Long non‐coding RNAs in cutaneous biology and proliferative skin diseases: Advances and perspectives

Abstract Advances in transcriptome sequencing have revealed that the genome fraction largely encodes for thousands of non‐coding RNAs. Long non‐coding RNAs (lncRNAs), which are a class of non–protein‐coding RNAs longer than approximately 200 nucleotides in length, are emerging as key epigenetic regulators of gene expression recently. Intensive studies have characterized their crucial roles in cutaneous biology and diseases. In this review, we address the promotive or suppressive effects of lncRNAs on cutaneous physiological processes. Then, we focus on the pathogenic role of dysfunctional lncRNAs in a variety of proliferative skin diseases. These evidences suggest that lncRNAs have indispensable roles in the processes of skin biology. Additionally, lncRNAs might be promising biomarkers and therapeutic targets for cutaneous disorders.


| LN CRNA CL A SS IFI C ATI ON AND REG UL ATORY MECHANIS MS
LncRNAs can be grouped according to the genomic location from which they are transcribed 5,20,24 (Figure 1). For example, enhancer lncRNAs originate from enhancer regions, and promoter-associated lncRNAs are transcribed in the opposite direction to the proteincoding transcript from regions in close proximity to a promoter. In addition, intergenic lncRNAs (lincRNAs) are transcribed from noncoding DNA sequences located between protein-coding genes, whereas intronic lncRNAs are transcribed from the introns of protein-coding genes. Sense lncRNAs and natural antisense lncRNAs are transcribed from the sense and antisense strands of protein-coding genes, respectively. Additionally, sense lncRNAs and natural antisense lncRNAs can overlap with one or several introns and/or exons of the sense sequence. Untranslated region (UTR) overlapping lncR-NAs are transcribed from DNA sequences overlapping the 3′UTR or 5′UTR region of a protein-coding gene in the sense strand ( Figure 1). and post-translation. For example, epigenetic control by lncRNAs, such as X inactivation, 25 genomic imprinting, 26 mediation of mRNA stability enhancement or decay 27 and neutralization of miRNAs, 28 has been reported. At the transcriptional level, lncRNAs play activator roles in enhancing or inhibiting protein-coding gene transcription. These ln-cRNAs are usually synthesized at enhancers. They influence the activity of enhancers or recruit protein complexes to enhancers, acting as cofactors that help remodel chromatin architecture and enhance kinase activity. 29 At the post-transcriptional level, lncRNAs regulate diverse processes, such as transport, translation, splicing or decay of mRNA and neutralization of miRNAs. [30][31][32] Recently, growing evidence indicates that lncRNAs exert important effects on the biological processes of cutaneous development as well as on the pathogenesis of skin diseases via multiple ways mentioned above.

| THE IND IS PEN SAB LE ROLE S OF LN CRNA S IN CUTANEOUS B I OLOGY
According to the physiological structural anatomy, the skin consists of three primary layers: epidermis, dermis and hypodermis. 33,34 F I G U R E 1 The classification of long non-coding RNA. Long non-coding RNAs (lncRNAs) can be grouped according to their transcribed genomic positions The epidermis, the top layer of skin, encompasses distinct layers of keratinocytes: ①the basal layer comprises self-renewing progenitor cells; ② the spinous layer, lying above the basal layer, contains upward migrating, differentiating keratinocytes; ③ the granular layer includes cells producing the substrates necessary to form the impermeable barrier; and ④ the stratum corneum comprises terminally differentiated enucleated lipid-embedded corneocytes (see Glossary) that have undergone cornification to form the outer skin surface. In addition, melanocytes, which reside in the bottom layer of the epidermis, produce melanin pigment to induce pigmentation and protect against UV light. The dermis, the middle layer of skin, is populated by macrophages, lymphocytes, mast F I G U R E 2 The structure of the epidermis and dermis. Epidermal skin encompasses distinct layers of keratinocytes, including the basal layer (stratum basale), the squamous cell layer (stratum spinosum), the granular layer (stratum granulosum) and the cornified layer (stratum corneum). Melanocytes reside in the bottom layer of the epidermis. Additionally, hair follicles extend from the deeper dermal tissue, through the basement membrane and epithelial layer and extend beyond the border of the skin F I G U R E 3 Schematic diagram of functional lncRNAs in cutaneous biology. LncRNAs function as promoters or suppressors in diverse skin physiological processes, including keratinocytes differentiation, melanocyte function, hair growth and wound healing. Red arrow indicates upregulation. Green arrow represents downregulation. Black arrow indicates promotion. represents suppression. means the abrogation of upstream suppression. ANCR, anti-differentiation ncRNA; ANRIL, antisense non-coding RNA in the INK4 locus; CREB, cAMP-responsive element-binding protein; DNMTs, DNA methyltransferases; DPPIV, dipeptidyl peptidase IV; E2F1, E2F transcription factor 1; EZH2, enhancer of zeste homolog 2; HIF-1α, hypoxia-inducible factor 1 subunit alpha; HOTAIR, HOX transcript antisense RNA; MiTF, microphthalmia-associated transcription factor; Prox1, Prospero homeobox 1; SPRIGHTLY, SPRY4 intronic transcript 1; STAU1, staufen double-stranded RNA binding protein 1; TGF-β1, transforming growth factor beta 1; TINCR, terminal differentiation-induced ncRNA; UCA1, urothelial cancer associated 1; WAKMAR1, wound and keratinocyte migration-associated lncRNA 1 cells, dendritic cells and fibroblasts. Additionally, hair follicle morphogenesis occurs via interactions between epidermal keratinocytes and dermal fibroblasts ( Figure 2).
Previous studies majorly focused on the modulatory effects of microRNAs on the processes of keratinocytes' proliferation 35,36 and differentiation, 37-39 melanocytes biology, [40][41][42] hair growth 43,44 and wound healing. 45,46 In recent years, emerging evidence has indicated that lncRNAs, another kind of non-coding RNAs, are also involved in these physiological processes (Figure 3；Table 1).

| LncRNAs regulate keratinocyte differentiation
The epidermis is a stratified surface epithelium that provides a barrier to the external environment. Physiologically, the human epidermis continuously renews itself approximately every 4 weeks by a process of keratinocyte migration, proliferation and differentiation.
During the course of epidermal differentiation, a subset of basal keratinocytes withdraws from the cell cycle, detaches from the basement membrane, moves outward from the basal membrane, migrates through the epidermis and undergoes terminal differentiation. A precise balance between the progenitor compartment and terminally differentiated layers is of great importance for the maintenance of the functional epidermis.
To determine the effects of lncRNA on the differentiation of keratinocyte, studies profiled lncRNA expression by microarray and validated the results by qRT-PCR in the differentiating human keratinocytes using a three-dimensional epidermal equivalent. In this model, lncRNA BC020554 was found to be downregulated upon keratinocyte differentiation. In contrast, lncRNA AK022798 was upregulated for early keratinocyte differentiation. 47 However, the biological functions of these differential lncRNAs remain to be elucidated.
Terminal differentiation-induced ncRNA (TINCR), a 3.7 kb nuclear and cytoplasmic intronic lncRNA, was found to control the human epidermal differentiation in vitro by a post-transcriptional mechanism in differentiated keratinocytes. 48 Research has revealed that TINCR interacted with a range of differentiation mRNAs through a 25-nucleotide "TINCR box" motif, which can better help them bind to the staufen double-stranded RNA binding protein 1 (STAU1) protein, increasing their stabilization in differentiated epidermal strata.
Anti-differentiation ncRNA (ANCR), another well-characterized 855 bp cytoplasmic intergenic lncRNA, was downregulated during the terminal differentiation of keratinocytes, adipocytes and osteoblasts in organotypic human tissues. 49 Contrary to TINCR, ANCR repressed keratinocyte differentiation by associating with the methyltransferase enhancer of zeste homolog 2 (EZH2) and acting as a guide for the polycomb repressive complex 2 (PRC2) chromatinmodifying complex that eventually leads to epigenetic silencing of target gene loci.
LncRNA H19, a 2322 bp nuclear intergenic lncRNA, promoted the keratinocyte differentiation process in primary human keratinocytes from fresh post-operative skin samples of children. 50 Mechanically, H19 acted as an endogenous "sponge," which bound directly to miR-130b-3p. This sponge directly decreased the activity of miR-130b-3p and consequently increased the expression of miR-130b-3p downstream target desmoglein-1 (Dsg1), resulting in the promotion of keratinocyte differentiation.
Summarily, these results indicate that lncRNAs have both prodifferentiation (such as TINCR and H19) and anti-differentiation (such as ANCR) effects on the processes of keratinocyte differentiation via diverse mechanisms. Further studies are needed to explore the precise roles of lncRNAs in regulating keratinocyte differentiation.

| LncRNAs modulate melanocyte functions
Melanocytes, which are located in the basal layer, make up 8% of the epidermis. They produce melanin pigment, which determines the colour of the skin and protects against UV radiation. 51 Previous studies have revealed that some non-coding RNAs, such as microRNAs, in melanocytes can alter and affect the synthesis of However, the roles of lncRNAs in modulating melanocyte prolifera- and human cutaneous melanocytes (MC). 54 Additionally, UCA1 can also antagonize UVB-induced pigmentation in PIG1 and MC cells.
Together, these results indicate that lncRNA can modulate melanocyte functions by promoting their proliferation (lncRNA SPRIGHTLY) and reducing their melanogenesis (lncRNA UCA1).

| LncRNAs affect hair growth
Hair follicles are one of the important skin appendages located in the dermal layer. Hair structure is complicated, containing an outer root sheath, an inner root sheath and a hair shaft. Hair follicle stem cells (HFSCs), which reside in a specialized region of the outer root sheath designated the bulge, are a vital cell resource of hair follicles and the epidermis. 55 The differentiation and proliferation of HFSCs in hair follicles are critical for normal hair homeostasis. Collectively, these studies show that lncRNAs contribute to key processes underlying hair growth, including the proliferation and differentiation of HFSCs (lncRNA PlncRNA-1) and hair follicle reconstruction (lncRNA H19, RP11-766N7.3 and HOTAIR) in DP cells.

| LncRNAs influence wound healing and cell proliferation
Wound healing is a fundamental and physiological process required to recover the integrity of the skin after injury, which is achieved through a series of dynamic and complicated processes including inflammation, angiogenesis, coagulation, tissue formation and remodelling. 58 Failure of these reparative processes leads to chronic impaired wounds, which often happen in patients with underlying disorders, such as diabetes mellitus. 59 However, the processes of physiological wound healing are intricate. In addition, efficient targeted treatments for chronic impaired wounds are still lacking. It is urgently needed to further explore the underlying molecular mechanism of physiological and pathological wound healing.
LOC105372576, which was also termed wound and keratinocyte migration-associated lncRNA 1 (WAKMAR1), was a nuclear-localized, critical pro-migratory lncRNA in human wound-edge keratinocytes in vitro and human wounds ex vivo. 60 Mechanistically, it exerted its pro-migratory functions through activation of E2F1 (E2F transcription factor 1) expression by sequestering DNMTs (DNA methyltransferases) and inhibiting methylation of E2F1 promoter.
These findings identify WAKMAR1 as a DNMT-associated lncRNA in promoting keratinocyte motility and re-epithelialization, providing human-specific mechanistic insights into skin wound healing.
lncRNA H19, which was increased in diabetic mouse when the whole blood was preserved by modified preservative fluid, promoted mice fibroblast activation and proliferation to improve wound healing. 61 Mechanically, H19 could bind to HIF-1α (hypoxia-inducible factor 1 subunit alpha) gene promoter region and increase its expression by recruiting EZH2-mediated histone methylation.
Antisense non-coding RNA in the INK4 locus (ANRIL), a 2659 bp nuclear and perinuclear cytoplasmic antisense lncRNA, was downregulated in the diabetic wound healing mouse model and high glucose-induced human lymphatic endothelial cells. 62 Further functional researches indicated that ANRIL could promote lymphangiogenesis during the diabetic wound healing process via sponging miR-181a to enhance Prox1 (Prospero homeobox 1) expression.
Together, these results indicated that lncRNAs play an essential functional role in human skin wound healing. Moreover, they also exerted encouraging effects on accelerating the impaired wound healing process in diabetes.

| THE CRITI C AL ROLE OF LN CRNA S IN CUTA N EO US PRO LI FE R ATI O N -R E L ATE D DISE A SE S
Recently, dysfunctional lncRNAs, which result in aberrant keratinocyte differentiation and disturbances of epidermal homeostasis, have also been implicated in the pathogenesis of several hyperproliferative skin diseases, such as cutaneous squamous cancer, melanoma, psoriasis, hypertrophic scar and haemangioma ( Figure 4).

| LncRNAs in cutaneous squamous cell carcinoma
Cutaneous squamous cell carcinoma (cSCC) is a malignant neoplasm of the skin characterized by an aberrant proliferation of keratinocytes. It is the second-most common metastatic skin cancer, with a worldwide increasing incidence. Understanding the potential pathology of cSCC will aid in development of effective treatments for cSCC.
To understand the role of lncRNAs in cSCC, Schapoor Hessam and his team performed a human-related lncRNA microarray and identified 1516 significantly upregulated and 2586 downregulated lncRNAs in cSCC biopsies comparing with non-lesional epithelial skin. 63 These results can serve as a template for further, larger functional, in-depth analyses regarding cSCC-associated lncRNAs.
In addition, TINCR, which was identified as an epidermal differentiation-related lncRNA, was downregulated in human squamous cell carcinoma specimens. 48 A new study from Minna Piipponen provided evidence that LINC00162, which was also named p38- downregulation of α2β1 and α5β1 integrin. 64,65 In summary, these findings improve the current knowledge that lncRNAs might serve as important mechanistic drivers in cSCC. Beyond this, these works also show significant potentials for

| LncRNAs in melanoma
Melanoma is the most lethal cutaneous cancer, with rapid progression and high metastasis potential and recurrence around the world.
It is very urgent to develop novel therapeutic strategy by figuring out the underlying pathogenesis of melanoma. Recently, thanks to the advanced developments of biological technology, several lncRNAs have been identified to play vital roles in melanomagenesis (Table 2). researchers showed that the brain-specific homeobox protein 3a (Brn3a) and the androgen receptor (AR) bound within and adjacent to SLNCR1's conserved region, respectively. SLNCR1, AR and Brn3a were specifically required for transcriptional activation of matrix metalloproteinase 9 (MMP9) and increased melanoma invasion. 70 These observations directly link AR to melanoma invasion, possibly explaining why males experience more melanoma metastases and have an overall lower survival in comparison with females.
HOX transcript antisense RNA (HOTAIR), a 2364 bp nuclear antisense lncRNA, was overexpressed in primary melanoma and matched lymph node metastatic tissues. 71 Interestingly, HOTAIR was also detected in the serum of selected metastatic patients. 72 Mechanically, HOTAIR promoted the proliferation, invasion and mi- assays revealed that PVT1 acted as a carcinogenic lncRNA by sponging tumour suppressor miR-26b. 80 Taken together, these studies indicate that dysfunctional ln-cRNAs have oncogenic effects on the pathology of melanoma.

| LncRNAs in psoriasis
Psoriasis is a multifactorial, hyperproliferative, chronic inflammatory skin disease that affects 1%-3% of the world's population.
Gene expression changes contribute to abnormal proliferation and differentiation of basal keratinocytes in psoriasis lesions. In addition to genes encoding proteins with characterized functions, emerging evidence indicates that lncRNAs also play a vital role in psoriasis.
At first, psoriasis susceptibility-related RNA gene induced by stress (PRINS) was found to be overexpressed in the uninvolved epi- nous sponge directly binding to miR-6731-5p and activating S100A7.
We speculate that the biological network of MSX2P1-miR-6731-5p-S100A7 might be a potential novel therapeutic target for the future treatment of psoriasis. 87 In summary, these sequencing results show the differential expression profiles of lncRNAs between healthy and psoriatic skin.
Moreover, functional studies indicate that lncRNAs are important contributors to key processes in psoriasis. Furthermore, these results also provide a novel basis for the development of diagnostic and treatment options for patients with psoriasis.

| LncRNAs in hypertrophic scar
Hypertrophic scar (HS), a pathological response to skin wound healing, is characterized by the invasive growth of fibroblasts and the excessive deposition of collagen. 88,89 It is a common and inevitable outcome of deep skin trauma or severe burn injury.
The overall incidence of hypertrophic scars for skin trauma is 40%-70%, whereas the incidence of burn scars is as high as 80%.

| LncRNAs in haemangioma
Haemangioma (HA), which is the most common benign vascular neoplasm of premature infants and infants with low birthweight, is resulted from abnormal proliferation of endothelial cells. 93 In summary, these studies indicate that dysfunctional lncRNAs might be vital contributors to the pathology of haemangioma.

| Biomarkers for the diagnosis and prognosis of skin diseases
The tissue-and disease-specific expression of lncRNAs makes them ideal biomarkers for diagnosis. For example, PICSAR is proved to be specifically expressed by tumour cells in actinic keratosis (UV-induced premalignant lesions), cSCC in situ and invasive cSCCs tissues but not by keratinocytes in normal skin in vivo, suggesting PICSAR as a specific biomarker for early diagnose of primary and invasive cSCC. 65 Besides their potential diagnostic implications, lncRNAs could also be used as molecular marker to predict prognosis of cutaneous disorders. Dysfunctional lncRNAs, such as FALEC, HEIH, SLNCR1 and HOTAIR, predict poor outcome in melanoma patients. 68,70,73,74 Additionally, HOTAIR can also be identified in the serum. The identification of lncRNAs in the blood might suggest its use as a non-invasive circulating marker for diagnosing cutaneous diseases or as a marker of relapses during the follow-up. 72 Moreover, lncRNAs can also act as biomarkers to predict the pathological stages of benign skin diseases. For example, the high level of lncRNA CASC9, SNHG16 and NEAT1 in haemangioma tissues predicts that haemangioma is on the proliferating phase rather than involuting phase. As the Liao's findings, the differential lncRNAs can also be used as biomarkers to monitor and predict the therapeutic responses to the treatment with adalimumab. 84 Overall, these results indicate that lncRNAs could serve as particularly useful biomarkers for diagnosis and prognosis of cutaneous disorders (Table 3). LNA gapmeR ASO has recently been indicated to trigger anti-multiple myeloma activities, providing proof of concept that the therapeutic potential of targeting lncRNAs. 109 Thus, targeting lncRNAs will be a promising therapeutic strategy for skin diseases.

| CON CLUS I ON S AND FUTURE PER S PEC TIVE
Cutaneous biology and skin disease-related lncRNAs are still an There are also some challenges of using lncRNAs as biomarkers. For instance, the amount of lncRNAs in plasma is generally low.
Therefore, unlike short non-coding RNAs, such as miRNAs, most ln-cRNAs are not detectable in plasma by standard methods, such as microarrays or quantitative PCR. More studies are required to examine whether lncRNAs are better predictive biomarkers than proteincoding genes or other non-coding RNAs (such as miRNAs).
Additionally, the full potential of using lncRNAs in skin disease therapy has not yet been fully explored now. Better understanding of precise biological functions of lncRNAs and better targeting technologies are required, which will advance the lncRNA therapy.
In future, more and more clinical trials targeting various lncRNAs are ongoing for the treatment with cutaneous diseases.

We appreciate Professor Chuanjian Lu, Professor Boudewijn
Burgering and Runyue Huang for their assistance in this article modification. This study was supported by the National Natural Science

CO N FLI C T O F I NTE R E S T
The authors have declared no conflicting interests.

AUTH O R CO NTR I B UTI O N S
LT, YL and HX wrote the manuscript; YL, XY and HX searched the literature; LT and GZ edited the paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.