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, 12 (1), 174

High-throughput RNA Sequencing From Paired Lesional- And Non-Lesional Skin Reveals Major Alterations in the Psoriasis circRNAome

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High-throughput RNA Sequencing From Paired Lesional- And Non-Lesional Skin Reveals Major Alterations in the Psoriasis circRNAome

Liviu-Ionut Moldovan et al. BMC Med Genomics.

Abstract

Background: Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes. It is one of the most prevalent chronic inflammatory skin conditions in adults worldwide, with a considerable negative impact on quality of life. Circular RNAs (circRNAs) are a recently identified type of non-coding RNA with diverse cellular functions related to their exceptional stability. In particular, some circRNAs can bind and regulate microRNAs (miRNAs), a group of RNAs that play a role in the pathogenesis of psoriasis. The aim of this study was to characterize the circRNAome in psoriasis and to assess potential correlations to miRNA expression patterns.

Methods: We used high-throughput RNA-sequencing (RNA-seq), NanoString nCounter technology and RNA chromogenic in situ hybridization (CISH) to profile the circRNA expression in paired lesional and non-lesional psoriatic skin from patients with psoriasis vulgaris. In addition, 799 miRNAs were profiled using NanoString nCounter technology and laser capture microdissection was used to study the dermis and epidermis separately.

Results: We found a substantial down-regulation of circRNA expression in lesional skin compared to non-lesional skin. We observed that this mainly applies to the epidermis by analyzing laser capture microdissected tissues. We also found that the majority of the circRNAs were downregulated independently of their corresponding linear host genes. The observed downregulation of circRNAs in psoriasis was neither due to altered expression levels of factors known to affect circRNA biogenesis, nor because lesional skin contained an increased number of inflammatory cells such as lymphocytes. Finally, we observed that the overall differences in available miRNA binding sites on the circRNAs between lesional and non-lesional skin did not correlate with differences in miRNA expression patterns.

Conclusions: We have performed the first genome-wide circRNA profiling of paired lesional and non-lesional skin from patients with psoriasis and revealed that circRNAs are much less abundant in the lesional samples. Whether this is a cause or a consequence of the disease remains to be revealed, however, we found no evidence that the loss of miRNA binding sites on the circRNAs could explain differences in miRNA expression between lesional and non-lesional skin.

Keywords: Genome-wide profiling; Inflammatory diseases; Non-coding RNA; Psoriasis; circRNA; microRNA.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Characterization of circular RNAs in lesional- and non-lesional skin using RNA-seq. (a-b) Expression levels of the top 50 most abundant circRNAs in non-lesional skin (a) and lesional skin (b). Each dot represents the expression in reads per million (RPM) for a circRNA in one individual sample represented by a unique color. Each line represents the mean RPM. Black arrows indicate top 10 alpha circRNAs and red arrows indicate circRNAs not present in circBase. (c-d) Histograms showing the number of host genes producing various numbers of unique high abundance circRNAs in non-lesional skin (c) and in lesional skin (d). (e-f) Pie charts showing the distribution of the numbers of exons annotated within the high abundance circRNAs in non-lesional skin (e) and lesional skin (f)
Fig. 2
Fig. 2
The circRNAome is massively downregulated in lesional- relative to non-lesional skin. (a) Venn diagram illustrating the overlap of circRNAs detected in the lesional- and the non-lesional skin. (b) Scatter plot showing that the average expression in reads per million (RPM) of the 298 unique high-abundance circRNAs is lower in lesional- relative to non-lesional skin. (c) Volcano plot of the 298 unique high-abundance circRNAs showing fold changes in circRNA expression in RPM between lesional- and non-lesional skin according to the levels of significance. The top 50 most abundant circRNAs are indicated in green. (d) Scatter plot of fold changes in RPM and fold changes in circular-to-linear (CTL) ratios of the high abundance circRNAs. It can be observed that most circRNAs were downregulated independently of their respective host genes (defined as those being present in between the blue lines)
Fig. 3
Fig. 3
Analyses of factors known to regulate circRNA biogenesis in lesional- relative to non-lesional skin. (a) RNA-seq analyses revealed that ADAR was the most differentially expressed of the analyzed factors. (b) NanoString nCounter analysis confirmed that ADAR is upregulated in lesional- relative to non-lesional skin. (c) Volcano plot of the 298 unique high-abundance circRNAs showing fold changes in circRNA expression in RPM between lesional- and non-lesional skin according to the levels of significance. One-hundred thirty-one circRNAs likely to have Alu-mediated biogenesis are indicated in green (flanked by IAEs within 2300 nucleotide regions flanking the BSJs). The numbers of circRNAs in each category are shown in the inserted table. There was no statistically significant association between downregulation of circRNAs (more than 2 fold) and the presence of flanking IAEs (P = 0.75, chi-squared test)
Fig. 4
Fig. 4
The downregulation of circRNAs, mainly observed in the epidermis, is unlikely to be caused by infiltrating lymphocytes. (a) Representative H&E staining of lesional- and non-lesional skin samples showing a relative abundance of lymphocytes in the dermis of the lesional skin. (b) RNA-seq data showed that T-cell specific genes were expressed at higher levels in the lesional skin- relative to non-lesional skin. (c-d) Following microdissection of the epidermis and NanoString nCounter analysis, five of seven circRNAs were shown to be significantly downregulated in the epidermis of the lesional skin (c), while this only applied to one of seven circRNAs analyzed in the dermis (d). (e-f) ADAR proved to be significantly upregulated both in the epidermis (e) and the dermis (f) of the lesional skin
Fig. 5
Fig. 5
RNA chromogenic in situ hybridization (CISH) for ciRS-7 in lesional- and non-lesional skin. (a-b) Overviews, with the areas shown in (c and d) indicated with a square. The ciRS-7 signal (red dots) is observed mainly in the epidermis of the non-lesional skin (a and c) and in the dermis of lesional skin (b and d). Scale bars are indicated in the lower-left corner
Fig. 6
Fig. 6
Characterization of miRNAs in lesional skin- and non-lesional skin using NanoString nCounter analysis. (a) Venn diagram illustrating the overlap between the miRNAs detected in lesional- and non-lesional skin. (b) The average expression of the 137 high abundance miRNAs is slightly lower in lesional- relative to non-lesional skin. (c) Volcano plot of the 137 high abundance miRNAs, with the top 50 most abundant miRNAs indicated in green along with the names of miRNAs previously shown to be differentially expressed in psoriasis
Fig. 7
Fig. 7
Changes in the amounts of available miRNA binding sites present on circRNAs do not correlate with changes in miRNA expression levels in lesional- relative to non-lesional skin. (a-b) For each miRNA, the sum of the number of binding sites for that particular miRNA was multiplied with the mean circRNA RPM (lesional) - mean circRNA RPM (non-lesional) for the circRNAs harboring binding sites for that particular miRNA. These values were plotted against either the fold change (mean miRNA counts (lesional)/mean miRNA counts (non-lesional)) for each high abundance miRNA with at least one binding site present on the high abundance circRNAs (a) or the absolute difference in expression level (mean miRNA counts (lesional) - mean miRNA counts (non-lesional)) for each high abundance miRNA with at least one binding site present on the high abundance circRNAs (b)

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