lncRNAs and miRNAs : key regulators in the pathogenesis of cSCC, senescence, and psoriasis

<p dir="ltr">The skin is the body's largest and outermost organ, serving as a dynamic interface between the internal environment and external stimuli. It plays a vital role in maintaining physiological balance by acting as a barrier against physical injury, pathogens, and environmental stressors. In addition to its protective functions, the skin contributes to pigmentation through melanin production and manages hydration via its barrier properties. Structurally, the skin consists of three layers: the epidermis, dermis, and hypodermis. Maintaining a delicate balance between keratinocyte growth and differentiation is essential for preserving epidermal integrity and function. This balance is regulated by a complex network of signalling pathways, transcription factors, epigenetic regulators, and non-coding RNAs. Disruption of these regulatory mechanisms can disturb skin homeostasis, leading to various pathological conditions. These include malignant diseases such as cutaneous squamous cell carcinoma (cSCC), benign inflammatory disorders like psoriasis, and contributions to aging and cellular senescence. Understanding the molecular frameworks that maintain skin homeostasis is crucial for developing targeted treatments for skin diseases.</p><p dir="ltr">In this thesis, I have examined the role of non-coding RNAs as key regulators in the development of cSCC, psoriasis, and cellular senescence.</p><p dir="ltr">Paper I: In this study, we investigated the role of miR-23b in cSCC and identified this microRNA as a tumour suppressor in the context of this disease. We demonstrated that miR-23b expression is downregulated in both cSCC and actinic keratosis (AK). Furthermore, we demonstrated that the MAPK signalling pathway regulates the expression of miR-23b, a key pathway in cSCC disease progression. In addition, we show that miR-23b suppresses the expression of a gene network associated with key oncogenic pathways, and its gene signature is enriched in human cSCCs. We observed that overexpression of miR-23b suppressed the ability of cSCC cells to form colonies and tumor spheroids. Additionally, we demonstrate that CRISPR/Cas9- mediated deletion of MIR23B leads to increased colony and tumor sphere formation in vitro. In vivo, we observed that miR-23b-overexpressing cSCC cells formed significantly smaller tumours upon injection into immunocompromised mice, with decreased cell proliferation and angiogenesis. Mechanistically, we verified Ras-related protein RRAS2 as a direct target of miR-23b in cSCC. We demonstrated that RRAS2 is overexpressed in cSCC and that interference with its expression impairs angiogenesis, as well as colony and tumor spheroid formation. These findings suggest that miR-23b acts as a tumor suppressor in cSCC, and its expression is decreased during the initiation and progression of cSCC.</p><p dir="ltr">Paper II: In this study, we identified a long non-coding RNA, CYDAER (RP11-295G20.2), which is significantly upregulated in keratinocytes from psoriatic skin. We demonstrate that CYDAER is mainly expressed in the suprabasal layers of the epidermis and is induced by the psoriasis-associated cytokine IL-17A. Functional studies show that CYDAER suppresses keratinocyte terminal differentiation, suggesting that its overexpression contributes to the impaired differentiation characteristic of psoriasis. These findings highlight CYDAER as a potential regulator of epidermal dysfunction in psoriasis.</p><p dir="ltr">Paper III: In this study, we show that the expression of a skin aging-associated microRNA (miR-383) increases during replicative senescence and stress-induced senescence of fibroblasts. Moreover, we demonstrate that overexpression of miR-383 induces a senescence-like phenotype in primary human fibroblasts, indicated by increased senescence-associated ß-galactosidase (SA-B-Gal) activity, altered expression of senescence markers, characteristic morphological changes, and reduced proliferative capacity. Conversely, we found that CRISPR-Cas9-mediated knockout of MIR383 delays the onset of the senescence-like phenotype and extends the replicative capacity of dermal fibroblasts. Transcriptome analysis of fibroblasts overexpressing miR-383 revealed that high levels of miR-383 decrease the expression of genes involved in repairing double-stranded DNA breaks. Furthermore, overexpression of miR-383 also leads to increased DNA damage, a hallmark of aging and senescence, in fibroblasts. Mechanistically, we demonstrate that miR-383 regulates the DNA damage response by directly targeting PCNA clamp-associated factor (PCLAF/KIAA0101), a key DNA repair regulator, resulting in increased DNA damage, enhanced nuclear localization of p21, and a senescence-like phenotype.</p><p dir="ltr">These findings identify miR-383 as a key regulator of the DNA damage response and a promoter of dermal fibroblast senescence, thereby enhancing our understanding of the molecular mechanisms that influence skin aging process.</p><p dir="ltr">Paper IV: In this study, we identified LINC01605 as an upregulated long non-coding RNA in cSCC compared to healthy skin. We demonstrate that LINC01605 expression is positively regulated by TGF-B, a key inducer of epithelial-to-mesenchymal transition (EMT) that is involved in cSCC progression. Functional studies showed that both siRNA-mediated knockdown and CRISPR-Cas9 knockout of LINC01605 in cSCC cell lines impair cell growth, colony formation, migration, and tumor spheroid formation. Loss of LINC01605 reduces metabolic activity, slows cell proliferation, increases epithelial cohesion, and suppresses EMT. RNA sequencing of LINC01605-knockout cells reveals downregulation of genes involved in cell-matrix adhesion, emphasizing its role in tumor cell adhesion dynamics. In vivo zebrafish xenograft models confirm increased cell aggregation after LINC01605 loss.</p><p dir="ltr">Together, these results suggest that elevated LINC01605 expression promotes cSCC progression by enhancing the plasticity of cancer cell proliferation, migration, and adhesion.</p><h3>List of scientific papers</h3><p dir="ltr">I. MicroRNA-23b Plays a Tumor-Suppressive Role in Cutaneous Squamous Cell Carcinoma and Targets Ras-Related Protein RRAS2. Chengxi Sun, KunalDas Mahapatra, <b>Jonathan Elton</b>, Chen Li, Winnie Fernando, Warangkana Lohcharoenkal, Jan Lapins, Bernhard Homey, Enikö Sonkoly, Andor Pivarcsi. Journal of Investigative Dermatology, 2023 Dec 1;143(12):2386-2396. <a href="https://doi.org/10.1016/j.jid.2023.05.026" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.jid.2023.05.026</a></p><p dir="ltr">II. RNA Sequencing Reveals the Long Non-Coding RNA Signature in Psoriasis Keratinocytes and Identifies CYDAER as a Long Non-Coding RNA Regulating Epidermal Differentiation. Jan Cedric Freisenhausen, Longlong Luo, Evelyn Kelemen, <b>Jonathan Elton</b>, Viktor Skoog, Andor Pivarcsi, Eniko Sonkoly. Experimental Dermatology, 2025 Feb;34(2):e70054. <a href="https://doi.org/10.1111/exd.70054" rel="noreferrer" target="_blank">https://doi.org/10.1111/exd.70054</a></p><p dir="ltr">III. miR-383, a skin ageing-associated microRNA, includes fibroblast senescence through targeting PCNA clamp-associated factor (PCLAF) and regulating DNA damage repair. Chen Li, <b>Jonathan Elton</b>, Ankit Srivastava, Claire Marionnet, Françoise Bernerd, Enikö Sonkoly, Andor Pivarcsi. [Manuscript]</p><p dir="ltr">IV. Investigation of the role of LINC01605 in Cutaneous Squamous Cell Carcinoma. <b>Jonathan Elton</b>, Zi Xin Ong, Finn Glockner, Longlong Luo, Eniko Sonkoly, and Andor Pivarcsi. [Manuscript]</p>