Abstract

   Sensitive skin (SS) is a condition of subjective cutaneous
   hyper-reactivity. The role of long non-coding RNAs (lncRNAs) in
   subjects with SS is unclear. Therefore, the aim of the present study
   was to provide a comprehensive profile of the mRNAs and lncRNAs in
   subjects with SS. Gene Ontology (GO) and Kyoto Encyclopedia of Genes
   and Genomes (KEGG) analysis presented the characteristics of associated
   protein-coding genes. In addition, a co-expression network of lncRNA
   and mRNA was constructed to identify potential underlying regulation
   targets; the results were verified by quantitative real-time PCR
   (qRT-PCR) and RNA-seq analyses in patients with SS and normal samples.
   Compared with the normal skin group, 266 novel lncRNAs and 6750
   annotated lncRNAs were identified in the SS group. A total of 71 lncRNA
   transcripts and 2615 mRNA transcripts were differentially expressed (P
   < 0.05). The heat signature of the SS samples could be distinguished
   from the normal skin samples, whereas the majority of the genes that
   were present in enriched pathways were those that participated in focal
   adhesion, PI3K-Akt signaling, and cancer-related pathways. Five
   transcripts were selected for qRT-PCR analysis and the results were
   consistent with RNA-seq. The results suggested that LNC_000265 may play
   a role in the epidermal barrier structure of patient with SS. The data
   suggest novel genes and pathways that may be involved in the
   pathogenesis of SS and highlight potential targets that could be used
   for individualized treatment applications.

   Keywords: sensitive skin, lncRNA, mRNA, RNA sequencing

INTRODUCTION

   Sensitive skin (SS) is a broad term used to describe a multitude of
   clinical findings that are attributed to different sensory perceptions,
   namely facial irritation, burning, stinging, tightness, tingling, pain,
   and pruritus [[38]1]. The sensitive skin syndrome (SSS) is considered
   as a state of hyperactivity to specific environmental stimuli that is
   caused from a single and/or a number of underlying pathologies [[39]2].
   The main disadvantage encountered during the diagnosis of the disease
   is the lack of an objective-screening test [[40]3]. The complex nature
   of the skin disease syndrome requires the use of a diagnostic algorithm
   and the need to test patients with multiple patch testing, prior to the
   establishment of a definite diagnosis, as it has been shown from the
   lack of association between different allergens in subjects with
   positive allergic reactions with regard to each allergen alone (SDS
   and/or lactic acid) [[41]3, [42]4]. Nevertheless, several studies have
   suggested a link between SS and disruption of the epidermal barrier
   function, resulting in the perception of skin discomfort [[43]5,
   [44]6]. Despite these promising findings, the molecular network that
   contributes to the development of SS has not been elucidated to date.

   Long non-coding RNAs (lncRNAs) are a class of RNA sequences that are
   more than 200 nt in length and are involved in the regulation of
   translation process, although they do not possess protein coding
   potential [[45]7]. A multitude of studies have shown that lncRNAs are
   involved in the regulation of developmental processes and in the
   progression of several human diseases [[46]8–[47]11], while their
   expression and localization varies among different cell types and
   subcellular compartments [[48]12–[49]17]. LncRNAs have been found
   crucial to genomic imprinting, dosage compensation, and
   pluripotency-regulation [[50]18, [51]19]. The rapid progress of RNA
   sequencing (RNA-seq) promoted the exploration and research of
   non-coding RNAs, and novel lncRNAs have been identified by different
   pipelines using RNA-seq data [[52]20, [53]21]. RNA-seq exhibits several
   advantages compared to the previously established methodologies that
   have been used for the evaluation of the complete set of transcripts in
   the cell, such as hybridization-based approaches and specialized
   microarrays. RNA-Seq has been successfully used to provide a ‘digital
   measurement’ of the gene expression difference between different
   tissues [[54]22]. Although no reports on the contribution of lncRNA in
   the development of SS have been reported, it has been suggested that
   these RNA sequences play a significant role in skin homeostasis and
   related skin diseases [[55]23, [56]24]. Several studies demonstrated
   the involvement of lncRNAs in the differentiation and maintenance of
   normal human keratinocytes and epidermal tissues [[57]25, [58]26]. For
   example, lncRNA such as anti-differentiation non-coding RNA (ANCR) and
   terminal differentiation-induced non-coding RNA (TINCR) are vital for
   epidermal stability [[59]27, [60]28]. Sonkoly et al. identified a novel
   lncRNA, namely psoriasis susceptibility-related RNA gene induced by
   stress (PRINS) that is involved in the susceptibility to psoriasis
   [[61]29]. The authors suggested that PRINS may play an important role
   in psoriasis by evidence derived from psoriasis patients and in vitro
   cell culture experiments [[62]29]. The use of bioinformatics methods
   has been adopted in the investigation of the genes involved in the
   development of atopic dermatitis and psoriasis. Notably, differentially
   expressed genes (DEGs) were associated with epidermis development and
   immune response in atopic dermatitis [[63]30]. Similarly, enrichment
   analysis of psoriasis- correlated modules revealed that pathways
   involved in short chain fatty acid metabolism, olfactory signaling, and
   regulation of leukocyte-mediated cytotoxicity were the main pathways in
   which the DEGs were identified [[64]31]. Of note is that more than 50%
   of the co-expressed genes in 18 psoriasis patients and 16 healthy
   controls were lncRNAs [[65]31].

   In view of the significant roles of lncRNA in the regulation and the
   differentiation of epidermal homeostasis and the disruption of the
   epidermal barrier function in SS, we hypothesized that lncRNAs may also
   take part in the pathogenesis of SS. The aim of the present study was
   to provide a more comprehensive and validated conclusion regarding the
   identification of differentially expressed lncRNAs in SS tissues.

RESULTS

Overview of RNA-Seq and mRNAs and lncRNAs identification

   The RefSeq (Build 37.3) and the GENCODE v19 databases were selected as
   the annotation references for mRNA and lncRNA analyses, respectively.