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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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Changes in miR-200a, ß-casein, E-cadherin and vimentin mRNA expression during mammary epithelial cell differentiation.(A and B) Mammary glands were collected at non-pregnancy (NP), days 7 (P7) and day 14 (P14) of pregnancy, and days 1 (L1) and day 7 (L7) of lactation (n = 3 animals). (C and D) EpH4 cells were treated with DIP for 0, 24, 48, or 72 h. Expression of miR-200a, ß-casein, E-cadherin and vimentin mRNA expression were analyzed by real-time PCR. All experiments were repeated three times.
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pone-0065127-g002: Changes in miR-200a, ß-casein, E-cadherin and vimentin mRNA expression during mammary epithelial cell differentiation.(A and B) Mammary glands were collected at non-pregnancy (NP), days 7 (P7) and day 14 (P14) of pregnancy, and days 1 (L1) and day 7 (L7) of lactation (n = 3 animals). (C and D) EpH4 cells were treated with DIP for 0, 24, 48, or 72 h. Expression of miR-200a, ß-casein, E-cadherin and vimentin mRNA expression were analyzed by real-time PCR. All experiments were repeated three times.

Mentions: To define the involvement of miR-200a in mammary gland development, mammary glands were collected from non-pregnant mice, mice at days 7 and 14 of pregnancy, and mice at days 1 and 7 of lactation. Real-time PCR analyses showed that miR-200a expression gradually increased from mid-pregnancy (P14) to lactation (Fig. 2A). On the other hand, there was no change in miR-23b expression. Expression of ß-casein (a mammary gland differentiation marker) and E-cadherin (an epithelial cell marker) was increased during the lactation stage (Fig. 2B). A positive correlation was observed for the expression of miR-200a. In contrast, expression of vimentin, a mesenchymal marker, was decreased during the lactation stage.


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

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

Changes in miR-200a, ß-casein, E-cadherin and vimentin mRNA expression during mammary epithelial cell differentiation.(A and B) Mammary glands were collected at non-pregnancy (NP), days 7 (P7) and day 14 (P14) of pregnancy, and days 1 (L1) and day 7 (L7) of lactation (n = 3 animals). (C and D) EpH4 cells were treated with DIP for 0, 24, 48, or 72 h. Expression of miR-200a, ß-casein, E-cadherin and vimentin mRNA expression 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-g002: Changes in miR-200a, ß-casein, E-cadherin and vimentin mRNA expression during mammary epithelial cell differentiation.(A and B) Mammary glands were collected at non-pregnancy (NP), days 7 (P7) and day 14 (P14) of pregnancy, and days 1 (L1) and day 7 (L7) of lactation (n = 3 animals). (C and D) EpH4 cells were treated with DIP for 0, 24, 48, or 72 h. Expression of miR-200a, ß-casein, E-cadherin and vimentin mRNA expression were analyzed by real-time PCR. All experiments were repeated three times.
Mentions: To define the involvement of miR-200a in mammary gland development, mammary glands were collected from non-pregnant mice, mice at days 7 and 14 of pregnancy, and mice at days 1 and 7 of lactation. Real-time PCR analyses showed that miR-200a expression gradually increased from mid-pregnancy (P14) to lactation (Fig. 2A). On the other hand, there was no change in miR-23b expression. Expression of ß-casein (a mammary gland differentiation marker) and E-cadherin (an epithelial cell marker) was increased during the lactation stage (Fig. 2B). A positive correlation was observed for the expression of miR-200a. In contrast, expression of vimentin, a mesenchymal marker, was decreased during the lactation stage.

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