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Epithelial cell differentiation regulated by MicroRNA-200a in mammary glands.

Nagaoka K, Zhang H, Watanabe G, Taya K - PLoS ONE (2013)

Bottom Line: Here, we found that one miRNA, miR-200a, was relatively highly expressed in epithelial cell-rich organs such as mammary glands, lung, and kidney in mice.However, knockdown of miR-200a prevented increases in ß-casein and E-cadherin mRNA expression.Protein analysis revealed that E-cadherin signal was decreased and ZEB1 (a marker of EMT) was increased following miR-200a knockdown.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan. nagaokak@cc.tuat.ac.jp

ABSTRACT
Mammary gland epithelial cells undergo periodic cycles of proliferation, differentiation, and involution. Many studies have reported that miRNAs, which are small, non-coding RNAs, influence a variety of biological processes during posttranscriptional regulation. Here, we found that one miRNA, miR-200a, was relatively highly expressed in epithelial cell-rich organs such as mammary glands, lung, and kidney in mice. In mammary glands, miR-200a expression increased during mid-pregnancy through lactation; its expression was stimulated by lactogenic hormone treatment of mammary epithelial cells. Lactogenic hormone also induced the expression of milk protein ß-casein mRNA (a marker of cell differentiation) and E-cadherin mRNA (a marker of epithelial cells). However, knockdown of miR-200a prevented increases in ß-casein and E-cadherin mRNA expression. Protein analysis revealed that E-cadherin signal was decreased and ZEB1 (a marker of EMT) was increased following miR-200a knockdown. Finally, in a three-dimensional culture system modeling lumen-containing mammary ducts, miR-200a knockdown decreased the cavity formation rate and suppressed claudin-3 and par-6b expression, indicating reduced epithelial cell polarity. These observations suggest that miR-200a is important for maintaining the epithelial cell phenotype, which contributes to lactogenic hormone induction of cellular differentiation in mammary glands.

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Effect of miR-200a knockdown on ß-casein, E-cadherin, vimentin, ZEB1 and Snail1 mRNA expression.EpH4 cells were transfected with anti-miR-200a or control antisense. Twenty-four hours after transfection, cells were treated with or without DIP for 72 h. After DIP treatment, expression of miR-200a (A), ß-casein (B), E-cadherin (C), vimentin (D), ZEB1 (E) and Snail1 (F) mRNA were analyzed by real-time PCR. All experiments were repeated three times.
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pone-0065127-g003: Effect of miR-200a knockdown on ß-casein, E-cadherin, vimentin, ZEB1 and Snail1 mRNA expression.EpH4 cells were transfected with anti-miR-200a or control antisense. Twenty-four hours after transfection, cells were treated with or without DIP for 72 h. After DIP treatment, expression of miR-200a (A), ß-casein (B), E-cadherin (C), vimentin (D), ZEB1 (E) and Snail1 (F) mRNA were analyzed by real-time PCR. All experiments were repeated three times.

Mentions: To investigate whether miR-200a controls mammary gland epithelial cell differentiation, we conducted loss-of-function experiments. Before performing the DIP treatment, we transfected oligoribonucleotide anti-sense miR-200a into EpH4 cells. At 24 h after transfection, we began DIP treatment for 72 h; at this time point, we confirmed the knockdown of miR-200a by real-time PCR (Fig. 3A). As shown in Fig. 3B, the expression of ß-casein mRNA after DIP treatment was decreased in transfection with anti-miR-200a compared to transfection with the anti-sense RNA control. Similar to ß-casein, the miR-200a knockdown decreased E-cadherin mRNA levels under the DIP treatment (Fig. 3C). No significant differences were observed in the vimentin, ZEB1 and Snail1 (EMT markers) expression levels (Fig. 3D–F).


Epithelial cell differentiation regulated by MicroRNA-200a in mammary glands.

Nagaoka K, Zhang H, Watanabe G, Taya K - PLoS ONE (2013)

Effect of miR-200a knockdown on ß-casein, E-cadherin, vimentin, ZEB1 and Snail1 mRNA expression.EpH4 cells were transfected with anti-miR-200a or control antisense. Twenty-four hours after transfection, cells were treated with or without DIP for 72 h. After DIP treatment, expression of miR-200a (A), ß-casein (B), E-cadherin (C), vimentin (D), ZEB1 (E) and Snail1 (F) mRNA were analyzed by real-time PCR. All experiments were repeated three times.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672172&req=5

pone-0065127-g003: Effect of miR-200a knockdown on ß-casein, E-cadherin, vimentin, ZEB1 and Snail1 mRNA expression.EpH4 cells were transfected with anti-miR-200a or control antisense. Twenty-four hours after transfection, cells were treated with or without DIP for 72 h. After DIP treatment, expression of miR-200a (A), ß-casein (B), E-cadherin (C), vimentin (D), ZEB1 (E) and Snail1 (F) mRNA were analyzed by real-time PCR. All experiments were repeated three times.
Mentions: To investigate whether miR-200a controls mammary gland epithelial cell differentiation, we conducted loss-of-function experiments. Before performing the DIP treatment, we transfected oligoribonucleotide anti-sense miR-200a into EpH4 cells. At 24 h after transfection, we began DIP treatment for 72 h; at this time point, we confirmed the knockdown of miR-200a by real-time PCR (Fig. 3A). As shown in Fig. 3B, the expression of ß-casein mRNA after DIP treatment was decreased in transfection with anti-miR-200a compared to transfection with the anti-sense RNA control. Similar to ß-casein, the miR-200a knockdown decreased E-cadherin mRNA levels under the DIP treatment (Fig. 3C). No significant differences were observed in the vimentin, ZEB1 and Snail1 (EMT markers) expression levels (Fig. 3D–F).

Bottom Line: Here, we found that one miRNA, miR-200a, was relatively highly expressed in epithelial cell-rich organs such as mammary glands, lung, and kidney in mice.However, knockdown of miR-200a prevented increases in ß-casein and E-cadherin mRNA expression.Protein analysis revealed that E-cadherin signal was decreased and ZEB1 (a marker of EMT) was increased following miR-200a knockdown.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan. nagaokak@cc.tuat.ac.jp

ABSTRACT
Mammary gland epithelial cells undergo periodic cycles of proliferation, differentiation, and involution. Many studies have reported that miRNAs, which are small, non-coding RNAs, influence a variety of biological processes during posttranscriptional regulation. Here, we found that one miRNA, miR-200a, was relatively highly expressed in epithelial cell-rich organs such as mammary glands, lung, and kidney in mice. In mammary glands, miR-200a expression increased during mid-pregnancy through lactation; its expression was stimulated by lactogenic hormone treatment of mammary epithelial cells. Lactogenic hormone also induced the expression of milk protein ß-casein mRNA (a marker of cell differentiation) and E-cadherin mRNA (a marker of epithelial cells). However, knockdown of miR-200a prevented increases in ß-casein and E-cadherin mRNA expression. Protein analysis revealed that E-cadherin signal was decreased and ZEB1 (a marker of EMT) was increased following miR-200a knockdown. Finally, in a three-dimensional culture system modeling lumen-containing mammary ducts, miR-200a knockdown decreased the cavity formation rate and suppressed claudin-3 and par-6b expression, indicating reduced epithelial cell polarity. These observations suggest that miR-200a is important for maintaining the epithelial cell phenotype, which contributes to lactogenic hormone induction of cellular differentiation in mammary glands.

Show MeSH
Related in: MedlinePlus