Limits...
Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocelular apoptosis.

M Rodrigues P, B Afonso M, L Simão A, M Borralho P, M P Rodrigues C, E Castro R - Sci Rep (2015)

Bottom Line: In fact, NF-κB overexpression or constitutive activation halted miR-21-dependent apoptosis by DCA while opposite results were observed upon NF-κB inhibition.In turn, DCA-induced oxidative stress resulted in caspase-2 activation and NF-κB/miR-21 inhibition, in a PIDD-dependent manner.These signalling circuits may constitute appealing targets for bile acid-associated liver pathologies.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal.

ABSTRACT
MicroRNAs (miRNAs/miRs) are key regulators of liver metabolism, while toxic bile acids participate in the development of several liver diseases. We previously demonstrated that deoxycholic acid (DCA), a cytotoxic bile acid implicated in the pathogenesis of non-alcoholic fatty liver disease, inhibits miR-21 expression in hepatocytes. Here, we investigated the mechanisms by which DCA modulates miR-21 and whether miR-21 contributes for DCA-induced cytotoxicity. DCA inhibited miR-21 expression in primary rat hepatocytes in a dose-dependent manner, and increased miR-21 pro-apoptotic target programmed cell death 4 (PDCD4) and apoptosis. Both miR-21 overexpression and PDCD4 silencing hampered DCA-induced cell death. Further, DCA decreased NF-κB activity, shown to represent an upstream mechanism leading to modulation of the miR-21/PDCD4 pathway. In fact, NF-κB overexpression or constitutive activation halted miR-21-dependent apoptosis by DCA while opposite results were observed upon NF-κB inhibition. In turn, DCA-induced oxidative stress resulted in caspase-2 activation and NF-κB/miR-21 inhibition, in a PIDD-dependent manner. Finally, modulation of the NF-κB/miR-21/PDCD4 pro-apoptotic pathway by DCA was also shown to occur in the rat liver in vivo. These signalling circuits may constitute appealing targets for bile acid-associated liver pathologies.

No MeSH data available.


Related in: MedlinePlus

DCA inhibits miR-21 expression in primary rat hepatocytes in a dose-dependent manner.Hepatocytes were isolated and plated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 24 h. (A) Real-Time RT-PCR analysis of miR-21 (n = 7). (B) Immunoblotting of PDCD4 (top; n = 7) and ratio between Wt and Mut miR-21 luciferase activity (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. Cells were co-transfected with a reporter vector consisting of a luciferase cDNA fused to the 3′ UTR of PDCD4, containing either a Wt or Mut miR-21 binding site. The cytomegalovirus-Renilla luciferase vector was used as an internal standard control. (C) Cell viability, measured by the ApoTox-GloTM Triplex assay (top; n = 5), cell death measured by the LDH assay (middle; n = 7) and caspase-3/7 activity (bottom; n = 5). (D) Apoptotic cells were detected by Hoechst staining. Representative images of control and 25, 50, 100 and and 200 μM DCA are shown. Bar, 30 μM. Arrows indicate apoptotic nuclei. Results are expressed as mean ± SEM fold change.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664913&req=5

f1: DCA inhibits miR-21 expression in primary rat hepatocytes in a dose-dependent manner.Hepatocytes were isolated and plated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 24 h. (A) Real-Time RT-PCR analysis of miR-21 (n = 7). (B) Immunoblotting of PDCD4 (top; n = 7) and ratio between Wt and Mut miR-21 luciferase activity (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. Cells were co-transfected with a reporter vector consisting of a luciferase cDNA fused to the 3′ UTR of PDCD4, containing either a Wt or Mut miR-21 binding site. The cytomegalovirus-Renilla luciferase vector was used as an internal standard control. (C) Cell viability, measured by the ApoTox-GloTM Triplex assay (top; n = 5), cell death measured by the LDH assay (middle; n = 7) and caspase-3/7 activity (bottom; n = 5). (D) Apoptotic cells were detected by Hoechst staining. Representative images of control and 25, 50, 100 and and 200 μM DCA are shown. Bar, 30 μM. Arrows indicate apoptotic nuclei. Results are expressed as mean ± SEM fold change.

Mentions: We first evaluated whether inhibition of miR-21 by DCA occurs in a dose-dependent manner. Our results show that primary rat hepatocytes incubated with 50 to 200 μM DCA for 24h decreased miR-21 expression between 20 to ~50% (at least p < 0.05) (Fig. 1A), comparing with controls. In the liver, miR-21 is generally associated with proliferative/anti-apoptotic functions, through targeting of PTEN and PDCD416293031. We next evaluated whether inhibition of miR-21 by DCA correlated with an increase in PTEN and/or PDCD4 protein levels. While PTEN expression was not significantly modulated (unpublished observations), DCA increased PDCD4 protein levels up to ~2-fold (at least p < 0.05) (Fig. 1B top). To evaluate whether DCA modulates PDCD4 expression via miR-21, cells were co-transfected with a luciferase reporter construct containing the wild-type miR-21 binding site within the PDCD4 3′UTR (Luc-PDCD4 Wt 3′UTR) or a mutated miR-21 binding site (Luc-PDCD4 Mut 3′UTR)32. In agreement with the previous results, PDCD4 luciferase activity increased up to 2-fold in cells incubated with >50 μM DCA (at least p < 0.05) (Fig. 1B bottom), suggesting that PDCD4 is modulated by DCA via miR-21.


Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocelular apoptosis.

M Rodrigues P, B Afonso M, L Simão A, M Borralho P, M P Rodrigues C, E Castro R - Sci Rep (2015)

DCA inhibits miR-21 expression in primary rat hepatocytes in a dose-dependent manner.Hepatocytes were isolated and plated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 24 h. (A) Real-Time RT-PCR analysis of miR-21 (n = 7). (B) Immunoblotting of PDCD4 (top; n = 7) and ratio between Wt and Mut miR-21 luciferase activity (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. Cells were co-transfected with a reporter vector consisting of a luciferase cDNA fused to the 3′ UTR of PDCD4, containing either a Wt or Mut miR-21 binding site. The cytomegalovirus-Renilla luciferase vector was used as an internal standard control. (C) Cell viability, measured by the ApoTox-GloTM Triplex assay (top; n = 5), cell death measured by the LDH assay (middle; n = 7) and caspase-3/7 activity (bottom; n = 5). (D) Apoptotic cells were detected by Hoechst staining. Representative images of control and 25, 50, 100 and and 200 μM DCA are shown. Bar, 30 μM. Arrows indicate apoptotic nuclei. Results are expressed as mean ± SEM fold change.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664913&req=5

f1: DCA inhibits miR-21 expression in primary rat hepatocytes in a dose-dependent manner.Hepatocytes were isolated and plated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 24 h. (A) Real-Time RT-PCR analysis of miR-21 (n = 7). (B) Immunoblotting of PDCD4 (top; n = 7) and ratio between Wt and Mut miR-21 luciferase activity (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. Cells were co-transfected with a reporter vector consisting of a luciferase cDNA fused to the 3′ UTR of PDCD4, containing either a Wt or Mut miR-21 binding site. The cytomegalovirus-Renilla luciferase vector was used as an internal standard control. (C) Cell viability, measured by the ApoTox-GloTM Triplex assay (top; n = 5), cell death measured by the LDH assay (middle; n = 7) and caspase-3/7 activity (bottom; n = 5). (D) Apoptotic cells were detected by Hoechst staining. Representative images of control and 25, 50, 100 and and 200 μM DCA are shown. Bar, 30 μM. Arrows indicate apoptotic nuclei. Results are expressed as mean ± SEM fold change.
Mentions: We first evaluated whether inhibition of miR-21 by DCA occurs in a dose-dependent manner. Our results show that primary rat hepatocytes incubated with 50 to 200 μM DCA for 24h decreased miR-21 expression between 20 to ~50% (at least p < 0.05) (Fig. 1A), comparing with controls. In the liver, miR-21 is generally associated with proliferative/anti-apoptotic functions, through targeting of PTEN and PDCD416293031. We next evaluated whether inhibition of miR-21 by DCA correlated with an increase in PTEN and/or PDCD4 protein levels. While PTEN expression was not significantly modulated (unpublished observations), DCA increased PDCD4 protein levels up to ~2-fold (at least p < 0.05) (Fig. 1B top). To evaluate whether DCA modulates PDCD4 expression via miR-21, cells were co-transfected with a luciferase reporter construct containing the wild-type miR-21 binding site within the PDCD4 3′UTR (Luc-PDCD4 Wt 3′UTR) or a mutated miR-21 binding site (Luc-PDCD4 Mut 3′UTR)32. In agreement with the previous results, PDCD4 luciferase activity increased up to 2-fold in cells incubated with >50 μM DCA (at least p < 0.05) (Fig. 1B bottom), suggesting that PDCD4 is modulated by DCA via miR-21.

Bottom Line: In fact, NF-κB overexpression or constitutive activation halted miR-21-dependent apoptosis by DCA while opposite results were observed upon NF-κB inhibition.In turn, DCA-induced oxidative stress resulted in caspase-2 activation and NF-κB/miR-21 inhibition, in a PIDD-dependent manner.These signalling circuits may constitute appealing targets for bile acid-associated liver pathologies.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal.

ABSTRACT
MicroRNAs (miRNAs/miRs) are key regulators of liver metabolism, while toxic bile acids participate in the development of several liver diseases. We previously demonstrated that deoxycholic acid (DCA), a cytotoxic bile acid implicated in the pathogenesis of non-alcoholic fatty liver disease, inhibits miR-21 expression in hepatocytes. Here, we investigated the mechanisms by which DCA modulates miR-21 and whether miR-21 contributes for DCA-induced cytotoxicity. DCA inhibited miR-21 expression in primary rat hepatocytes in a dose-dependent manner, and increased miR-21 pro-apoptotic target programmed cell death 4 (PDCD4) and apoptosis. Both miR-21 overexpression and PDCD4 silencing hampered DCA-induced cell death. Further, DCA decreased NF-κB activity, shown to represent an upstream mechanism leading to modulation of the miR-21/PDCD4 pathway. In fact, NF-κB overexpression or constitutive activation halted miR-21-dependent apoptosis by DCA while opposite results were observed upon NF-κB inhibition. In turn, DCA-induced oxidative stress resulted in caspase-2 activation and NF-κB/miR-21 inhibition, in a PIDD-dependent manner. Finally, modulation of the NF-κB/miR-21/PDCD4 pro-apoptotic pathway by DCA was also shown to occur in the rat liver in vivo. These signalling circuits may constitute appealing targets for bile acid-associated liver pathologies.

No MeSH data available.


Related in: MedlinePlus