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. 2020 Dec 5;21(23):9288.
doi: 10.3390/ijms21239288.

miR-155 Contributes to Normal Keratinocyte Differentiation and Is Upregulated in the Epidermis of Psoriatic Skin Lesions

Lucian Beer  1 , Polina Kalinina  2 , Martin Köcher  2 , Maria Laggner  3   4 , Markus Jeitler  5 , Salman Abbas Zadeh  2 , Dragan Copic  3   4 , Erwin Tschachler  2 , Michael Mildner  2
Affiliations

miR-155 Contributes to Normal Keratinocyte Differentiation and Is Upregulated in the Epidermis of Psoriatic Skin Lesions

Lucian Beer et al. Int J Mol Sci. .

Abstract

The role of microRNAs (miRNAs) during keratinocyte (KC) differentiation and in skin diseases with epidermal phenotypes has attracted strong interest over the past few years. However, combined mRNA and miRNA expression analyses to elucidate the intricate mRNA-miRNA networks of KCs at different stages of differentiation have not been performed yet. In the present study, we investigated the dynamics of miRNA and mRNA expression during KC differentiation in vitro and in normal and psoriatic epidermis. While we identified comparable numbers of up- and downregulated mRNAs (49% and 51%, respectively), miRNAs were predominantly upregulated (76% vs 24%) during KC differentiation. Further bioinformatics analyses suggested an important inhibitory role for miR-155 in KC differentiation, as it was repressed during KC differentiation in normal skin but strongly upregulated in the epidermis of psoriatic skin lesions. Mimicking the inflammatory milieu of psoriatic skin in vitro, we could show that the pro-inflammatory cytokines IL17, IL1β and INFγ synergistically upregulated miR-155 expression in KCs. Forced over-expression of miR-155 in human in vitro skin models specifically reduced the expression of loricrin (LOR) in KCs, indicating that miR-155 interferes with the establishment of a normal epidermal barrier. Together, our data indicate that downregulation of miR-155 during KC differentiation is a crucial step for epidermal barrier formation. Furthermore, its strong upregulation in psoriatic lesions suggests a contributing role of miR-155 in the altered keratinocyte differentiation observed in psoriasis. Therefore, miR-155 represents as a potential target for treating psoriatic skin lesions.

Keywords: epidermal keratinocytes; epidermis; keratinocyte differentiation; mRNA; miRNA; psoriasis; skin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Dynamic regulation of mRNA expression during keratinocyte differentiation. (A) Samples are displayed based on their mRNA expression with respect to the first two components and are clustered according to their differentiation state (P proliferating monolayer; D, differentiated monolayer; SE, skin equivalent). All three conditions can be separated from each other. (B) Venn diagrams showing the overlap of up- and downregulated miRNAs with changes in expression between the three conditions. The area of the circles corresponds to the number of differentially expressed miRNAs. The greatest alterations were observed in mRNA expression between P keratinocytes and SE. (C) One cluster (green) characterizes mRNAs repressed during differentiation, (D) one cluster (violet) shows mRNAs upregulated during keratinocyte differentiation in monolayer and SE and (E) one cluster (red) shows mRNAs exclusively upregulated in SE. KEGG pathway analysis shows mRNAs of the first cluster are significantly associated with the cell cycle, while genes of the second and third clusters are associated with the epidermal differentiation and cell adhesion, respectively. ECM, extracellular matrix. n = 3.
Figure 2
Figure 2
Dynamic regulation of miRNA expression during keratinocyte differentiation. (A) Samples are displayed with respect to the first two components and are clustered according to their differentiation state (P proliferating monolayer; D, differentiated monolayer; SE, skin equivalent). P cells can be clearly separated from D and SE indicating differences in global miRNA expression patterns. Differentiated keratinocytes cultures in monolayers and in SE cannot be clearly separated in the PCA plot. (B) Venn diagrams showing the overlap of up- and downregulated miRNAs with significant changes in expression between the three conditions. The area of the circles corresponds to the number of differentially expressed miRNAs. The greatest alterations were observed in miRNA expression between P keratinocytes and SE. (C) One cluster (green) characterizes miRNAs repressed during differentiation, (D) one cluster (violet) shows miRNAs upregulated during keratinocyte differentiation in monolayer and SE and (E) one cluster (red) shows miRNAs exclusively upregulated in SE. KEGG pathway analysis shows that miRNAs targeted mRNAs of cluster are significantly associated with cell cycle, while genes of the second and third clusters are associated with the cytoskeleton and cell adhesion, respectively. n = 3.
Figure 3
Figure 3
Top 10 up- and downregulated miRNAs during epidermal KC differentiation. The heatmap displays the top 10 up- and downregulated (upregulated red; downregulated blue) miRNAs differentially regulated between the three conditions. P, proliferating; D, differentiated; SE, skin equivalent. n =3.
Figure 4
Figure 4
miRNA–mRNA interaction during epidermal differentiation. The interaction network displays the top 10 downregulated miRNAs during epidermal differentiation and targeted upregulated mRNAs. miR-155 encounters a central role in the network with 47 target genes.
Figure 5
Figure 5
miR-155 overexpression represses loricrin expression. To study the effect of miR-155 on epidermal differentiation, we performed overexpression of miR-155 in skin equivalents (SE). While miR-155 overexpressing SE showed a normal morphological structure in H&E staining (A), we found (B) that the expression of loricrin (LOR) (white arrow head) was strongly reduced compared to control samples. There were no changes in the (C) KRT5 and (D) KRT10 expression. (E) Biotin permeability assay shows that miR-155 over-expression did not disturb the inside-out epidermal permeability barrier. Isotype control is shown in (F). The dashed line represents the border between the stratum granulosum and stratum corneum. Scale bar = 40 µm. n = 3.
Figure 6
Figure 6
miRNA and mRNA alterations in psoriasis. (A) Samples are displayed based on their mRNA expression with respect to the first two components (L lesional skin; H healthy skin). The two conditions are clearly separated from each other. (B) The heatmap displays 141 (upregulated red; downregulated blue) significantly regulated miRNAs. (C) Target genes of the upregulated miRNAs in psoriasis lesions are associated with the cell cycle process and epithelial cell differentiation. (D) mRNA expression characteristics of the same samples are displayed in the PCA. (E) The heatmap shows 1987 (upregulated red; downregulated blue) differentially expressed mRNAs. (F) Upregulated genes in psoriasis skin are associated with the cell cycle processes. n = 3 for (A–C); n = 4 for (D–F).
Figure 7
Figure 7
miR-155 is induced by inflammatory cytokines and TLR3 activation. (A) miR-155 expression is upregulated in psoriasis lesions (P) compared to normal skin (NS) based on our PCR analysis; n =4. (B) Validation data from the Genevestigator platform using expression values from five independent studies. (C) PCR analysis reveals significant downregulation of loricrin in psoriasis lesions (P) compared to normal skin (NS); n =3. (D) Stimulation of TLR3 induced miR-155 expression in vitro, while other TLRs did not affect miR-155 expression; n = 3. (E) The combination of IL17 and INFγ as well as IL17, INFγ and IL1β induced miR-155 expression in proliferating keratinocytes, while the individual components alone did not alter miR-155 expression; n = 3. (F) The interaction network displays miRNA-155, which is one of the top 10 upregulated miRNAs in psoriasis lesions, and their 26 downregulated, targeted mRNAs. * p < 0.05; ** p < 0.01; *** p < 0.001.
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