Cell lines and cell culture
Human osteosarcoma cell line U2OS and human embryonic kidney (HEK) 293T cells were maintained in Dulbecco`s modified Eagle medium (DMEM) (Life Technologies Corporation, California, USA) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Israel). 143B and 143B/Luc were cultivated in MEM/EBSS basic medium (HyClone, Utah, USA) supplemented with 10% FBS (Biological Industries, Israel). All cell lines were maintained in an incubator at 37℃ in an atmosphere containing 5% CO2. All cells were repeatedly screened for mycoplasma and maintained in culture for < 6 months after receipt.
Cells were transfected to knock down the expression of METTL1 and AMO-miR-26a-5p using Lipofectamine TM 3000 Transfection Reagent (Cat# L3000-015; Invitrogen, California, USA). METTL1, FTH1, FTH1-mut plasmid and miR-26a-5p mimic were purchased from Gene Pharma (China) to increase the expression of the plasmid. Briefly, for siRNAs, 2 × 105 cells were seeded in a 6-well plate and they will be 70% confluent at the time of transfection 6 µL Lipofectamine TM 3000 reagent and 20 nM siRNA (Gene Pharma, Shanghai, China) were diluted in 125 µL Opti-MEM (Cat# 31985-070; gibco, Grand Island, USA) medium respectively. After Mix and incubating for 2 min separately, these two regents were then mixed and incubated for another 10 min and then added to cells. For plasmids, cells were transfected with 500 ng plasmid using LipofectamineTM 3000 Transfection Reagent according to the manufacturers' protocols. Subsequent experimental measurements were performed 24 h after transfection.
The sequences used are as following:
METTL1 siRNA sense: 5’-CCAAAGGAUAAGAAAGAAATT-3’
METTL1 siRNA antisense: 5’-UUUCUUUCUUAUCCUUUGGTT-3’
hsa-miR-26a-5p mimics sense: 5’-UUCAAGUAAUCCAGGAUAGGCU-3’
hsa-miR-26a-5p mimics antisense: 5’-AGCCUAUCCUGGAUUACUUGAA-3’
Matrigel invasion assay was performed using 24 well plates inserted by 24 mm Transwell® chambers (Corning #3412, USA) precoated with Matrigel (BD Biosciences, San Jose, CA). 5 × 104 cells were resuspended in 200 µL Serum-free medium, seeded into the upper chamber, and medium containing 10% FBS was added into the bottom chamber subsequently. After incubation at 37℃ for 24 h, cells on the underside of the membrane were immobilized and stained with 0.1% crystal violet (Beyotime Biotechnology, China) for 15 min and counted using light microscopy (ECLIPSE TS100, Nikon).
Cells transfected with targeted siRNA or plasmid were seeded in 6 well plates with a concentration of 1,500 cells per well and suspended in the media with 10% FBS. The indicated cells cultured in a humidiଁed atmosphere containing 5% CO2 at a constant temperature of 37℃ to form colonies for 14 days. Colonies were stained with 0.1% crystal violet for 20 min after fixation with 10% formaldehyde for 5 min. Different colony morphologies were captured under a light microscope (ECLIPSE TS100, Nikon).
Cell Counting Kit-8 (CCK8) assay
Cell viability and growth were determined using CCK8 assay in 96 well plates. Cells were transfected with the relevant plasmids culturing for 24 h, followed by incubation with 10 µL CCK8 for 90 min. Absorbance was read at 450 nm using a spectrophotometer (Tecan, Männedorf, Switzerland).
Cells were plated into 6 well plates at a density of 2.5 × 105 cells/mL. When the confluence of cells reached 70%, a wound was created by scraping the cells with a 200 µL pipette tip. Cells were washed with phosphate-buffered saline (PBS) and then transfected with siRNAs or plasmid. Images were captured at 0 h, 24 h, and 48 h after wounding with standard light microscopy (ECLIPSE TS100, Nikon, Japan). The wound area was measured using ImageJ software (National Institutes of Health, USA).
Alkaline hydrolysis and aniline cleavage sequencing (AlkAniline-seq)
The m7G AlkAniline-Seq sequencing service was provided by CLOUDSEQ (Shanghai cloud-seq biomart, http://www.cloud-seq.com.cn). Alkaline hydrolysis of poly (A) -enriched mRNA fragments were used. Cells were incubated with thermosensitive phosphatase (NewEnglandBiolabs, Inc. USA) to dephosphorylate RNA sheet pairs, followed by incubation and lysis in 1 M aniline. RNA libraries were constructed using NEBNext® MultiplexSmallRNALibraryPrepSetforIllumina ® (NewEnglandBiolabs, Inc., USA) according to the supplier's instructions, and library quality control and quantification were performed using the BioAnalyzer2100 system (Agilent Technologies, USA). High-throughput sequencing was performed using an IlluminaHiSeq sequencer.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted using TRIZOL reagent (Life Technologies Corporation) followed the manufacturer's protocol. 500 ng total RNA was reverse-transcribed into 10 µL cDNA using High Capacity cDNA Reverse Transcription Kit (Cat#00676299; Thermo Fisher Scientific, Waltham, USA). qRT-PCR analysis was needed 1 µL cDNA, 1 µL forward primer, 1 µL reverse primer, and SYBR Green PCR Master (Cat#31598800; Roche) by a 7500 Fast Real-Time instrument (Applied Biosystems, Foster City, USA). Gene expression was normalized to endogenous GAPDH. The miRNA amplified transcript level was normalized to U6. Primer sequences can be found in Additional file file: Table S1.
Ethynyl-2-deoxyuridine (EdU) staining assay
EdU Apollo DNA in vitro kit (Ribobio, Guangzhou, China) was used to detect cell proliferation. Cells were plated into 24 well plates (NEST, Hong Kong, China) at a density of 2 × 105. Briefly, cells were fixed with 4% paraformaldehyde (m/v) for 30 min and followed by incubation of 30 µM/mL EdU at 37℃ for 90 min. After permeabilized in 0.5% Triton X-100, the Apollo staining solution was added into the cell culture medium for 30 min in the dark. Finally, the cells were incubated with 20 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. The EdU index (%) was the average ratio of the number of EdU positive cells over total cells in five randomly selected areas under the confocal laser scanning microscope (FV10i, Olympus, Tokyo, Japan).
Tissue microarrays (TMAs) and Immunohistochemistry (IHC) analysis
Osteosarcoma tissue microarrays were purchased from the Bioaitech Company (Xi’an, China), comprised of 11 normal bone tissues, 70 malignant osteosarcoma cores. The slide was baked at 60℃ for 30 min and then followed by antigen retrieval with tris-EDTA buffer (pH 9.0), medium heat for 10 min to boil, cease-fire for 5 min, and washed with PBS for 5 min × 3 times. Endogenous peroxidase was blocked with 3% H2O2-methanol at RT for 25 min and washed with PBS for 5 min × 3 times. The sections were added to normal non-immune animal serum at RT for 10 min and then removed the serum and added different primary antibodies FTH1 (#4393, CST), 4-HNE (bs6313R, Bioss), Ki67 (27309-1-AP, Protein tech), METTL1 (14994-1-AP, protein tech) at 4℃ overnight. Then it was washed with 0.1% tween-20 PBS for 5 min × 3 times. Biotin-labeled sheep anti-mouse/rabbit IgG was added and incubated in a 37℃ wet box for 30 min followed by washing with 0.1 % tween-20 PBS for 5 min × 3 times. DAB working solution was incubated for 5 min and stopped by distilled water washing. After hematoxylin re-staining, washing and differentiation, the slide returned to be blue with full washing followed with regular dehydration transparent and being sealed by neutral gum. The percentage of positive cells were counted in 5 (×400) highpower fields (upper, lower, left, right, and middle) under the microscope, and the mean values were then calculated.
RNA immunoprecipitation (RIP)-qPCR
RIP assay was carried out in 143B cells using Magna RIP Kit (17-700, Millipore, MA) following the manufacturer’s instructions. In brief, a sufficient number of 143B cells (more than 2 × 107 cells per sample) were lysed by RIP lysis buffer, magnetic beads pre-coated with 5 µg m7G antibody or mouse IgG (Millipore) were incubated with sufficient cell lysates at 4℃ overnight. The mixture was digested with proteinase K before the immunoprecipitated RNAs were extracted, purified and subjected to qPCR. The RNA levels were normalized to the input RNA levels (10%).
RNA stability assay
Cells were cultured in 6 well plates and transfected with desired constructs as described above. After 24 h transfection, cells were treated with actinomycin D (Act D, 10 µg/mL, Cat# GC16866, GLPBIO) for 0 h, 3 h, 6 h and 9 h before collection. Total RNAs were isolated for qRT-PCR analysis.
Cells were cultured in a 6 well plate, after 24 h transfection, cell precipitation was collected, the intracellular ferrous iron level using an iron assay kit from ScienCell (8448, California, USA). The absorbance was finally measured at 590 nm.
Luciferase reporter assay
The luciferase reporter assay was performed with the Dual-Luciferase (Promega E2920) according to the manufacturer's protocol. HEK-293T cells seeded in 24 well plates were transfected with a SV40-firefly-Luciferase-MCS fused the wild-type FTH1, miR-26a-5p mimics or NC were co-transfected. Firefly luciferase activity was normalized to Renilla luciferase activity to reflect transfection efficiency.
Lipid peroxidation assay
The relative lipid peroxidation level in cells was assessed using the Image-iT® Lipid Peroxidation Kit (molecular probes, C10445). Cells were treated with 5 µM C11-BODIPY for 30 min harvested, washed twice with PBS and resuspended in 500 µL PBS. Oxidation of the polyunsaturated butadienyl portion of the dye results in a shift of the fluorescence emission peak from 590 nm to 510 nm.
Xenograft tumorigenesis model
BABL/c female nude mice were purchased from Beijing Vital River Laboratory Animal Technology Limited Company (Beijing, China) and randomized into two groups. All animal experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of Harbin Medical University. Mice were injected subcutaneously with 5 × 106 143B cells to form subcutaneous xenografts. Mouse tumor growth was monitored by measuring tumor length as well as width. Mice weights in each group were recorded accordingly. When tumors grew to a volume of 200 mm3, the mice were divided randomly into six groups (n = 5/group) and treated with normal saline, 1 mg/kg Dox (Doxorubicin hydrochloride, MCE, HY15142), 1 mg/kg Fer-1 (Ferrostatin-1, MCE, HY100579, ferroptosis inhibitor), vehicle by daily intraperitoneal administration, body weights of mice in each group during treatment were also recorded accordingly, the animals were sacrificed four weeks after 143B cell transplantation. Xenograft sampling was performed, and tumor volume was calculated by the standard formula: (length × width2)/2. Before xenograft sampling, the xenografts were anesthetized with isoflurane gas, scanned with an IVIS in vivo imaging system (IVIS LuminaIII, MA, USA), and the total photon radiation at each tumor site was quantified using Living Image software. Mice were euthanized for xenograft sampling.
Transmission electron microscopy
After the cells were pretreated accordingly, the cells were mixed and centrifuged for 250 revolutions for 5 min. Discard the culture medium, add 1 mL PBS culture medium, and resuspend the cells. Next, the samples were horizontally centrifuged at 3000 rpm for 20 min. After the completion of centrifugation, PBS was discarded, and 2.5% glutaraldehyde was added to fix the cells. Cells were dehydrated with an ethanol concentration gradient and embedded in resin. They were then sectioned with an ultramicrotome, stained with uranyl acetate-lead citrate double staining, and finally observed under a transmission electron microscope.
Cell samples were washed using PBS, and then the total proteins were extracted using 1×RIPA lysis buffer (Beyotime, Shanghai, China). The lysates were fully crushed by ultrasonic and cleared by high-speed centrifugation at 13,500g for 15 min. The extracted total proteins were quantified by BCA Protein Assay Kit (Beyotime, Shanghai, China), then equal amounts of proteins were separated by SDS-PAGE and transferred to NC membrane. After the membranes were blocked with 5% w/v non-fat milk for 1h, the proteins were probed at 4℃ overnight using different primary antibodies METTL1 (14994-1-AP, proteintech), FTH1 (#4393, CST), GPX4 (A13309, Abclonal), GAPDH (AC002, Abclonal), β-Tubulin (AC021, Abclonal). On the next day, the membranes were incubated with a secondary antibody (RS23910, ImmunoWay) for 1 h at room temperature. Results were detected using Odessey Clex (LI-COR, America), followed by further analysis.
Hematoxylin and Eosin Staining (H&E) staining
The H&E staining Kit (G1120, Solarbio) was used for H&E staining, paraffin sections were stained with hematoxylin for 5 min and differentiated with differentiation solution for 30 s, followed by soaking into tap water for 15 min. And then the sections were stained with eosin, followed by dehydrated with gradient alcohol and cleared with xylene. Images were acquired using a Leica microscope and Leica image software.
All experiment results were at least repeated three times and expressed as means ± SEM. Statistical analyses were performed using GraphPad Prism 8.0 and Student’s T-test was used for two-group comparisons. p < 0.05 was considered statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001.