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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

mir-21 overexpression counteracts DCA-induced apoptosis.Primary rat hepatocytes were transfected with a miR-21 precursor (Pre-miR-21) or control (Pre-miR-C) and treated with 100 μM DCA or no addition for 24 h. (A) Real-Time RT-PCR analysis of miR-21 expression (n = 7). (B) Immunoblotting of PDCD4 (n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. (C) Cell viability (top), cytotoxicity (middle) and caspase-3/7 activity (bottom) measured by the ApoToxGloTM Triplex assay (n = 5). Results are expressed as mean ± SEM fold change.
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f2: mir-21 overexpression counteracts DCA-induced apoptosis.Primary rat hepatocytes were transfected with a miR-21 precursor (Pre-miR-21) or control (Pre-miR-C) and treated with 100 μM DCA or no addition for 24 h. (A) Real-Time RT-PCR analysis of miR-21 expression (n = 7). (B) Immunoblotting of PDCD4 (n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. (C) Cell viability (top), cytotoxicity (middle) and caspase-3/7 activity (bottom) measured by the ApoToxGloTM Triplex assay (n = 5). Results are expressed as mean ± SEM fold change.

Mentions: To evaluate the functional relevance of miR-21 during DCA-induced cell death, we transfected primary rat hepatocytes with a miR-21 precursor, both in the presence or absence of DCA. As expected, miR-21 was significantly overexpressed in cells transfected with Pre-miR-21 (p < 0.001), when compared to cells transfected with Pre-miR-Control (Fig. 2A). In Pre-miR-Control-transfected cells, DCA inhibited miR-21 expression by 60% (p < 0.01). DCA also counteracted miR-21 overexpression in Pre-miR-21-transfected cells, although to a lesser extent. miR-21 overexpression alone reduced PDCD4 levels by almost 40% (p < 0.01) (Fig. 2B). In these conditions, the ability of DCA to increase PDCD4 protein expression was significantly compromised, comparing with DCA-treated Pre-miR-Control cells. As previously, no significant changes were observed in PTEN protein levels following miR-21 overexpression (unpublished observations), further substantiating PDCD4 as a prime target modulated by DCA via miR-21 in primary rat hepatocytes. Surprisingly, although miR-21 overexpression alone significantly increased cell viability by ~20% (p < 0.05) (Fig. 2C top), effects on cytotoxicity (Fig. 2C middle) and caspase-3 activity (Fig. 2C bottom) were almost absent. Nevertheless, miR-21 overexpression almost completely abrogated DCA-mediated reduction in cellular viability; increase in cytotoxicity; and caspase-3 activation. These results further confirm that the miR-21/PDCD4 pathway mediates DCA-induced apoptosis.


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)

mir-21 overexpression counteracts DCA-induced apoptosis.Primary rat hepatocytes were transfected with a miR-21 precursor (Pre-miR-21) or control (Pre-miR-C) and treated with 100 μM DCA or no addition for 24 h. (A) Real-Time RT-PCR analysis of miR-21 expression (n = 7). (B) Immunoblotting of PDCD4 (n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. (C) Cell viability (top), cytotoxicity (middle) and caspase-3/7 activity (bottom) measured by the ApoToxGloTM Triplex assay (n = 5). Results are expressed as mean ± SEM fold change.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: mir-21 overexpression counteracts DCA-induced apoptosis.Primary rat hepatocytes were transfected with a miR-21 precursor (Pre-miR-21) or control (Pre-miR-C) and treated with 100 μM DCA or no addition for 24 h. (A) Real-Time RT-PCR analysis of miR-21 expression (n = 7). (B) Immunoblotting of PDCD4 (n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. (C) Cell viability (top), cytotoxicity (middle) and caspase-3/7 activity (bottom) measured by the ApoToxGloTM Triplex assay (n = 5). Results are expressed as mean ± SEM fold change.
Mentions: To evaluate the functional relevance of miR-21 during DCA-induced cell death, we transfected primary rat hepatocytes with a miR-21 precursor, both in the presence or absence of DCA. As expected, miR-21 was significantly overexpressed in cells transfected with Pre-miR-21 (p < 0.001), when compared to cells transfected with Pre-miR-Control (Fig. 2A). In Pre-miR-Control-transfected cells, DCA inhibited miR-21 expression by 60% (p < 0.01). DCA also counteracted miR-21 overexpression in Pre-miR-21-transfected cells, although to a lesser extent. miR-21 overexpression alone reduced PDCD4 levels by almost 40% (p < 0.01) (Fig. 2B). In these conditions, the ability of DCA to increase PDCD4 protein expression was significantly compromised, comparing with DCA-treated Pre-miR-Control cells. As previously, no significant changes were observed in PTEN protein levels following miR-21 overexpression (unpublished observations), further substantiating PDCD4 as a prime target modulated by DCA via miR-21 in primary rat hepatocytes. Surprisingly, although miR-21 overexpression alone significantly increased cell viability by ~20% (p < 0.05) (Fig. 2C top), effects on cytotoxicity (Fig. 2C middle) and caspase-3 activity (Fig. 2C bottom) were almost absent. Nevertheless, miR-21 overexpression almost completely abrogated DCA-mediated reduction in cellular viability; increase in cytotoxicity; and caspase-3 activation. These results further confirm that the miR-21/PDCD4 pathway mediates DCA-induced apoptosis.

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