Ocular surface MicroRNA associated with chronic ocular graft versus host disease

Abstract

Chronic ocular GvHD occurs in 30–60% of patients after stem cell transplantation. Its pathogenesis surmounts to a cycle of chronic inflammation and fibrosis, leading to dry eye that is often severe, and in cases refractive to treatment. There is no gold standard test nor distinct sign or symptom. Instead, a diagnosis is made through a set of criteria that place heavy reliance on clinical findings. This need for subspecialist input can delay diagnosis, treatment and lead to unsatisfactory outcomes and patient suffering.The objective of this study was to identify microRNA associated with disease signature and severity in the conjunctival epithelial cells (CECs) of patients with chronic ocular GvHD. Through an exploration of their predicted gene targets this study sought to develop a deeper understanding of the epigenetic signalling pathways involved in disease as well as identifying urgently needed biomarkers of diagnostic and therapeutic potential.Consecutive patients with a diagnosis of chronic ocular GvHD as per the National Institute of Health and/or the International Chronic Ocular Graft‐versus‐Host Disease diagnostic criteria were recruited along with healthy age and gender matched controls. CECs were collected from the ocular surface via impression cytology. MicroRNA were isolated and then analysed via Affymetrix Genechip miRNA 3.0 microarray. MicroRNA with a fold change of >2 or <−2 and an FDR p value of <0.05 were retained for further analysis. A LASSO regression model was applied in order to identify microRNA associated with disease signature and severity (SAS v. 9.4). Computational predictive methods were then used to identify high confidence microRNA target interactions (MTIs) using R studio (R version 4.1.3).Nine patients, five male (56%) four female (44%) with an average age of 38.6 yrs (±11.3) participated. Differentially expressed microRNAs were identified (n = 94, FDR <0.05, fold change ±2). A panel of microRNAs associated with disease signature (n = 3) and 50 MTIs were identified. Of interest was the upregulation of HDAC4, an inflammation and pain mediator and NOTCH2 which is overexpressed in dry eye models. In addition, SCARF1, which is needed for immunotolerance, PCDH1 needed for epithelial repair, NTS which is a regulator of pain and inflammation, and IHH which is needed for corneal healing were all downregulated. An additional panel of microRNA were identified terms of disease severity (n = 3), along with 27 MTIs. Those downregulated gene targets which were of particular interest included CDH1, which is involved in healing and maintaining the integrity of the corneal epithelium, NFIC, which suppresses the expression of pro‐inflammatory mediators and ZNF148 whose suppressed expression is associated with immune mediated inflammatory pathways.Overall, there is an urgent need for biomarkers in chronic ocular GvHD. The results from this study provide novel microRNA panels and associated target genes which have potential diagnostic and therapeutic value, along with providing an innovative perspective into the molecular mechanisms underpinning disease development and severity.