LncRNA-599547 contributes the inductive property of dermal papilla cells in cashmere goat through miR-15b-5p/Wnt10b axis.

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.

CircRNA-1926 Promotes the Differentiation of Goat SHF Stem Cells into Hair Follicle Lineage by miR-148a/b-3p/CDK19 Axis.

Circular RNAs (CircRNAs) are a type of non-coding RNAs, which contain a covalently closed loop structure without 5' to 3' free ends. CircRNAs play essential roles in the regeneration of secondary hair follicle (SHF) and cashmere growth in goats. CircRNA-1926 was previously identified in SHF of cashmere goats, but its potential roles are unclear. In this study, we confirmed the expression of circRNA-1926 in SHF bulge of nine cashmere goats with a significantly higher level at anagen than that of telogen. Through the use of both overexpression and siRNA interference, we showed that circRNA-1926 promoted the differentiation of SHF stem cell into hair follicle lineage in cashmere goats which was evaluated via indictor genes Keratin 7 and Keratin 17. Using RNA pull-down, we found that circRNA-1926 bound with miR-148a/b-3p. Additionally, our data indicated that circRNA-1926 promoted the expression of the CDK19 gene. Using dual-luciferase reporter assays, it was revealed that circRNA-1926 positively regulated the CDK19 expression through miR-148a/b-3p. The results from this study demonstrated that circRNA-1926 contributes the differentiation of SHF stem cells into hair follicle lineages in cashmere goats via sponging miR-148a/b-3p to enhance CDK19 expression.

LncRNA-599554 sponges miR-15a-5p to contribute inductive ability of dermal papilla cells through positive regulation of the expression of Wnt3a in cashmere goat

BACKGROUND: Long non-coding RNAs (lncRNAs), as post-transcriptional regulators, were thought to function in the inductive property of dermal papilla cells (DPCs) in cashmere goat. Previously, lncRNA-599554 was identified in secondary hair follicle (SHF) of cashmere goat, but its functional significance is unknown. RESULTS: In the present investigation, we verified that lncRNA-599554 had significantly higher expression at the anagen dermal papilla of cashmere goat SHF than that at telogen. Based on overexpression and knockdown techniques, we found that lncRNA-599554 contributes the inductive property of DPCs of cashmere goat, which was assessed by detecting the changes in the expression of several typical indictor genes in DPCs including ET-1, SCF, Versican, ALP, Lef1 and Ptc-1. Based on RNA pull-down assay, we verified that lncRNA-599554 directly interacted with chi-miR-15a-5p. Also, we showed that lncRNA-599554 positively regulated the Wnt3a expression in DPCs but which did not appear to involve its modulating of promoter methylation. Based on the use of Dual-luciferase reporter assays, our data indicated that lncRNA-599554 regulated the Wnt3a expression through chi-miR-15a-5p-mediated post-transcriptional level. CONCLUSIONS: We showed that lncRNA-599554 contributes the inductive property of DPCs in cashmere goat which might be achieved through sponging chi-miR-15b-5p to promote the Wnt3a expression. The results from the present investigation provided a novel insight into the functional mechanism of lncRNA-599554 in the SHF regeneration of cashmere goat along with the formation and growth of cashmere fiber.

Identification and molecular analysis of a lncRNA-HOTAIR transcript from secondary hair follicle of cashmere goat reveal integrated regulatory network with the expression regulated potentially by its promoter methylation.

The HOTAIR transcript is transcribed from the antisense strand within the HOXC gene cluster, and it is thought to play a role in regulating the inductive capacity of dermal papilla cells during the reconstruction of hair-follicle. In the current investigation, we firstly isolated and characterized a lncRNA-HOTAIR transcript from the secondary hair follicle of cashmere goat. Also, we analyzed its transcriptional pattern and methylation level of HOTAIR gene promoter in secondary hair follicle of cashmere goat during anagen and telogen stages. Nucleotide composition analysis indicated that the contents of Adenine (A) and Thymine (T) are higher than that of Guanine (G) and Cytosine (C) in lncRNA-HOTAIR transcript of cashmere goat with the highest frequency distribution of AG nucleotide pair (8.06%). The regulatory network analysis showed a directly or indirectly complex regulatory relationships between lncRNA-HOTAIR of cashmere goat and its potential target molecules: miRNAs, mRNAs and proteins. Also, we showed that lncRNA-HOTAIR was properly transcribed at both anagen and telogen stages of secondary hair follicle of cashmere goat with the anagen being significantly higher than telogen in its expression, which suggest that lncRNA-HOTAIR transcript might be involved in the reconstruction of secondary hair follicle with the formation and growth of cashmere fiber. Taken together with methylation analysis of HOTAIR gene promoter, our data suggest that the promoter methylation of HOTAIR gene most likely is involved in its transcriptional suppression in secondary hair follicle of cashmere goat.

LncRNAs in Secondary Hair Follicle of Cashmere Goat: Identification, Expression, and Their Regulatory Network in Wnt Signaling Pathway.

Long noncoding RNAs (lncRNAs) are a novel class of eukaryotic transcripts. They are thought to act as a critical regulator of protein-coding gene expression. Herein, we identified and characterized 13 putative lncRNAs from the expressed sequence tags from secondary hair follicle of Cashmere goat. Furthermore, we investigated their transcriptional pattern in secondary hair follicle of Liaoning Cashmere goat during telogen and anagen phases. Also, we generated intracellular regulatory networks of upregulated lncRNAs at anagen in Wnt signaling pathway based on bioinformatics analysis. The relative expression of six putative lncRNAs (lncRNA-599618, -599556, -599554, -599547, -599531, and -599509) at the anagen phase is significantly higher than that at telogen. Compared with anagen, the relative expression of four putative lncRNAs (lncRNA-599528, -599518, -599511, and -599497) was found to be significantly upregulated at telogen phase. The network generated showed that a rich and complex regulatory relationship of the putative lncRNAs and related miRNAs with their target genes in Wnt signaling pathway. Our results from the present study provided a foundation for further elucidating the functional and regulatory mechanisms of these putative lncRNAs in the development of secondary hair follicle and cashmere fiber growth of Cashmere goat.

A lncRNA-H19 transcript from secondary hair follicle of Liaoning cashmere goat: Identification, regulatory network and expression regulated potentially by its promoter methylation.

The H19 transcript (imprinted maternally expressed transcript) is well-known as long noncoding RNA (lncRNA), and it is thought to be associated with the inductive capacity of dermal papilla cells for hair-follicle reconstruction. In this study, we isolated and characterized a lncRNA-H19 transcript from the secondary hair follicle of Liaoning cashmere goat. Also, we investigated its transcriptional pattern and methylation status of H19 gene in secondary hair follicle of this breed during different stages of hair follicle cycle. Nucleotide composition analysis indicated that guanine (G) and cytosine (C) are the dominant nucleotides in the lncRNA-H19 transcript of Liaoning cashmere goat with the highest frequency distribution (11.25%) of GG nucleotide pair. The regulatory network showed that lncRNA-H19 transcript appears to have remarkably diverse regulatory relationships with its related miRNAs and the potential target genes. In secondary hair follicle, the relative expression of lncRNA-H19 transcript at the anagen phase is significantly higher than that at both telogen and catagen phases suggesting that lncRNA-H19 transcript might play essential roles in the formation and growth of cashmere fiber of goat. Methylation analysis indicated that the methylation of the promoter region of H19 gene most likely participates in its transcriptional suppression in secondary hair follicle of Liaoning cashmere goat.