Additional file 1 of Hsa_circ_0003258 promotes prostate cancer metastasis by complexing with IGF2BP3 and sponging miR-653-5p
Creators: name: Yu, Yu-Zhong
name: Lv, Dao-Jun
name: Wang, Chong
name: Song, Xian-Lu
name: Xie, Tao
name: Wang, Tao
name: Li, Zhi-Min
name: Guo, Jia-Ding
name: Fu, Du-Jiang
name: Li, Kang-Jin
name: Wu, Ding-Lan
name: Chan, Franky Leung
name: Feng, Ning-Han
name: Chen, Zhe-Sheng
name: Zhao, Shan-Chao
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Statistics
subject: FOS: Mathematics

description: Additional file 1: Supplementary Figure 1. The expression and function of hsa_circ_0003258 in PCa. (A). The heatmap showing some of the differentially expressed circRNAs between normal and prostate cancer patient plasma samples. (B). The representative images of FISH analysis of hsa_circ_0003258 in normal and PCa tissues. (C). The protein coding potential of hsa_circ_0003258. (D). The melting curve of hsa_circ_0003258 amplified product using the divergent primers. (E-H). Transwell assay detecting the migratory capacity of PCa cells after transfecting with siRNAs or vector plasmid. (I-J). Colony formation assays and CCK8 assays were utilized to evaluate the proliferation of PCa cells after knockdown of hsa_circ_0003258 in DU145 and C4–2 cells. ns: Not Significant. Supplementary Figure 2. Hsa_circ_0003258/ miR-653-5p/Arhgap5 pathway in C4-2. (A). The heatmap of differentially expressed mRNAs in DU145/sh-nc and DU145/sh- hsa_circ_0003258 cells. Each sample was mixed in three replicates. (B). Transwell assay detecting the migratory capacity of PCa cells after SCH772984 treatment for 48 h in DU145 Lv- hsa_circ_0003258 cells. (C). The potential binding miRNAs predicted by CircInteractome database. (D). Lysates from C4–2 cells was subjected to biotinylated-hsa_circ_0003258 pull-down assay and the expression levels of hsa_circ_0003258 were measured by qRT-PCR. (E). The expression levels of the top three candidate miRNAs predicted by Circinteractome database quantified by qRT-PCR after biotinylated-hsa_circ_00032583 pull-down assay in C4–2 cells. (F). The relative expression of hsa_circ_0003258 detected by qRT-PCR after the transfection of miR-653-5p mimic or inhibitor. (G-H). The qRT-PCR and Western blot showing the ARHGAP5 mRNA and protein level change of the predicated targets after silencing or overexpression of hsa_circ_0003258. (I-J). The qRT-PCR and Western blot showing the ARHGAP5 mRNA and protein level change of the predicated targets after silencing or overexpression of miR-653-5p. ns: Not Significant; * P &lt; 0.05; ** P &lt; 0.01; *** P &lt; 0.001. Supplementary Figure 3. MiR-653-5p and ARHGAP5 promote metastasis of PCa. (A). Cell migration observed in transwell assays after overexpression or silencing of miR-653-5p in C4–2 cells. (B). Transwell assay was performed after transfection with indicated vectors, lv- hsa_circ_0003258, mimic-nc or mimic-miR-653-5p in C4–2. (C). Transwell assay detected the migratory capacity of C4–2 cells after silencing ARHGAP5. ns: Not Significant; ** P &lt; 0.01; *** P &lt; 0.001; **** P &lt; 0.0001. Supplementary Figure 4. The RNA-binding protein IGF2BP3 binds to hsa_circ_0003258 in C4-2. (A). UP Western blot analysis of IGF2BP3 levels in pulldown assays using a biotinylated antisense oligomer targeting the junction of hsa_circ_0003258 in C4–2. Down RIP assay showing the association of IGF2BP3 with Hsa_circ_0003258in C4–2. Relative enrichment representing RNA levels associated with IGF2BP3 relative to an input control. IgG antibody served as a control. (B). IF and FISH assay showing hsa_circ_0003258 colocalized with IGF2BP3 protein in the cytoplasm in C4–2. (C). The m6A modification site of hsa_circ_0003258 predicted by SRAMP website tools. ** P &lt; 0.01. Supplementary Figure 5. Hsa_circ_0003258 binds to IGF2BP3 and enhances its interaction with HDAC4. (A-B). Flow chart illustrates the criteria of identifying of HDAC4 as the target of hsa_circ_0003258. (C). The qRT-PCR showing the mRNA level change of the predicated targets after silencing of hsa_circ_0003258 in C4–2 cells. (D). RIP assay showing the association of IGF2BP3 with HDAC4 in C4–2 cells. Relative enrichment representing RNA levels associated with HDAC4 compared to an input control. IgG antibody served as a control. ns: Not Significant; * P &lt; 0.05; ** P &lt; 0.01; ***P &lt; 0.001. Supplementary Figure 6. The expression and function of HDAC4 in PCa cells and the images of lung metastasesin mice. (A). HDAC4 protein and mRNA expression in the normal prostate epithelial cell line (RWPE-1) and five PCa cell lines detected by Western blot and qRT-PCR. (B). Transwell assay detected the migratory capacity of C4–2 cells after silencing HDAC4. (C). Transwell assay detected the migratory capacity after silencing the two signaling pathways. (D). Images of lungs derived from DU145 cells transfected with nc and sh- hsa_circ_0003258. (E). The luciferase images and the gross observation of lung metastases in mice injected with DU145/sh-nc and DU145/sh-hsa_circ_0003258 cells (n = 5). * P &lt; 0.05; ** P &lt; 0.01; *** P &lt; 0.001.