Human Antigen R -mediated modulation of Transforming Growth Factor Beta 1 expression in retinal pathological milieu.

The fate and stability of messenger RNA (mRNA), from transcription to degradation is regulated by a dynamic shuttle of epigenetic modifications and RNA binding proteins in maintaining healthy cellular homeostasis and disease development. While Transforming Growth Factor Beta 1 (TGFβ1) has been implicated as a key regulator for diabetic retinopathy, a microvascular complication of diabetes, the RNA binding proteins post-transcriptionally regulating its expression remain unreported in the ocular context. Further, dysfunction of TGFβ1 signalling is also strongly associated with angiogenesis, inflammatory responses and tissue fibrosis in many eye conditions leading to vision loss. In this study, computational and molecular simulations were initially carried out to identify Human Antigen R (HuR) binding sites in mRNA and predict the structural stability of these RNA-protein interactions. These findings were further validated through experiments utilizing Cobalt Chloride (CoCl) as a hypoxia mimetic agent in human retinal microvascular endothelial cells (HRMVEC). analysis revealed that HuR preferentially binds to the 5'-UTR of and displayed more stable interaction than the 3'UTR. Consistent with analysis, RNA immunoprecipitation demonstrated a robust association between HuR and mRNA specifically under hypoxic conditions. Further, silencing of HuR significantly reduced TGFβ1 protein expression upon CoCl treatment. Thus, for the first time in ocular pathological milieu, direct evidence of HuR- mRNA interaction under conditions of hypoxia has been reported in this study providing valuable insights into RNA binding proteins as therapeutic targets for ocular diseases associated with TGFβ1 dysregulation.