A total of 30 EOC tissue specimens and 30 normal epithelial ovarian tissue specimens were collected from patients who underwent surgical resection at the Department of Gynaecology, Xiangya Hospital, Central South University, from 2017 to 2019. Two pathologists independently confirmed the tumour specimens, which were stored at − 80 °C until analysis. This study was approved by the Ethics Committee of Xiangya Hospital, Central South University, and all specimens were handled and anonymized according to ethical and legal standards. Written informed consent was obtained from all patients.
The cell lines CAOV3, OVCAR3, and SKOV3 were obtained from American Type Culture Collection (ATCC, USA), the OVSAHO cell line was obtained from Japanese Collection of research bioresources cell bank, the SNU119 cell line was obtained from Korean cell bank, the A2780 cell line and the normal human ovarian epithelial cell line IOSE80 were purchased from Yubo Biotech (Shanghai), and the immortalized normal human fallopian tube epithelial cell line FTE 187 was obtained from Jinsong Liu’s Laboratory. ES-2 is a cell line of ovarian clear cell adenocarcinoma, OVSAHO, SUN119, OVCAR3 and CAOV3 cells are high-grade ovarian serous adenocarcinoma, A2780 is a cell line of ovarian endometrioid adenocarcinoma, and SKOV3 is a cell line of well-differentiated adenocarcinoma. All cells were cultured in Dulbecco’s modified Eagle’s medium (HyClone, Logan, UT, USA) supplemented with 10% foetal bovine serum (FBS) and penicillin/streptomycin (100 U/mL). Cells were incubated at 37 °C in a 5% CO2 atmosphere.
Lentivirus construction and transfection of shHCG18, miR-29 mimics and shTRAF4/5
Short hairpin RNAs targeting HCG18 (shHCG18; 5′-UUGGCUUCAGUCCUGUUCAUCAG-3′) and a negative control (shNC; 5′-AAUUCUCCGAACGUGUCACGU-3′) were purchased from GenePharma Company (Shanghai, China) and subcloned into the pLKO.1 vector. The pLKO.1-shHCG18 plasmid was then transfected into HEK293T cells with the psPAX2 packaging plasmid and pMD2.G envelop plasmid lentivirus. Cells (2 × 106 cells per well) were cultured in 6-well plates to 80–90% confluence and then transfected with lentivirus (titre: 5 × 107 TU/mL) at a multiplicity of infection (MOI) of 50 mixed with 5 μg/mL polybrene (Sigma–Aldrich, St. Louis, MO, United States) for 48 h. Mimics of miR-29a-3p and miR-29b-3p and negative control, TRAF4/5 overexpression and TRAF4/5 knockdown shRNAs and their negative control were purchased from GenePharma Company (Shanghai, China) and transiently transfected into the indicated cell lines. All cell transfection assays were performed using Lipofectamine 3000 (Invitrogen, Eugene, OR, USA).
EOC cells (3 × 103/well) were seeded into 96-well plates and assayed at 0 h, 24 h, 48 h, and 72 h after incubation with 25 μL of 4 mg/mL MTT solution (Solarbio, Beijing, China). For transfection, cells were first transfected with shHCG18, miR-29 mimics, shTRAF4/5, or vectors in 100 mm dishes and then split into 96-well plates. After incubating with MTT solution at 37 °C for 4 h, the solution was removed, dissolved in 150 μL dimethyl sulfoxide and subjected to 15 min of shaking. Absorbance was determined using a microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA) at 490 nm.
Colony formation assay
After being treated with shHCG18, miR-29 mimics, shTRAF4/5, or vectors, cells were subjected to trypsinization, and 1500 viable cells were subcultured in six-well plates. After culture for 2 weeks, the media were removed, and cells were fixed in 95% ethanol for 20 min. Then, the cells were stained with 0.5% crystal violet for 20 min. The colonies were subsequently quantified.
Scratch wound healing assays
EOC cells were incubated in 6-well plates to ~ 80% confluence, and a scratch in the cell layer was generated using a pipette tip. PBS was then used to wash the plates to remove the scraped cells. Images were obtained 24 h later.
Cell invasion assay
EOC cells harvested in serum-free medium were seeded in upper Transwell chambers (pore size, 6 μm; Corning Inc., Corning, NY, USA) after treatment with shHCG18, miR-29 mimics, shTRAF4/5, or vectors. Regular medium supplemented with 10% FBS was added to the bottom chamber. After incubation at 37 °C for 24 h, cells on the upper membrane were removed using a cotton swab, and the cells on the bottom surface of the membrane were fixed in 4% paraformaldehyde. Then, the cells were stained at room temperature with crystal violet for 15 min. Light microscopy was employed to quantify the number of cells to assess cell invasion.
Total RNA extraction and real-time PCR
Total RNA was isolated using TRIzol reagent (Invitrogen), and the concentration was measured using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A TaqMan® miRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) was used for miRNA qPCR, with cDNA synthesized from 5 ng of total RNA. For the other genes, random primers from the RT Master Mix kit (Takara, Dalian, China) were used to synthesize cDNAs from total RNA. SYBR Green Real-Time PCR Master Mix (Toyobo, Osaka, Japan) and the ABI 7500 Sequence Detection system (Life Technologies, Grand Island, NY, USA) were used to perform qRT–PCR. Relative expression levels were normalized to that of GAPDH mRNA or U6. All experiments were performed in triplicate.
Total proteins were extracted from EOC cell lines using RIPA buffer, and a BCA protein assay kit was used to determine the corresponding concentrations. Protein (25 μg) was isolated using 8% SDS–PAGE and subsequently transferred to PVDF membranes. BSA (1%) in TBS buffer was used to block the membranes, and primary antibodies were incubated at 4 °C. The membrane was then washed with 1 × TBST and incubated with a secondary antibody conjugate in 1 × TBS at room temperature for 1 h. The membrane was washed with 1 × TBST, and protein expression was determined using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). The loading control was β-actin detected on the same blot. The primary antibodies were purchased from Cell Signaling Technology as follows: β-actin (8H10D10), Vimentin (D21H3), E-cadherin (24E10), MMP-2 (D4M2N), MMP-9 (D6O3H), ZEB1 (D80D3), NF-κB p65 (D14E12), Slug (C19G7), TWIST1 (#46702), Snail (C15D3), Phospho-NF-κB p65 (Ser536) (93H1), acetyl-NF-κB p65 (Lys310) (D2S3J), AKT (pan) (C67E7), phospho-AKT Substrate (RXXS*/T*) (110B7E), β-tubulin (9F3), lamin A/C (4C11), TRAF4 (D1N3A), and TRAF5 (D3E2R).
Bioinformatics and dual-luciferase reporter assay
The binding sites between HCG18 and the 3′ UTRs of TRAF4/5 mRNA with miR-29a/b were predicted using starBase (http://starbase.sysu.edu.cn). A QuikChange Mutagenesis kit (Stratagene) was then used to generate the mutations. The 3′-UTR sequences of HCG18, TRAF4 or TRAF5 containing wild type or mutated binding sites were subcloned into the pRL-TK luciferase reporter. All constructs were sequenced to verify integrity. EOC cells were transfected with 300 ng of firefly luciferase reporter and 25 ng of Renilla luciferase plasmid plus 900 ng of empty vector or miRNA mimic plasmid. Twenty-four hours after transfection, luciferase assays were performed using a Dual Luciferase Reporter Assay kit (Promega), and the ratio of firefly to Renilla luciferase activity was determined.
For NF-κB transcriptional activity, cells were cotransfected with 100 ng of the pNFκB reporter luciferase plasmid. Renilla plasmid (Promega) containing 5 ng pRL-TK was also transfected as a control signal. All transfections were performed using Lipofectamine 3000 (Invitrogen). The signals were detected using a Dual Luciferase Reporter Assay Kit (Promega).
Female BALB/c-nude mice (20–22 g, 6 weeks old) were purchased from the Animal Experiment Centre of the Chinese Academy of Sciences (Shanghai, China) and used as xenograft animal models (n = 16). Animals were raised in specific pathogen-free conditions, and all animal experiments were approved by the animal ethics committee of Xiangya Hospital, Central South University. Mice were randomly divided into 2 groups, and CAOV3 cells, (5 × 106) stably expressing shHCG18 or shNC, were injected subcutaneously into the right flank of nude mice under aseptic conditions. Then, we measured tumour size using callipers every 4 days, and after 24 days, mice were sacrificed. Then, xenograft tumour tissues were harvested for immunohistochemical staining. Then, the remaining tissues were stored in a − 80 °C freezer for analysis of mRNA and protein levels.
Tumour tissue sections were blocked in 4% non-fat milk containing 1% Triton X-100, incubated with Ki67 primary antibody (1:200, ab16667, Abcam) at 4 °C overnight and then washed with PBS 3 times. Next, after incubation with the biotinylated secondary antibody for 30 min at room temperature, the sections were stained with freshly prepared 3,3′-diaminobenzidine (DAB), followed by counterstaining.
Data are shown as the mean and standard deviation (SD). Student’s t-test was used to compare differences between two groups for continuous variables. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used for multiple comparisons. All analyses were performed using GraphPad Prism 6 (GraphPad Software, Inc.) P < 0.05 was considered statistically significant.
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