Animal ethics statement
Four-week-old BALB/c female nude mice weighing 15–20 g (Shanghai Jihui Laboratory Animal Care Co., Ltd.) were used in this study. All animal experiments were approved by The Ethics Committee of the Fourth Hospital of Hebei Medical University. Experiments were conducted according to the Guide for Care and Use of Laboratory Animals (8th Edition).
Strand-specific RNA-seq library preparation and high-throughput RNA-Seq
Total RNA was extracted from OC and paired adjacent noncancerous OC tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Tissues were treated with DNase I (DNA-free kit, Ambion, Texas, USA) twice at 37 °C for 0.5 h each time. Total RNA (~ 3 μg) from each sample was treated with the VAHTS total RNA-seq (H/M/R) Library Prep Kit for Illumina (Vazyme Biotech Co., Ltd, Nanjing, China) to remove ribosomal RNA while retaining other RNA types (including mRNAs and ncRNAs). RNAs were purified using RNase R (Epicenter; 40 U) at 37 °C for 3 h, followed by treatment with TRIzol. Subsequently, an RNA-seq library using the KAPA Stranded RNA-Seq Library Prep Kit (Illumina) was constructed and subjected to deep sequencing with Illumina HiSeq 4000 (Aksomics, Inc., Shanghai, China).
Cell culture
We purchased the human ovarian epithelial cell line, IOSE80, from Shanghai Zhen Biotechnology Co., Ltd. (Shanghai, China) and two OC cell lines, SKOV3 and OV90, from American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were cultured in RPMI-1640 (cat no. SH30809.01; Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS, cat no. SH30084.03; Hyclone) and 5% CO2 at 37 °C.
Cell transfection
Circ-PHC3 small interfering RNA (si-circ-PHC3, 5’-CUCAGAUUUAAGGACCAUAUU-3’), miR-497-5p inhibitor (5’-ACAAACCACAGUGUGCUGCUG-3’), miR-497-5p mimic (5’-CAGCAGCACACUGUGGUUUGUAAACCACAGUGUGCUGCUGUU-3’), SOX9 overexpression vector (SOX9) and the respective negative controls (NC) were synthesized by GenePharma (Shanghai, China). Cell transfection experiments were performed at ~ 80% confluence using Lipofectamine 3000 (cat no. L3000-015; Invitrogen).
Quantitative real-time polymerase chain reaction (qPCR)
Total RNA was extracted from cancer tissue and cells using TRIzol reagent (Invitrogen). cDNA was synthesized and amplified using the TaqMan miRNA Reverse Transcription Kit (Thermo Fisher Scientific) and qPCR conducted with the TaqMan™ MicroRNA Assay Kit (Applied Biosystems, Foster City, CA, USA). We applied the 2−ΔΔCT method to detect related fold changes in expression, with GAPDH and U6 as the internal references. The following primers were utilized: circ-PHC3 forward, 5'-GGCTGCTGTACAGTC-3', reverse, 5'-GTGAGGTGGTGGTG-3'; miR-497-5p forward, 5'-CCTTCAGCAGCACACTGTGG-3', reverse 5'-CAGTGCAGGGTCCGAGGTAT-3'; SOX9 forward, 5'-TGAAGATGACCGACGAGCAGGAGAAG-3', reverse, 5'-CTTCCTCGCTCTCCTTCTTCAG-3'; U6 forward, 5'-CTCGCTTCGGCAGCACA-3', reverse 5'-AACGCTTCACGAATTTGCGT-3'; GAPDH forward 5'-AATGGGCAGCCGTTAGGAAA-3', reverse 5'-TGAAGGGGTCATTGATGGCA-3'.
SKOV3 and OV90 cells subjected to various treatments were used to generate cell suspensions. To this end, we transferred 200 cells to 6-well plates, which were cultured in an incubator for 10 d at 37 °C prior to morphological analysis. The culture medium was changed every 3 days. Prior to the end of the experiment, our team observed images of cells under a light microscope (Primovert; Carl Zeiss, Jena, Germany), which were subsequently washed twice using phosphate-buffered saline (PBS). Next, 500 µL Giemsa dye was added to individual wells. Cells were stained for 10–20 min followed by three washes with double-distilled water. Images were captured using a digital camera.
CCK-8 assay
Cells were incubated in 10% CCK-8 solution diluted with normal culture medium at 37 °C, which resulted in a color change. Cell proliferation rates at 1, 2, and 3 d were assessed following transfection via measurement of absorbance at 570 nm using a microplate reader.
Transwell migration assay
Transwell chambers (Corning, NY, USA) were utilized for transwell migration and matrigel invasion assays conducted following standard protocols (BD Biosciences, Bedford, MA, USA). In brief, 200 μL serum-free medium including 5 × 104 treated cells was transferred to the upper chambers and 600 μL complete medium added to the lower chambers. After incubation of cells for 1 day, images were obtained under an inverted light microscope (Primovert; Carl Zeiss, Jena, Germany). Migrated cells were quantified from more than two random fields of view.
Single OV90 and SKOV3 cells were resuspended in serum-free DMEM medium. Following cell sorting, 200 cells/well in 200 μL serum-free medium supplemented with 20 ng/mL bFGF and 20 ng/mL EGF were cultured in 96-well plates at a density of 10 wells/group, with medium changes every two days. Five regions were randomly selected per well with the aid of a camera-equipped microplate reader (Leica, Wetzlar, Germany). The sphere percentage was expressed as sphere number/200.
Dual-luciferase reporter assay
The putative miR-497-5p binding site was cloned into SOX9 3'-UTR and wild-type (WT) or mutant (MUT) circ-PHC3 into psi-CHECK vector (Promega, Madison, WI USA) downstream of firefly luciferase 3'-UTR or circ-PHC3 as the primary luciferase signal using Renilla luciferase as the normalization signal, and the constructs designated SOX9-Wt/circ-PHC3-Wt and SOX9-Mut/circ-PHC3-Mut, respectively. The psi-CHECK vector normalized the Renilla luciferase signal to compensate for variations between harvested efficiencies and transfections. Lipofectamine 2000 (Invitrogen Life Technologies) was employed for transfection into HEK293 cells. We examined Renilla and firefly luciferase activities one day after transfection using the Dual-Luciferase Reporter Assay System (Promega, Mannheim, Germany) and a luminometer (Molecular Devices, San Jose, CA, USA). Relative Renilla luciferase activity was calculated following the manufacturer's instructions.
In vivo experiments
Animal experiments were performed in accordance with established procedures (8). To validate the nude mouse model of OC, stable lentiviral-mediated circ-PHC3-silenced (sh-circ-PHC3) (1 × 106) SKOV3 cells or NC in 100 μL PBS were injected into flanks of BALB/C nude mice, and tumor volumes and weights were measured. Each treatment group contained six mice.
For analysis of tumor metastasis, stable lentiviral-mediated circ-PHC3-silenced (sh-circ-PHC3) (2 × 105) SKOV3 cells or NC in 100 μL PBS were injected into tail veins of 4 week-old female nude mice. After 4 weeks, lung metastasis was evaluated using an in vivo bioluminescence imaging system. The number of metastatic foci in lung tissues was assessed via hematoxylin and eosin staining.
Immunofluorescence and immunohistochemistry
Tumor tissue samples were fixed in 10% formalin solution, followed by embedding in paraffin. Sections (5 μm) were subsequently stained with Ki-67 for evaluation of proliferation. Samples were examined under an Axiophot light microscope (Zeiss, Oberkochen, Germany) and images captured using a digital camera.
Statistical analysis
Data are shown as means ± standard deviation (SD). Statistical analyses were performed in GraphPad Prism (La Jolla, USA) to assess significant differences among groups. P-values ≤ 0.05 were regarded as statistically significant. Two-tailed Student’s t-tests were used to determine significant differences between two groups, and one-way ANOVA with post hoc Bonferroni test was used to determine significant differences among three or more groups.