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miR-150 Deficiency Protects against FAS-Induced Acute Liver Injury in Mice through Regulation of AKT.

Chen W, Han C, Zhang J, Song K, Wang Y, Wu T - PLoS ONE (2015)

Bottom Line: Although miR-150 is implicated in the regulation of immune cell differentiation and activation, it remains unknown whether miR-150 is involved in liver biology and disease.Luciferase reporter assays in hepatocytes transfected with the Akt1 or Akt2 3'-UTR reporter constructs (with or without mutation of miR-150 binding site) established Akt1 and Akt2 as direct targets of miR-150.Tail vein injection of lentiviral particles containing pre-miR-150 enhanced Jo2-induced liver injury in miR-150 KO mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Tulane University School of Medicine,1430 Tulane Avenue SL-79, New Orleans, Louisiana, United States of America.

ABSTRACT
Although miR-150 is implicated in the regulation of immune cell differentiation and activation, it remains unknown whether miR-150 is involved in liver biology and disease. This study was performed to explore the potential role of miR-150 in LPS/D-GalN and Fas-induced liver injuries by using wild type and miR-150 knockout (KO) mice. Whereas knockout of miR-150 did not significantly alter LPS/D-GalN-induced animal death and liver injury, it protected against Fas-induced liver injury and mortality. The Jo2-induced increase in serum transaminases, apoptotic hepatocytes, PARP cleavage, as well as caspase-3/7, caspase-8, and caspase-9 activities were significantly attenuated in miR-150 KO mice. The liver tissues from Jo2-treated miR-150 KO mice expressed higher levels of Akt1, Akt2, total Akt, as well as p-Akt(Ser473) compared to the wild type livers. Pretreatment with the Akt inhibitor V reversed Jo2-induced liver injury in miR-150 KO mice. The primary hepatocytes isolated from miR-150 KO mice also showed protection against Fas-induced apoptosis in vitro (characterized by less prominent PARP cleavage, less nuclear fragmentation and less caspase activation) in comparison to hepatocytes from wild type mice. Luciferase reporter assays in hepatocytes transfected with the Akt1 or Akt2 3'-UTR reporter constructs (with or without mutation of miR-150 binding site) established Akt1 and Akt2 as direct targets of miR-150. Tail vein injection of lentiviral particles containing pre-miR-150 enhanced Jo2-induced liver injury in miR-150 KO mice. These findings demonstrate that miR-150 deficiency prevents Fas-induced hepatocyte apoptosis and liver injury through regulation of the Akt pathway.

No MeSH data available.


Related in: MedlinePlus

The role of miR-150 in Fas-induced primary hepatocyte apoptosis.Primary hepatocytes were isolated from eight-week-old male WT mice and miR-150 KO mice. (A) The expression of miR-150 in hepatocytes was quantified by qRT-PCR. Data are expressed as mean ± SD. (B) Hepatocytes were treated with Jo2 (0.5 μg/mL) plus CHX (10 μg/mL) for 4 hours. The cell lysates were obtained for Western blotting to detect PARP cleavage. (C) Representative Hoechst staining (200×) of WT and miR-150 KO hepatocytes 0 and 4 hours after treatment with 0.5 μg/mL Jo2 plus 10 μg/mL CHX. Arrows indicate fragmented nucleus. (D) Quantitative analysis for apoptotic cells under Hoechst staining. Data are expressed as mean ± SD, *p<0.05. (E) Cell viability as assessed by trypan blue exclusion 4 hours after treatment with 0.5 μg/mL Jo2 plus 10 μg/mL CHX or with an equal volume of 1×PBS plus DMSO as control (Con). The data are expressed as mean ± SD, *p<0.05. (F) Caspase-3/7, caspase-8, caspase-9 activities in WT and miR-150 KO hepatocytes 4 hours after treatment with 0.5 μg/mL Jo2 plus 10 μg/mL CHX or with an equal volume of 1×PBS plus DMSO as control (Con). The results are expressed as mean ± SD of fold changes over WT hepatocytes.
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pone.0132734.g006: The role of miR-150 in Fas-induced primary hepatocyte apoptosis.Primary hepatocytes were isolated from eight-week-old male WT mice and miR-150 KO mice. (A) The expression of miR-150 in hepatocytes was quantified by qRT-PCR. Data are expressed as mean ± SD. (B) Hepatocytes were treated with Jo2 (0.5 μg/mL) plus CHX (10 μg/mL) for 4 hours. The cell lysates were obtained for Western blotting to detect PARP cleavage. (C) Representative Hoechst staining (200×) of WT and miR-150 KO hepatocytes 0 and 4 hours after treatment with 0.5 μg/mL Jo2 plus 10 μg/mL CHX. Arrows indicate fragmented nucleus. (D) Quantitative analysis for apoptotic cells under Hoechst staining. Data are expressed as mean ± SD, *p<0.05. (E) Cell viability as assessed by trypan blue exclusion 4 hours after treatment with 0.5 μg/mL Jo2 plus 10 μg/mL CHX or with an equal volume of 1×PBS plus DMSO as control (Con). The data are expressed as mean ± SD, *p<0.05. (F) Caspase-3/7, caspase-8, caspase-9 activities in WT and miR-150 KO hepatocytes 4 hours after treatment with 0.5 μg/mL Jo2 plus 10 μg/mL CHX or with an equal volume of 1×PBS plus DMSO as control (Con). The results are expressed as mean ± SD of fold changes over WT hepatocytes.

Mentions: Hepatocytes are sensitive to Fas-mediated apoptosis due to their high constitutive expression of Fas[36]. To determine the contribution of hepatocyte miR-150 in Fas-mediated apoptosis, primary hepatocytes were isolated from WT mice and miR-150 KO mice; the cultured hepatocytes were treated with Jo2 plus CHX to determine parameters of apoptosis. As expected, qRT-PCR analysis showed that miR-150 is expressed in the hepatocytes isolated from the wild type mice, but not in hepatocytes isolated from miR-150 KO mice (Fig 6A). Jo2 treatment induced less prominent PARP cleavage in miR-150 KO hepatocytes compared to wild type hepatocytes (Fig 6B). Likewise, Jo2 treatment induced less prominent nuclear fragmentation in miR-150 KO hepatocytes compared to wild type hepatocytes (analyzed by Hoechst staining, Fig 6C and 6D). The viability of hepatocytes was higher in miR-150 KO hepatocytes than in the WT hepatocytes after Jo2 treatment (Fig 6E). Furthermore, Jo2-induced increase of caspase-3/7, caspase-8, caspase-9 activities was less prominent in miR-150 KO hepatocytes compared to wild type hepatocytes (Fig 6F). These findings provide direct evidence for miR-150 in hepatocytes for protection against Fas-induced apoptosis.


miR-150 Deficiency Protects against FAS-Induced Acute Liver Injury in Mice through Regulation of AKT.

Chen W, Han C, Zhang J, Song K, Wang Y, Wu T - PLoS ONE (2015)

The role of miR-150 in Fas-induced primary hepatocyte apoptosis.Primary hepatocytes were isolated from eight-week-old male WT mice and miR-150 KO mice. (A) The expression of miR-150 in hepatocytes was quantified by qRT-PCR. Data are expressed as mean ± SD. (B) Hepatocytes were treated with Jo2 (0.5 μg/mL) plus CHX (10 μg/mL) for 4 hours. The cell lysates were obtained for Western blotting to detect PARP cleavage. (C) Representative Hoechst staining (200×) of WT and miR-150 KO hepatocytes 0 and 4 hours after treatment with 0.5 μg/mL Jo2 plus 10 μg/mL CHX. Arrows indicate fragmented nucleus. (D) Quantitative analysis for apoptotic cells under Hoechst staining. Data are expressed as mean ± SD, *p<0.05. (E) Cell viability as assessed by trypan blue exclusion 4 hours after treatment with 0.5 μg/mL Jo2 plus 10 μg/mL CHX or with an equal volume of 1×PBS plus DMSO as control (Con). The data are expressed as mean ± SD, *p<0.05. (F) Caspase-3/7, caspase-8, caspase-9 activities in WT and miR-150 KO hepatocytes 4 hours after treatment with 0.5 μg/mL Jo2 plus 10 μg/mL CHX or with an equal volume of 1×PBS plus DMSO as control (Con). The results are expressed as mean ± SD of fold changes over WT hepatocytes.
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pone.0132734.g006: The role of miR-150 in Fas-induced primary hepatocyte apoptosis.Primary hepatocytes were isolated from eight-week-old male WT mice and miR-150 KO mice. (A) The expression of miR-150 in hepatocytes was quantified by qRT-PCR. Data are expressed as mean ± SD. (B) Hepatocytes were treated with Jo2 (0.5 μg/mL) plus CHX (10 μg/mL) for 4 hours. The cell lysates were obtained for Western blotting to detect PARP cleavage. (C) Representative Hoechst staining (200×) of WT and miR-150 KO hepatocytes 0 and 4 hours after treatment with 0.5 μg/mL Jo2 plus 10 μg/mL CHX. Arrows indicate fragmented nucleus. (D) Quantitative analysis for apoptotic cells under Hoechst staining. Data are expressed as mean ± SD, *p<0.05. (E) Cell viability as assessed by trypan blue exclusion 4 hours after treatment with 0.5 μg/mL Jo2 plus 10 μg/mL CHX or with an equal volume of 1×PBS plus DMSO as control (Con). The data are expressed as mean ± SD, *p<0.05. (F) Caspase-3/7, caspase-8, caspase-9 activities in WT and miR-150 KO hepatocytes 4 hours after treatment with 0.5 μg/mL Jo2 plus 10 μg/mL CHX or with an equal volume of 1×PBS plus DMSO as control (Con). The results are expressed as mean ± SD of fold changes over WT hepatocytes.
Mentions: Hepatocytes are sensitive to Fas-mediated apoptosis due to their high constitutive expression of Fas[36]. To determine the contribution of hepatocyte miR-150 in Fas-mediated apoptosis, primary hepatocytes were isolated from WT mice and miR-150 KO mice; the cultured hepatocytes were treated with Jo2 plus CHX to determine parameters of apoptosis. As expected, qRT-PCR analysis showed that miR-150 is expressed in the hepatocytes isolated from the wild type mice, but not in hepatocytes isolated from miR-150 KO mice (Fig 6A). Jo2 treatment induced less prominent PARP cleavage in miR-150 KO hepatocytes compared to wild type hepatocytes (Fig 6B). Likewise, Jo2 treatment induced less prominent nuclear fragmentation in miR-150 KO hepatocytes compared to wild type hepatocytes (analyzed by Hoechst staining, Fig 6C and 6D). The viability of hepatocytes was higher in miR-150 KO hepatocytes than in the WT hepatocytes after Jo2 treatment (Fig 6E). Furthermore, Jo2-induced increase of caspase-3/7, caspase-8, caspase-9 activities was less prominent in miR-150 KO hepatocytes compared to wild type hepatocytes (Fig 6F). These findings provide direct evidence for miR-150 in hepatocytes for protection against Fas-induced apoptosis.

Bottom Line: Although miR-150 is implicated in the regulation of immune cell differentiation and activation, it remains unknown whether miR-150 is involved in liver biology and disease.Luciferase reporter assays in hepatocytes transfected with the Akt1 or Akt2 3'-UTR reporter constructs (with or without mutation of miR-150 binding site) established Akt1 and Akt2 as direct targets of miR-150.Tail vein injection of lentiviral particles containing pre-miR-150 enhanced Jo2-induced liver injury in miR-150 KO mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Tulane University School of Medicine,1430 Tulane Avenue SL-79, New Orleans, Louisiana, United States of America.

ABSTRACT
Although miR-150 is implicated in the regulation of immune cell differentiation and activation, it remains unknown whether miR-150 is involved in liver biology and disease. This study was performed to explore the potential role of miR-150 in LPS/D-GalN and Fas-induced liver injuries by using wild type and miR-150 knockout (KO) mice. Whereas knockout of miR-150 did not significantly alter LPS/D-GalN-induced animal death and liver injury, it protected against Fas-induced liver injury and mortality. The Jo2-induced increase in serum transaminases, apoptotic hepatocytes, PARP cleavage, as well as caspase-3/7, caspase-8, and caspase-9 activities were significantly attenuated in miR-150 KO mice. The liver tissues from Jo2-treated miR-150 KO mice expressed higher levels of Akt1, Akt2, total Akt, as well as p-Akt(Ser473) compared to the wild type livers. Pretreatment with the Akt inhibitor V reversed Jo2-induced liver injury in miR-150 KO mice. The primary hepatocytes isolated from miR-150 KO mice also showed protection against Fas-induced apoptosis in vitro (characterized by less prominent PARP cleavage, less nuclear fragmentation and less caspase activation) in comparison to hepatocytes from wild type mice. Luciferase reporter assays in hepatocytes transfected with the Akt1 or Akt2 3'-UTR reporter constructs (with or without mutation of miR-150 binding site) established Akt1 and Akt2 as direct targets of miR-150. Tail vein injection of lentiviral particles containing pre-miR-150 enhanced Jo2-induced liver injury in miR-150 KO mice. These findings demonstrate that miR-150 deficiency prevents Fas-induced hepatocyte apoptosis and liver injury through regulation of the Akt pathway.

No MeSH data available.


Related in: MedlinePlus