Journal: Frontiers in Cell and Developmental Biology

Article Title: CircFAM120B Blocks the Development of Colorectal Cancer by Activating TGF-Beta Receptor II Expression via Targeting miR-645

doi: 10.3389/fcell.2021.682543

Figure Lengend Snippet: CircFAM120B was downregulated in colorectal cancer (CRC) tissues and cells. (A,B) A microarray profile revealed that circFAM120B was a differently expressed circRNA that was significantly downregulated in CRC tissues ( n = 10). (C) The expression of circFAM120B in tumor tissues ( n = 50) and normal tissues ( n = 50) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). (D) The expression of circFAM120B in LoVo, HCT15, and NCM460 cells was detected by qRT-PCR. (E,F) LoVo and HCT15 cells were exposed to actinomycin D to test the stability of circFAM120B. * P < 0.05.

Article Snippet: Colorectal cancer cell lines, including LoVo and HCT15, were purchased from ProCell Co., Ltd. (Wuhan, China) and maintained in matched special medium (ProCell Co., Ltd.).

Techniques: Microarray, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircFAM120B Blocks the Development of Colorectal Cancer by Activating TGF-Beta Receptor II Expression via Targeting miR-645

doi: 10.3389/fcell.2021.682543

Figure Lengend Snippet: CircFAM120B overexpression weakened cell proliferation, migration/invasion and glycolysis. In circFAM120B-overexpressed LoVo and HCT15 cells, (A–C) cell proliferation was assessed by CCK-8 assay and colony formation assay. (D,E) Cell migration and invasion were determined by transwell assay (magnification: 100×). (F,G) Extracellular acidification rate (ECAR) was measured by glycolysis stress test to evaluate glycolysis metabolism. (H,I) Lactate production and glucose consumption were quantified to assess glycolysis progression using corresponding kits. (J,K) Glycolysis-related markers, HK2 and LDHA, were quantified by western blot to observe glycolysis. * P < 0.05.

Article Snippet: Colorectal cancer cell lines, including LoVo and HCT15, were purchased from ProCell Co., Ltd. (Wuhan, China) and maintained in matched special medium (ProCell Co., Ltd.).

Techniques: Over Expression, Migration, CCK-8 Assay, Colony Assay, Transwell Assay, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircFAM120B Blocks the Development of Colorectal Cancer by Activating TGF-Beta Receptor II Expression via Targeting miR-645

doi: 10.3389/fcell.2021.682543

Figure Lengend Snippet: MiR-645 was a target of circFAM120B. (A,B) Biotinylated-circFAM120B probe could significantly enrich the level of miR-645. (C) Wild-type circFAM120B sequence containing miR-645 binding sites was mutated to generate mutant-type circFAM120B sequence for dual-luciferase reporter assay. (D,E) Dual-luciferase reporter assay was performed to further validate the relationship between miR-645 and circFAM120B. (F) RIP assay was performed to validate the relationship between miR-645 and circFAM120B. (G) The expression of miR-645 in tumor tissues ( n = 50) and normal tissues ( n = 50) was detected by qRT-PCR. (H) The expression of miR-645 in LoVo, HCT15, and NCM460 cells was detected by qRT-PCR. (I) The expression of miR-645 in LoVo and HCT15 cells with circFAM120B overexpression was detected by qRT-PCR. * P < 0.05.

Article Snippet: Colorectal cancer cell lines, including LoVo and HCT15, were purchased from ProCell Co., Ltd. (Wuhan, China) and maintained in matched special medium (ProCell Co., Ltd.).

Techniques: Sequencing, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircFAM120B Blocks the Development of Colorectal Cancer by Activating TGF-Beta Receptor II Expression via Targeting miR-645

doi: 10.3389/fcell.2021.682543

Figure Lengend Snippet: CircFAM120B blocked CRC progression in vitro by targeting miR-645. LoVo and HCT15 cells were transfected with oe-circFAM120B alone or oe-circFAM120B + miR-645, with vector or oe-circFAM120B + miR-NC as a control. (A) The expression of miR-645 in these transfected cells was detected by qRT-PCR. (B,C) Cell proliferation was assessed by CCK-8 assay. (D) Cell proliferation was assessed by colony formation assay. (E,F) Cell migration and invasion were monitored by transwell assay (magnification: 100×). (G,H) Glycolysis was evaluated by ECAR using glycolysis stress test. (I,J) Lactate production and glucose consumption were quantified to monitor glycolysis. (K,L) The expression of HK2 and LDHA was quantified by western blot. * P < 0.05.

Article Snippet: Colorectal cancer cell lines, including LoVo and HCT15, were purchased from ProCell Co., Ltd. (Wuhan, China) and maintained in matched special medium (ProCell Co., Ltd.).

Techniques: In Vitro, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Migration, Transwell Assay, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircFAM120B Blocks the Development of Colorectal Cancer by Activating TGF-Beta Receptor II Expression via Targeting miR-645

doi: 10.3389/fcell.2021.682543

Figure Lengend Snippet: TGFBR2 was a target of miR-645. (A) Wild-type TGFBR2 3′UTR sequence containing miR-645 binding sites was mutated to generate mutant-type TGFBR2 3′UTR sequence for dual-luciferase reporter assay. (B,C) Dual-luciferase reporter assay was conducted to verify the interaction between miR-645 and TGFBR2. (D) RIP assay was conducted to verify the interaction between miR-645 and TGFBR2. (E,F) The expression of TGFBR2 suppressed by miR-645 restoration was detected by qRT-PCR and western blot. (G,H) The expression of TGFBR2 in tumor tissues ( n = 50) and normal tissues ( n = 50) was detected by qRT-PCR and western blot. (I,J) The expression of TGFBR2 in LoVo, HCT15 and NCM460 cells was detected by qRT-PCR and western blot. * P < 0.05.

Article Snippet: Colorectal cancer cell lines, including LoVo and HCT15, were purchased from ProCell Co., Ltd. (Wuhan, China) and maintained in matched special medium (ProCell Co., Ltd.).

Techniques: Sequencing, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircFAM120B Blocks the Development of Colorectal Cancer by Activating TGF-Beta Receptor II Expression via Targeting miR-645

doi: 10.3389/fcell.2021.682543

Figure Lengend Snippet: miR-645 promoted CRC progression in vitro by mediating TGFBR2. LoVo and HCT15 cells were transfected with anti-miR-645 or anti-miR-645 + si-TGFBR2, with anti-miR-NC or anti-miR-645 + si-NC as the corresponding control. (A,B) The expression of TGFBR2 was examined using qRT-PCR and western blot. (C–E) Cell proliferation was investigated using CCK-8 assay and colony formation assay. (F,G) Cell migration and invasion were monitored by transwell assay (magnification: 100×). (H,I) ECAR was measured by glycolysis stress test. (J,K) Lactate production and glucose consumption were detected using the corresponding kits. (L,M) The expression of HK2 and LDHA was measured by western blot. * P < 0.05.

Article Snippet: Colorectal cancer cell lines, including LoVo and HCT15, were purchased from ProCell Co., Ltd. (Wuhan, China) and maintained in matched special medium (ProCell Co., Ltd.).

Techniques: In Vitro, Transfection, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Colony Assay, Migration, Transwell Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircFAM120B Blocks the Development of Colorectal Cancer by Activating TGF-Beta Receptor II Expression via Targeting miR-645

doi: 10.3389/fcell.2021.682543

Figure Lengend Snippet: CircFAM120B activated the expression of TGFBR2 by targeting miR-645. In LoVo and HCT15 cells transfected with oe-circFAM120B, vector, oe-circFAM120B + miR-645 or oe-circFAM120B + miR-NC, the expression of TGFBR2 was detected by (A,B) qRT-PCR and (C) western blot. * P < 0.05.

Article Snippet: Colorectal cancer cell lines, including LoVo and HCT15, were purchased from ProCell Co., Ltd. (Wuhan, China) and maintained in matched special medium (ProCell Co., Ltd.).

Techniques: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrated Chromatin Accessibility and Transcriptome Landscapes of 5-Fluorouracil-Resistant Colon Cancer Cells

doi: 10.3389/fcell.2022.838332

Figure Lengend Snippet: Identification and functional analysis of differentially expressed transcripts associated with 5-FU resistance in HCT15 cells. (A) Volcano plot representing the expression levels of all expressed mRNAs and lncRNA between the parental and 5-FU-resistant HCT15 cells. 1,649 downregulated mRNAs and lncRNAs are shown as blue dots, whereas 1,384 upregulated mRNAs and lncRNAs are shown as red dots. Filtered by absolute log 2 (FC) > 2, p -value < 0.05, and average FPKM >50, 57 significantly down- or upregulated mRNAs and lncRNAs are labeled in different colors based on log 2 fold change (FC). (B) Heatmap of 57 critical mRNAs and lncRNAs was labeled in the volcano plot (A) , which are listed in a descending order based on log2(FC). The numeric values were log2(FPKM+1). (C) Heatmap showing the expression levels of 142 differentially expressed miRNAs (DEMs) in HCT15-FR cells compared to the parental cells. The expression values were normalized per row (Z-score). (D) Gene set enrichment analysis (GSEA) of HCT15-FR versus HCT15 cells was performed on DEG lists ranked by the log 2 (FC) using the R package “clusterProfiler” with HALLMARK and KEGG gene sets downloaded from MSigDB. Gene ratio refers to the ratio of the number of genes enriched in each term to the total number of genes in the term. (E and F) Representative GSEA plots upregulated p53 (E) and downregulated mTOR (E) signaling pathways using “WebGestalt” for DEGs ranked by the log 2 (FC).

Article Snippet: The HCT15 and 5-fluorouracil-resistant HCT15 (HCT15-FR) cell lines were purchased from the iCell Bioscience Inc. (Shanghai, China) and cultured in RPMI-1640 medium (KEL Biotech, China) with 10% fetal bovine serum (KEL Biotech, China), 2 mM L-glutamine (Gibco, United States), 0.1 M sodium pyruvate (Gibco, United States), 50 units/mL penicillin, and 50 μg/ml streptomycin (Sangon Biotech, China).

Techniques: Functional Assay, Expressing, Labeling, Protein-Protein interactions

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrated Chromatin Accessibility and Transcriptome Landscapes of 5-Fluorouracil-Resistant Colon Cancer Cells

doi: 10.3389/fcell.2022.838332

Figure Lengend Snippet: Differentially expressed epigenetic regulators and competing endogenous RNA (ceRNA). (A) Venn diagram illustrating the overlap among the known histone-modifying enzymes and chromatin remodeling regulators and DEGs. (B) Bar plot showing average expression levels (FPKM) of histone-modifying enzymes chromatin remodeling regulators identified in (A) , including histone acetyltransferase (purple), methyltransferase (orange), demethylase (brown), deacetylase (blue), and chromatin remodeling regulators (red). (C and D) Sankey diagram for the ceRNA network in HCT15-FR cells. Each rectangle represents an lncRNA, miRNA, or mRNA, and the connection degree of each RNA was displayed based on the size of the rectangle.

Article Snippet: The HCT15 and 5-fluorouracil-resistant HCT15 (HCT15-FR) cell lines were purchased from the iCell Bioscience Inc. (Shanghai, China) and cultured in RPMI-1640 medium (KEL Biotech, China) with 10% fetal bovine serum (KEL Biotech, China), 2 mM L-glutamine (Gibco, United States), 0.1 M sodium pyruvate (Gibco, United States), 50 units/mL penicillin, and 50 μg/ml streptomycin (Sangon Biotech, China).

Techniques: Expressing, Histone Deacetylase Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrated Chromatin Accessibility and Transcriptome Landscapes of 5-Fluorouracil-Resistant Colon Cancer Cells

doi: 10.3389/fcell.2022.838332

Figure Lengend Snippet: Chromatin accessibility landscape of HCT15 and HCT15-FR cells. (A) Fragment size distribution of ATAC-seq libraries obtained from each sample. Clear modulation of signals indicates mono-, di-, and tri-nucleosomes. (B) Proportion of ATAC-seq peaks to genomic features: promoters-TSS, TES, exon, intron, intergenic, 3′ UTR, 5′ UTR, and non-coding regions. TES, transcription end site. Promoter-TSS, peak summit located within upstream -1 Kb to +100 bp downstream of the transcription start site (TSS). (C) The normalized read density (top) and heatmaps (bottom) showing ATAC-seq signal across a genomic window within a 4-Kb region flanking the TES (from -2 Kb to +2 Kb). (D) Distribution of differentially accessible regions (DARs) over chromosomes in HCT15-FR cells compared to HCT15. The red and blue colors refer to hyper- and hypo-accessible regions, respectively.

Article Snippet: The HCT15 and 5-fluorouracil-resistant HCT15 (HCT15-FR) cell lines were purchased from the iCell Bioscience Inc. (Shanghai, China) and cultured in RPMI-1640 medium (KEL Biotech, China) with 10% fetal bovine serum (KEL Biotech, China), 2 mM L-glutamine (Gibco, United States), 0.1 M sodium pyruvate (Gibco, United States), 50 units/mL penicillin, and 50 μg/ml streptomycin (Sangon Biotech, China).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrated Chromatin Accessibility and Transcriptome Landscapes of 5-Fluorouracil-Resistant Colon Cancer Cells

doi: 10.3389/fcell.2022.838332

Figure Lengend Snippet: Genome-wide identification and analysis of differentially accessible regions (DARs). (A) Distance to nearest TSS of all accessible regions, differentially open, or closed regions. (B) Heatmaps indicating the normalized ATAC-seq signals (RPKM) in parental and 5-FU-resistant HCT15 cells over DARs. The top panel shows read signals over the 4,937 hyper-accessible regions, while the bottom panel shows read signals over the 4,931 hypo-accessible regions. Signals across a genomic window of ±1.5 Kb surrounding the center of DARs are shown in descending order. (C) Profiles of normalized tag density within a 3-Kb region flanking the hyper- (top) and hypo-accessible (bottom) regions. (D) Pie chart showing the annotation of hyper- (top) and hypo-accessible (bottom) sites within the indicated genomic regions: Promoters-TSS, TES, exon, intron, intergenic, 3′ UTR, 5′ UTR, and non-coding regions. TES, transcription end site. Promoter-TSS, peak summit located upstream -1 Kb to +100 bp downstream of the TSS. (E) Venn diagram illustrating the overlap among DEGs, DELs, differentially accessible regions nearest mRNAs, and lncRNAs. (F) Box plots for expression levels of DEGs and DELs relative to hyper- (left) and (right) hypo-accessible regions identified in (E) . p -values were calculated by Wilcoxon’s signed-rank test in R, **** p < 0.0001. (G) Correlation analysis between DARs and their nearest DEGs and DELs. Blue dots represent mRNAs or lncRNAs that are differentially expressed and associated with the changes in chromatin accessibility. The top- and bottom-ranked 15 DEGs or DELs are labeled and shown in different colors according to the log 2 (FC). Pearson’s correlation coefficient (r) and the corresponding p -value were calculated by R. (H and I) The New WashU Epigenome Browser tracks showing ATAC-seq (red) and RNA-seq (blue) signals of representative upregulated (H) IL33 and H19 , and downregulated (I) MGST1 and LINC01012 . Hyper- and hypo-accessible regions are shaded in red and blue, respectively. Blue arrows indicate the TSS and direction of transcription.

Article Snippet: The HCT15 and 5-fluorouracil-resistant HCT15 (HCT15-FR) cell lines were purchased from the iCell Bioscience Inc. (Shanghai, China) and cultured in RPMI-1640 medium (KEL Biotech, China) with 10% fetal bovine serum (KEL Biotech, China), 2 mM L-glutamine (Gibco, United States), 0.1 M sodium pyruvate (Gibco, United States), 50 units/mL penicillin, and 50 μg/ml streptomycin (Sangon Biotech, China).

Techniques: Genome Wide, Expressing, Labeling, RNA Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrated Chromatin Accessibility and Transcriptome Landscapes of 5-Fluorouracil-Resistant Colon Cancer Cells

doi: 10.3389/fcell.2022.838332

Figure Lengend Snippet: Transcription factor (TF) motif enrichment analysis of DARs in 5-FU-resistant HCT15 cells. (A,B) Top 30 enriched known TF motifs of hyper- (A) and hypo-accessible (B) regions. The AP-1, FOX, and KLF family members are shaded blue, red, and green, respectively. The polygonal chain shows the percentages of target sequences with motif, and the p -value was calculated using the cumulative binomial distribution in HOMER v4.10. (C and D) Predicted transcription factor binding sites (TFBSs) of representative up- (C) and downregulated (D) DEGs that positively correlated to chromatin accessibility changes.

Article Snippet: The HCT15 and 5-fluorouracil-resistant HCT15 (HCT15-FR) cell lines were purchased from the iCell Bioscience Inc. (Shanghai, China) and cultured in RPMI-1640 medium (KEL Biotech, China) with 10% fetal bovine serum (KEL Biotech, China), 2 mM L-glutamine (Gibco, United States), 0.1 M sodium pyruvate (Gibco, United States), 50 units/mL penicillin, and 50 μg/ml streptomycin (Sangon Biotech, China).

Techniques: Binding Assay

Journal: British Journal of Cancer

Article Title: FAT1: a potential target for monoclonal antibody therapy in colon cancer

doi: 10.1038/bjc.2016.145

Figure Lengend Snippet: mAb198.3 efficacy in colon cancer xenografts. ( A ) Preventive model. Athymic mice were engrafted subcutaneously with HCT15 cells and treated intravenously two times a week with 12 mg mAb per kg of mAb198.3 or isotype control mAb, or untreated. ( B ) Therapeutic model. mAb198.3 was administered repeatedly in athymic mice bearing either HCT15 or HT29 xenograft of ∼100 mm 3 . Median tumour volumes of each mAb-treated and -untreated mouse group are shown.

Article Snippet: HCT15 or HT29 human colon carcinoma cells (5 × 10 6 ) were injected subcutaneously into the right flank of nude athymic mice (Harlan Laboratories; 8 mice per group).

Techniques: Control