The long-noncoding RNA MALAT1 regulates TGF-β/Smad signaling through formation of a lncRNA-protein complex with Smads, SETD2 and PPM1A in hepatic cells.

Recent studies have demonstrated the implication of long noncoding RNAs (lncRNAs) in a variety of physiological and pathological processes. However, the majority of lncRNAs are functionally unknown. The current study describes that the lncRNA MALAT1 regulates TGF-β/Smad signaling pathway through formation of a lncRNA-protein complex containing Smads, SETD2 and PPM1A. Our data show that this lncRNA-proteins complex facilitates the dephosphorylation of pSmad2/3 by providing the interaction niche for pSmad2/3 and their specific phosphatase PPM1A, thus terminating TGF-β/Smad signaling in hepatic cells. Based on these mechanistic studies, we performed further experiments to determine whether depletion of MALAT1 would augment cellular TGF-β/Smad signaling. We observed that MALAT1 depletion enhanced TGF-β/Smad signaling response, as reflect by amplification of Smad-mediated differentiation of induced pluripotent stem (iPS) cells to hepatocytes. Our experimental results demonstrate an important role of MALAT1 for regulation of TGF-β/Smad signaling in hepatic cells. Given the diverse functions of TGF-β/Smad pathway in various physiological and pathogenic processes, our results described in the current study will have broad implications for further understanding the role of MALAT1 in TGF-β/Smad pathway in human biology and disease

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

<div><p>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 <i>in vitro</i> (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 <i>Akt1</i> or <i>Akt2</i> 3’-UTR reporter constructs (with or without mutation of miR-150 binding site) established <i>Akt1</i> and <i>Akt2</i> as direct targets of <i>miR-150</i>. Tail vein injection of lentiviral particles containing pre-<i>miR-150</i> 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.</p></div

The levels of miR-150 and Akt in WT and miR-150 KO mice.

<p>(A) Mature miR-150 sequence was aligned among mouse, human, rat, dog and gorilla. (B) miR-150 expression in different tissues from WT and miR-150 KO mice. Total RNA was isolated from eight-week-old male WT and miR-150 KO mice. 1μg of total RNA was used for reverse transcription followed by qRT-PCR analysis. Relative expression of miR-150 was normalized to U6. Data are expressed as mean ± SD. (C) The levels of miR-150 and miR-221 in WT and miR-150 KO livers with or without Jo2 treatment. WT and miR-150 KO mice were intraperitoneally injected with or without 0.5 μg/g of body weight Jo2 (n = 6–8 per group). The livers were harvested 4 hours after Jo2 injection. The levels of miR-150 and miR-221 in the livers were determined by qRT-PCR (Data are expressed as mean ± SD, *<i>p</i><0.05). (D) Western blot for Akt1, Akt 2, total Akt, p-Akt(Ser473), 4E-BP1 and p-4E-BP1(Thr37/46) (with GAPDH as the loading control). The blots shown in this figure were from two individual mice for each group. Quantifications of relative protein levels are shown at the right panel (the data are expressed as mean ± SD, *<i>p</i><0.05).</p

miR-150 targets <i>Akt1</i> and <i>Akt2</i> in hepatocyte.

<p>(A) Primary hepatocytes isolated from WT mice and miR-150 KO mice were treated with Jo2 (0.5 μg/mL) for 4 hours and the cell lysates were subjected to Western blotting for Akt1, Akt2 and total Akt. Quantifications of relative Akt1, Akt 2 and total Akt protein levels are shown at the right panel. Data are expressed as mean ± SD *<i>p</i><0.05. (B) The 3’-UTRs of <i>Akt1</i> and <i>Akt2</i> contain predicted miR-150 binding sites. Mutations were generated on the potential targets sequences as indicated. (C) Luciferase reporter assay. Hepatocytes were isolated from WT mice and cotransfected with miR-150 mimic plus the reporter plasmid containing the 3’-UTR of <i>Akt1</i>, <i>Akt2</i> or their mutants. The experiments were repeated three times. Data are expressed as mean ± SD, **<i>p</i><0.01.</p

The levels of miR-150 and Akt in WT and miR-150 KO mice.

<p>(A) Mature miR-150 sequence was aligned among mouse, human, rat, dog and gorilla. (B) miR-150 expression in different tissues from WT and miR-150 KO mice. Total RNA was isolated from eight-week-old male WT and miR-150 KO mice. 1μg of total RNA was used for reverse transcription followed by qRT-PCR analysis. Relative expression of miR-150 was normalized to U6. Data are expressed as mean ± SD. (C) The levels of miR-150 and miR-221 in WT and miR-150 KO livers with or without Jo2 treatment. WT and miR-150 KO mice were intraperitoneally injected with or without 0.5 μg/g of body weight Jo2 (n = 6–8 per group). The livers were harvested 4 hours after Jo2 injection. The levels of miR-150 and miR-221 in the livers were determined by qRT-PCR (Data are expressed as mean ± SD, *<i>p</i><0.05). (D) Western blot for Akt1, Akt 2, total Akt, p-Akt(Ser473), 4E-BP1 and p-4E-BP1(Thr37/46) (with GAPDH as the loading control). The blots shown in this figure were from two individual mice for each group. Quantifications of relative protein levels are shown at the right panel (the data are expressed as mean ± SD, *<i>p</i><0.05).</p

Effect of Akt inhibitor V on Fas-induced liver injury and hepatocyte apoptosis.

<p>(A) WT and miR-150 KO mice were injected intraperitoneally with vehicle or Akt inhibitor V (1mg/kg of body weight) 30 minutes before PBS administration (n = 3 each group). Mice were sacrificed 3 hours after PBS administration. Western blot showed that Akt inhibitor V treatment inhibited the phosphorylation of Akt. (B) WT and miR-150 KO mice were injected intraperitoneally with vehicle or Akt inhibitor V (1mg/kg of body weight) 30 minutes before Jo2 (0.5μg/g body weight) administration (n = 6 each group). Mice were sacrificed 3 hours after Jo2 administration. H&E staining (100×, scale bar 20μm) of formalin-fixed, paraffin-embedded liver tissues. (C) Serum levels of ALT and AST in mice with or without Akt inhibitor V pretreatment (data are expressed as mean ± SD **<i>p</i><0.01). (D) TUNEL staining of the liver tissues from WT and miR-150 KO mice 3 hours after Jo2 injection (with or without Akt inhibitor V pretreatment). Representative photographs (100×, scale bar 20μm) are shown at the left panel; quantification of TUNEL-positive cells is shown at the right panel (the data are expressed as mean ± SD; *<i>p</i><0.05, **<i>p</i><0.01). (E) Immunostaining for cleaved caspase-3 in liver tissues from mice with or without Akt inhibitor V pretreatment (100×, scale bar 20μm). Quantification of cleaved caspase-3 positive cells is shown at the right panel (the data are expressed as mean ± SD; *<i>p</i><0.05, **<i>p</i><0.01). (F) Caspase-3/7, caspase-8, and caspase-9 activities in liver tissue from mice with or without Akt inhibitor V pretreatment (the data are expressed as mean ± SD; *<i>p</i><0.05, **<i>p</i><0.01).</p

miR-150 deficiency protects against Fas-induced caspase and PARP cleavage.

<p>WT and miR-150 KO mice were intraperitoneally injected with 0.5μg/g of body weight Jo2 (n = 6 or 8 per group, respectively). The animals were sacrificed 0, 2 and 4 hours after Jo2 injection. (A) Caspase activity in liver tissue 4 hours after Jo2 injection. The levels of caspase-3/7, caspase-8, and caspase-9 activities in miR-150 KO livers were significantly lower compared to the WT livers (the data are expressed as mean ± SD) **<i>p</i><0.01. (B) Western blotting analysis to detect PARP and caspases cleavage. (C) Formalin-fixed and paraffin-embedded sections (5 μm thick) were stained with antibody against cleaved caspase-3 (100×, scale bar 20μm). miR-150 KO livers showed fewer numbers of caspase- 3-positive hepatocytes compared to the WT livers (4 hours after Jo2 treatment). Quantification of cleaved caspase-3 positive hepatocytes is shown at the right panel (data are expressed as mean ± SD, **<i>p</i><0.01).</p

miR-150 KO fails to protect mice against LPS/D-GalN induced liver injury.

<p>(A) WT and miR-150 KO mice were intraperitoneally injected with LPS/GalN (30ng/g body weight LPS in combination with 800 μg/g body weight of D-GalN) (n = 12 per group). Survival rates are shown after LPS/GalN treatment. There was no difference between these two groups. (B) H&E staining (100×, scale bar 20μm). WT and miR-150 KO mice were intraperitoneally injected with LPS/GalN (30ng/g body weight LPS in combination with 800 μg/g body weight of D-GalN) (n = 6 per group). Mice were sacrificed 0 and 4 hours after LPS/D-GalN injection. (C) Serum levels of ALT and AST at 4 hours after LPS/D-GalN treatment. Data expressed as mean ±SD.</p