Restriction enzymes (BamHI and EcoRI) and T4 ligase were purchased from Sangon Biotech Co., Ltd. Rabbit polyclonal or monoclonal antibodies against human cytokeratin 7 (cat. no. ab181598), Bax (cat. no. ab32503), Bcl-2 (cat. no. ab196495), PI3K (cat. no. ab86714), optineurin (OPTN; cat. no. ab151240), Akt1/2/3 (cat. no. ab184136), phosphorylated (p)-Akt1 (T308; cat. no. ab105731), excision repair cross-complementation group 1 (ERCC1; cat. no. ab129267) and β-actin (cat. no. ab8227) were purchased from Abcam. Rabbit polyclonal antibodies against human caspase-3 p17 (cat. no. sc-271,028) and EEF1D (cat. no. abs136494) were purchased from Santa Cruz Biotechnology, Inc., and Absin Bioscience, Inc., respectively. Lipofectamine® 3000 transfection reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The HRP-conjugated secondary antibody (goat anti-mouse or goat anti-rabbit IgG, cat. no. PV-6000) were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. FITC-labeled goat anti-rabbit IgG (H + L) secondary antibody (cat. no. A0562) and Hoechst 33342 (cat. no. C1022) were purchased from Beyotime Institute of Biotechnology. LY294002 (PI3K inhibitor; cat. no. ab120243) and MK-2206 (Akt inhibitor; cat. no. SF2712) were purchased from Abcam and Beyotime Institute of Biotechnology, respectively.
Human ovarian cancer cell line SKOV3 (cat. no. HTB-77), human embryonic kidney cell line 293 (cat. no. CRL-3216) and Escherichia coli strain TOP10 (cat. no. PTA-5669) were purchased from the American Type Culture Collection (ATCC). The DDP-resistant human ovarian cancer cell line SKOV3/DDP was purchased from Shanghai Suran Biotechnology Co. Ltd., China. The pLJM1, Δ8.91 and pVSVG plasmids were provided by Wuhan Miaoling Biotechnology Co., Ltd., China. The pLentiCRISPR, pL-SSA-puro, pSPAX2 and pMD2G plasmids were provided by Shanghai GenePharma Co., Ltd. The lentiviral vector systems used in the experiment were all third-generation products. SKOV3, SKOV3/DDP and 293 cells were cultured in DMEM from Gibco with 10% FBS from Gibco in 5% CO2 at 37 °C, and then digested with 0.4% trypsin once they reached the logarithmic growth phase for subsequent experiments.
Fresh primary ovarian tumor tissues classified as serous ovarian adenocarcinoma (IV grade) according to the World Health Organization criteria  were collected from 5 patients with ovarian cancer with progressive disease/stable disease (PD/SD) who underwent surgery debulking at The First Affiliated Hospital of Anhui Medical University (Hefei, China) between January and December 2019. These patients had received DDP or carboplatin chemotherapy. Prior to tissue deposition, all patients signed written informed consent forms confirming their donation of tissue for research purposes according to the Declaration of Helsinki. All methods were carried out in accordance with relevant guidelines and regulations (eg. Helsinki declaration). This study was approved by Biomedical Ethics Committee of Anhui Medical University (approval no. 20170213; Hefei, China). Fresh primary ovarian tumor tissues were slowly washed with PBS 2–3 times, and the tumor tissue was cut into small pieces (~ 1 mm3), transferred to a 50 ml sterile centrifuge tube with 0.4% trypsin and digested at 37 °C for 40–50 min. Then, the tumor tissue fragment was centrifuged at 100 x g for 5 min under 4 °C, and the pellet was resuspended in DMEM with 10% FBS containing 0.1 mg/l EGF, 0.1 mg/l insulin-like growth factor and 0.1 mg/l β-estradiol, then cultured in 5% CO2 at 37 °C. After 48 h, the unattached tissues were washed with PBS, and the adherent cells were further cultured until they reached 70–80% confluence, cells were then digested with 0.4% trypsin and subcultured for subsequent experiments. The ovarian cancer cells were identified and analyzed using immunofluorescence staining of cytokeratin 7.
Knockdown (KD) of EEF1D gene
Design and screening of EEF1D small interfering (si)RNA
For EEF1D knockdown (KD), EEF1D siRNA was designed and screened based on the EEF1D gene sequence, and scrambled siRNA served as the control. The sequences were as follows: EEF1D siRNA, 5′-GGUUCGACAAGUUCAAAUATT-3′; and scrambled RNA, 5′-UUCUCCGAACGUGUCACGUTT-3′. EEF1D siRNA and scrambled siRNA were synthesized by Shanghai GenePharma Co., Ltd. EEF1D siRNA or scrambled RNA was transiently transfected into SKOV3 and SKOV3/DDP cells using Lipofectamine® 3000 transfection reagent. According to the open reading frame of EEF1D gene sequence queried from PubMed, PCR primers with low GC content and high specificity were designed and selected with Primer-Blast (Primer 6.0 Software, Canada). The forward (F) and reverse (R) primers of EEF1D were 5′-GTATCTCCCATGCGCCAAGT-3′ and 5′-ATCCAGCAGGATGGAGGACT-3′, respectively. Transfection efficacy was determined via reverse transcription (RT) PCR, with β-actin serving as the control gene.
Construction of pLJM1-EEF1D short hairpin (sh) RNA plasmids
The EEF1D shRNA and scrambled shRNA sequences were designed according to the aforementioned siRNA or scrambled RNA in which EcoRI and BamHI restriction recognition sites were designed at the end of these sequences (Table S1). The EEF1D shRNA and scrambled shRNA synthesized by Sangon Biotech Co., Ltd., were ligated with the lentiviral vector pLJM1. The pLJM1-EEF1D shRNA and pLJM1-Scrambled shRNA plasmids were transformed into E. coli TOP10. The recombinant plasmids were purified, further identified by restriction endonucleases and sequenced.
Lentiviral infection and establishment of stably transfected cell lines
The recombinant lentiviral expression vector (pLJM1-EEF1D shRNA and pLJM1-Scrambled shRNA) and the lentiviral packaging plasmid (Δ8.91 and pVSVG) were transfected into 293 cells using Lipofectamine 3000. The detailed protocol was as follows. 293 T cells were seeded into wells of 6-well plates at 2.5 ml (7.5 × 104 cells/ml) /well for culturing in DMEM with 10% FBS at 37 °C for 24 h. The medium was then replaced with serum-free medium, and 3 μg/well pLJM1 and 9 μg/well packaging vectors (Δ8.91 and pVSVG) mixed with Lipofectamine 3000 were added. After culturing for 6 h, the medium containing the transfection mixture was replaced by DMEM containing 10% at 2 ml/well and cell culturing was continued for 72 h. The titers (TU/ml) of virus solution were determined by calculating the number of cells with fluorescence in the maximum dilution multiplication well. After being centrifuged at 4000 rpm at 4 °C and filtered with 0.45 μm syringe filter, the virus stock solution was ultracentrifuged at 20,000 rpm for 2 h, then the supernatant was removed and resuspended in 1 ml of DMEM medium. Last, the viral supernatant was divided into small aliquots and placed at − 80 °C.
SKOV3 and SKOV3/DDP cells were inoculated into a 6-well plate until they reached 70% confluency in 5% CO2 at 37 °C. Then, the lentivirus was added to the culture medium to infect the cells with a multiplicity of infection (MOI) of 20 in 5% CO2 at 37 °C, meanwhile polybrene was added to the culture medium at a final concentration of 6 μg/ml. The infection was repeated once a day, and its efficiency was observed and identified using a fluorescence microscope with GFP reporter at 72 h post-infection. Using puromycin selection, infected cells were subcultured 4–5 times to obtain a monoclonal cell population, in which EEF1D gene KD was tested via RT-PCR and Western blotting. Each experiment for the electrophoresis of PCR products and Western bloting of proteins was performed in triplicate with two independent sets.
For EEF1D KD in human primary ovarian cancer cell (POCC), the method was the same as aforementioned for SKOV3 and SKOV3/DDP cells, but cells were transiently infected with the lentivirus and infected cells were not selected by puromycin and were not subcultured.
Knockout (KO) of EEF1D gene
For EEF1D knockout (KO), the single guide RNAs (sgRNAs) were designed according to the specificity and efficiency. The exon 2 region of EEF1D was selected to be targeted via CRISPR/Cas9 genome editing. The target sequence of the gene was 5′-GGCCACGGCCCCACAGACCC-3′, in which the last CC bases were knocked out. The guide (g) RNA of EEF1D was constructed into the vector (site of restriction enzyme BsmBI) to obtain the pLentiCRISPR-EEF1D plasmid. For effective screening of positive cells, SSA vectors were constructed, onto which the target sequence containing the gRNA was constructed to obtain the pL-SSA-puro-EEF1D plasmid. Recombinant lentivirus vectors were produced by transfecting 293 cells with the aforementioned two plasmids and packaging mix plasmids (pSPAX2 and pMD2G) using Lipofectamine 3000, according to the manufacturer’s protocols.
SKOV3/KO and SKOV3/DDP cells in the optimal growth state were infected with the aforementioned lentiviral particles. After the cell culture and selection of single colonies, the genome was extracted from a growing monoclonal cell mass, in which the CRISPR/Cas9 edited site was amplified via PCR and constructed onto a TA vector for sequencing validation. The primers of PCR amplified target sequence were as follows: F, 5′-GGTTGTCCCTAGGACTGTGAG-3′ and R, 5′-GCCCCAGGAAAGACAAAAACT-3′. EEF1D expression in cells was validated via Western blotting. To avoid an off-target effect, potential off-target regions were selected and subjected to PCR and Sanger sequence analysis.
KD of OPTN gene
For OPTN KD in SKOV3/DDP/KD line, OPTN shRNA and scrambled shRNA sequences were designed in which EcoRI and BamHI restriction recognition sites were designed at the end of these sequences (Table S2). The detailed protocol was the same as aforementioned KD of EEF1D gene. However, cells were transiently infected with the lentivirus and infected cells were not selected by puromycin and were not subcultured.
Measurement of cell viability and apoptosis
SKOV3, SKOV3/KD, SKOV3/KO, SKOV3/DDP, SKOV3/DDP/KD and SKOV3/DDP/KO cells were seeded into 96-well plates in sextuplicate at a density of 5 × 103 cells/well and incubated with DDP at the following concentrations: 0 (as control), 12.5, 25 and 50 μmol/l for 48 h. Cell viability was measured via a water-soluble tetrazolium 1 (WST-1) assay with a cell viability and cytotoxicity assay kit (Beyotime Institute of Biotechnology), according to the manufacturer’s instructions. The viability of POCC and transfected cells (EEF1D shRNA and scrambled shRNA) was measured using the same aforementioned methods for the other cell lines. In addition, the aforementioned cell lines were also seeded into 96-well plates in sextuplicate at a density of 5 × 103 cells/well, and incubated with the medium containing various drugs (0 as control, 25 μmol/l DDP, 25 μmol/l DDP + 15 μmol/l LY294002 and 25 μmol/l DDP + 10 μmol/l MK-2206) for 48 h. Cell viability was measured according to the aforementioned method. The SKOV3/DDP/KD cells transiently infected with OPTN shRNA and scrambled shRNA were also seeded into 96-well plates in sextuplicate at a density of 5 × 103 cells/well, and incubated with the medium containing various drugs (0 as control and 25 μmol/l DDP) for 48 h, in which cell viabilities were measured according to the aforementioned method. Cell viability was calculated using the following formula: Cell viability ratio (%) = (the experimental group A450 nm value/control group A450 nm value) × 100%. Each experiment was performed in triplicate independently.
Apoptosis rates of SKOV3, SKOV3/KD, SKOV3/KO, SKOV3/DDP, SKOV3/DDP/KD and SKOV3/DDP/KO cells treated with DDP at 25 μmol/l were measured via flow cytometry (EPICSRXL-MCL; Beckman Coulter, Inc.) using FITC-conjugated Annexin-V and PI from Sigma-Aldrich (Merck KGaA). Saline was used as the control. The experimental operating procedure was performed according to the manufacturer’s protocols. The results of flow cytometry were analyzed using FlowJo v10.6.2 (FlowJo LLC). Cells that stained positive for Annexin-V were counted as apoptotic. The cell apoptosis rates of POCC and cells transiently infected with the lentivirus (EEF1D shRNA and scrambled shRNA) were measured the same as aforementioned cell lines. The experiment was performed in triplicate (cell lines) or duplicate (POCC) in two independent sets.
Animal xenograft tumor model
A total of 50 healthy female nude mice inbred strain (BALB/cAnN-nu/nu), 14–17 g body weight and 3–4 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (no. 11400700209297). All animal experiments were carried out following the protocol approved by the Institutional Animal Care and Use Committee of Anhui Medical University (approval no. LLSC20170064). All methods and experimental protocols were carried out in accordance with relevant guidelines and following the regulations of the protocols of the Institutional Animal Care and Use Committee. The study was carried out in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. All mice were kept in a specific pathogen-free environment in a sterile room at 22 ± 2 °C, 40–60% relative humidity and a 12 h light/dark cycle with a nutritionally adequate diet and free access to food and drinking water at the Anhui Provincial Center for Medical Experimental Animals. During animal experiments, animal suffering was minimized as much as possible. All nude mice were euthanized at the end of the experiments. All mice were acclimated for 1 week, and then each nude mouse was inoculated subcutaneously in the right armpit with 0.2 ml SKOV3/DDP cell suspension at 1 × 107 cells/ml. After 2 weeks, 24 mice with similar tumor sizes (~ 100 mm3) were selected from the aforementioned group of mice, and then randomly divided into four groups (control, DDP, DDP + scrambled shRNA and DDP + EEF1D shRNA) with six mice in each group. The mice were intraperitoneally injected with DDP at 10 μmol/kg body weight once every week for 4 weeks (in the control group, DDP was replaced with saline), meanwhile, DDP + scrambled shRNA and DDP + EEF1D shRNA groups were injected with 100 μl scrambled shRNA lentivirus and EEF1D shRNA lentivirus, respectively, around the tumor tissues once every week. The tumor volumes were measured with Vernier calipers every 5 days and calculated according to the following formula: V = (1/2) ab2. V, tumor volume; a, the largest diameter of tumor; b, the minimum diameter of tumor. After 45 days, all nude mice were euthanized according to Guideline for Euthanasia of Laboratory Animal in China (T/CALAS 31–2017, Chinese Society of Experimental Zoology Group Standards). Then xenograft tumors were removed, weighed, collected and frozen in liquid nitrogen for subsequent experiments.
Morphological observation of xenograft tumor tissues and TUNEL assay
The tumor specimens were fixed with 4% paraformaldehyde solution at room temperature for 24 h, dehydrated with alcohol, embedded in paraffin and prepared into normal tissue sections. These xenograft tumor tissues were stained with H&E at room temperature for 5 min (H) + 3 s (E) and subsequently observed and analyzed under an optical microscope. The ovarian cancer cell apoptosis of xenograft tumor tissues was analyzed using a TUNEL assay (Roche Diagnostics GmbH), following the manufacturer’s instructions. Apoptosis was quantified using ImageJ software (version 1.44; National Institutes of Health). The apoptosis index (AI) was calculated according to the following formula: AI = (the number apoptotic cells / the total number of cells) × 100%.
The aforementioned ovarian cancer cells were harvested, in which the total RNAs were extracted with TRIzol® reagent and RNA extraction buffer according to the manufacturer’s instructions (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.). RNA was reverse-transcribed to cDNA using above TRIzol transcription kit following the manufacturer’s instructions. The reverse transcription reaction conditions were 42 °C for 1 h and 70 °C for 5 min. The cDNA amplification in PCR instrument (Veriti 96-Well Thermal Cycler, Thermo Fisher Scientific, Inc.). The reaction conditions were as follows: 94 °C pre-denaturation 5 min; 94 °C × 30 s, 55 °C × 30 s, 72 °C × 45 s (30 cycles). β-actin was used as the housekeeping gene, primer pairs of which (designed by Primer 6.0 and synthesized by Sangon Biotech Co., Ltd.) were used for the PCR: F, 5′-ATCCAGGCTGTGCTATCCCT-3′ and R, 5′-TTGCCAATGGTGATGACCTG-3′.PCR products were observed by 1.5% agarose gel electrophoresis, in which the intensities were semi-quantified using Quantity One software version 29.0 (Bio-Rad Laboratories, Inc.). All reactions were performed in triplicate, and two independent experiments were run.
Xenograft tumor tissues were lysed in RIPA lysis buffer, and proteins were resolved via SDS-PAGE [5% stacking gel, 10% lower gel (w/v); 22.5 μg protein in 15 μl loaded per lane] and electrotransfered onto PVDF membranes, which were performed using a standard protocol . Protein concentration was measured using a bicinchoninic acid protein assay kit (cat. no. P0012; Beyotime Institute of Biotechnology). Western blotting assays were performed using primary antibodies (1:500 or 1:1000) specific for EEF1D, PI3K, OPTN, Akt, p-Akt, Bcl-2, Bax, caspase-3, cleaved caspase-3, ERCC1 and β-actin. Membranes were then incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit IgG secondary antibodies (1:10,000). The proteins were detected with an ECL system followed by exposure in ChemiScope 6300 Fluorescence and Chemiluminescence Imaging system (Clinx Science Instruments Co., Ltd.), in which digital images were captured and the intensities were semi-quantified using Quantity One software version 29.0 (Bio-Rad Laboratories, Inc.). β-actin was used as the loading control. Each experiment was performed in two independent sets.
All results are presented as the mean ± standard deviation. The differences among groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test with SPSS software (version 20.0; SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference.