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

Joseph R, Srivastava OP, Pfister RR - Mol. Vis. (2014)

Bottom Line: However, when the β-actin gene was silenced, its expression was significantly decreased but showed no effect on HuR gene expression.When the β-actin or HuR gene was individually silenced, the motility and proliferation of corneal fibroblasts were significantly reduced.The results show that downregulation of the HuR gene results in decreased β-actin gene expression, which in turn results in decreased motility and proliferation of corneal fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL.

ABSTRACT

Purpose: In an earlier study, we showed that human antigen R (HuR) and β-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 β-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 β-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, β-actin expression was significantly downregulated. This was further confirmed at the translational level with immunohistochemical-confocal analysis. However, when the β-actin gene was silenced, its expression was significantly decreased but showed no effect on HuR gene expression. When the β-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 β-actin gene expression, which in turn results in decreased motility and proliferation of corneal fibroblasts. We conclude that decreased β-actin expression in normal corneal stroma clearly disrupts the cytoskeletal structure and functions, including keratocyte motility and wound healing.

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Analysis of cell migration after gene silencing (n = 4), using a modified Boyden chamber. Cell migration is shown with arrows. A: Migration of normal corneal fibroblast. B: Migration of cells after treatment with scrambled siRNA. C: Migration of cells after treatment of corneal fibroblast with GAPDH siRNA. D: Migration of cells after treatment of normal corneal fibroblast with human antigen R (HuR) siRNA. E: Migration of cells after treatment of normal corneal fibroblast with β-actin siRNA. F: Bar graph showing cell migration after different treatments (A to E). Cells were counted from six different frames. All the treatments were performed in tetraplicate, and standard deviations were calculated.
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f4: Analysis of cell migration after gene silencing (n = 4), using a modified Boyden chamber. Cell migration is shown with arrows. A: Migration of normal corneal fibroblast. B: Migration of cells after treatment with scrambled siRNA. C: Migration of cells after treatment of corneal fibroblast with GAPDH siRNA. D: Migration of cells after treatment of normal corneal fibroblast with human antigen R (HuR) siRNA. E: Migration of cells after treatment of normal corneal fibroblast with β-actin siRNA. F: Bar graph showing cell migration after different treatments (A to E). Cells were counted from six different frames. All the treatments were performed in tetraplicate, and standard deviations were calculated.

Mentions: Next we analyzed the functional significance of the loss of the HuR gene and whether it leads to a loss in the cytoskeletal function such as corneal fibroblast migration. Cell migration is an important biologic process mainly generated by the β-actin cytoskeleton [3]. The modified Boyden chamber assay was used to determine if targeted deletion of the HuR or β-actin gene affected migration of normal corneal fibroblasts. Figure 4A shows the migrated cells (pointed arrows denote the cells) in the corneal fibroblasts, and Figure 4B,C,D,E shows cell migration following silencing of scrambled siRNA, and siRNA of the GAPDH gene, HuR gene, or β-actin gene, respectively. Cells migrated per image (Figure 4F) were counted using Image-J software, incorporating multiple images and standard deviation analysis. Together, the results show that scrambled siRNA treatment had no effect on cell migration, which was identical to that of normal cell migration (Figure 4A,B). GAPDH gene silencing resulted in reduced cell migration compared to the scrambled siRNA. Silencing the HuR gene significantly decreased cell migration, whereas β-actin gene silencing abolished the migration. Although it is known that β-actin is an essential component of cell migration [15], the role of HuR in cell migration is shown for the first time in this report.


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

Joseph R, Srivastava OP, Pfister RR - Mol. Vis. (2014)

Analysis of cell migration after gene silencing (n = 4), using a modified Boyden chamber. Cell migration is shown with arrows. A: Migration of normal corneal fibroblast. B: Migration of cells after treatment with scrambled siRNA. C: Migration of cells after treatment of corneal fibroblast with GAPDH siRNA. D: Migration of cells after treatment of normal corneal fibroblast with human antigen R (HuR) siRNA. E: Migration of cells after treatment of normal corneal fibroblast with β-actin siRNA. F: Bar graph showing cell migration after different treatments (A to E). Cells were counted from six different frames. All the treatments were performed in tetraplicate, and standard deviations were calculated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016806&req=5

f4: Analysis of cell migration after gene silencing (n = 4), using a modified Boyden chamber. Cell migration is shown with arrows. A: Migration of normal corneal fibroblast. B: Migration of cells after treatment with scrambled siRNA. C: Migration of cells after treatment of corneal fibroblast with GAPDH siRNA. D: Migration of cells after treatment of normal corneal fibroblast with human antigen R (HuR) siRNA. E: Migration of cells after treatment of normal corneal fibroblast with β-actin siRNA. F: Bar graph showing cell migration after different treatments (A to E). Cells were counted from six different frames. All the treatments were performed in tetraplicate, and standard deviations were calculated.
Mentions: Next we analyzed the functional significance of the loss of the HuR gene and whether it leads to a loss in the cytoskeletal function such as corneal fibroblast migration. Cell migration is an important biologic process mainly generated by the β-actin cytoskeleton [3]. The modified Boyden chamber assay was used to determine if targeted deletion of the HuR or β-actin gene affected migration of normal corneal fibroblasts. Figure 4A shows the migrated cells (pointed arrows denote the cells) in the corneal fibroblasts, and Figure 4B,C,D,E shows cell migration following silencing of scrambled siRNA, and siRNA of the GAPDH gene, HuR gene, or β-actin gene, respectively. Cells migrated per image (Figure 4F) were counted using Image-J software, incorporating multiple images and standard deviation analysis. Together, the results show that scrambled siRNA treatment had no effect on cell migration, which was identical to that of normal cell migration (Figure 4A,B). GAPDH gene silencing resulted in reduced cell migration compared to the scrambled siRNA. Silencing the HuR gene significantly decreased cell migration, whereas β-actin gene silencing abolished the migration. Although it is known that β-actin is an essential component of cell migration [15], the role of HuR in cell migration is shown for the first time in this report.

Bottom Line: However, when the β-actin gene was silenced, its expression was significantly decreased but showed no effect on HuR gene expression.When the β-actin or HuR gene was individually silenced, the motility and proliferation of corneal fibroblasts were significantly reduced.The results show that downregulation of the HuR gene results in decreased β-actin gene expression, which in turn results in decreased motility and proliferation of corneal fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL.

ABSTRACT

Purpose: In an earlier study, we showed that human antigen R (HuR) and β-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 β-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 β-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, β-actin expression was significantly downregulated. This was further confirmed at the translational level with immunohistochemical-confocal analysis. However, when the β-actin gene was silenced, its expression was significantly decreased but showed no effect on HuR gene expression. When the β-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 β-actin gene expression, which in turn results in decreased motility and proliferation of corneal fibroblasts. We conclude that decreased β-actin expression in normal corneal stroma clearly disrupts the cytoskeletal structure and functions, including keratocyte motility and wound healing.

Show MeSH
Related in: MedlinePlus