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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-induced oxidative stress contributes for miR-21 inhibition and apoptosis.Cells were isolated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 16 h (A) Dose-dependent modulation of ROS levels by DCA (n = 5). (B) ROS levels (n = 3). Cells were pre-treated with 5 mM NAC for 1 h and then incubated with 100 μM DCA for 24 h (or no addition). (C) Cell viability (top) and caspase-3/7 activity (bottom) measured by the ApoTox-GloTM Triplex assay (n = 5). (D) Real-Time RT-PCR analysis of miR-21 (n = 5). Results are expressed as mean ± SEM fold change.
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f6: DCA-induced oxidative stress contributes for miR-21 inhibition and apoptosis.Cells were isolated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 16 h (A) Dose-dependent modulation of ROS levels by DCA (n = 5). (B) ROS levels (n = 3). Cells were pre-treated with 5 mM NAC for 1 h and then incubated with 100 μM DCA for 24 h (or no addition). (C) Cell viability (top) and caspase-3/7 activity (bottom) measured by the ApoTox-GloTM Triplex assay (n = 5). (D) Real-Time RT-PCR analysis of miR-21 (n = 5). Results are expressed as mean ± SEM fold change.

Mentions: DCA induces oxidative stress and DNA damage in hepatocytes8394041. In our model, primary rat hepatocytes incubated with 25–200 μM DCA for 16 h increased ROS production in a dose-dependent manner up to ~3.4-fold (p < 0.01) (Fig. 6A). Of note, NF-κB activity is modulated in response to oxidative stress2742. To investigate whether triggering of oxidative stress by DCA represents an NF-κB and/or miR-21 upstream mechanism, cells were incubated with NAC, a well-known antioxidant, in the presence or absence of DCA. NAC treatment almost completely abrogated DCA-induced ROS production (p < 0.05) (Fig. 6B) and -inhibition of cell viability (p < 0.05) (Fig. 6C top), while DCA-induced caspase-3 activity was no longer significant (Fig. 6C bottom). Importantly, DCA-inhibition of miR-21 was also completely abrogated by NAC (Fig. 6D), highlighting the role of oxidative stress during modulation of miR-21 by DCA.


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-induced oxidative stress contributes for miR-21 inhibition and apoptosis.Cells were isolated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 16 h (A) Dose-dependent modulation of ROS levels by DCA (n = 5). (B) ROS levels (n = 3). Cells were pre-treated with 5 mM NAC for 1 h and then incubated with 100 μM DCA for 24 h (or no addition). (C) Cell viability (top) and caspase-3/7 activity (bottom) measured by the ApoTox-GloTM Triplex assay (n = 5). (D) Real-Time RT-PCR analysis of miR-21 (n = 5). Results are expressed as mean ± SEM fold change.
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Related In: Results  -  Collection

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f6: DCA-induced oxidative stress contributes for miR-21 inhibition and apoptosis.Cells were isolated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 16 h (A) Dose-dependent modulation of ROS levels by DCA (n = 5). (B) ROS levels (n = 3). Cells were pre-treated with 5 mM NAC for 1 h and then incubated with 100 μM DCA for 24 h (or no addition). (C) Cell viability (top) and caspase-3/7 activity (bottom) measured by the ApoTox-GloTM Triplex assay (n = 5). (D) Real-Time RT-PCR analysis of miR-21 (n = 5). Results are expressed as mean ± SEM fold change.
Mentions: DCA induces oxidative stress and DNA damage in hepatocytes8394041. In our model, primary rat hepatocytes incubated with 25–200 μM DCA for 16 h increased ROS production in a dose-dependent manner up to ~3.4-fold (p < 0.01) (Fig. 6A). Of note, NF-κB activity is modulated in response to oxidative stress2742. To investigate whether triggering of oxidative stress by DCA represents an NF-κB and/or miR-21 upstream mechanism, cells were incubated with NAC, a well-known antioxidant, in the presence or absence of DCA. NAC treatment almost completely abrogated DCA-induced ROS production (p < 0.05) (Fig. 6B) and -inhibition of cell viability (p < 0.05) (Fig. 6C top), while DCA-induced caspase-3 activity was no longer significant (Fig. 6C bottom). Importantly, DCA-inhibition of miR-21 was also completely abrogated by NAC (Fig. 6D), highlighting the role of oxidative stress during modulation of miR-21 by DCA.

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