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Quercetin Decreases Claudin-2 Expression Mediated by Up-Regulation of microRNA miR-16 in Lung Adenocarcinoma A549 Cells.

Sonoki H, Sato T, Endo S, Matsunaga T, Yamaguchi M, Yamazaki Y, Sugatani J, Ikari A - Nutrients (2015)

Bottom Line: Both LY-294002 and U0126 inhibited promoter activity of claudin-2, but quercetin did not.The stability of claudin-2 mRNA was decreased by quercetin.An inhibitor of miR-16 rescued quercetin-induced decrease in the claudin-2 expression.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196, Japan. 116023@gifu-pu.ac.jp.

ABSTRACT
Claudin-2 is highly expressed in human lung adenocarcinoma tissues and cells. Knockdown of claudin-2 decreases cell proliferation and migration. Claudin-2 may be a novel target for lung adenocarcinoma. However, there are no physiologically active substances of foods which decrease claudin-2 expression. We here found that quercetin, a flavonoid present in fruits and vegetables, time- and concentration-dependently decreases claudin-2 expression in lung adenocarcinoma A549 cells. In the present study, we examined what regulatory mechanism is involved in the decrease in claudin-2 expression by quercetin. Claudin-2 expression was decreased by LY-294002, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, and U0126, a MEK inhibitor. These drugs inhibited the phosphorylation of Akt and ERK1/2, which are downstream targets of PI3-K and MEK, respectively. In contrast, quercetin did not inhibit the phosphorylation. Both LY-294002 and U0126 inhibited promoter activity of claudin-2, but quercetin did not. The stability of claudin-2 mRNA was decreased by quercetin. Quercetin increased the expression of microRNA miR-16. An inhibitor of miR-16 rescued quercetin-induced decrease in the claudin-2 expression. These results suggest that quercetin decreases claudin-2 expression mediated by up-regulation of miR-16 expression and instability of claudin-2 mRNA in lung adenocarcinoma cells.

No MeSH data available.


Related in: MedlinePlus

Effect of quercetin on polyadenylation. (A) A549 cells were incubated in the absence (control) and presence of 50 μM quercetin or 10 μM cordycepin for 6 h. The expression level of mRNA was quantified by real-time PCR using primers for claudin-2 and β-actin, and was represented relative to values at control; (B) After reverse transcription using Olig-dT-3 sites adaptor primer, 3′-RACE PCR was carried out. The PCR products were visualized with ethidium bromide. n = 3–4. ** p < 0.01 is significantly different from control.
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nutrients-07-04578-f006: Effect of quercetin on polyadenylation. (A) A549 cells were incubated in the absence (control) and presence of 50 μM quercetin or 10 μM cordycepin for 6 h. The expression level of mRNA was quantified by real-time PCR using primers for claudin-2 and β-actin, and was represented relative to values at control; (B) After reverse transcription using Olig-dT-3 sites adaptor primer, 3′-RACE PCR was carried out. The PCR products were visualized with ethidium bromide. n = 3–4. ** p < 0.01 is significantly different from control.

Mentions: The stability of mRNAs is regulated by polyadenylation and mRNAs with long poly (A) are more stable than those with short poly (A) [32]. We examined the effect of cordycepin, an inhibitor of polyadenylation, on the expression level of claudin-2 mRNA. Both quercetin and cordycepin decreased the expression level of claudin-2 mRNA (Figure 6A). In the same samples, 3′-UTR of claudin-2 is amplified by PCR reaction in control and quercetin-treated cells (Figure 6B). In contrast, the short and faint band was detected in the cordycepin-treated cells.


Quercetin Decreases Claudin-2 Expression Mediated by Up-Regulation of microRNA miR-16 in Lung Adenocarcinoma A549 Cells.

Sonoki H, Sato T, Endo S, Matsunaga T, Yamaguchi M, Yamazaki Y, Sugatani J, Ikari A - Nutrients (2015)

Effect of quercetin on polyadenylation. (A) A549 cells were incubated in the absence (control) and presence of 50 μM quercetin or 10 μM cordycepin for 6 h. The expression level of mRNA was quantified by real-time PCR using primers for claudin-2 and β-actin, and was represented relative to values at control; (B) After reverse transcription using Olig-dT-3 sites adaptor primer, 3′-RACE PCR was carried out. The PCR products were visualized with ethidium bromide. n = 3–4. ** p < 0.01 is significantly different from control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488803&req=5

nutrients-07-04578-f006: Effect of quercetin on polyadenylation. (A) A549 cells were incubated in the absence (control) and presence of 50 μM quercetin or 10 μM cordycepin for 6 h. The expression level of mRNA was quantified by real-time PCR using primers for claudin-2 and β-actin, and was represented relative to values at control; (B) After reverse transcription using Olig-dT-3 sites adaptor primer, 3′-RACE PCR was carried out. The PCR products were visualized with ethidium bromide. n = 3–4. ** p < 0.01 is significantly different from control.
Mentions: The stability of mRNAs is regulated by polyadenylation and mRNAs with long poly (A) are more stable than those with short poly (A) [32]. We examined the effect of cordycepin, an inhibitor of polyadenylation, on the expression level of claudin-2 mRNA. Both quercetin and cordycepin decreased the expression level of claudin-2 mRNA (Figure 6A). In the same samples, 3′-UTR of claudin-2 is amplified by PCR reaction in control and quercetin-treated cells (Figure 6B). In contrast, the short and faint band was detected in the cordycepin-treated cells.

Bottom Line: Both LY-294002 and U0126 inhibited promoter activity of claudin-2, but quercetin did not.The stability of claudin-2 mRNA was decreased by quercetin.An inhibitor of miR-16 rescued quercetin-induced decrease in the claudin-2 expression.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196, Japan. 116023@gifu-pu.ac.jp.

ABSTRACT
Claudin-2 is highly expressed in human lung adenocarcinoma tissues and cells. Knockdown of claudin-2 decreases cell proliferation and migration. Claudin-2 may be a novel target for lung adenocarcinoma. However, there are no physiologically active substances of foods which decrease claudin-2 expression. We here found that quercetin, a flavonoid present in fruits and vegetables, time- and concentration-dependently decreases claudin-2 expression in lung adenocarcinoma A549 cells. In the present study, we examined what regulatory mechanism is involved in the decrease in claudin-2 expression by quercetin. Claudin-2 expression was decreased by LY-294002, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, and U0126, a MEK inhibitor. These drugs inhibited the phosphorylation of Akt and ERK1/2, which are downstream targets of PI3-K and MEK, respectively. In contrast, quercetin did not inhibit the phosphorylation. Both LY-294002 and U0126 inhibited promoter activity of claudin-2, but quercetin did not. The stability of claudin-2 mRNA was decreased by quercetin. Quercetin increased the expression of microRNA miR-16. An inhibitor of miR-16 rescued quercetin-induced decrease in the claudin-2 expression. These results suggest that quercetin decreases claudin-2 expression mediated by up-regulation of miR-16 expression and instability of claudin-2 mRNA in lung adenocarcinoma cells.

No MeSH data available.


Related in: MedlinePlus