LncRNA H19 Overexpression Activates Wnt Signaling to Maintain the Hair Follicle Regeneration Potential of Dermal Papilla Cells.

Ningxia Zhu,Keng Huang,Le Chen,Bozhi Cai,En Lin,Congcong Fu,Changmin Lin,Yang Liu,Yang Zeng,Yanping Yuan,Bin Xia,Guiyuan Cao,Huan Zhang

doi:10.3389/fgene.2020.00694

Abstract

Androgenetic alopecia (AGA) is a common hair loss disorder resulting in seriously abnormal social interaction and psychological disorders. Transplantation with autologous dermal papilla cells represents a prospective therapy. However, the ability of dermal papilla cells to induce hair follicle development is lost upon cell culturing. Long non-coding RNAs (lncRNAs) are an important class of genes involved in various biological functions, are aberrantly expressed in disease and may play roles in the regulation of Wnt signaling, a critical pathway in maintaining the hair follicle-inducing capability of dermal papilla cells. Examination of dermal papilla cells by lncRNA microarray revealed that H19 was highly expressed in early passage dermal papilla cells compared with late-passage dermal papilla cells. In this study, we constructed H19-overexpressing dermal papilla cells to examine the role of H19 on hair follicle inductivity. Dermal papilla cells infected with lentivirus encoding H19 maintained their cell shape, and continued to display both multiple-layer aggregation and hair follicle-inducing ability upon prolonged culture. H19 exerted these effects through inducing miR-29a to activate Wnt signaling by directly downregulating the expression of Wnt suppressors, including DKK1, Kremen2, and sFRP2, thereby forming a novel regulatory feedback loop between H19 and miR-29a to maintain hair follicle- inducing potential. These results suggest that lncRNA H19 maintains the hair follicle-inducing ability of dermal papilla cells through activation of the Wnt pathway and could be a target for treatment of androgenetic alopecia.

Highlights

MATERIALS AND METHODSAndrogenetic alopecia (AGA) is an extremely common type of hair loss affecting the appearance and mental state in both men and women (Wang et al, 2010; Vincenzi et al, 2019)
In our previous long non-coding RNA (lncRNA) by microarray profiling (Lin et al, 2014) we reported that five transcripts from the H19 gene were up-regulated in early passage dermal papilla (DP) cells (DP4), which were hair follicle (HF)-inducible
High levels of lncRNAH19 were located in the nucleus of DP4 cells (Figure 1B)

Summary

MATERIALS AND METHODS

Androgenetic alopecia (AGA) is an extremely common type of hair loss affecting the appearance and mental state in both men and women (Wang et al, 2010; Vincenzi et al, 2019). To evaluate the influence of H19 on the Wnt/β-catenin signaling pathway, a luciferase reporter plasmid pGL4.49[luc2P/TCF-LEF RE/Hygro] (Promega, United States) and pcDNA3.1/pcDNA3.1H19 as well as an internal control pGL4.74[hRluc/TK] vector (Promega, United States) were co-transfected into the passage 8 (DP8) cells at 70% confluence. Total RNA, including lncRNA/miRNA fractions, was isolated from DP cells or dorsal skins, with a miRNeasy Mini Kit (QIAGEN, Beijing, China) and the quality of the RNA was assessed with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, United States) at 260 and 280 nm (A260/280). Total protein from DP cells, following H19 transient transfection, and dorsal skin tissues from nude mice were extracted. Skin tissue sections were incubated with blocking solution containing 10% donkey serum for 30 min at 37◦C and probed overnight at 4◦C with a diluted primary antibody, followed by a secondary antibody for 1 h. All experiments were repeated at least three times, and for each experiment, samples were analyzed in triplicate

RESULTS

H19 Overexpression Activates Wnt Signaling by Upregulating MiR-29a-3p in DP Cells

DISCUSSION

CONCLUSION