MicroRNA‐145‐5p regulates fibrotic features of recessive dystrophic epidermolysis bullosa skin fibroblasts

Abstract

Summary Background Recessive dystrophic epidermolysis bullosa (RDEB) is a skin fragility disorder caused by mutations in the COL7A1 gene encoding type VII collagen, a cutaneous basement membrane component essential for epidermal–dermal adhesion. Hallmarks of the disease are unremitting blistering and chronic wounds with severe inflammation and fibrosis. MicroRNAs (miRNAs) are post‐transcriptional regulators of gene expression also implicated in fibrotic processes. However, the role of miRNAs in RDEB fibrosis is almost unexplored. Objectives Our study aimed to identify miRNAs deregulated in primary RDEB skin fibroblasts (RDEBFs) and to characterize their function in RDEB fibrosis. Methods Real‐time quantitative polymerase chain reaction (qRT‐PCR) was used to screen RDEBFs for expression levels of a group of miRNAs deregulated in hypertrophic scars and keloids, pathological conditions with abnormal wound healing and fibrosis. Contractility, proliferation and migration rate were evaluated by different in vitro assays in RDEBFs transfected with a miR‐145‐5p inhibitor. Expression levels of fibrotic markers and miR‐145‐5p targets were measured using qRT‐PCR and western blot. Results The miR‐143/145 cluster was upregulated in RDEBFs compared with fibroblasts from healthy subjects. RDEBFs transfected with a miR‐145‐5p inhibitor showed attenuated fibrotic traits of contraction, proliferation and migration, accompanied by reduced expression of the contractile proteins α‐smooth muscle actin and transgelin. These effects were associated with upregulation of Krüppel‐like factor 4 transcriptional repressor and downregulation of Jagged1, a known inducer of fibrosis. Conclusions Our results highlight the profibrotic role of miR‐145‐5p and its regulatory networks in RDEB, shedding light on novel disease pathomechanisms and targets for future therapeutic approaches. What's already known about this topic? Recessive dystrophic epidermolysis bullosa (RDEB) is a highly disabling genetic skin disease caused by mutations in the collagen VII gene and characterized by unremitting blistering and defective wound healing, leading to inflammation and fibrosis. MicroRNAs (miRNAs) are post‐transcriptional regulators of gene expression in health and disease, and their deregulation has been implicated in fibrotic skin conditions. To date, only miR‐29 has been associated with injury‐driven fibrosis in RDEB. What does this study add? In patients with RDEB, miR‐145‐5p is overexpressed in RDEB skin fibroblasts (RDEBFs), where it plays a profibrotic role, as its inhibition reduces RDEBF fibrotic traits (contraction, proliferation and migration). miR‐145‐5p inhibition in RDEBFs determines the reduction of contractile markers α‐smooth muscle actin and transgelin through upregulation of Krüppel‐like factor 4, a transcriptional repressor of contractile proteins, and downregulation of Jagged1 (JAG1), an inducer of fibrosis. What is the translational message? Our findings expand the knowledge on miRNA‐driven pathomechanisms implicated in RDE...

Epidermolysis Bullosa-Associated Squamous Cell Carcinoma: From Pathogenesis to Therapeutic Perspectives

“…Collagen VII (COL7) deficiency in recessive dystrophic epidermolysis bullosa (RDEB) skin and extracutaneous tissues favors squamous cell carcinoma (SCC) development. The figure summarizes literature findings on relevant pro-tumorigenic processes triggered by COL7 loss in RDEB keratinocytes, fibroblasts and lymphoid organs [42,44,49,52,57,58,59,61,62,63,64,67,84]. Red up arrows indicate increase/up-regulation, green down arrows indicate decrease/down-regulation.…”

“…In RDEB patients, fibrosis is a regular and severe disease complication resulting from the impaired healing of chronic wounds [41]. Dermal fibroblasts in RDEB patients are continuously exposed to the detrimental effects of several pro-inflammatory cytokines and growth factors that alter the transcriptional profile [42], and force fibroblasts to remain into the “myofibroblast state”. Indeed, myofibroblasts chronically reside in the dermis of RDEB patients and contribute to ECM stiffness, an event fueling the fibrotic process in a self-renewing cycle.…”

“…Finally, microRNAs (miRNAs), a class of small non-coding RNAs with pleiotropic functions, are emerging as novel players in RDEB fibrosis [42,67], and potential regulators in tumor stroma cancerization. Deregulation of miRNAs expression and activity has been demonstrated to play a significant role in a variety of human diseases, including fibrotic skin disorders and SCC [68,69], but their involvement in RDEB and its complications are almost unexplored.…”

“…Recently, we demonstrated the pro-fibrotic role of miR-145-5p in primary skin fibroblasts from RDEB patients (RDEBFs). In RDEBFs, miR-145-5p is up-regulated as compared to cells from healthy subjects, and its inhibition determinates the reduction of typical fibrotic behaviors (i.e., contractile force, proliferation and migration) and fibrotic markers by direct and indirect modulation of multiple and partially overlapping signaling cascades, such as the NOTCH pathway [42].…”

Epigenetic and metabolic regulation of epidermal homeostasis

“…In the context of epidermolysis bullosa (EB) – a rare genetic disorder of the skin discussed in more detail below – the role of miRNAs in disease pathogenesis is beginning to surface. To date, three miRNAs have been described to modulate EB‐associated complications such as fibrosis (miR‐29b, 86 , 87 miR‐145 88 , 89 ) and cancer (miR‐10b 90 ). The repetitive destabilization of the extracellular matrix that accompanies recessive dystrophic EB (RDEB) upon injury results in progressive soft tissue fibrosis with debilitating consequences, such as tumour development.…”