Smad7 is less understood and varies in different biological settings. For instance, nuclear Smad7 functioning via TGF-β-independent mechanism acted as a nuclear coactivator essential for myogenic differentiation in myoblasts[34], while some studies indicated a nuclear corepressor role of Smad7 as to its interaction with histone proteins and transcription factors, such as SIRT1, HDAC1, and E2F[35-37]. In our study, nuclear Smad7 was also found to serve as a transcriptional suppressor of Snail gene expression through ProT-regulated nuclear distribution and stabilization. In the presence of ProT, high levels of Smad7 were detected by immunoblotting and nuclear localization by immunostaining, but notably the increased Smad7 expression was not observed on transcriptional levels. This led us to further investigating the regulatory relation between nuclear Smad7 and ProT. Knowing that histone acetyltransferase p300 acetylates Smad7 on lysines 64 and 70, preventing the ubiquitination of these lysine residual and leading to protein stability[38], we generated a mutant Smad7 that lacked acetylated lysines, and demonstrated that ProT failed to exert its suppressive effect on SNAI1 expression via mutant Smad7. In fact, ProT enhancing protein acetylation has been found in various diseases, including STAT3 in polycystic kidney disease[39], NF-kB in emphysema[40], P53 in cancer cell lines[41], as well as histone acetylation[11]. We previously reported that ProT and Smad7 acetylation was associated with emphysema progression[15]. Here we further identified that the regulation required nuclear ProT expression which then triggered Smad7 nuclear localization and transcriptional suppression of Snail gene.
四、研究方法
Human specimens
Clinical specimens were collected from lung cancer patients who underwent surgery at the Thoracic Division, Department of Surgery, National Cheng Kung University (NCKU) Hospital, Tainan, Taiwan. Informed consent was obtained from all subjects, and the experimental protocol was approved by the Human Experiment and Ethics Committee, NCKU Hospital. All clinical samples were obtained with the approval of the Institutional Review Board of NCKU Hospital.
Cell culture and treatments
Human lung cancer cell lines A549 and H1299 were obtained from American Type Culture Collection (ATCC). Human 293T embryonic kidney cells were obtained from the
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National RNAi Core Facility, Academia Sinica, Taiwan. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C. Where indicated, cells were treated with 10 ng/ml of recombinant TGF-β1 (PeproTech) in DMEM with 2% FBS, for the indicated time.
Plasmids and lentivirus vector
Lentivirus vectors pWPXL-ProT and pWPXL-GFP were constructed by replacing the coding region of pWPXL-puro plasmid with human ProT coding sequences. The human Smad7 and Smad7(K64R/K70R) expression vectors were derived from pLKO.1-puro plasmid. The plasmid pcDNA3.1-ProT used in this study has been described previously [15]. For knockdown experiment, lentiviral vector pLKO.1-puro expressing short hairpin RNA specific for human ProT (TRCN0000135421) and luciferase (Luc) (TRCN0000072246) were obtained from the National RNAi Core Facility, Academia Sinica, Taiwan. Lentiviral particles expressing lentivirus vector encoding ProT and GFP, Smad7, and Smad7(K64R/K70R) were produced by transient transfection in 293T cells, along with the packaging plasmid psPAX2 and the VSV-G expression plasmid pMD2G as previously described [42].
Migration assay
Cells were pretreated with TGF-β1 for 24 h and seeded for migration assay. For Transwell assays, cells were placed onto the upper chamber of Transwell filter with 8 μm pores (Thermo Fisher Scientific) and bottom well contained regular growth media. After incubating for 6 h, migrated cells were fixed with methanol and stained with 0.1% Giemsa.
Migration was quantified by cell counts in three random fields (×100 magnification) per sample. Data represent 3 independent experiments in each group, performed in triplicate.
For wound healing assays, confluent cells were wounded using a 200-μl pipette tip and cultured in DMEM with 2% of FBS in the presence of mitomycin C (5 μg/ml). After incubating for 8 h, cells were visualized under a light microscope. Cell migration was calculated by measuring final wound area compared to initial area.
RNA extraction and real-time PCR
Total RNA were isolated using TRIzol Reagent (Invitrogen) according to the manufacturer’s instruction and 2 μg RNA was used for cDNA synthesis performed with High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Gene expression analysis was evaluated by real-time PCR using QuantiNova SYBR green PCR kit (Qiagen)
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in triplicates and performed on Roter-Gene Q (Qiagen) according to manufacturer’s recommendations. The relative expression levels of mRNA were normalized to GAPDH and fold change was calculated using the 2^-(ΔΔCt) method. The following primers were used for real-time RT-PCR: human SNAI1 (5’-TCGGAAGCCTAACTACAGCGA-3’
and 5’-AGATGAGCATGGCAGCGAC-3’); human TWIST1
(5’-GTCCGCAGTCTTACGAGGGAG-3’ and 5’-TGGAGGACCTGGTAGAGGAA-3’);
human ZEB1 (5’-CAGCTTGATACCTGTGAAGGG-3’ and
5’-TATCTTGTGGTCGTGTGGGACT-3’); human SMAD7
(5’-CCCCATCACCTTAGCCGACTCTGC-3’ and 5’-CCCAGGGGCCAGATAATT-3’);
human GAPDH (5’-ACTTCAACAGCACACCCACT-3’ and
5’-GCCAAATTCGTTGTCATACCAG-3’).
Immunoblotting and immunoprecipitation
Cells were homogenized in RIPA lysis buffer containing protease inhibitor cocktail and protein was quantified using a BCA assay (Thermo Fisher Scientific) according to manufacturer's instructions. Total 20-40 μg of protein content were separated on handmade 10-12% SDS-PAGE gels, transferred onto PVDF membranes, and blocked with 5% semi-skimmed milk. Membranes were incubated overnight at 4 °C with primary antibodies against E-cadherin (BD), N-cadherin (BD), Snail (Cell signaling), Twist1 (Sigma), acetylated-lysine (Cell Signaling), Flag epitope (Sigma), ProT (clone 2F11;
ascites fluid) and β-actin (abcam). For immunoprecipitation, anti-Flag M2 gel (Sigma) was used to detect the Flag-conjugated protein. HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG (Cell signaling) were used as secondary antibodies where appropriate, and protein-antibody complexes were visualized by the ECL system (Millipore) and the Biospectrum AC imaging system (UVP).
Chromatin immunoprecipitation (ChIP)
Cells were transduced with lentiviral vectors encoding different targeted proteins and treated with or without TGF-β1. ChIP was performed as described previously [40]. Briefly, proteins were crosslinked with 1% formaldehyde, washed with cold PBS, and cells were lysed with SDS lysis buffer. The cell lysate was sonicated to shear DNA to lengths between 500 and 1,000 bp, and followed by immunoprecipitation with anti-Flag-M2 affinity gel (Sigma-Aldrich), or protein A plus G sepharose (Santa Cruz) combined with anti-Smad2 (Genetex) or anti-IgG antibodies (Santa Cruz). The immunoprecipitates were washed and eluted for the protein/DNA complexes and reverse crosslinks of the complexes to free DNA. DNA purification using the phenol-chloroform-extracted method and PCR was performed to detect the binding sites for Smad7 and Smad2 within the Snail1
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promoters with 5’-CGCTCCGTAAACACTGGATAA-3’ and 5’-GAAGCGAGGAAAGGGACAC-3’ primers. The PCR products were separated by 1%
agarose gel electrophoresis.
Histology and immunostaining
Human and mice lung tissue sections were prepared by fixation in formalin fixed and embedding in paraffin. For histological staining, the sections were processed with hematoxylin and eosin (H&E) staining after deparaffinization and rehydration. For immunohistochemistry. after deparaffinization and rehydration, tissue sections were blocked with BSA, followed by incubation with primary antibodies at 4oC overnight.
Primary antibodies included monoclonal antibodies against ProT (clone 2F11, ascites fluid)[43], Smad7 (R&D), and Snail (Abcam). This was followed by incubation with secondary antibodies, horseradish peroxidase (HRP)-conjugated goat mouse or anti-rabbit IgG (Jackson) at room temperature for 2 h. The reactivity was visualized with aminoethyl carbazole (AEC, red color, Zymed) and counterstained with hematoxylin.
Secondary antibodies used for immunofluorescence staining were Alexa-fluor 594 goat anti-mouse IgG (Invitrogen) and nuclei were stained with DAPI (Sigma) under a dark room-temperature-humidified environment. Images were captured using a constant exposure time by Olympus microscope. Photographs from immunohistochemistry was digitally processed to obtain the integrated optical density (IOD) and data was analyzed using MetaMorph.
In vivo models
Cells were transduced with lentiviral vectors, selected for stable clones and 4x106 cells were inoculated subcutaneously onto the right flank of male NOD/SCID mice (8 weeks old, approximately 22 g). Tumor growth was measured every 3 to 4 days using Vernier calipers. The tumor volume was calculated as length × width2 × 0.45. Ethical endpoint criteria were based on tumor size (>20 mm in diameter in any dimension) or acute weight loss. Tumors and lungs were collected at the time of euthanasia. All procedures were approved and monitored by the Institutional Animal Care and Use Committee of NCKU, Taiwan.
Statistical analysis
Statistical analyses were performed using GraphPad Prism software. All data were presented as mean ± SEM from at least three independent replicates for each experiment.
Statistical significance was determined by Student’s T-test or one-way ANOVA. A p
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value of ≤0.05 was considered to indicate a statistically significant difference (*p<0.05,
**p<0.01 and ***p<0.001).