2.1 Patients and tissues

Fifty-three OC patients were recruited from Chongming Hospital Affiliated to Shanghai University of Medicine and Health Sciences, and they did not receive radiotherapy or chemotherapy prior to surgery. The tumor tissue specimens as well as the adjacent normal tissues were collected, and these tissue materials were placed in liquid nitrogen for rapid freezing. The study was approved by the Ethics Committee of Chongming Hospital Affiliated to Shanghai University of Medicine and Health Sciences (Ethics number: 2023-23-015) and was performed in accordance with the 1964 Declaration of Helsinki. All patients with OC provided written informed consent.

2.2 Cell culture

Human normal ovarian epithelial IOSE80 cells and A2780 OC cells were purchased from the China Strain Collection (Shanghai, China), and SKOV3 OC cells were purchased from Shanghai Aolu Biotechnology Co., Ltd. (Shanghai, China). All cell lines were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) enriched with 1% penicillin/streptomycin (Invitrogen) and 10% fetal bovine serum (FBS, Gibco, Tokyo, Japan) at 37 °C and 5% CO2.

2.3 Cell transfection

All plasmids and oligonucleotides were provided by Genechem (Shanghai, China), including circ_0001068 small interference RNA (siRNA) pool (si-circ_0001068), miR-149-5p mimic (miR-149-5p), miR-149-5p inhibitor (anti-miR-149-5p), FXYD5 siRNA pool (si-FXYD5), FXYD5 overexpression vector (pc-DNA3.0-FXYD5) and the corresponding control vectors (si-NC, miR-NC, anti-miR-NC and pc-DNA3.0). The Lipofectamine 2000 (Thermo Fisher, Shanghai, China) was used as the transfection reagent to transfect the above oligonucleotide or/and vector into A2780 and SKOV3 cells as described by the producer. Briefly, A2780 and SKOV3 cells were plated in 24-well culture plates at the appropriate amount to yield 50–60% confluence after overnight culture. At the time of transfection, a mixture of oligonucleotide or/and vector, Opti-MEM and Lipofectamine 2000 was prepared and added into each well. After 8 h incubation, the media were changed with fresh grown media, and cells were cultured for another 48 h before further analyses.

2.4 Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from tissue samples or cultured cells using Trizol reagent (#15596018CN, Thermo Fisher). Nuclear and cytoplasmic fractions were prepared using the NE-PER™ Kit (#78833, Invitrogen) before RNA extraction by Trizol reagent. For circ_0001068 and FXYD5 expression analyses, total RNA was reverse transcribed (RT) using a complementary DNA (cDNA) synthesis kit (#K1622, Thermo Fisher), and SYBR Green reagent (#A46110, Thermo Fisher) was used for PCR amplification. For miR-149-5p analysis, a TaqMan miRNA RT Kit (#4366596, Thermo Fisher) was used for miRNA-specific cDNA synthesis, and the TaqMan miRNA assay (#4440888, Thermo Fisher) was used for RNA quantification. Relative mRNA expression was quantified by the 2−ΔΔCt method after normalization to the amount of β-actin or U6. The primer sequences were shown in Table 1.

Table 1 Primers sequences used for qRT-PCR2.5 Colony formation assay

Transfected A2780 and SKOV3 cells were seeded in 6-well cell culture plates (Thermo Fisher) at 200 cells/well in triplicate and then cultured using standard protocols at 37 °C and 5% CO2. The media were changed with fresh mediums every three days. After 2 weeks, the cells were fixed and stained with 0.5% crystal violet (Sangon, Shanghai, China). The number of formed colonies (more than 50 cells) was counted, and the results were analyzed from three independent biological replicates.

2.6 Transwell assay

Transwell plates coated (for invasion) or uncoated (for migration) Matrigel (Unikon Biotechnology, Beijing, China) were employed for the detection of cell invasion and migration. Transfected OC cells re-suspended in serum-free medium were seeded into the upper chambers (1 × 105 cells/well for invasion, 4 × 104 cells/well for migration), and the lower chambers were added with 600 µL growth media containing 20% FBS. 24 h later, the migrated or invaded cells on the lower surface of the membrane were observed under a microscope (BX53, Olympus, Tokyo, Japan) and quantified by ImageJ (National Institutes of Health, Bethesda, MD, USA) after staining with 0.5% crystal violet.

2.7 Measurement of glucose uptake, lactate production, glutamate and glutamine levels

OC cells after 48 h transfection were lysed by 0.2% Triton X-100 (Sangon) and then centrifuged for 15 min. Then, 50 µL of supernatant was collected to measure glucose consumption, lactate production, glutamate production and glutamine production levels, as per the recommended protocols, using the glucose assay kit, lactate assay kit (Jinpanbio, Shanghai, China), glutamate assay kit and glutamine assay kit (Amyjet, Wuhan, China).

2.8 Western blot assay

Total protein was extracted from cell samples as well as tissue samples using RIPA lysis buffer purchased from Biolab Technology (Beijing, China), and the extracted protein was quantified using the BCA Protein Assay Kit (Sigma, Shanghai, China). About 50 µg protein samples were separated on SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes (Mingyangkehua, Beijing, China). The membranes were blocked in 5% skim milk and then incubated (overnight at 4 °C) with primary antibodies, including anti-proliferating cell nuclear antigen (anti-PCNA, 1:1000, ab29, Abcam, Cambridge, UK), anti-matrix metalloprotein 9 (anti-MMP9, 1:1000, ab76003, Abcam), anti-hexokinase 2 (anti-HK2, 1:1000, ab209847, Abcam), anti-Recombinant Glutaminase 2 (anti-GLS2, 1:1000, ab113509, Abcam), anti-FXYD5 (PRO-1366, ProSpec), and anti-beta-Actin (anti-β-actin, 1:5000, ab8226, Abcam). Following secondary antibody incubation, protein signals were visualized using Western Blotting Substrate (Thermo Fisher) and Fluorochem M imaging system (Protein Simple, San Jose, CA, USA). Blots were quantified by AlphaView Software (Protein Simple). All western blot assays were performed with three independent replicates (in triplicate).

2.9 Dual-luciferase reporter assay

In accordance with the standard protocols [24], the circ_0001068 fragments containing the predicted wild-type (WT, CUCGGUC) or mutant (MUT, GAGCCAG) binding sites for miR-149-5p and the FXYD5 3ʹUTR segments harboring the predicted WT (CUCGGUC) miR-149-5p binding sites or miss-matched target sequence (GAGCCAG) were synthesized by Thermo Fisher and inserted into the pmirGLO luciferase reporter vector (Youbio, Chongqing, China), respectively, to generate reporter constructs including circ_0001068-WT, circ_0001068-MUT, FXYD5-3’UTR-WT and FXYD5-3’UTR-MUT. Each reporter construct was transfected into OC cells along with miR-NC mimic or miR-149-5p mimic using Lipofectamine 2000 as the transfection reagent. After 24 h, the Dual-Luciferase Reporter Assay System (Thermo Fisher) was used to measure the luciferase intensities (firefly and Renilla). The ratio of firefly to Renilla was determined.

2.10 RNA pull-down assay

Cells (A2780 and SKOV3) were lysed in RIPA buffer and then incubated (overnight at 4 °C) with biotin-labeled miRNA mimic Bio-miR-149-5p or negative control Bio-miR-NC (both from Thermo Fisher) and streptavidin magnetic beads (Beyotime, Shanghai, China). After that, RNA was extracted from beads using Trizol reagent (Thermo Fisher), and circ_0001068 enrichment levels were detected by RT-qPCR.

2.11 In vivo xenograft tumor formation assay

For xenograft generation, female BALB/c nude mice (age: 6–7 weeks, SYXK: 20100526) were purchased from Shanghai Slaughter Laboratory Animal Co., Ltd., (Shanghai, China). A2780 OC cells stably expressing sh-NC or sh-circ_0001068 (4 × 106 cells/mouse) were inoculated into the right flanks of nude mice (5 mice/group) by subcutaneous injection. Tumor size was measured every week (tumor volume = length×width2 × 0.5) by a caliper. At the fifth week, the nude mice were euthanized, and the subcutaneous tumors were removed and weighed. All animal experiments were conducted strictly in line with the guidelines approved by the Ethics Committee of Chongming Hospital Affiliated to Shanghai University of Medicine and Health Sciences and performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals. The maximum size of xenograft tumors permitted by the Institutional Review Board is 1.5 cm3. In this study, all of subcutaneous xenograft tumors were less than 1 cm3 in size at the end of animal experiments.

2.12 Immunohistochemistry (IHC)

Xenograft specimens were fixed with paraformaldehyde solution, followed by paraffin embedding and sectioning into 4 μm thickness. After dehydration, rehydration, antigen retrieval and endogenous peroxidase blockade, sections were incubated overnight at 4 °C with primary antibody (anti-Ki-67, 1:200, ab16667, Abcam). Signals were developed with 3,3’-diaminobenzidine working solution (Thermo Fisher) and counterstained with hematoxylin (Thermo Fisher). Under the BX53 microscope, images were microscopically photographed.

2.13 Statistical analysis

Data were presented as mean ± SD from three independent biological replicates and analyzed using GraphPad Prism 8.0 software. Differences were analyzed using Student’s t-test (two groups) and one-way or two-way analysis of variance (more than two groups). Statistical significance was considered at P < 0.05.