Abstract

The lncRNA-599547 (619-nt in length) is identified in secondary hair follicle (SHF) of cashmere goat, but its functional roles in regulating the inductive property of dermal papilla cells (DPCs) remains unknown. We found that lncRNA-599547 had significantly higher expression in dermal papilla of cashmere goat SHF at anagen than its counterpart at telogen. The overexpression of lncRNA-599547 led to a significant increase of ALP and LEF1 expression in DPCs (p < 0.05), whereas, the siLncRNA-1 mediated silencing of lncRNA-599547 significantly down-regulated the expression of ALP and LEF1 in DPCs (p < 0.05). Based on biotin-labeled RNA pull-down assay, we found that lncRNA-599547 directly interacted with chi-miR-15b-5p in DPCs. Based on both overexpression and silencing analysis of lncRNA-599547, our results indicate that lncRNA-599547 promotes the expression of Wnt10b in DPCs but without modulating its promoter methylation level. Using the mRNA-3′UTR fragments of goat Wnt10b containing the predicted binding sites of chi-miR-15b-5p in Dual-luciferase Reporter Assays, we show that lncRNA-599547 modulates the expression of Wnt10b at the chi-miR-15b-5p mediated post-transcriptional level. Taken together, our results indicate that lncRNA-599547 sponges miR-15b-5p to positively regulate the expression of Wnt10 gene, and thereby contributes the inductive property of DPCs in cashmere goat.

摘要

lncRNA-599547（长度为 619-nt）在羊绒山羊的次级毛囊 (SHF) 中被鉴定，但其在调节真皮乳头细胞 (DPC) 的诱导特性中的功能作用仍然未知。我们发现，lncRNA-599547 在生长期的绒山羊 SHF 真皮乳头中的表达明显高于其在休止期的对应物。lncRNA-599547 过表达导致 DPCs 中 ALP 和 LEF1 的表达显着增加（p  < 0.05），而 siLncRNA-1 介导的 lncRNA-599547 沉默显着下调 DPCs 中 ALP 和 LEF1 的表达（p < 0.05)。基于生物素标记的 RNA pull-down 分析，我们发现 lncRNA-599547 直接与 DPC 中的 chi-miR-15b-5p 相互作用。基于 lncRNA-599547 的过表达和沉默分析，我们的结果表明 lncRNA-599547 促进了 DPC 中 Wnt10b 的表达，但不调节其启动子甲基化水平。使用山羊 Wnt10b 的 mRNA-3'UTR 片段，在 Dual-luciferase Reporter Assays 中包含预测的 chi-miR-15b-5p 结合位点，我们发现 lncRNA-599547 调节 chi-miR-15b-处 Wnt10b 的表达5p 介导的转录后水平。总之，我们的结果表明，lncRNA-599547 海绵状 miR-15b-5p 正向调节 Wnt10 基因的表达，从而有助于绒山羊 DPCs 的诱导特性。