Ethics statement

Our team employed BALB/c nude female mice aged 4 weeks with 15 ∼ 20 g weight (SLARC, Shanghai, China). Animal Research Committee in the Shanghai First People’s Hospital, affiliated with Shanghai Jiao Tong University (SJTU), approved animal protocols.

Tissue chips and FISH

We gained OC tissue chips (n = 10) from the Shanghai First People’s Hospital affiliated with SJTU. Geneseed Biotech (CA, USA) provided particular probes for circ-TFRC (Dig-5′-GATCGTCTTTTTCTTGATTGGTTCTTCTGTG-3′-Dig). For FISH analyses, the technician captured signals and counterstained nuclei applying DAPI (Yeasen Biotechnology, Shanghai, China) for 15 min. Technician obtained photos utilizing Zeiss LSM 700 confocal microscope (Carl Zeiss GmbH; Oberkochen, Germany).

Cell culture

Our team obtained 4 human OC cells (OVCAR-3, SKOV3, A2780, and ES-2) and 1 normal epithelial cell line (IOSE80) from Chinese Academy of Sciences Cell Bank (Shanghai, China). The cells were cultured in DMEM (Gibco, MD, USA) using 10% fetal bovine serum (FBS) (Gibco) and 50 µg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2.

Bioinformatics analysis

We used the Encyclopedia of RNA Interactomes database to evaluate interactions among miRNA, mRNA, and circRNA.

RNA overexpression or interference

miR-615-3p inhibitor, miR-615-3p mimic, siRNA against circ-TFRC (si-circ-TFRC, 5′-CTGTGTGGCAGTTCAGAATGATG-3′) were purchased from GenePharma (Shanghai, China). and IGF2 overexpression vector (constructed the IGF2 cDNA sequence into the cDNA3.1 vector) were used for transfections.

Cell migration assay

This study used 24-well Transwell chambers (BD Biosciences, NJ, USA) for cell migration analyses. We plated A2780 and SKOV3 cells (1 × 105) into upper chamber and 500 µL of DMEM with 20% FBS into lower chamber. We cultured cells for 1 day at 37 °C before being fixed to the bottom chamber cells for 0.5 h with 4% paraformaldehyde. Subsequently, technician stained cells with 0.1% crystal violet (Shanghai Yisheng Biotechnology, Shanghai, China). We obtained photographs using Zeiss Axio Observer D1 microscope.

5-ethynyl-20-deoxyuridine analysis

The technician employed 5-ethynyl-20-deoxyuridine (EdU) assay kits (Thermo Fisher Scientific) to analyze cell activities. We maintained SKOV3 and A2780 cells (1 × 105) in six-well plates for 2 days in prior putting EdU to wells for 2 h. We fixed the cells using 4% formaldehyde.

Cell counting kit-8 assay

The technician incubated A2780 and SKOV3 cells in 10% cell counting kit-8 (CCK8) diluted in a normal culture medium at 37 °C. The proliferation rates were recorded after 0, 1, 2, and 3 days of transfection. Absorbance was recorded by employing microplate reader.

RT-qPCR

RNA from tumor tissues and cells were isolated using TRIzol kit (Thermo Fisher Scientific). Total RNAs (500 ng) were reverse transcribed to cDNA with a PrimeScript Reverse Transcriptase Kit (Takara, Dalian, China). Further, miR-615-3p, circ-TFRC and IGF2 were detected by quantitative reverse transcription PCR (qRT-PCR) using a Bulge-Loop TM miRNA qRT-PCR Starter Kit (Applied RiboBio Biotechnology, Guangzhou, Guangdong, China) and a SYBR Green PCR Kit (Takara), respectively. Our team utilized 2−ΔΔCT approach to capture fold changes in relative expression. Primers that employed in current study were:

circ-TFRC:5′-GATCAAGCTAGATCAGC-3′ (forward) and 5′-GCTGAACCGGGTATATGAC-3′ (reverse); miR-615-3p:5′-CCGCGCATCTGGAGGTAAGAAG-3′ (forward) and 5′-AGTGCAGGGTCCGAGGTATT-3′ (reverse);

IGF2:5′-GTGGCATCGTTGAGGAGTG-3′ (forward) and 5′-CACGTCCCTCTCGGACTTG-3′ (reverse);

U6:5′-CTCGCTTCGGCAGCACA-3′ (forward) and 5′-AACGCTTCACGAATTTGCGT-3′ (reverse);

GAPDH:5′-AATGGGCAGCCGTTAGGAAA-3′ (forward) and reverse: 5′-TGAAGGGGTCATTGATGGCA-3′ (reverse).

Dual-luciferase reporter assay

The technician cloned putative miR-615-3p-binding sites of the wild type (wt) or mutant (mut) 3’ UTR IGF2 and circ-TFRC into psi-CHECK (Promega, WI, USA) vector downstream. Firefly luciferase 3’ UTR or circ-TFRC was primary luciferase signal. We named these clones IGF2-Wt/circ-TFRC-Wtand IGF2-Mut/circ-TFRC-Mut. We employed the psi-CHECK vector with the Renilla luciferase signal for normalization to compensate for the variances between harvested efficiencies and transfections. This study involved HEK293 cell transfections applying Lipofectamine 2000. We detected firefly luciferase and Renilla actions 1 day after transfection.

Tumor xenograft formation

Wt or 2 × 106 sh-circ-TFRC SKOV3 cells were tail vein injection into the mouse’s right flank to calculate tumor sizes each five days for 1 month utilizing Vernier caliper. Tumor volume was computed utilizing formula: length × width2 × 0.5. The relative expression of Ki67 was measured employing the IH method. Each group have 5 mice.

We stably transfected luminescence-labeled SKOV3 cells with negative control (NC) for metastasis analyses. Technician suspended sh-circ-TFRC, which we injected into each nude mouse tail vein. After four weeks, our team validated lung metastasis by applying bioluminescence imaging system. The technician counted lung tissue metastatic foci after hematoxylin and eosin staining. Each group have 5 mice.

Statistics analysis

We represented data by mean ± standard deviation (SD). GraphPad Prism (GraphPad, CA, USA) was employed to gain variances among groups. P value ≤ 0.05 indicated a statistics significance.