc.)of the above two lncRNAs might play a role in regulating cellular inflammation through inflamma⁃tion⁃related signaling pathways such as JAK⁃STAT,NF⁃κB and MAPK.【Conclusion】The up⁃regulated lncRNA TCONS⁃72171 and lncRNA TCONS⁃62349 may play an important regulatory role in bovine mammary epithelial cells with LPS⁃induced inflammation,and the results may lay the foundation for an in⁃depth study of the molecular regulatory mechanism of dairy cow mastitis.