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.


Cells proliferation WST1 assay
Cells were cultured in serum-free medium for 24 hours to synchronize cell cycle. 2×10 3 cells were seeded into each well of 96-well plates in 200μl culture medium containing 1% fetal bovine serum. The medium was removed at indicated time points; 90μl serum-free medium mixed with 10μl WST-1 (Roche) was added to each well. Four hours after incubation, absorbance of each well was measured by an ELISA plate reader at wavelength of 450nm.

Luciferase activity assay
Twenty-four hours after p3TP-lux plasmid (Addgene) transfection, cells were incubated with TGF-β1 (5ng/mL) for additional 24 hours. The cells were then collected and analyzed by using the Dual-Luciferase reporter assay system (Promega, Madison, WI) as per user manual.
Luciferase activity was measured by centro XS3LB960 microplate fluorescence reader (Berthold Tech, Ontario Canada). The pRL-TK plasmid with constitutive expression of Renilla luciferase was co-transfected with p3TP-lux as internal control.

Quantitative real-time PCR
Total RNA was prepared using Trizol reagent (Invitrogen). Reverse-transcription and quantitative PCR were performed by using SuperScript II Reverse Transcriptase (Invitrogen) and SYBR Green Supermix reagent (Qiagen, Valencia, CA).

Immunoprecipitation (IP)
The cells cultured in 100-mm dishes were fixed by 1% paraformaldehyde and then lysed in 1 ml RIPA buffer (1% Triton X-100, 0.1% sodium dodecyl sulfate, 50mM Tris-HCl pH 7.4, 150mM sodium chloride, 0.5% sodium deoxycholate, 1mM EDTA) containing phosphatase and protease cocktail inhibitors. Pre-cleared supernatants were rotated with 1μg antibodies and 50μl protein A/G-plus agarose beads at 4°C overnight; then samples were collected by centrifugation at 4°C 5000 rpm for 5 min, followed by five times wash with RIPA buffer; pellets were suspended in 2× SDS-PAGE sample loading buffer and subjected to Western blotting.

DNA pull-down assay
The cells in 100-mm dishes were lysed in RIPA buffer containing phosphatase and protease inhibitors. The samples were precleared with Streptavidin-agarose resin (Thermo) for 1 hour.
The precleared supernatant was then incubated with 1μg biotinylated double-stranded SBE (Smad Binding Element) and 10μg poly (dI-dC) at 4°C for 24 hours. DNA-protein complex was collected by streptavidin-agarose resin incubation at 4°C for 1 hour. The complex was washed, boiled and subjected to West blotting.

Western blotting
The logarithmically growing cells were lysed in RIPA buffer with phosphatase and protease inhibitors. Centrifuged cell lysates were denatured at 95°C 5 min and subjected to SDS-PAGE gel. The separated proteins were transferred onto nitrocellulose membranes (BioRad, Hercules, CA); the membranes were blocked in 5% non-fat milk for 1 hour, followed by primary incubation with primary antibodies at 4°C overnight. IRDye 800CW or 680LT labeled antibody (1:10000) were used as secondary antibodies. The membranes were scanned and quantified with The Odyssey® Infrared Imaging System (LI-COR Biosciences, Lincoln, Ne).

Fluorescent in situ hybridization (FISH)
Probe set containing 48 different oligonucleotides for MALAT1 was designed and synthesized by Biosearch Technologies (Novato, CA) based on principles described by Raj et al (2). Cells on cover slide were fixed with 1% formaldehyde for 10 minutes at room temperature and permeabilized with 70% ethanol at 4°C overnight, followed by washing with Wash buffer (10% formamide, 2× saline-sodium citrate). Then, the cells were incubated with FISH probe and primary antibody diluted in Hybridization buffer (10% Dextran sulfate, 10% deionized formamide) in a dark humidified chamber at 37°C overnight. After washing with Wash buffer twice, the cells were incubated with secondary antibody in hybridization buffer for 1 hour at 37°C, followed by triple washings with Wash buffer, DAPI (5 ng/mL) counterstaining, and mounting with anti-fluorescence medium.

Immunofluorescence (IF)
Cells growing on coverslips (70% confluence) were washed twice with cold phosphate buffered saline and fixed in 2% paraformaldehyde for 10 min, followed by penetrating with 0.1% Triton X-100 and blocking with 5% BSA. Subsequently, the cells were incubated with primary antibodies at 4 o C overnight, followed by three washings and incubation with Alexa488 or Alexa594 labeled secondary antibodies. After counterstaining with DAPI (0.1μg/ml) for 1min, the coverslips were applied with mounting medium for fluorescence; images were captured using fluorescence microscope.