Downregulation of β-actin and its regulatory gene HuR affect cell migration of human corneal fibroblasts

Purpose: In an earlier study, we showed that human antigen R (HuR) and beta-actin expression levels were downregulated in fibroblasts isolated from human keratoconus stroma compared to normal corneal stroma. To further extend the finding, we determined whether HuR expression affects beta-actin gene expression and in turn affects corneal fibroblast migration and wound healing. Methods: Stromal keratocytes from normal human corneas were cultured in the presence of serum. Cells were transfected with siRNA specific for beta-actin or HuR. SiRNAs specific for GAPDH or a scrambled sequence were used as positive and negative controls (siCTR) for transfection, respectively. The effects of gene silencing were analyzed at the transcriptional and translational levels. Specific proteins were immunohistochemically localized using confocal imaging. The effects of gene silencing on cell migration and cell proliferation were analyzed using a modified Boyden chamber and with a wound healing assay, respectively. Results: Reverse-transcription PCR (RT-PCR) and western blot analyses showed that when the HuR gene was silenced, beta-actin expression was significantly downregulated. This was further confirmed at the translational level with immunohistochemical-confocal analysis. However, when the beta-actin gene was silenced, its expression was significantly decreased but showed no effect on HuR gene expression. When the beta-actin or HuR gene was individually silenced, the motility and proliferation of corneal fibroblasts were significantly reduced. Conclusions: The results show that downregulation of the HuR gene results in decreased beta-actin gene expression, which in turn results in decreased motility and proliferation of corneal fibroblasts. We conclude that decreased beta-actin expression in normal corneal stroma clearly disrupts the cytoskeletal structure and functions, including keratocyte motility and wound healing.