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Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells.

Mikami T, Yoshida K, Sawada H, Esaki M, Yasumura K, Ono M - Biol. Res. (2015)

Bottom Line: Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent.Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet.In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion.

View Article: PubMed Central - PubMed

Affiliation: Department of Histology and Cell Biology, Yokohama City University School of Medicine, Yokohama, Kanagawa-ken, Japan. zeong3@mac.com.

ABSTRACT

Background: The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen.

Results: Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion.

Conclusions: The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.

No MeSH data available.


Related in: MedlinePlus

Efficiency of siRNAs targeting ROCKs and RhoA. a The effectiveness of each siRNA knockdown was evaluated by quantitative polymerase chain reaction analysis. The relative quantities of ROCK1, ROCK2, and RhoA mRNAs were compared between each experiment. TopROCK1 expression was reduced to about 60 and 40 % that of the control using ROCKs-#1 or ROCKs#2 siRNAs. Middle Both combinations of ROCK siRNAs reduced ROCK2 mRNA levels to 40–50 % that of the control. Bottom Each RhoA siRNA reduced RhoA mRNA levels by about 40 % that of the negative control. Data represent the mean ± SEM of 3 independent experiments. NC negative control. Each graph indicates significant difference (*p < 0.01, n = 3). b Immunoblots showing protein expression of ROCK1, ROCK2, RhoA, and β-actin following the incubation of TE-10 cells with siRNA for each target. The expression of each protein was well suppressed, even after 24 h after RNAi treatment. Bars indicate the mean ± SEM of experiments performed in duplicate (RhoA) or triplicate (ROCKs)
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Fig4: Efficiency of siRNAs targeting ROCKs and RhoA. a The effectiveness of each siRNA knockdown was evaluated by quantitative polymerase chain reaction analysis. The relative quantities of ROCK1, ROCK2, and RhoA mRNAs were compared between each experiment. TopROCK1 expression was reduced to about 60 and 40 % that of the control using ROCKs-#1 or ROCKs#2 siRNAs. Middle Both combinations of ROCK siRNAs reduced ROCK2 mRNA levels to 40–50 % that of the control. Bottom Each RhoA siRNA reduced RhoA mRNA levels by about 40 % that of the negative control. Data represent the mean ± SEM of 3 independent experiments. NC negative control. Each graph indicates significant difference (*p < 0.01, n = 3). b Immunoblots showing protein expression of ROCK1, ROCK2, RhoA, and β-actin following the incubation of TE-10 cells with siRNA for each target. The expression of each protein was well suppressed, even after 24 h after RNAi treatment. Bars indicate the mean ± SEM of experiments performed in duplicate (RhoA) or triplicate (ROCKs)

Mentions: Small interfering RNAs (siRNAs) targeting either ROCK1 or ROCK2 reduced the expression of the respective ROCK isoform mRNAs by approximately 40–60 %, compared to negative control cells transfected with a non-silencing siRNA (Fig. 4a). A combination of both siRNAs reduced the expression of ROCK1 and ROCK2 proteins by 70–90 % (Fig. 4b; Additional files 16, 17). Furthermore, knockdown of both ROCK1 and ROCK2 led to an estimated 20 % reduction in the migration distance of the wound edge (Fig. 5a). In cells transfected with ROCK siRNAs, the migration distance of the stratified region toward the rear of the wounded edge also significantly decreased by 20–30 % relative to negative control cells (Fig. 5a). In ROCK-knockdown cells, some cells along the leading edge assumed irregular shapes, and the lamellipodia became obscure, which was similar to the results observed with cells treated with ROCK inhibitors (Fig. 5b; Additional file 18, and the movies in Additional files 19 and 20).Fig. 4


Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells.

Mikami T, Yoshida K, Sawada H, Esaki M, Yasumura K, Ono M - Biol. Res. (2015)

Efficiency of siRNAs targeting ROCKs and RhoA. a The effectiveness of each siRNA knockdown was evaluated by quantitative polymerase chain reaction analysis. The relative quantities of ROCK1, ROCK2, and RhoA mRNAs were compared between each experiment. TopROCK1 expression was reduced to about 60 and 40 % that of the control using ROCKs-#1 or ROCKs#2 siRNAs. Middle Both combinations of ROCK siRNAs reduced ROCK2 mRNA levels to 40–50 % that of the control. Bottom Each RhoA siRNA reduced RhoA mRNA levels by about 40 % that of the negative control. Data represent the mean ± SEM of 3 independent experiments. NC negative control. Each graph indicates significant difference (*p < 0.01, n = 3). b Immunoblots showing protein expression of ROCK1, ROCK2, RhoA, and β-actin following the incubation of TE-10 cells with siRNA for each target. The expression of each protein was well suppressed, even after 24 h after RNAi treatment. Bars indicate the mean ± SEM of experiments performed in duplicate (RhoA) or triplicate (ROCKs)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4556056&req=5

Fig4: Efficiency of siRNAs targeting ROCKs and RhoA. a The effectiveness of each siRNA knockdown was evaluated by quantitative polymerase chain reaction analysis. The relative quantities of ROCK1, ROCK2, and RhoA mRNAs were compared between each experiment. TopROCK1 expression was reduced to about 60 and 40 % that of the control using ROCKs-#1 or ROCKs#2 siRNAs. Middle Both combinations of ROCK siRNAs reduced ROCK2 mRNA levels to 40–50 % that of the control. Bottom Each RhoA siRNA reduced RhoA mRNA levels by about 40 % that of the negative control. Data represent the mean ± SEM of 3 independent experiments. NC negative control. Each graph indicates significant difference (*p < 0.01, n = 3). b Immunoblots showing protein expression of ROCK1, ROCK2, RhoA, and β-actin following the incubation of TE-10 cells with siRNA for each target. The expression of each protein was well suppressed, even after 24 h after RNAi treatment. Bars indicate the mean ± SEM of experiments performed in duplicate (RhoA) or triplicate (ROCKs)
Mentions: Small interfering RNAs (siRNAs) targeting either ROCK1 or ROCK2 reduced the expression of the respective ROCK isoform mRNAs by approximately 40–60 %, compared to negative control cells transfected with a non-silencing siRNA (Fig. 4a). A combination of both siRNAs reduced the expression of ROCK1 and ROCK2 proteins by 70–90 % (Fig. 4b; Additional files 16, 17). Furthermore, knockdown of both ROCK1 and ROCK2 led to an estimated 20 % reduction in the migration distance of the wound edge (Fig. 5a). In cells transfected with ROCK siRNAs, the migration distance of the stratified region toward the rear of the wounded edge also significantly decreased by 20–30 % relative to negative control cells (Fig. 5a). In ROCK-knockdown cells, some cells along the leading edge assumed irregular shapes, and the lamellipodia became obscure, which was similar to the results observed with cells treated with ROCK inhibitors (Fig. 5b; Additional file 18, and the movies in Additional files 19 and 20).Fig. 4

Bottom Line: Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent.Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet.In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion.

View Article: PubMed Central - PubMed

Affiliation: Department of Histology and Cell Biology, Yokohama City University School of Medicine, Yokohama, Kanagawa-ken, Japan. zeong3@mac.com.

ABSTRACT

Background: The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen.

Results: Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion.

Conclusions: The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.

No MeSH data available.


Related in: MedlinePlus