Additional file 3 of SF3B3-regulated mTOR alternative splicing promotes colorectal cancer progression and metastasis
Creators: name: Xu, Tong
name: Li, Xichuan
name: Zhao, Wennan
name: Wang, Xue
name: Jin, Leixin
name: Feng, Zhiqiang
name: Li, Huixiang
name: Zhang, Mingzhe
name: Tian, Yiqing
name: Hu, Ge
name: Yue, Yuan
name: Dai, Xintong
name: Shan, Changliang
name: Zhang, Weihua
name: Zhang, Chunze
name: Zhang, Youcai
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Cancer

description: Additional file 3. Supplementary figures and tables. Figure S1. SF3B3 is upregulated in human CRC and regulated by H3K27ac. (A) SF3B7 transcript levels in normal and CRC tissues. TCGA-COAD and TCGA-READ datasets (normal, n=51; cancer, n=383) were obtained from Xena (http://xena.ucsc.edu/). Data are shown as mean ± SD. (B) mRNA levels of SF3b family members in CRC tissues from TCGA-COAD and TCGA-READ datasets in cBioPortal (https://www.cbioportal.org/). (C) mRNA levels of SF3b family members in normal and CRC tissues after integrating the data of normal colon tissues from GTEx datasets with TCGA datasets. Analysis was performed by GEPIA2 (http://gepia2.cancer-pku.cn/). (D) KEGG pathway analysis of SF3B3 positively and negatively co-expressed genes that were identified using cBioPortal (https://www.cbioportal.org/). (E) Correlation analysis between SF3B3 and MKI67 mRNA levels in TCGA datasets from Xena (http://xena.ucsc.edu/). (F) Potential epigenetic factors for SF3B3 gene using a parameter of 1-kb regulatory potential decay in Cistrome DB Toolkit (http://dbtoolkit.cistrome.org). (G) Visualization of H3K27ac enrichment in the promoter region of SF3B3 gene using UCSC Genome Browser (http://genome.ucsc.edu/). (H) Genome browser view of H3K27ac occupancy in SF3B3 promoter region. ChIP-Seq data from three CRC cell lines (GSE83968, GSE96069, and GSE71510) were retrieved from GEO datasets (http://www.ncbi.nlm.nih.gov/geo). (I) SF3B3 mRNA levels in LoVo and HT29 cells treated with 40 μM curcumin for 24 h. FOXM1 was used as positive control for H3K27ac target. Data are shown as mean ± SD. Figure S2. SF3B3 promotes proliferation and metastasis in vitro. (A) qRT-PCR quantification of SF3B3 mRNA expression as well as representative western blots of SF3B3 in 7 CRC cell lines and normal human colon mucosal epithelial cell line (NCM460). (B) qRT-PCR analysis of SF3B3 mRNA as well as representative western blots of SF3B3. LoVo and HT29 cells were transfected with siRNAs and collected at 48 h for mRNA detection or at 72 h for protein detection. SW480 cells were transfected with SF3B3-overexpressing plasmids for 72 h. (C) Growth curves and (D) colony formation of LoVo and HT29 cells after SF3B3 knockdown by siRNAs (siNC vs siSF3B3#2), or after SF3B3 knockdown by siRNAs for 12 h followed with re-expression of SF3B3 (siSF3B3#2+EV vs siSF3B3#2+SF3B3) (empty vector, EV; SF3B3 overexpressing plasmid, SF3B3). (E) Growth curves and colony formation of SW480 cells after transfection with SF3B3 overexpressing plasmids. (F) Wound healing assays and (G) Transwell assays were used to measure cell migration and invasion abilities. LoVo and HT29 cells were treated with siRNAs (siNC vs siSF3B3#2) to knockdown SF3B3. For SF3B3 re-expression study, cells were treated with siRNAs (siNC vs siSF3B3#2) for 12 h, followed by transfection with EV or SF3B3 overexpressing plasmids (siSF3B3#2+EV vs siSF3B3#2+SF3B3). Scale bars, 100 μm. (H) Statistical analysis of Wound healing and Transwell assay results. Data are shown as mean ± SD. (I) Representative immunofluorescence staining images of E-cadherin (green), VIM (red) and DAPI (blue) in CRC cells using a confocal microscope with 100× oil immersion lens. Figure S3. Silencing of SF3B3 impedes CRC proliferation and metastasis in vivo. qRT-PCR and western blot analyses of stably SF3B3-knockdown LoVo (A) and HT29 (B) cells. LoVo cells were infected with shSF3B3#1 or shSF3B3#2 lentivirus, whereas HT29 cells were infected with shSF3B3#2 lentivirus. (C) Body weights of LoVo cell xenograft nude mice. The stably SF3B3-knockdown LoVo cells (LoVo-shSF3B3#1) were subcutaneously injected into flank region of nude mice. (D) Representative IHC images of E-cadherin and VIM proteins in LoVo-shNC and LoVo-shSF3B3#1 xenografts. Scale bars, 25 μm. (E) Representative images of lung, H&amp;E staining for lung tissues, and statistical analysis of metastatic nodules in lung (n=5/group). Control and SF3B3-knockdown HT29 cells (shSF3B3#2) were injected into nude mice via the tail vein. Scale bars, 200 μm. Data are shown as mean ± SD. (F) Representative IHC images of E-cadherin and VIM proteins in metastasis lung and liver tissues. Scale bars, 200 μm. Figure S4. SF3B3 regulates mitochondria-mediated apoptosis in CRC cells. (A) Cell viability of SF3B3-knockdown CRC cells co-treated with apoptosis-, necrosis- or ferroptosis-inhibitors. LoVo and HT29 cells were transfected with siRNAs for 24 h, followed by treatment with 20 μM ZVF (Z-VAD-FMK), 10 μM NSA (necrosulfonamide), 1 μM Lip-1 (liproxstatin-1) or 1 μM Fer-1 (ferrostatin-1) for 48 h. (B) Cell viability of SF3B3-knockdown CRC cells co-treated with ROS inhibitor. LoVo and HT29 cells were transfected with siRNAs for 24 h, followed by treatment with 10 mM NAC (N-acetylcysteine) for 48 h. (C) Representative western blots of apoptosis-related proteins. LoVo and HT29 cells were transfected with siRNAs for 24 h, followed by 10 mM NAC for 48 h. Data are shown as mean ± SD. Figure S5. SF3B3 regulates lipogenesis in CRC cells via SREBF1-FASN signaling. (A) Heatmap and (B) volcano plot displaying the significantly differential expression genes from RNA-seq of LoVo cells after transfection with siRNAs (mixture of siSF3B3#1 and siSF3B3#2) for 48 h. (C) Transcript levels of lipogenesis-related genes from RNA-seq of control and SF3B3-knockdown LoVo cells. (D) Transcript levels of SREBF2 in RNA-seq analysis of SF3B3-knockdown LoVo cells, as well as qRT-PCR analysis of SREBF2 mRNA in CRC cells after transfection with siRNAs for 48 h. (E) Triglyceride levels in SF3B3-knockdown CRC cells after transfection with siRNAs for 72 h. (F) Nile red staining (Red) images and analysis of SF3B3-knockdown CRC cells after transfection with siRNAs for 72 h. Nuclei (blue) were stained with DAPI. Scale bars, 50 μm. (G) Representative fluorescence images of ROS (green) staining. CRC cells were transfected with siRNAs for 24 h, followed by treatment with 10 μM palmitate for 48 h. Scale bars, 50 μm. (H) Concentrations of individual fatty acids in LoVo cells after transfection with siRNAs (mixture of siSF3B3#1 and siSF3B3#2) for 72 h. The data are calculated based on lipidomic study. (I) Correlation analysis of the mRNA expression between SF3B3 and lipogenesis-related genes based on TCGA-COAD and TCGA-READ datasets using GEPIA2. (J) qRT-PCR quantification of the mRNA expression of SREBF1 and its target genes. (K) Representative western blots of p-S6K, SREBF1, ACLY, and FASN. LoVo and HT29 cells were transfected with siRNAs (mixture of siSF3B3#1 and siSF3B3#2) for 12 h, followed by transfection with combined siTSC1 and siTSC2 for 60 h. Data are shown as mean ± SD. Figure S6. SF3B3 regulates mTOR splicing and autophagy. (A) mRNA levels of mTOR in SF3B3-knockdown LoVo cells from RNA-seq data. (B) mRNA expression of mTOR was quantified by qRT-PCR in CRC cells after transfection with siRNAs for 48 h. (C) KEGG pathway enrichment analysis of SF3B3-regualted AS events from RNA-seq data. Differentially alternative splicing patterns were determined using the rMATS tool. (D) Representative western blots of mTORα and mTORβ in the protein extracts of HEK293T cells using previous reported N-terminal mTOR antibody (sc-517464). (E) Cell viability and representative western blots of mTOR and SREBF1 of SF3B3-knockdown CRC cells after mTOR (+exon8 or -exon8) overexpression. LoVo and HT29 cells were transfected with siSF3B3#1 for 12 h, followed by transfection with overexpressing plasmids for full length mTOR (+exon 8) or mTOR variant 3 (-exon 8) for 60 h. (F) Exon 8 skipping of mTOR was examined by 3% agarose gel electrophoresis of PCR products in SW480 cells. Representative western blots of mTORα and mTORβ in SW480 cells using the mTOR antibody (sc-517464). SW480 cells were transfected with SF3B3-overexpressing plasmids for 72 h. Data are shown as mean ± SD. (G) RNA-IP analysis of the enrichment of mTOR in SF3B3 protein-antibody-beads system in LoVo cells. (H) Representative immunofluorescence images of LC3B (green) and DAPI (blue) in SF3B3-knockdown CRC cells using a confocal microscope with 63× oil immersion lens. LoVo and HT29 cells were transfected with siRNAs for 72 h. (I) Representative western blots of LC3B in SF3B3-knockdown CRC cells after forced expression of mTOR. LoVo and HT29 cells were transfected with siRNAs for 12 h, followed by transfection with mTOR-overexpressing plasmids for 60 h. (J) Representative western blots of LC3 in SF3B3-knockdown CRC cells after treatment with autophagy inhibitors. LoVo and HT29 cells were transfected with siRNAs for 24 h, followed by treatment with 20 μM CQ or 80 nM BafA1 for 48 h. (K) mTOR isoform structures identified in TCGA datasets (illustrated by GEPIA2). Correlation analysis between the SF3B3 mRNA levels and full-length mTOR-001 based on BRCA-, CESC- and LIHC-TCGA datasets using GEPIA2. (L) Exon 8 skipping of mTOR was examined by 3% agarose gel electrophoresis of PCR products in SF3B3-knockdown MCF7, Hela and Huh7 cells transfected with siRNAs for 48 h. (M) Representative western blots of mTOR and LC3 in SF3B3-knockdown MCF7, Hela and Huh7 cells transfected with siRNAs for 72 h. Data are shown as mean ± SD. Figure S7. Effect of SF3B3 knockdown and mTOR inhibitor on CRC. (A) The clinical information of three donor patients investigated in this study. (B) qRT-PCR verification of SF3B3 knockdown in two PDX murine models. PDX1 models were intratumorally treated with shSF3B3#1 lentivirus, whereas PDX2 models were treated with shSF3B3#2 lentivirus. (C) Representative H&amp;E staining and TUNEL staining (green) of two PDXs. Scale bars, 50 μm. (D) Cell viability of LoVo and HT29 cells after transfection with siRNAs for 24 h, followed by rapamycin or everolimus treatment at the indicated concentrations for 48 h. (E) Viability and qRT-PCR validation of SF3B3-knockdown CRC tumor organoids. Digested organoids were transduced with shSF3B3 lentivirus for 6 h, followed by reconstitution in matrigel for 7 days. (F) Body weights of PDX mice. Xenograft tumors were transplanted into left and right flanks of each nude mouse to establish PDXs. Mice were intratumorally injected with shNC lentivirus on the left flank and shSF3B3 lentivirus (mixture of shSF3B3#1 and shSF3B3#2) on the right flank. After that, mice were orally administered vehicle (40%PEG400, 5% Tween-80 and 5% DMSO) or everolimus (5 mg/kg, every two days for 3 weeks). (G) Representative H&amp;E staining as well as immunohistochemistry images of mTOR and Ki67 in PDX3 tumor tissues. Data are shown as mean ± SD. Figure S8. SF3B3 regulates alternative splicing of DHCR24 and apoptotic genes. (A) mRNA and protein levels of DHCR24. CRC cells were transfected with siRNAs, and collected at 48 h for qRT-PCR and at 72 h for western blots. (B) Minigene detection showed the effects of SF3B3 knockdown on DHCR24 splicing in CRC cells. The minigene contains exons 3-4 of DHCR24. LoVo cells were transfected with DHCR24 minigene together with SF3B3 siRNAs for 48 h. (C) Detection of cell viability and (D) ROS in stably SF3B3-knockdown CRC cells transfected with DHCR24 overexpressing plasmid for 72 h. Data are shown as mean ± SD. (E) Alternative splicing of apoptotic genes was examined by 1-3% agarose gel electrophoresis of PCR products in CRC cells. LoVo and HT29 cells were transfected with siRNAs for 48 h. Table S5. Sequences of siRNAs. Table S6. Sequences for plasmid construction. Table S7. Sequences of shRNAs. Table S8. Primer sequences for qRT-PCR or PCR. Table S9. List of antibodies used in this study.