Understanding the role of regulatory RNAs in human skin wound healing

Abstract

Human skin wound healing is characterized by four phases in a timely manner, including hemostasis, inflammation, proliferation, and remodeling. Various cell types are involved in the biological process. Keratinocytes that constitute around 95% of epidermal cells recruit immune cells by secreting pro-inflammatory cytokines/chemokines and undergo re-epithelialization in the proliferation phase. Ribonucleic acids (RNAs) without protein-coding capacity, defined as noncoding RNAs, consist of the majority of transcription output, are indispensable for multiple biological processes and are critical during disease contexts. Due to their cell and context specificity, noncoding RNAs present a therapeutic potential. However, revealing their underlying mechanism in the skin wound healing is the prerequisite. In this thesis, we identified and comprehensively characterized the role of long noncoding RNAs (lncRNAs) (Paper I, III, IV) and microRNAs (miRNAs) (Paper II) in human skin wound healing.

Paper I identified Wound And Keratinocyte Migration-Associated lncRNA 1 (WAKMAR1), and it was upregulated during wound healing but deficient in nonhealing wounds. WAKMAR1 silencing inhibited keratinocyte migration and re-epithelialization of human ex vivo wounds, whereas its overexpression promoted cell migration. Moreover, we revealed that the WAKMAR1-regulated network composed of pro-migratory genes was driven by E2F Transcription Factor 1 (E2F1). Further mechanistic investigation showed that WAKMAR1 enhanced E2F1 expression by hijacking DNA methyltransferases (DNMTs) and reducing methylation at the E2F1 promoter. This study demonstrates that WAKMAR1 is essential for keratinocyte migration and re-epithelialization of human ex vivo wounds, and its deficiency may be associated with delayed healing.

Paper II aimed to identify clinically relevant miRNAs and develop an open database for future studies in skin wound healing. We performed the comprehensive and integrative small and long RNA sequencing analysis in human skin, normal wounds collected at different healing phases, and venous ulcers (VUs). We found 17 VU-relevant miRNAs, whose targets were overrepresented in the VU-specific signature. The upregulated miRNAs in VU were predicted to promote inflammatory response but impair cell proliferation, but the downregulated miRNAs might be needed for cell proliferation and migration. We tested the combined effects of miR-34a-5p, miR-424-5p, and miR-516-5p upregulated in VU. Simultaneous overexpression of miR-34a-5p and miR-424-5p had stronger inhibitory effects on keratinocyte proliferation and migration, whereas the combination of miR-34a-5p and miR-516b-5p promoted the expression of the pro-inflammatory Chemokine (C-C Motif) Ligand 20 (CCL20). Overall, our study identifies VU-relevant miRNAs and demonstrates that their abnormal expression may contribute to the pathogenesis of nonhealing wounds.

Paper III investigated the role of the HOXC13 Antisense RNA (HOXC13-AS) in epidermal differentiation. LncRNA HOXC13-AS was specifically expressed in human skin and downregulated in the early phases of wound healing. We analyzed our single-cell RNA sequencing in the human skin and found that HOXC13-AS was highly expressed in the differentiated keratinocytes. Furthermore, we showed that HOXC13-AS was decreased by the epidermal growth factor receptor (EGFR) signaling pathway but gradually increased during keratinocyte differentiation. Transcriptomic analysis and functional assays indicated that HOXC13-AS promoted keratinocyte differentiation using differentiation models in vitro and organotypic epidermis. Mechanistically, we revealed that HOXC13-AS physically interacted with COPI Coat Complex Subunit Alpha (COPA) which is essential for the retrograde transport from the Golgi to the endoplasmic reticulum (ER). HOXC13-AS hijacked COPA, which interfered with the retrograde transport, promoting ER stress and keratinocyte differentiation. Rescue assays confirmed that the role of HOXC13-AS in keratinocyte differentiation was dependent on COPA. Overall, this study demonstrates HOXC13-AS as a molecule of importance for epidermal differentiation.

Paper IV focused on lncRNA SNHG26, which plays a key role in the transition from inflammation to proliferation during wound healing. SNHG26 was upregulated during wound healing, and Snhg26 knockout mice showed delayed re-epithelialization. By single-cell RNA sequencing analysis, we found decreased migratory but increased inflammatory keratinocyte progenitors in the wound edge of Snhg26 deficient mice. Moreover, we confirmed that SNHG26 enhanced cell proliferation and migration but inhibited inflammatory response in human keratinocytes and ex vivo wounds. Mechanistically, we demonstrated that Interleukin Enhancer Binding Factor 2 (ILF2) physically interacted with SNHG26 using RNA pulldown and RNA immunoprecipitation (RIP). Chromatin immunoprecipitation (ChIP) and chromatin isolation by RNA purification (ChIRP) sequencing showed that SNHG26 guided ILF2 from the inflammatory genomic loci to the Laminin Subunit Beta 3 (LAMB3) genomic locus, switching the gene network and facilitating the inflammatory-to-proliferative state transition of keratinocyte progenitors. This study provides compelling evidence for SNHG26 being a crucial regulator for human skin wound healing.