sh-CD164, scramble control sequence (sh-NC), miR-506-3p mimics and inhibitor, and CD164 (pcDNA-CD164), were brought from RiboBio Co., Ltd. (China). EGF, bFGF, B27, methylcellulose, and ferroptosis inducer Erastin was obtained from Sigma-Aldrich (Zhou et al. 2019). Antibodies for ABCG2, C-Myc, Bmi1, Sox2, Vimentin, E-cadherin, CD164, and β-Catenin were purchased from Proteintech (China).
Cells lines and treatment
Human cervical cancer cell lines Hela (CCL-2, ATCC, USA) and C33A (HTB-31, ATCC, USA), and normal cervical epithelial cells HcerEpic (AC340374, ATCC, USA) were obtained from American Type Culture Collection (ATCC, USA) was cultured in DMEM (Gibco, USA) containing 10% FBS (Gibco, USA) at 37 °C incubator with 5% CO2. To activate ferroptosis, cells were treated with Erastin at a dose of 5 μg/ml for 8 h after indicated cell transfection.
The Synthesis and Characterization of ZON
ZON were synthesized by refluxing the precursor zinc acetate dihydrate (0.1 M) in ethane-1,2-diol and triglycol at 180 °C and 220 °C, respectively. The time of the reaction was for 2–3 h in the presence of sodium acetate (0.01 M). The solution was put on a magnetic stirrer at 80 °C for 1.5 h and centrifuged at 8000 rpm for 15 min and rinsed with deionized water and ethyl alcohol 3 times. Finally, the samples were dried at 80 °C overnight. ZON dose was dissolved in deionized water till the complete dissolution. Size, morphology, and elemental composition were observed and measured by a transmission electron microscope (TEM, JEOL, Japan), while the surface zeta potential measurements were also measured by a zeta potential analyzer (Malvern Device, UK) (Nabil et al. 2020).
Cell counting kit 8 (CCK-8)
Cell viability was detected using CCK-8 kit (Thermo). Hela and C33A cells were digested and seeded in 96-well plate. After seeding for 0, 24, 48, 72, and 96 h, CCK-8 reagent was added into each well to incubate for another 1 h. Then, the optical density (OD) at 450 nm was detected by a microplate reader (BioTech, USA).
Colony formation assay
Hela and C33A cells were digested and seeded in 6-well plate (1000 cells per well), and cultured for 2 weeks. The visible colonies were fixed with methanol, followed by staining with 0.2% crystal violet for twelve minutes, and were captured by a digital camera.
Hela and C33A cells were transfected with sh-circFoxo3, and/or circFoxo3 overexpressing vector for 48 h, digested, and collected for detection of apoptosis using a Annecin-V/PI Apoptosis Detection Kit (Thermo) in accordance with manufacturer’s description.
Cell migration and invasion
Cell migration was determined by Transwell and wound healing experiment. For Transwell assay, cells were suspended in FBS-free medium and seeded in top chambers, and the lower chambers contained complete medium. After incubation for 24 h, the cells migrated to the lower side of top chambers were stained with 0.2% crystal violet and captured. For wound healing experiment, cell monolayer was scratched by a 200 μl pipette, washed with PBS, and cultured in FBS-free media. The migration ability was evaluated by calculating cell movement into the scratched area drawn at 0–24 h.
Hela and C33A cells were subjected to indicated treatment, digested and seeded in 96-well ultra-low-attachment plates (Corning) at a density of 500 cells per well for 10 days. The culture medium consists of DMEM/F12 medium (Hyclone), 10 ng/mL EGF, 20 ng/mL bFGF, B27 (1:50), and 20% methylcellulose. The spheres were photographed by microscope (Leica, Germany).
Western blotting assay was performed following a standard procedure. In short, total protein was extracted from Hela and C33A cells after treatment, separated in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), shifted to NC membranes. Then, the membranes were soaked in blocking solution (Sigma-Aldrich) for 15 min, incubated with indicated primary antibodies at 4 °C for one night, and subsequently hatched with corresponding secondary antibodies at room temperature for 45 min. The protein bands were then reacted with enhanced chemiluminescent solution (ECL, Millipore, USA).
Quantitative real-time PCR (qRT-PCR)
For quantification of miR-506-3p and CD164, total RNA was extracted by Trizol reagent and reverse-transcribed using a cDNA reverse transcription kit (Thermo). For miR-506-3p quantification, the RNA was subjected to an All-in-One™ miRNA cDNA Synthesis Kit (GeneCopoeia, USA). qRT-PCR was conducted using a SYBR kit (Thermo). The relative expression were calculated using the 2−△△CT method. The CD164 expression was normalized by GAPDH. The miR-506-3p expression was normalized by U6 small nuclear RNA. The primers were as following:
miR-506-3p, sense 5′-ACACTCATAAGGCACCCTTC-3′,
CD164, sense 5′-ACCCGAACGTGACGACTTTAG-3′,
GAPDH, sense 5′-CTTTGGTA TCGTGGAAGGACTC-3′,
U6, sense 5′-GCTTCGGCAGCACATATACTAAAA T-3′,
antisense 5′-CGCTTCACGAA TTTGCGTGTCA T-3′.
Identification of ferroptosis
To identify the occurrence of ferroptosis, we detected the levels of glutathione (GSH), malondialdehyde (MDA), ROS and intracellular iron using GSH assay kit (Beyotime, China), MDA assay kit (Beyotime), C11-BODIPY (Beyotime), and Iron Assay Kit (Beyotime) following the manufacturer’s protocols.
Dual luciferase reporter gene assay
Recombinant vectors of pMIR-CD164 wild-type (WT), pMIR-CD164 mutant (MUT), were synthesized by RiboBio (China). Hela and C33A cells were transfected by WT or MUT luciferase reporter vectors along with miR-506-3p mimics for 24 h. Activity of luciferase was measured using dual luciferase reporter assay system (Promega) in accordance with manufacturer’s description.
Mice xenograft model
6-week aged BALB/C nude mice were ordered from Vital River Laboratory (China) and randomly divided into three groups. C33A and Hela cells (5 × 106) were planted into the right flank of mice. Tumor size ((length × width2)/2) and mice body weight were measured every 5 days for 30 days. The mice were then sacrificed and tumors were collected for immunohistochemical (IHC) analysis and western blotting experiment. This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Shanxi Provincial People’s Hospital.
Tumor tissue were fixed in 4% polyformaldehyde (PFA), embedded with paraffin, and sliced into 4-μm sections. Subsequently, the tissue slides went through dewaxing, rehydration, antigen repair, blocking, incubation with antibody against Ki67 (Proteintech) and HRP-conjugated secondary antibody (Proteintech). The labelled sections were then stained by DAB solution and hematoxylin (Beyotime). The positive staining was photographed by microscope (Leica) and calculated by Image J.
All results were presented as the mean ± standard deviation (SD), and analyzed using an SPSS 20.0 software. The statistical differences between two or multiple groups were calculated by Student’s t tests or one-way analysis of variance (ANOV A) followed by Tukey’s post hoc test. The p values lower than 0.05 was considered as statistically significant.