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

Figures

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  Fig 1. MALAT1 binds to pSmad2/3 specifically. A, RNA immunoprecipitation assays of multiple lncRNAs with antibodies targeting Smad2, Smad3, pSmad2 or pSmad3 in Hep3B treated with TGF-β1 for 1 hour. The precipitated lncRNAs were assessed by qRT-PCR. B, RNA immunoprecipitation assays of MALAT1 with indicated antibodies in Hep3B, PLC/PRF/5 and SK-Hep1 cells treated with TGF-β1 for 1 hour. The precipitated lncRNA was assessed by qRT-PCR. C, Co-localization of pSmad2 or pSmad3 with MALAT1 in the nuclei of Hep3B cells as demonstrated by FISH in combination with IF staining (scale bar 10 μm). All data are shown as means ± SD (n = 3).

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  Fig 2. SETD2 mediates the binding of MALAT1 and pSmads. A, Smad2 and Smad3 bind to SETD2 protein. Hep3B cells co-transfected with HA-SETD2 and flag-Smad2/3 expression plasmids were treated with TGF-β1 for 1 hour. Cells were harvested and subjected to immunoprecipitation assays. B, RNA immunoprecipitation of MALAT1 by using anti-Smad2/3 or anti-SETD2 antibodies in Hep3B cells (with siSETD2 or siControl transfection) treated with 5ng/mL TGF-β1 or vehicle for 2 hours. The precipitated MALAT1 was assessed by qRT-PCR. C. qRT-PCR results indicated that SETD2 knockdown did not affect MALAT1 level in Hep3B cells. The data are presented as means ± SD (n = 3; **p < 0.01; ns, no statistical significance). D, Smad2 linker region mutations decrease its capability to form protein complex with PPM1A and SETD2 proteins. PLC/PRF/5 cells co-transfected with SETD2 and flag-Smad2 or flag-Smad2EPSM (T220V, S245A, S250A and S255A, Erk/Pro-directed kinase Site Mutated Smad2) expression plasmids were incubated with TGF-β1 for 1 hour. The treated cells were then harvested and subjected to immunoprecipitation and immunoblotting with indicated antibodies.

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  Fig 3. Depletion of MALAT1 enhances the effect of TGF-β. A, The levels of MALAT1 were assessed by FISH (upper) and qRT-PCR (lower) in Hep3B, SK-Hep1 and PLC/PRF/5 cells stably transfected with pGFP-V-RS-shMALAT1 or scramble control plasmid (scale bar 10 μm). B, Smad reporter activity in Hep3B, SK-Hep1 and PLC/PRF/5 cells with or without MALAT1 depletion. Twenty-four hours after transfection with p3TP-lux reporter plasmid, MALAT1 depleted and control cells were stimulated with 5ng/ml TGF-β1 or vehicle for additional 24 hours. The cell lysates were obtained for dual-luciferase activity assay. The data are presented as means ± SD (n = 3; *p < 0.05, **p < 0.01). C-E, DNA pull-down assay. Equal amount of cell lysates from MALAT1 depleted or control Hep3B (C), PLC/PRF/5 (D) or SK-Hep1 (E) cells stimulated with TGF-β1 (5 ng/ml for 2 hours) were pulled down with biotinylated SBE (Smads binding elements) DNA probe[40, 41], which were followed by immunoblotting with indicated antibodies. F-H, Cell proliferation assay. Equal numbers of MALAT1 depleted or control Hep3B (F), PLC/PRF/5 (G) or SK-Hep1 (H) cells were seeded into 96-well plates. The cells were treated with TGF-β1 (1 ng/ml) or vehicle…

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  Fig 4. Depletion of MALAT1 reduces Smad2/3 dephosphorylation. A, B, Time course of Smad3 phosphorylation. MALAT1 depleted or control Hep3B cells were treated with 5 ng/ml of TGF-β1 for the indicated time periods. Phospho-Smad3 and total Smad3 were analyzed by Western blotting. Line graph demonstrates relative intensity ratio of phospho-Smad3 to total Smad3, the relative level of phospho-Smad3 at 1-hour time point was set as 1. C-H, Time course of Smad2 dephosphorylation in cells post TGF-β1 treatment. MALAT1 depleted or control Hep3B cells (C, D), MALAT1 depleted or control PLC/PRF/5 cells (E, F) and SETD2 knockdown and control Hep3B cells (G, H) were treated with 5 ng/ml of TGF-β1 for 1 hour, followed by TGF-β1 washout and simultaneous addition of 10 μM TGF-βR Inhibitor SB431542 and 10 μM proteasome inhibitor MG-132. The line graphs (D, F and H) show relative ratio of pSmad2 level over total Smad2 (the value at time point 0 was set as 1). The band intensities from representative Western blot results were quantified by Odyssey software (LI-COR Bio, Lincoln NE).

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  Fig 5. MALAT1 facilitates the SETD2-pSmad2/3-PPM1A complex formation. A-C, MALAT1 depletion prevents SETD2-pSmad2/3-PPM1A complex formation. MALAT1 depleted or control Hep3B (A) or PLC/PRF/5 (B, C) cells were transfected with flag-Smad2/3, PPM1A or SETD2 expression plasmids (as indicated at the top of each panel) and the cells were treated with TGF-β1 for 1–2 hours. The cell lysates were then subjected to IP and Western blotting analyses using indicated antibodies. D, SETD2 depletion prevents pSmad2/3 binding to PPM1A. Cell lysates from SETD2-depleted and control PLC/PRF/5 cells (co-transfected with PPM1A and flag-Smad3 expression plasmids) were subjected to co-immunoprecipitation using anti-flag antibody followed by Western blotting for SETD2 and PPM1A. E, RNA immunoprecipitation of MALAT1 by using anti-PPM1A antibodies in Hep3B cells with or without TGF-β1 treatment (5ng/ml for 1 hour); precipitated MALAT1 was assessed by qRT-PCR. The data are shown as mean ± SD (n = 3; ns, no statistical significance). F, Co-immunoprecipitation of PPM1A and SETD2 in PLC/PRF/5 cells (co-transfected with SETD2 and PPM1A expression plasmids) with or without TGF-β1 treatment. G, A diagram illustrating…

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  Fig 6. MALAT1 depletion enhances TGF-β- and Activin A-induced iPS cell differentiation to hepatocytes. A, A schematic representation of three-step induction from iPS cells to hepatocyte-like cells. B, Immunofluorescence staining for iPS cell markers (OCT-4A, SOX2, MYC) and hepatocyte markers (ALB, AFP, HNF-4a and TAT) before and after hepatic lineage induction (scale bar = 1mm). C and D, Quantitative RT-PCR analyses for the hepatocyte markers ALB, AFP, HNF-4a, TAT, CK-18, CYP3A4, CYP7A1, G-6P, and TDO2 in cells with or without MALAT1 depletion. iPS cells were transfected with MALAT1-specific 2’-OME-gamper ASO (phosphorothioated 2' O-methyl modified antisense oligonucleotide) or scramble control ASO; 24 hours later the cells were treated with Activin A (C) or TGF-β (D) for hepatocytes lineage induction as described in the Methods section. The data are shown as mean ± SD (n = 3).