The gene expression data were obtained from The Cancer Genome Atlas (TCGA) dataset for ovarian serous cystadenocarcinoma. Pathways that were substantially enriched in both SOCS7 high- and low- expression groups were identified by Gene set enrichment analysis (GSEA). The survival data were downloaded from the Kaplan Meier plotter website (http://www.kmplot.com/), using the best available cut-off value, including the GSE9891 (probe ID: 214015_at) and GSE26193 (probe ID: 226572_at) datasets for HGSOC [Grades 2 and 3; International Federation of Gynecology and Obstetrics (FIGO) stages III and IV].
Two cohorts of patients with HGSOC (Grades 2 and 3; FIGO stages III and IV) who underwent surgery in Obstetrics and Gynecology Hospital of Fudan University from 2012 to 2021 were included for the study. None of these patients had received chemotherapy or radiotherapy before surgery. Cohort 1 includes fresh 30 tumor and paired normal tissues and cohort 2 includes paraffin-embedded 68 tumor and 15 normal tissues. Approval of this study was obtained from the Ethics Committee of Fudan University.
Immunohistochemistry (IHC) evaluation
Paraffin-embedded HGSOC tissue sections in cohort 2 were used for IHC studies. The IHC staining analysis was performed using anti-SOCS7 (bs-20151R), anti-HuR (bs-2651R), or anti-FOXM1 (bs-2687R; all from Bioss Antibodies Inc. Woburn, MA, USA) primary antibodies, followed by secondary antibody incubation (D-3004; Shanghai Long Island Biotechnology). Immunoreactivity was scored using the H-score system based on percentage of positively stained cells (0, < 5%; 1, 5%-25%; 2, 25%-50%; 3, 50%-75%; 4, > 75%) and staining intensity (0, negative; 1, weak; 2, moderate; 3, strong), which ranged from 0–12. HGSOC patients were divided into low expression (H-score < 4) or high-expression (H-score ≥ 4) group.
HGSOC cell lines were fixed by using 4% formaldehyde and then permeabilized by using 0.5% Triton X-100 in PBS. Subsequently, they were blocked for 30 min by using 1% bovine serum albumin in PBS, and the cells were further incubated with anti-SOCS7 (ab224589; Abcam), anti-HuR (ab200342; Abcam), Alexa Fluor 555-labeled Donkey Anti-Mouse IgG (H + L) (A0460, Beyotime Biotechnology, Nanjing, China), or Alexa Fluor 488-labeled Goat Anti-Rabbit IgG (H + L) (A0423, Beyotime Biotechnology) antibody before nuclear staining with DAPI (C1002, Beyotime Biotechnology). The stained cells were visualized and examined by using laser scanning confocal microscopy (Leica Microsystems Inc., Buffalo Grove, IL, USA).
Human normal ovarian epithelial cell line IOSE80 and human HGSOC cell lines SNU119, CAOV3, OVCAR3, COV318, and OVCAR4 were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in 10% fetal bovine serum-supplemented RPMI-1640 medium in the presence of Penicillin–Streptomycin Solution (100X; SolarbioScience & Technology Co., Ltd., Shanghai, China) in 5% CO2 at 37 °C.
SOCS7 and HuR shRNA and scrambled shRNA lentivirus were purchased from Applied Biological Materials Company (Vancouver, Canada). Lentivirus production and purification were conducted as we previously described . Full-length coding sequences of human HuR or FOXM1 genes were cloned into the expression vector pLVX-Puro (Addgen, Cambridge, MA, USA) for the construction of HuR or FOXM1 overexpression plasmids. Blank pLVX-Puro vector served as a negative control. Transfection was carried out based on Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the protocols from the manufacturer (Life Technologies Corporation, Carlsbad, CA, USA). Following the transfection, the HGSOC cells were incubated for 48 h and subsequently transduced with the recombined expression vectors.
Cell viability assay
Approximately 3 × 103 CAOV3, OVCAR3, or COV318 cells/well were seeded in separate 96-well plates and then transduced with indicated lentiviral plasmids. Cell viability was measured at different time points (0, 24, 48, and 72 h) post-transduction, by using CCK-8 (cell counting kit-8; SAB Biotech, College Park, MD, USA). The absorbance at 450 nm was measured using a microplate reader.
Analysis of cell cycle
Approximately 3 × 105 CAOV3, OVCAR3, or COV318 cells/well were seeded in separate 6-well plates and then transduced with indicated lentiviral plasmids. At 48 h post-transduction, the cells were collected and incubated with 1 mg/ml RNase A (Sigma) and 50 μg/ml propidium iodide (Sigma) for 30 min at 25 °C for cell cycle analysis. Subsequently, the fluorescence intensities were determined by Accuri C6 flow cytometry (BD Biosciences, San Diego, CA, USA).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated from HGSOC tissues and cell lines by TRIzol reagent (Life Technologies, Grand Island, NY, USA), and reversely transcribed into cDNA by following the First Strand cDNA Synthesis Protocols (New England Biolabs, Ipswich, MA, USA). SYBR Green PCR Master Mix (Agilent Technologies, Santa Clara, CA, USA) was used for carrying out qRT-PCR, based on an ABI7300 System (Applied Biosystems, Carlsbad, CA, USA). The following primers were used for qRT-PCR: GAPDH-F:5ʹ-AATCCCATCACCATCTTC-3ʹ, GAPDH-R:5ʹ-AGGCTGTTGTCATACTTC-3ʹ; SOCS7-F:5ʹ-GGTTTGTGGCTTCCTGATG-3ʹ, SOCS7-R:5ʹ-GTGGGCTGTGTTTATGGTG-3ʹ; HuR-F:5ʹ-TCTGCGCTTGGCCTTAGTC-3ʹ, HuR-R:5ʹ-AACCGTCTTCGGGTGCTTC-3ʹ; FOXM1-F:5ʹ-AATGGCAAGGTCTCCTTC-3ʹ, FOXM1-R:5ʹ-AGCAGTGGCTTCATCTTC-3ʹ. The 2−ΔΔCT method was applied for determining the fold changes of target genes at the transcript level.
Western blot assay
Total protein was isolated and collected from HGSOC cell lines with radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotech, Santa Cruz, CA, USA). The isolated proteins with equal quantity were further separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE; 10% or 12%), followed by transferring onto separate polyvinylidene fluoride membranes (Beijing Solarbio Science& Technology, Beijing, China). The antibodies used for immunoblotting were the following: GAPDH (Cell Signaling Technology, Beverly, MA, USA), SOCS7 (ab224589; Abcam, Cambridge, MA, USA), HuR (ab238528; Abcam), Cyclin D1 (ab16663; Abcam), Survivin (ab76424; Abcam), CDC25B (ab124819; Abcam), and FOXM1 (ab207298; Abcam). After incubating with horseradish peroxidase-conjugated secondary antibody (Beyotime Institute of Biotechnology), the signals were further examined based on an enhanced chemiluminescence system (Bio-Rad, Richmond, CA, USA).
Immunoprecipitation and liquid chromatography/mass spectrometry (LC/MS) analyses
Full-length coding sequence of human SOCS7 was cloned into a pCMV-FLAG vector. The 293 T cells expressing FLAG-tagged SOCS7 were constructed by transfection with Lipofectamine 2000 (Invitrogen) according to the instructions from the manufacturer and lysed with lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris pH7.5, and 1 mM EDTA) supplemented with protease inhibitor cocktail (Sigma-Aldrich, MO, USA). The cell lysates were then incubated with anti-FLAG beads (Sigma-Aldrich) overnight incubated at 4 °C. After washing with lysis buffer five times, the immunoprecipitated protein complex was eluted by using FLAG peptide (Sigma-Aldrich) for 1 h at 4 °C. Protein samples were then resolved by SDS-PAGE for Coomassie Blue staining. Protein bands were excised from the gel and subjected to in-gel digestion with trypsin, and the resulting peptides were dissolved in a solution comprising 0.1% trifluoroacetic acid and 2% acetonitrile for analysis with Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled with Easy nLC (Thermo Fisher Scientific, Waltham, MA, USA) as previously described. The raw mass files generated by the Orbitrap Fusion instrument were processed using Proteome Discoverer software (Thermo Scientific, version 1.4) integrated with the MASCOT (Matrix Science, London, UK; version 2.3.2) search engine for protein identification. Data were searched against the Human UniProtKB/Swiss-Prot database. Proteins and peptides with a false discovery rate less than 1% were selected.
Co-immunoprecipitation (co-IP) and ubiquitination assay
Cell lysates were prepared with lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris pH7.5, and 1 mM EDTA) supplemented with protease inhibitor cocktail (Sigma-Aldrich), incubated with normal IgG (ab172730; Abcam), anti-HuR (ab200342; Abcam), or anti-SOCS7 (ab224589; Abcam) antibody, followed by further incubating with Protein A/G PLUS-Agarose beads (sc-2003; Santa Cruz Biotechnology) at 4 °C for 2 h. Then, the immunocomplex was three-time washed by the lysis buffer for Western blot analysis with antibodies against SOCS7 (ab224589; Abcam), HuR (ab200342; Abcam), and ubiquitin (ab7780; Abcam).
RNA immunoprecipitation (RIP) assay
Magna RNA immunoprecipitation (RIP) RNA-Binding Protein Immunoprecipitation kit (Millipore) was used for the RIP assay following the manufacturer’s instructions. Cells transduced with indicated lentiviral plasmids were prepared using RIP lysis buffer, the lysed cells were centrifuged at 12,000 × g for 10 min at 4˚C, and the RNA–protein complexes were incubated with anti-HuR (ab200342; Abcam) or anti-IgG antibody (ab172730; Abcam) at 4 °C for 1 h. Once incubation was complete, agarose beads and 50 µl of protein A/G were added and cells were incubated for a further 60 min at 4˚C. Subsequently, the precipitated beads were washed with RIP-wash buffer for 10 min at 4 °C and then RIP-lysis buffer for 5 min at 4 °C. The RNA in the immunoprecipitated complex and the RNA in the previously saved input fraction were released by incubating cells at 65˚C for 2 h with 200 mM NaCl and 20 µg proteinase K, which reversed the cross-linking. The amount of FOXM1 mRNA bound by HuR was determined by qRT-PCR.
Plasmid construction and dual-luciferase reporter assay
The wide-type and mutant FOXM1 3' UTR reporter plasmid was constructed by cloning PCR-amplified sequences from the 3' UTR of FOXM1 cDNA into a pGL3-basic firefly luciferase reporter plasmid (Promega, Madison, WI, USA). CAOV3 cells transduced with HuR-expressing vector were seeded into 24-well plates and transfected with 400 ng of FOXM1 3' UTR reporter plasmid (WT or Mutant). To normalize the transfection efficiency, cells were cotransfected with 50 ng of pRL-TK containing renilla luciferase. After 48 h, cells were washed with PBS and lysed using passive lysis buffer. Luciferase activity was assessed using a Dual-Luciferase Reporter Assay system (Promega) following the manufacturer’s instructions.
Measurement of mRNA stability
Cells transduced with the indicated lentiviral plasmids were incubated with 5 μg/ml transcription inhibitor actinomycin D (Sigma, St. Louis, MO, USA). Total RNA was isolated at time intervals of 0, 2, 4 and 6 h following actinomycin D addition. FOXM1 mRNA was determined using qRT-PCR, and the relative amount of FOXM1 mRNA at 0 h following actinomycin D treatment was set to 100%.
Tumor xenografts in animals
CAOV3 cells stably expressing pLVX-Puro-SOCS7 or blank pLVX-Puro were subcutaneously injected into male nude mice (6-week-old; n = 6 per group). At the 33rd day post-inoculation, tumors were collected, weighed, photographed, and analyzed by qRT-PCR, Western blot and immunofluorescence staining by using anti-Ki67 antibody (ab15580; Abcam). All the animal studies were approved by the Ethics Committee of Fudan University.
Data were presented in the form of ‘mean ± standard deviation’, and validated by three independent experiments or multiple independent mice trials, as indicated. Experimental results were analyzed with GraphPad Prism Software Version 8.4.2 (GraphPad Software Inc., La Jolla, CA, USA). Two-tailed Student's t-test and analysis of variance were applied for two groups comparisons and multiple groups comparisons, respectively. Wilcoxon test and nonparametric Kruskal–Wallis test were both utilized for evaluating the differences of tumor tissues and controls. Pearson's or Spearman's correlation was used for measuring association between expression levels of SOCS7/FOXM1, FOXM1/HuR or SOCS7/HuR. A statistical significance was considered when the P value was less than 0.05.