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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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Related in: MedlinePlus

Gene expression analysis after gene silencing. A: Detection of the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, and human antigen R (HuR) genes after gene silencing using RT–PCR. In the analyses, cells were examined 24 h after the treatments. In (A), (I): RT–PCR analysis of β-actin gene expression in corneal fibroblasts after gene silencing using siScrambled, siGAPDH, siβ-actin, or small interfering human antigen R (siHuR). (II): RT–PCR analysis of HuR gene expression in corneal fibroblasts after gene silencing using siScrambled, siGAPDH, siβ-actin, or siHuR. B: Western blot analysis of β-actin expression using the anti-β-actin antibody in corneal fibroblasts after gene silencing using siScrambled, siGAPDH, siβ-actin or siHuR. Vimentin expression was used as a control. β-Actin expression was downregulated on either β-actin gene silencing or that of HuR gene. C: A bar graph shows the percent of β-actin levels after the gene silencing. D: Real-time PCR analysis of the β-actin gene after the following treatments: siScrambled (siCTR), siGAPDH, siβ-actin, and siHuR.
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f1: Gene expression analysis after gene silencing. A: Detection of the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, and human antigen R (HuR) genes after gene silencing using RT–PCR. In the analyses, cells were examined 24 h after the treatments. In (A), (I): RT–PCR analysis of β-actin gene expression in corneal fibroblasts after gene silencing using siScrambled, siGAPDH, siβ-actin, or small interfering human antigen R (siHuR). (II): RT–PCR analysis of HuR gene expression in corneal fibroblasts after gene silencing using siScrambled, siGAPDH, siβ-actin, or siHuR. B: Western blot analysis of β-actin expression using the anti-β-actin antibody in corneal fibroblasts after gene silencing using siScrambled, siGAPDH, siβ-actin or siHuR. Vimentin expression was used as a control. β-Actin expression was downregulated on either β-actin gene silencing or that of HuR gene. C: A bar graph shows the percent of β-actin levels after the gene silencing. D: Real-time PCR analysis of the β-actin gene after the following treatments: siScrambled (siCTR), siGAPDH, siβ-actin, and siHuR.

Mentions: Our previous results showed downregulation in the expression of β-actin and HuR genes in keratoconus corneal stroma relative to normal corneas [12]. In the present study, we used RNAi-mediated gene silencing for β-actin or HuR in normal corneal fibroblasts to understand the respective gene functions. The GAPDH gene and scrambled sequence (which do not affect any targeted genes) were used as positive and negative controls for transfection, respectively. The sequences of the siRNA duplex were selected from the coding region of the desired target mRNAs, and the transfection was performed as described in the Methods section. The desired mRNA levels were analyzed using RT–PCR at 24 h post-transfection. Figure 1A(I) shows β-actin gene expression using β-actin primers after transfection of human corneal fibroblasts with the scrambled siRNA, siGAPDH, siβ-actin, or siHuR. Expression of the β-actin gene was downregulated after the HuR gene was silenced, which suggested that HuR has a regulatory role in β-actin gene expression. Additionally, β-actin gene silencing also showed downregulation of the β-actin gene, which was used as the control. Figure 1A(II) shows HuR gene expression using HuR primers after transfection of human corneal fibroblasts with siRNA for scrambled, GAPDH, β-actin, or HuR. Expression of the HuR gene was downregulated on HuR gene silencing, whereas β-actin gene silencing had no effect on HuR expression. Figure 1B shows western blot analysis of β-actin expression after transfection with the siRNA of scrambled, GAPDH, β-actin, and HuR. The western blot results show the reduced expression of β-actin and HuR relative to vimentin (used as the control). For visualization of vimentin with anti-vimentin antibody, the blot that was used for β-actin visualization was stripped and used again for the vimentin visualization. RT–PCR analysis showed that after 24 h, GAPDH gene silencing had no effect, and the expression remained at the same level (data not shown), which was confirmed with the western blot analysis. Figure 1C shows the percentage of β-actin expression levels after they normalized to vimentin and calculated as described in the Methods section. Relative to the expression of vimentin, the levels of β-actin expression were 24% and 18% lower after HuR and β-actin gene silencing, respectively. This suggests that the loss of HuR results in downregulation of the β-actin gene. There was no change in the β-actin expression levels after the GAPDH gene or negative control (siCTR) was silenced. Figure 1D shows the real-time PCR analysis after 72 h of siRNA treatments for the siCTR, GAPDH, HuR, and β-actin genes. The y-axis in the figure shows the relative fold expression, and the x-axis shows the β-actin primer used for the real-time PCR analysis. β-actin gene expression decreased by 80–90% after the β-actin and HuR genes were silenced, which was similar to the RT–PCR analysis. Whereas GAPDH gene silencing suppressed β-actin gene expression after 72 h of gene silencing (Figure 1D).


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)

Gene expression analysis after gene silencing. A: Detection of the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, and human antigen R (HuR) genes after gene silencing using RT–PCR. In the analyses, cells were examined 24 h after the treatments. In (A), (I): RT–PCR analysis of β-actin gene expression in corneal fibroblasts after gene silencing using siScrambled, siGAPDH, siβ-actin, or small interfering human antigen R (siHuR). (II): RT–PCR analysis of HuR gene expression in corneal fibroblasts after gene silencing using siScrambled, siGAPDH, siβ-actin, or siHuR. B: Western blot analysis of β-actin expression using the anti-β-actin antibody in corneal fibroblasts after gene silencing using siScrambled, siGAPDH, siβ-actin or siHuR. Vimentin expression was used as a control. β-Actin expression was downregulated on either β-actin gene silencing or that of HuR gene. C: A bar graph shows the percent of β-actin levels after the gene silencing. D: Real-time PCR analysis of the β-actin gene after the following treatments: siScrambled (siCTR), siGAPDH, siβ-actin, and siHuR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Gene expression analysis after gene silencing. A: Detection of the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, and human antigen R (HuR) genes after gene silencing using RT–PCR. In the analyses, cells were examined 24 h after the treatments. In (A), (I): RT–PCR analysis of β-actin gene expression in corneal fibroblasts after gene silencing using siScrambled, siGAPDH, siβ-actin, or small interfering human antigen R (siHuR). (II): RT–PCR analysis of HuR gene expression in corneal fibroblasts after gene silencing using siScrambled, siGAPDH, siβ-actin, or siHuR. B: Western blot analysis of β-actin expression using the anti-β-actin antibody in corneal fibroblasts after gene silencing using siScrambled, siGAPDH, siβ-actin or siHuR. Vimentin expression was used as a control. β-Actin expression was downregulated on either β-actin gene silencing or that of HuR gene. C: A bar graph shows the percent of β-actin levels after the gene silencing. D: Real-time PCR analysis of the β-actin gene after the following treatments: siScrambled (siCTR), siGAPDH, siβ-actin, and siHuR.
Mentions: Our previous results showed downregulation in the expression of β-actin and HuR genes in keratoconus corneal stroma relative to normal corneas [12]. In the present study, we used RNAi-mediated gene silencing for β-actin or HuR in normal corneal fibroblasts to understand the respective gene functions. The GAPDH gene and scrambled sequence (which do not affect any targeted genes) were used as positive and negative controls for transfection, respectively. The sequences of the siRNA duplex were selected from the coding region of the desired target mRNAs, and the transfection was performed as described in the Methods section. The desired mRNA levels were analyzed using RT–PCR at 24 h post-transfection. Figure 1A(I) shows β-actin gene expression using β-actin primers after transfection of human corneal fibroblasts with the scrambled siRNA, siGAPDH, siβ-actin, or siHuR. Expression of the β-actin gene was downregulated after the HuR gene was silenced, which suggested that HuR has a regulatory role in β-actin gene expression. Additionally, β-actin gene silencing also showed downregulation of the β-actin gene, which was used as the control. Figure 1A(II) shows HuR gene expression using HuR primers after transfection of human corneal fibroblasts with siRNA for scrambled, GAPDH, β-actin, or HuR. Expression of the HuR gene was downregulated on HuR gene silencing, whereas β-actin gene silencing had no effect on HuR expression. Figure 1B shows western blot analysis of β-actin expression after transfection with the siRNA of scrambled, GAPDH, β-actin, and HuR. The western blot results show the reduced expression of β-actin and HuR relative to vimentin (used as the control). For visualization of vimentin with anti-vimentin antibody, the blot that was used for β-actin visualization was stripped and used again for the vimentin visualization. RT–PCR analysis showed that after 24 h, GAPDH gene silencing had no effect, and the expression remained at the same level (data not shown), which was confirmed with the western blot analysis. Figure 1C shows the percentage of β-actin expression levels after they normalized to vimentin and calculated as described in the Methods section. Relative to the expression of vimentin, the levels of β-actin expression were 24% and 18% lower after HuR and β-actin gene silencing, respectively. This suggests that the loss of HuR results in downregulation of the β-actin gene. There was no change in the β-actin expression levels after the GAPDH gene or negative control (siCTR) was silenced. Figure 1D shows the real-time PCR analysis after 72 h of siRNA treatments for the siCTR, GAPDH, HuR, and β-actin genes. The y-axis in the figure shows the relative fold expression, and the x-axis shows the β-actin primer used for the real-time PCR analysis. β-actin gene expression decreased by 80–90% after the β-actin and HuR genes were silenced, which was similar to the RT–PCR analysis. Whereas GAPDH gene silencing suppressed β-actin gene expression after 72 h of gene silencing (Figure 1D).

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