Korean J Physiol Pharmacol.  2019 Mar;23(2):151-159. 10.4196/kjpp.2019.23.2.151.

Prediction of itching diagnostic marker through RNA sequencing of contact hypersensitivity and skin scratching stimulation mice models

Affiliations
  • 1Department of Physiology, Chung-Ang University College of Medicine, Seoul 06974, Korea. haena@cau.ac.kr, akdongyi01@cau.ac.kr
  • 2Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV 89557, USA.
  • 3Department of Medicine, Chung-Ang University College of Medicine, Seoul 06974, Korea.

Abstract

Pruritus (itching) is classically defined as an unpleasant cutaneous sensation that leads to scratching behavior. Although the scientific criteria of classification for pruritic diseases are not clear, it can be divided as acute or chronic by duration of symptoms. In this study, we investigated whether skin injury caused by chemical (contact hypersensitivity, CHS) or physical (skin-scratching stimulation, SSS) stimuli causes initial pruritus and analyzed gene expression profiles systemically to determine how changes in skin gene expression in the affected area are related to itching. In both CHS and SSS, we ranked the Gene Ontology Biological Process terms that are generally associated with changes. The factors associated with upregulation were keratinization, inflammatory response and neutrophil chemotaxis. The Kyoto Encyclopedia of Genes and Genomes pathway shows the difference of immune system, cell growth and death, signaling molecules and interactions, and signal transduction pathways. Il1a , Il1b and Il22 were upregulated in the CHS, and Tnf, Tnfrsf1b, Il1b, Il1r1 and Il6 were upregulated in the SSS. Trpc1 channel genes were observed in representative itching-related candidate genes. By comparing and analyzing RNA-sequencing data obtained from the skin tissue of each animal model in these characteristic stages, it is possible to find useful diagnostic markers for the treatment of itching, to diagnose itching causes and to apply customized treatment.

Keyword

Cytokines; Pruritus; RNA sequence analysis; Transient receptor potential channels; Wound healing

MeSH Terms

Animals
Biological Processes
Chemotaxis
Classification
Cytokines
Dermatitis, Contact*
Gene Expression
Gene Ontology
Genome
Hypersensitivity
Immune System
Interleukin-6
Mice*
Models, Animal
Neutrophils
Pruritus*
RNA*
Sensation
Sequence Analysis, RNA*
Signal Transduction
Skin*
Transcriptome
Transient Receptor Potential Channels
Up-Regulation
Wound Healing
Cytokines
Interleukin-6
RNA
Transient Receptor Potential Channels

Figure

  • Fig. 1 Overview of RNA-sequencing based characterization. (A) The top 10 Gene Ontology Biological Process terms associated with the commonly upregulated genes. The p-value were computed by Fisher's exact test and corrected by Bonferroni procedure. The vertical dash line denotes the significance level of α = 0.05. (B) Heatmaps of genes differentially expressed between CHS and SSS. Each column in the heatmap denotes one mouse while each row denotes one Kyoto Encyclopedia of Genes and Genomes pathway. Red represents relatively increased gene expression while blue represents down-regulation. A t-test was used to prioritize the pathways differentially expressed between suitable control and case samples (adjusted p < 0.05). The pathway expression score was computed by the FAIME algorithm. CHS, contact hypersensitivity group; SSS, skin-scratching stimulation group; VT, vehicle control group; NT, non-treated control group.

  • Fig. 2 Bubble chart representing the relationship of the gene expression between CHS and SSS. (A) Changes in the genes associated with inflammation. (B) Changes in all of the Trp channel genes. X-axis, log2FC between NT and SSS; Y-axis, log2FC between VT and CHS. The bubble size was the sum of absolute value of log2FC for representative of the genes attributed to expression levels. CHS, contact hypersensitivity group; SSS, skin-scratching stimulation group; FC, fold change; NT, non-treated control group; VT, vehicle control group.

  • Fig. 3 Boxplot of candidate genes related pruritus. Y-axis represented the expression level by transcripts per million (TPM). Last two plots showed two genes with housekeeping profile. CHS, contact hypersensitivity group; SSS, skin-scratching stimulation group; NT, non-treated control group; VT, vehicle control group.

  • Fig. 4 Network analysis of candidate itching related genes. The differentially expressed genes (13 genes) which identified knowledge and RNA-sequencing data were uploaded. The nodes were genes and linear edges indicated molecular action for association in published data sets and line shape indicates the predicted mode of action.

  • Fig. 5 In-vivo data. (A) Changes in the body weight. The body weight was increased generally and there was no difference between each group. The final body weight was measured after fasting the day before the sacrifice. (B) Changes in scratching behavior VT and CHS shown scratching behavior. There was difference compared with before and after stimulation, statistically, in CHS (*p < 0.05). Olive oil, materials of AOO, itself may cause contact allergy. Some differences may exist between NT and VT. CHS, contact hypersensitivity group; SSS, skin-scratching stimulation group; NT, non-treated control group; VT, vehicle control group; AOO, a mixture of acetone and olive oil.


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