Up-regulation of microRNA 506 leads to decreased Cl -/HCO 3 - anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis

Jesús M. Banales, Elena Sáez, Miriam Úriz, Sarai Sarvide, Aura D. Urribarri, Patrick Splinter, Pamela S. Tietz Bogert, Luis Bujanda, Jesús Prieto, Juan F. Medina, Nicholas F La Russo

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Abstract

Cl -/HCO3- anion exchanger 2 (AE2) participates in intracellular pH homeostasis and secretin-stimulated biliary bicarbonate secretion. AE2/SLC4A2 gene expression is reduced in liver and blood mononuclear cells from patients with primary biliary cirrhosis (PBC). Our previous findings of hepatic and immunological features mimicking PBC in Ae2-deficient mice strongly suggest that decreased AE2 expression might be involved in the pathogenesis of PBC. Here, we tested the potential role of microRNA 506 (miR-506) - predicted as candidate to target AE2 mRNA - for the decreased expression of AE2 in PBC. Real-time quantitative polymerase chain reaction showed that miR-506 expression is increased in PBC livers versus normal liver specimens. In situ hybridization in liver sections confirmed that miR-506 is up-regulated in the intrahepatic bile ducts of PBC livers, compared with normal and primary sclerosing cholangitis livers. Precursor-mediated overexpression of miR-506 in SV40-immortalized normal human cholangiocytes (H69 cells) led to decreased AE2 protein expression and activity, as indicated by immunoblotting and microfluorimetry, respectively. Moreover, miR-506 overexpression in three-dimensional (3D)-cultured H69 cholangiocytes blocked the secretin-stimulated expansion of cystic structures developed under the 3D conditions. Luciferase assays and site-directed mutagenesis demonstrated that miR-506 specifically may bind the 3′untranslated region (3′UTR) of AE2 messenger RNA (mRNA) and prevent protein translation. Finally, cultured PBC cholangiocytes showed decreased AE2 activity, together with miR-506 overexpression, compared to normal human cholangiocytes, and transfection of PBC cholangiocytes with anti-miR-506 was able to improve their AE2 activity. Conclusion: miR-506 is up-regulated in cholangiocytes from PBC patients, binds the 3′UTR region of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions. In view of the putative pathogenic role of decreased AE2 in PBC, miR-506 may constitute a potential therapeutic target for this disease.

Original languageEnglish (US)
Pages (from-to)687-697
Number of pages11
JournalHepatology
Volume56
Issue number2
DOIs
StatePublished - Aug 2012

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Chloride-Bicarbonate Antiporters
Biliary Liver Cirrhosis
MicroRNAs
Up-Regulation
Epithelium
Liver
Secretin
Protein Biosynthesis
Messenger RNA
Cytophotometry
Antiporters
Intrahepatic Bile Ducts
Sclerosing Cholangitis
Bicarbonates
Site-Directed Mutagenesis
Luciferases
Immunoblotting

ASJC Scopus subject areas

  • Hepatology

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Up-regulation of microRNA 506 leads to decreased Cl -/HCO 3 - anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis. / Banales, Jesús M.; Sáez, Elena; Úriz, Miriam; Sarvide, Sarai; Urribarri, Aura D.; Splinter, Patrick; Tietz Bogert, Pamela S.; Bujanda, Luis; Prieto, Jesús; Medina, Juan F.; La Russo, Nicholas F.

In: Hepatology, Vol. 56, No. 2, 08.2012, p. 687-697.

Research output: Contribution to journalArticle

Banales, JM, Sáez, E, Úriz, M, Sarvide, S, Urribarri, AD, Splinter, P, Tietz Bogert, PS, Bujanda, L, Prieto, J, Medina, JF & La Russo, NF 2012, 'Up-regulation of microRNA 506 leads to decreased Cl -/HCO 3 - anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis', Hepatology, vol. 56, no. 2, pp. 687-697. https://doi.org/10.1002/hep.25691
Banales, Jesús M. ; Sáez, Elena ; Úriz, Miriam ; Sarvide, Sarai ; Urribarri, Aura D. ; Splinter, Patrick ; Tietz Bogert, Pamela S. ; Bujanda, Luis ; Prieto, Jesús ; Medina, Juan F. ; La Russo, Nicholas F. / Up-regulation of microRNA 506 leads to decreased Cl -/HCO 3 - anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis. In: Hepatology. 2012 ; Vol. 56, No. 2. pp. 687-697.
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abstract = "Cl -/HCO3- anion exchanger 2 (AE2) participates in intracellular pH homeostasis and secretin-stimulated biliary bicarbonate secretion. AE2/SLC4A2 gene expression is reduced in liver and blood mononuclear cells from patients with primary biliary cirrhosis (PBC). Our previous findings of hepatic and immunological features mimicking PBC in Ae2-deficient mice strongly suggest that decreased AE2 expression might be involved in the pathogenesis of PBC. Here, we tested the potential role of microRNA 506 (miR-506) - predicted as candidate to target AE2 mRNA - for the decreased expression of AE2 in PBC. Real-time quantitative polymerase chain reaction showed that miR-506 expression is increased in PBC livers versus normal liver specimens. In situ hybridization in liver sections confirmed that miR-506 is up-regulated in the intrahepatic bile ducts of PBC livers, compared with normal and primary sclerosing cholangitis livers. Precursor-mediated overexpression of miR-506 in SV40-immortalized normal human cholangiocytes (H69 cells) led to decreased AE2 protein expression and activity, as indicated by immunoblotting and microfluorimetry, respectively. Moreover, miR-506 overexpression in three-dimensional (3D)-cultured H69 cholangiocytes blocked the secretin-stimulated expansion of cystic structures developed under the 3D conditions. Luciferase assays and site-directed mutagenesis demonstrated that miR-506 specifically may bind the 3′untranslated region (3′UTR) of AE2 messenger RNA (mRNA) and prevent protein translation. Finally, cultured PBC cholangiocytes showed decreased AE2 activity, together with miR-506 overexpression, compared to normal human cholangiocytes, and transfection of PBC cholangiocytes with anti-miR-506 was able to improve their AE2 activity. Conclusion: miR-506 is up-regulated in cholangiocytes from PBC patients, binds the 3′UTR region of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions. In view of the putative pathogenic role of decreased AE2 in PBC, miR-506 may constitute a potential therapeutic target for this disease.",
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T1 - Up-regulation of microRNA 506 leads to decreased Cl -/HCO 3 - anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis

AU - Banales, Jesús M.

AU - Sáez, Elena

AU - Úriz, Miriam

AU - Sarvide, Sarai

AU - Urribarri, Aura D.

AU - Splinter, Patrick

AU - Tietz Bogert, Pamela S.

AU - Bujanda, Luis

AU - Prieto, Jesús

AU - Medina, Juan F.

AU - La Russo, Nicholas F

PY - 2012/8

Y1 - 2012/8

N2 - Cl -/HCO3- anion exchanger 2 (AE2) participates in intracellular pH homeostasis and secretin-stimulated biliary bicarbonate secretion. AE2/SLC4A2 gene expression is reduced in liver and blood mononuclear cells from patients with primary biliary cirrhosis (PBC). Our previous findings of hepatic and immunological features mimicking PBC in Ae2-deficient mice strongly suggest that decreased AE2 expression might be involved in the pathogenesis of PBC. Here, we tested the potential role of microRNA 506 (miR-506) - predicted as candidate to target AE2 mRNA - for the decreased expression of AE2 in PBC. Real-time quantitative polymerase chain reaction showed that miR-506 expression is increased in PBC livers versus normal liver specimens. In situ hybridization in liver sections confirmed that miR-506 is up-regulated in the intrahepatic bile ducts of PBC livers, compared with normal and primary sclerosing cholangitis livers. Precursor-mediated overexpression of miR-506 in SV40-immortalized normal human cholangiocytes (H69 cells) led to decreased AE2 protein expression and activity, as indicated by immunoblotting and microfluorimetry, respectively. Moreover, miR-506 overexpression in three-dimensional (3D)-cultured H69 cholangiocytes blocked the secretin-stimulated expansion of cystic structures developed under the 3D conditions. Luciferase assays and site-directed mutagenesis demonstrated that miR-506 specifically may bind the 3′untranslated region (3′UTR) of AE2 messenger RNA (mRNA) and prevent protein translation. Finally, cultured PBC cholangiocytes showed decreased AE2 activity, together with miR-506 overexpression, compared to normal human cholangiocytes, and transfection of PBC cholangiocytes with anti-miR-506 was able to improve their AE2 activity. Conclusion: miR-506 is up-regulated in cholangiocytes from PBC patients, binds the 3′UTR region of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions. In view of the putative pathogenic role of decreased AE2 in PBC, miR-506 may constitute a potential therapeutic target for this disease.

AB - Cl -/HCO3- anion exchanger 2 (AE2) participates in intracellular pH homeostasis and secretin-stimulated biliary bicarbonate secretion. AE2/SLC4A2 gene expression is reduced in liver and blood mononuclear cells from patients with primary biliary cirrhosis (PBC). Our previous findings of hepatic and immunological features mimicking PBC in Ae2-deficient mice strongly suggest that decreased AE2 expression might be involved in the pathogenesis of PBC. Here, we tested the potential role of microRNA 506 (miR-506) - predicted as candidate to target AE2 mRNA - for the decreased expression of AE2 in PBC. Real-time quantitative polymerase chain reaction showed that miR-506 expression is increased in PBC livers versus normal liver specimens. In situ hybridization in liver sections confirmed that miR-506 is up-regulated in the intrahepatic bile ducts of PBC livers, compared with normal and primary sclerosing cholangitis livers. Precursor-mediated overexpression of miR-506 in SV40-immortalized normal human cholangiocytes (H69 cells) led to decreased AE2 protein expression and activity, as indicated by immunoblotting and microfluorimetry, respectively. Moreover, miR-506 overexpression in three-dimensional (3D)-cultured H69 cholangiocytes blocked the secretin-stimulated expansion of cystic structures developed under the 3D conditions. Luciferase assays and site-directed mutagenesis demonstrated that miR-506 specifically may bind the 3′untranslated region (3′UTR) of AE2 messenger RNA (mRNA) and prevent protein translation. Finally, cultured PBC cholangiocytes showed decreased AE2 activity, together with miR-506 overexpression, compared to normal human cholangiocytes, and transfection of PBC cholangiocytes with anti-miR-506 was able to improve their AE2 activity. Conclusion: miR-506 is up-regulated in cholangiocytes from PBC patients, binds the 3′UTR region of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions. In view of the putative pathogenic role of decreased AE2 in PBC, miR-506 may constitute a potential therapeutic target for this disease.

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