Cell lines and transfection
Human PCa cells (PC3, DU145) were obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. PC3, DU145 cells were cultured in Minimal Essential Medium (MEM) supplemented with 10% FBS purchased from GIBCO(America) . The siRNA of circRNAs, miRNA mimic, inhibitor, and corresponding control oligonucleotides were purchased from Ruibo (Guangzhou, China). The transfection of siRNA, miRNA mimics, miRNA inhibitor and plasmids were performed using jetPRIME (France). Lentivirus shRNAs for METTL3 and circRPS6KC1 were purchased from Genechem (Shanghai, China). CircRPS6KC1 overexpression plasmid was constructed by RiboBio(Guangzhou, China) The transfection procedure was conducted according to the manufacturer’s instructions. The sequences of siRNA are available in Supplementary Table S1.
Clinical tissue samples
Twenty four pairs of prostate cancer tissues and normal tissues were obtained from The First Affiliated Hospital of Zhejiang University School of Medicine. All experiments involving human samples and clinical data were approved by the Accreditation Committee of The First Affiliated Hospital of Zhejiang University School of Medicine.
CircRNA array sequence and RNA sequence
After transfecting METTL3 and negative control siRNA into PC3 cell, the total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, USA). The circRNA array sequence was performed at Arraystar (America). Differential gene expression analysis was performed between siMETTL3 group and siNC group with the screening threshold of fold change > 1.5 and P-value < 0.05. The public datasets GSE140927 were obtained from the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) database. Differential gene expression analysis was conducted using the limma R package with log fold change > 0.8 and P-value < 0.05 as the screening criterion. The downstream miRNAs of circRPS6KC1 were predicted through Arraystar annotation. The downstream target genes of miR-761 were predicted through TargetScan (http://www.targetscan.org/). RNA sequence was performed by Oebiotech(Shanghai, China). The expressions of FOXM1 in prostate cancer and normal prostate tissue from TCGA and GTEx databases and the correlation analysis of FOXM1 and PCNA in TCGA database were analysed by GEPIA2 (http://gepia2.cancer-pku.cn/).
RNA extraction and quantitative real-time PCR
Total RNA from PCa cells, tumor tissues were extracted using the TRIzol Reagent (Thermo, USA). Cytoplasmic & Nuclear RNA Purification Kit (Norgen, Canada) was applied for cytoplasmic and nuclear RNA. PrimeScript TM RT Master Mix (Takara, Japan). Mir-X TM miRNA First-Strand Synthesis Kit (Takara, Japan) were performed for mRNA, circRNA and miRNA reverse transcription. The real-time PCR reactions were conducted with ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). The relative mRNA, circRNA and miRNA expression levels were normalized to β-actin or U6, calculated by the 2−△△CT method. The sequences of primers are listed in Supplementary Table S2.
RNase R treatment
RNase R (Servicebio, China) was used to digest linear RNA. RNAs extracted from PC3 and DU145 cells were divided into two groups for RNase R treatment and control. The sample was incubated for 30 min at 37 °C with 2 U/μg of RNase R.
Western blot
The previous study had reported the specific protocol for western blot. The antibodies we used contained: β-actin(Cat No: 81115-1-RR, proteintech, USA), FOXM1(Cat No: 13147-1-AP), p21 (Cat. No. 10355–1-AP, proteintech, USA), CDK2(Cat No: 10122-1-AP, proteintech, USA), METTL3(Cat No: 15073-1-AP, proteintech, USA) , YTHDF1(Cat No : 17479-1-AP, proteintech, USA).
Colony formation assay and cell counting kit 8 (CCK-8) assay
The previous study had reported the concrete protocol for colony formation assay and cell counting kit 8 assay[8].
Senescence associated β-galactosidase staining(SA-β-gal staining)
Senescence β-Galactosidase Staining Kit wae purchased from Beyotime(Shanghai, China). PC3 and DU145 cell were cultured cultured in 24-well plates. After washing with PBS and fixation, dispense the working fluid according to the protocol. Incubate overnight at 37ºC. The SA-β-gal positve cells were obtained via optical microscope.
Cell cycle flow cytometry and EdU assays
Cell cycle flow cytometry was performed using Cell Cycle Staining Kit (MultiSciences, Hangzhou, China). EdU assays were perform using BeyoClick™ EdU Cell Proliferation Kit(Beyotime, Shanghai), cuture prostate cells in 96 well plates and prepare the EdU solution. Incubate for around 2 hours. After washing, fixating and Permeabilizating cells, incubate with click additive Solution for 30mins and Hoechst solution for 10mins. Acquire the result using inverted fluorescence microscope.
Methylated RNA immunoprecipitation(MeRIP)
RNA immunoprecipitation (RIP) was conducted using Magna MeRIP™ m6A kit (Millipore, USA). The previous study had reported the concrete protocol for MeRIP assays[16].The sequences of MeRIP primers are listed in Supplementary Table S3.
RNA immunoprecipitation(RIP)
RNA immunoprecipitation was performed using Magna RIP™ RNA-binding protein immunoprecipitation kit (Millipore, USA). The previous study had reported the concrete protocol for RIP assays[16].
CircRNA pull-down
The previous study had reported the detailed protocol for circRNA pull-down assays[16]. The biotin-labeled probe for circRPS6KC1 was constructed from RiboBio (Guangzhou, China). The sequences of probes were list in Supplementary Table S3
Dual-luciferase reporter assays
The wild type circRPS6KC1 pmirGLO plasmid, mutant type circRPS6KC1 pmirGLO plasmid for miR-761 binding site, mutant type circRPS6KC1 pmirGLO plasmid for m6a site and wild type FOXM1 3’UTR pmirGLO plasmid, mutant type FOXM1 3’UTR pmirGLO plasmid for miR-761 binding sites, the pRL-TK plasmid and PNCA promoter pGL3 plasmid were synthesized and constructed into the dual-luciferase reporter from RiboBio (Guangzhou, China).The luciferase activities were analysed via the Dual-Luciferase Assay kit (Promega, USA) following the manufacturer’s protocol.
Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation was performed with the ChIP kit(Millipore, USA)
The previous study had reported the detailed protocol for circRNA pull-down assays[17]. The primers sequence for ChIP-qPCR were listed in Supplementary Table S2
Animal experiments and immunohistochemistry staining (IHC)
The previous study had reported the detailed protocol for subcutaneous xenograft tumor models and immunohistochemistry staining[8].
Statistical analysis
GraphPad Prism 9 was used for statistical analysis. Student’s t-test and Wilcoxon test were applied for P-value test between the test and control groups. P value lower than 0.05 showed the statistical significance.