Cell culture
HepG2, Huh7 and Hep3B cell lines were purchased from the Cell Culture Center of Chinese Academy of Medical Sciences (Beijing, China). They were cultured in Dulbecco’s Modified Eagle’s Medium and Minimum Essential Medium (Gibco Life Technologies) containing 10% fetal bovine serum (FBS, Gibco Life Technologies) and maintained at 37°C in a moist atmosphere with 5% CO2.
Animals
The Atf6fl/fl C57BL/6 mice were purchased from the Jackson Laboratory (ME, USA). Alb-cre C57BL/6 mice were obtained from the Model Animal Rearch Center of Nanjing University (Nanjing, China). Atf6fl/fl mice and Alb-cre mice hybridized and generated liver-specifically Atf6 deficient Atf6Δhep mice. Wild type C57BL/6 mice and BALB/c nude mice were purchased from the Center of Medical Experimental Animals, Chinese Academy of Medical Sciences (Beijing, China). Due to the great gender differences in the incidence of liver cancer, male mice were chosen to perform the experiments in this study. The animal feeding and experimental protocols has been approved by the Animal Care and Use Committees and Ethics Committee of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.
Plasmids and transfection
ATF6, ATF6(1–7), PPM1H and RPS6KB1 coding sequence were inserted into Lv-ef1a-IRES-GFP or p3XFLAG-CMV™-14 vectors to construct the overexpression plasmids. shATF6 and shPPM1H sequences were inserted into pGPU6/GFP/Neo vectors to establish the knockdown plasmids (Generay Biotechnology, Shanghai, China). PPM1H promoters were inserted into pGL3-basic to construct luciferase reporter plasmids. The target plasmid of interest was transfected into cells using Lipofectamine™ 3000 reagent (Invitrogen, USA).
Antibodies
Antibodies against ATF6 (polyclonal rabbit, cat no.DF6009), RPS6KB1 (polyclonal rabbit, cat no.AF6226), p-RPS6KB1 (phospho-RPS6KB1, Thr389/Thr412; polyclonal rabbit, cat no.AF3228), Flag-tag (polyclonal rabbit, cat no. T0053) were purchased from Affinity (OH, USA). Antibodies against E-cadherin (monoclonal mouse, cat no. ab231303), N-cadherin (monoclonal rabbit, cat no. ab76011) were purchased from Abcam (Cambridge, UK). Antibodies against p-SMAD1(phospho-SMAD1, Ser463/465; polyclonal rabbit, cat no. 13820T), Flag-tag (polyclonal mouse, cat no. 8146) were purchased from Cell Signaling Technology (MA, USA). Anti-PPM1H (polyclonal rabbit, cat no. CSB-PA892345LA01HU) antibody was purchased from CUSABIO (Wuhan, China).
Human subjects
134 paraffin-embedded tissues and basic information of HCC patients were collected from the Affiliated Hospital of Qingdao University between 2010 and 2015. All treatment with the tissues had acquired informed consent of the patients.
Stable cell lines establishment and nude mouse tumorigenicity assay
Lv-ATF6, Lv-PPM1H, or control plasmids were co-transfected with lentivirus packaging plasmids (delta8.91 and pVSVG) respectively into 293T cells planted in 6-well plates. The supernatant culture medium containing corresponding lentivirus were collected 72h after transfection and used to infect Hep3B cells planted in 6-well plates. Cells stably expressing ATF6 or PPM1H were selected by EGFP using flow cytometry. By geneticin (G418) selection of Hep3B cell transiently transfected with pGPU6/GFP/Neo-sh-PPM1H plasmid, stable sh-PPM1H cell line was established. 5×106 cells were subcutaneously injected into male BALB/c nude mice aged 4 weeks. Xenogeneic tumors were collected 4 weeks later.
DEN/CCl4-induced HCC mouse model
Adeno-associated virus AAV8-PPM1H or AAV8-control were generated by Vigene Bioscience Company (Jinan, China). Wild type or Atf6Δhep C57BL/6 mice were injected with 25mg/kg DEN (Sigma Aldrich, USA) intraperitoneally 2 weeks after birth. Then 5mL/kg CCl4 (Sigma Aldrich, USA) were injected intraperitoneally twice a week for 24 weeks to induce HCC, and 1×1012 vg/animal (vg, viral genome equivalents) AAV8-PPM1H or AAV8-control were injected into mice via tail vein 8 weeks after birth. 3 days after the final injection of CCl4, mice were sacrificed, and livers were extracted.
Dual luciferase assay
HepG2 cells planted in 24-well plates were co-transfected with PPM1H luciferase reporter plasmids and ATF6 or control plasmids. The luciferase activity was analyzed 48h after transfection using Dual-Luciferase Reporter Assay System (Promega, USA).
Quantitative real-time PCR (qRT-PCR)
Total RNA of cells or mouse liver tissues were extracted (Ultrapure RNA Kit, CWBIO, Beijing, China) and reverse-transcribed (ReverTra Ace® qPCR RT Master Mix with gDNA Remover, Toyobo life science, Shanghai, China) into cDNA, then qRT-PCR was performed using MagicSYBR Mixture (CWBIO, Beijing, China). Primer sequence was listed in Table. S1.
MTT assay
2x103 cells planted in 96-well plate were transfected with plasmids or a certain concentration of recombinant protein. 20 µL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma Aldrich, USA) was added. After 4 hours of incubation, OD value at 490nm was detected to reflect the proliferation of HCC cells.
Transwell migration and invasions
For cell migration assay, 2x105 HepG2 or Huh7 cells suspended in serum-free medium were directly planted in transwell upper chamber after transfection of target plasmids and the chamber was kept in cell culture medium supplemented with 20% serum. While for the cell invasion assay, cells were seeded in matrigel basement membrane matrix in transwell chamber. After 48 hours of incubation, cells were fixed with 4% neutral formaldehyde for 20 minutes, followed by staining with 0.1% crystal violet for 10 minutes and washing with PBS. The migration and invasion were observed via an inverted microscope. For the RBS6KB1 or SMAD1 blocking, PF-4708671 (MedChemExpress, Shanghai, China) or LDN-193189 (MedChemExpress, Shanghai, China) was added to the culture medium at a final concentration of 10µM and 0.5µM respectively.
Western blot
Total protein extracted from cells or mouse liver tissues with RIPA lysis buffer (CWBIO, Beijing, China) containing protease inhibitors (phenylmethylsulfonyl fluoride, PMSF; VWR, USA) were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore; Billerica, MA, USA). After blocking in 5% non-fat powdered milk, the membranes were incubated with specific primary antibodies at 4℃ overnight, following HRP-conjugated secondary antibodies incubation at room temperature for 1 hour. Chemiluminescence images were taken by Tanon-5200Multi (Shanghai, China) using Pierce™ ECL western blotting substrate (Thermo Fisher Scientific, Inc., USA).
Co-Immunoprecipitation (Co-IP)
Total protein dissolved in RIPA lysis buffer (CWBIO, Beijing, China) were incubated with specific antibodies or isotype nonspecific negative control antibodies at 4℃ overnight with rotation. Protein A/G plus-agarose (Santa Cruz Biotechnology, Inc.CA, USA) was added to incubate for another 2 hours. Then wash the agarose beads with cold PBS for 3 times and centrifugate at 4℃, 3000 rpm for 5 minutes each time to harvest the immunoprecipitated protein. Immunoblotting was performed to examine the interest proteins. For the in vitro phosphatase assay, the agarose beads were diluted with 20% glycerol PBS containing protease inhibitors and preserved in − 80°C.
Protein purification of prokaryotic expressed PTD-ATF6 and PPM1H
PTD (protein transduction domain of trans activator of transcription) is a short of peptide (YGRKKRRQRRR) that mediates protein entry into cell14, PTD-ATF6 or PPM1H coding sequence were inserted into pET-28a + plasmids, after transferred into E. coli strain Transetta (DE3) (Transgen biotech, China), the recombinant proteins were induced by IPTG, the supernatant was subjected to Ni affinity chromatography after ultrasonic lysis of the bacteria solution (6×His-Tagged Protein Purification Kit - Soluble Protein, CWBIO, Beijing, China). Then proteins were concentrated using an ultrafiltration centrifuge tube (Millipore, USA) with a filter pore size of 30 kDa. The purified His-PPM1H was used to perform in vitro phosphatase assay. PTD-ATF6 was applied to incubate cells at a certain concentration, and the culture medium was changed after 48 hours.
In vitro phosphatase assay
Immunoprecipitated p-RPS6KB1 was incubated with increasing dosages of PPM1H protein purified from bacteria at 37℃ for 30 minutes. The mixtures were then subjected to western blot using specific antibodies against p-RPS6KB1 or total RPS6KB1.
PPM1H structural model construction and substrates screening
The 3D structure of PPM1H was predicted by Modeler software using homologous multi-templates 4jnd, 4ds8, 2isn. Then the model was optimized for molecular dynamics using AMBER14 software through two steps. Firstly, the protein was kept in a free state and to maintain the minimum energy; secondly, the long-range electrostatic energy was calculated using the PME method. The stability of the constructed model was evaluated based on the (skeletal atom rms fluctuation) RMSD value and the Tip3P water box model.
The pathway proteins of PI3K-Akt and BMP/TGFβ’s docking analysis used Rosetta software. The protein in which the PPM1H is stably docked and is screened based on the energy list of the PPM1H prediction model combined with each pathway protein. The spatial combined conformation is further simulated by software.
Immunohistochemistry (IHC)
Tissue slides of HCC patients and mice were incubated with PPM1H antibody. The immunohistochemistry process was carried out by Servicebio (Wuhan, China).
RNA-sequencing (RNA-seq)
Total RNA of Atf6fl/fl or Atf6Δhep C57BL/6 mice liver tissues were used to perform RNA-sEq. The process was completed by Igenecode (Beijing, China).
Statistical analysis
Statistical analysis was performed in GraphPad 8.0 software. Each statistical result was determined by three independent experiments. A paired t-test test and Wilcoxon signed rank test were used.