Journal: International Journal of Molecular Sciences

Article Title: Transcriptomic Profiling Reveals Isoform-Specific Regulatory Roles of miR-196A and miR-196B in Colorectal Cancer Cells

doi: 10.3390/ijms27093959

Figure Lengend Snippet: Generation and functional validation of miR-196 knockout SW48 cells. ( A ) Sequence alignment of miR-196A and miR-196B showing a high degree of similarity between the two miRNA isoforms. ( B ) Quantitative RT-PCR analysis of miR-196 expression in parental SW48 cells and in miR-196A-KO or miR-196B-KO cells. Relative expression levels were normalized to an internal control RNA and presented as fold change compared with control cells. ( C ) Cell proliferation assay showing the relative growth rates of control, miR-196A-KO, and miR-196B-KO SW48 cells. Data represent the mean ± SD from independent experiments. Statistical significance is indicated (* p < 0.05, *** p < 0.001).

Article Snippet: The human colorectal cancer cell line SW48 (ATCC Cat. No. CCL-231) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Functional Assay, Biomarker Discovery, Knock-Out, Sequencing, Quantitative RT-PCR, Expressing, Control, Proliferation Assay

Journal: International Journal of Molecular Sciences

Article Title: Transcriptomic Profiling Reveals Isoform-Specific Regulatory Roles of miR-196A and miR-196B in Colorectal Cancer Cells

doi: 10.3390/ijms27093959

Figure Lengend Snippet: Identification of differentially expressed genes following deletion of miR-196 isoforms in SW48 cells. ( A ) Volcano plot showing the distribution of differentially expressed genes (DEGs) in miR-196 knockout cells compared with control SW48 cells. Significantly up-regulated and down-regulated genes are highlighted based on predefined statistical thresholds. ( B ) Summary of DEGs identified in the transcriptomic analysis, showing the numbers of significantly up-regulated and down-regulated genes in miR-196 knockout cells relative to control cells. The vertical dashed lines indicate the fold-change thresholds, and the horizontal dashed line represents the statistical significance threshold (adjusted p -value cutoff). The grey shaded area indicates genes that are not significantly differentially expressed. ( C ) Venn diagram illustrating the overlap of DEGs between miR-196A-KO and miR-196B-KO cells. A subset of genes was commonly regulated by both miR-196 isoforms, while additional genes were uniquely altered in each knockout condition.

Article Snippet: The human colorectal cancer cell line SW48 (ATCC Cat. No. CCL-231) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knock-Out, Control

Journal: International Journal of Molecular Sciences

Article Title: Transcriptomic Profiling Reveals Isoform-Specific Regulatory Roles of miR-196A and miR-196B in Colorectal Cancer Cells

doi: 10.3390/ijms27093959

Figure Lengend Snippet: Functional enrichment analysis of differentially expressed genes following miR-196A or miR-196B deletion. ( A ) Functional categorization of differentially expressed genes (DEGs) identified in miR-196A-KO cells compared with control SW48 cells. The pie chart shows the percentage distribution of genes associated with major biological processes. The percentages may exceed 100% because individual genes can be associated with multiple functional categories and are therefore counted in more than one category. ( B ) Distribution of significantly up-regulated and down-regulated genes in each functional category in miR-196A-KO cells. ( C ) Expression profiles of representative genes altered in miR-196A-KO cells. Each point represents normalized RNA-seq expression values from independent samples. ( D ) Functional classification of DEGs identified in miR-196B-KO cells relative to control cells. The percentages may exceed 100% because individual genes can be associated with multiple functional categories and are therefore counted in more than one category. ( E ) Numbers of significantly up-regulated and down-regulated genes in each functional category in miR-196B-KO cells. ( F ) Expression patterns of representative genes affected by miR-196B deletion.

Article Snippet: The human colorectal cancer cell line SW48 (ATCC Cat. No. CCL-231) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Functional Assay, Control, Expressing, RNA Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Transcriptomic Profiling Reveals Isoform-Specific Regulatory Roles of miR-196A and miR-196B in Colorectal Cancer Cells

doi: 10.3390/ijms27093959

Figure Lengend Snippet: Hierarchical clustering and reproducibility assessment of RNA-seq data from miR-196 knockout SW48 cells. ( A – C ) Heatmap visualization of differentially expressed genes between miR-196 knockout cells and parental SW48 cells using fold-change thresholds of >1.5 ( A ), >2.0 ( B ), and >3.0 ( C ). Hierarchical clustering reveals distinct expression patterns associated with miR-196 deletion. ( D ) Pearson correlation heatmap showing strong correlations among biological replicates and experimental groups. Color intensity represents the Pearson correlation coefficient (r), ranging from −1 to 1, where values closer to 1 indicate stronger similarity between samples. The numerical values in each cell correspond to the correlation coefficients. ( E ) Pairwise scatter plot matrix demonstrating high concordance in gene expression profiles across samples. The red line represents the linear regression fit. Asterisks (***) indicate statistical significance ( p < 0.001).

Article Snippet: The human colorectal cancer cell line SW48 (ATCC Cat. No. CCL-231) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: RNA Sequencing, Knock-Out, Expressing, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Transcriptomic Profiling Reveals Isoform-Specific Regulatory Roles of miR-196A and miR-196B in Colorectal Cancer Cells

doi: 10.3390/ijms27093959

Figure Lengend Snippet: Functional enrichment analysis of genes altered following miR-196 deletion. Gene Ontology (GO) and KEGG pathway enrichment analyses were performed using differentially expressed genes identified from RNA-seq analysis of miR-196A-KO and miR-196B-KO SW48 cells. ( A ) Bubble plot showing significantly enriched GO terms and KEGG pathways in miR-196A-KO cells. Functional categories are grouped into biological process (BP), cellular component (CC), molecular function (MF), and KEGG pathways. Bubble size represents the number of genes associated with each term, and color intensity indicates the −log10( p -value). ( B ) Bubble plot showing enriched GO terms and KEGG pathways in miR-196B-KO cells using the same criteria. ( C ) Distribution of up-regulated and down-regulated genes contributing to each enriched functional category in miR-196A-KO cells. ( D ) Distribution of up-regulated and down-regulated genes contributing to each enriched functional category in miR-196B-KO cells.

Article Snippet: The human colorectal cancer cell line SW48 (ATCC Cat. No. CCL-231) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Functional Assay, RNA Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Transcriptomic Profiling Reveals Isoform-Specific Regulatory Roles of miR-196A and miR-196B in Colorectal Cancer Cells

doi: 10.3390/ijms27093959

Figure Lengend Snippet: Differential gene expression patterns in miR-196A-KO and miR-196B-KO SW48 cells. Radar charts display the top ten most highly upregulated and downregulated genes in each knockout condition. Left panels show average normalized expression values (log2); right panels show fold change relative to parental SW48 cells (SW48-vector). ( A ) Top ten upregulated genes in miR-196A-KO cells, including RIMS2 , ADGRL2 , and LAMA2 . ( B ) Top ten upregulated genes in miR-196B-KO cells, including PCCA , LAMA2 , and AKAP12 . ( C ) Top ten downregulated genes in miR-196A-KO cells, including KRT14 , KLK11 , and KRT16 . ( D ) Top ten downregulated genes in miR-196B-KO cells, including KRT16 , KRT14 , and KRT19 . ( E ) qPCR validation of selected miR-196-associated genes ( NT5E , PRRX1 , KITLG , CLDN4 , and FLG ) in parental SW48 and miR-196 isoform knockout cells, showing isoform-dependent differences in gene expression. Data are presented as mean ± SD ( n = 3).

Article Snippet: The human colorectal cancer cell line SW48 (ATCC Cat. No. CCL-231) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Gene Expression, Knock-Out, Expressing, Plasmid Preparation, Biomarker Discovery

Journal: JCI Insight

Article Title: The critical role of GRP78/BiP MARylation in ER stress of KRAS-mutant colorectal cancer

doi: 10.1172/jci.insight.182809

Figure Lengend Snippet: The analysis of overall survival ( A ), progression-free survival ( B ), disease-free survival ( C ), and disease-specific survival ( D ) analysis of patients with KRAS-WT and -mutant CRC based on the Cbioportal database. ( E ) The proportion of each mutation subtype in KRAS-mutant CRC. ( F ) The proportion of patients with KRAS-WT and -mutant patients in each subtype of CRC. ( G ) The mutation event frequency of various genes in KRAS-WT and -mutant CRC. ( H ) Differentially expressed genes in patients with KRAS-WT and -mutant CRC. ( I ) ART1 mRNA levels in KRAS-WT and -mutant CRC tissues. P < 0.05 by t test. ( J ) The positive intensity of ART1 in KRAS-WT and -mutant CRC tissues detected by immunohistochemistry. Original magnification, ×20 (top) and ×40 (bottom). ( K ) The expression of ART1 protein in KRAS-WT (SW48, Caco2, HT-29) and -mutant CRC cell lines (LoVo, HCT-116, SW480) observed by Western blot. Immunohistochemical detection of the expression of GRP78/BiP ( L ), HSC70 ( M ), and CHOP ( N ) in KRAS-WT and -mutant CRC tissues. Original magnification, ×200 (left) and ×400 (right). ( O ) The expression of GRP78/BiP, HSC70, and CHOP proteins in KRAS-WT and -mutant CRC cell lines detected by Western blot. ( P ) Transmission electron microscopy shows the morphology of the ER (arrows) in KRAS-WT and -mutant CRC cell lines. Original magnification, ×25,000.

Article Snippet: Human CRC cell lines SW48 (KRAS WT), HT-29 (KRAS WT), SW480 (G12V), and HCT116 (G13D) were obtained from American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Immunohistochemistry, Expressing, Western Blot, Immunohistochemical staining, Transmission Assay, Electron Microscopy

Journal: Cancer Reports

Article Title: Transmembrane Protein TMEM59L Modulates 5‐ FU Resistance via PTPRN ‐Mediated DNA Damage Repair in Colorectal Cancer

doi: 10.1002/cnr2.70448

Figure Lengend Snippet: TMEM59L regulates colorectal cancer cells proliferation, migration, and invasion. (A) Western blotting confirmed expression of TMEM59L in different CRC cell lines. (B) Western blotting detects the knockdown of TMEM59L by shRNA and overexpression of TMEM59L by plasmid. (C) Downregulation of TMEM59L suppresses cell proliferation in HCT116 cells; overexpression of TMEM59L in SW480 promotes cell proliferation. (D) The function of TMEM59L on the migration and invasion ability of CRC cells was detected by Transwell assay. (E) E‐cadherin and Vimentin were evaluated through immunofluorescence staining in TMEM59L knockdown and overexpression CRC cells.

Article Snippet: Five human CRC cell lines (NCI‐H716, HCT116, COLO 320DM, SW48, SW480) were sourced from the American Type Culture Collection (ATCC, USA), while a 5‐fluorouracil‐resistant HCT116 subline (HCT116/FU, BNCC342640) was obtained from BNCC (China).

Techniques: Migration, Western Blot, Expressing, Knockdown, shRNA, Over Expression, Plasmid Preparation, Transwell Assay, Immunofluorescence, Staining

Journal: Cancer Reports

Article Title: Transmembrane Protein TMEM59L Modulates 5‐ FU Resistance via PTPRN ‐Mediated DNA Damage Repair in Colorectal Cancer

doi: 10.1002/cnr2.70448

Figure Lengend Snippet: TMEM59L was elevated in 5‐FU resistance CRC cell lines and reduced 5‐FU sensitivity. (A) The expression of TMEM59L in non‐responder and responder groups treated by 5‐FU ( n = 279 vs. 379, p = 0.0002), oxaliplatin ( n = 173 vs. 265, p = 0.012) and Capecitabine ( n = 62 vs. 47, p = 0.000019), respectively. (B) TMEM59L level in CRC cell lines and corresponding 5‐FU resistant cells was examined by western blot. (C, D) CCK‐8 assays of TMEM59L downregulation and upregulation on sensitivity of HCT116 and SW480 cells to 5‐FU; the half maximal inhibitory concentration (IC50) was calculated using GraphPad software. Data represent the mean ± SD ( n = 3), * p < 0.05 vs. HCT116 group, $ p < 0.05 vs. SW480 group.

Article Snippet: Five human CRC cell lines (NCI‐H716, HCT116, COLO 320DM, SW48, SW480) were sourced from the American Type Culture Collection (ATCC, USA), while a 5‐fluorouracil‐resistant HCT116 subline (HCT116/FU, BNCC342640) was obtained from BNCC (China).

Techniques: Expressing, Western Blot, CCK-8 Assay, Concentration Assay, Software

Journal: Cancer Reports

Article Title: Transmembrane Protein TMEM59L Modulates 5‐ FU Resistance via PTPRN ‐Mediated DNA Damage Repair in Colorectal Cancer

doi: 10.1002/cnr2.70448

Figure Lengend Snippet: Silencing of TMEM59L enhanced DNA damage and 5‐FU sensibility in colorectal cancer cells and drug‐resistant CRC cell lines. (A, B) γ‐H2AX foci formation in HCT116 and SW480 cells was detected by immunofluorescence 48 h after treatment with 5‐FU (25 μg/mL). (C) Intracellular ROS levels in HCT116 and SW480 cells treated with 5‐FU for 48 h were detected by reactive oxygen species detection kit. (D) Effect of TMEM59L on apoptosis in CRC cells induced by 5‐FU (25 μg/mL) treatment for 48 h was determined by flow cytometric analysis. (E) Downregulation of TMEM59L reduced colony formation of HCT116/FU and SW480/FU cells.

Article Snippet: Five human CRC cell lines (NCI‐H716, HCT116, COLO 320DM, SW48, SW480) were sourced from the American Type Culture Collection (ATCC, USA), while a 5‐fluorouracil‐resistant HCT116 subline (HCT116/FU, BNCC342640) was obtained from BNCC (China).

Techniques: Immunofluorescence

Journal: Cancer Reports

Article Title: Transmembrane Protein TMEM59L Modulates 5‐ FU Resistance via PTPRN ‐Mediated DNA Damage Repair in Colorectal Cancer

doi: 10.1002/cnr2.70448

Figure Lengend Snippet: TMEM59L regulated 5‐FU induced DNA damage and ROS through PTPRN. (A) Physical interactions with TMEM59L in GeneMANIA website. (B) Correlation analysis between TMEM59L and PTPRN in COAD from GEPIA database. (C) PTPRN expression in COAD and READ from the TCGA database analyzed by the GEPIA database. (D) Higher PTPRN expression was related to poorer OS in CRC patients from TCGA through Kaplan–Meier Plotter database. (E) PTPRN partially reversed the effect of TMEM59L on 5‐FU induced ROS of HCT116 and SW480 cells. (F) PTPRN partially reversed the effect of TMEM59L on 5‐FU induced DNA damage of HCT116 and SW480 cells.

Article Snippet: Five human CRC cell lines (NCI‐H716, HCT116, COLO 320DM, SW48, SW480) were sourced from the American Type Culture Collection (ATCC, USA), while a 5‐fluorouracil‐resistant HCT116 subline (HCT116/FU, BNCC342640) was obtained from BNCC (China).

Techniques: Expressing