Journal: Frontiers in Immunology

Article Title: FASN promotes the stemness of cancer stem cells and protects colorectal cancer cells from ferroptosis by inhibiting the activation of SREBP2

doi: 10.3389/fimmu.2025.1611375

Figure Lengend Snippet: Effects of FASN knockdown on CSC characteristics in CRC cells. (A) Representative images of conventional culture (adherent cells) and 3D spheroidization culture (tumor spheres) of HCT116 cells. (B) Real-time PCR analysis of FASN mRNA expression levels in adherent cells and tumor spheres. (C) Flow cytometry was used to identify the CD133+CD44+ cells and CD133-CD44- cells sorted from HCT116 cells and perform quantitative analysis. (D) Real-time PCR analysis of FASN mRNA expression levels in different types of sorted cells. (E) Observation of the formation of spheroids in vitro after transfection with FASN-silencing plasmids and quantitative analysis. (F) Images of tumors from vaccinated colorectal cancer cells transfected with the sh-FASN plasmid after 4 weeks, n=6. (G–I) Changes in tumor volume, weight, and tumor suppression rate within 4 weeks of vaccination. (J) Flow cytometry was used to detect the numbers of CD133+CD44+ cells and CD133-CD44- cells and perform quantitative analysis. * P < 0.05, ** P < 0.01 and *** P < 0.001 between the indicated groups.

Article Snippet: CRC cell lines (CACO-2, HCT116, SW480) and normal human intestinal epithelial cells (HIEC-6) were purchased from Procell Life Science & Technology Co., Ltd. All cell lines were officially authenticated, with confirmed mycoplasma-free status and cell line identity (STR profiling).

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, In Vitro, Transfection, Plasmid Preparation

Journal: Military Medical Research

Article Title: Genome-wide enhancer RNA profiling adds molecular links between genetic variation and human cancers

doi: 10.1186/s40779-024-00539-2

Figure Lengend Snippet: eRNAQTL rs3094296 promotes eRNA ENSR00000155786 expression mediated by TF HOXA5. a Workflow to identify the candidate variant by integrating eRNAQTL analysis and CRC GWAS data, the target gene was selected by combining co-expression analysis and eQTL result in CRC. b Manhattan plot of eRNAQTL results in 154 CRC samples. The red line shows the FDR < 0.05 threshold. c Manhattan plot for associations between eRNAQTLs and CRC risk from the Chinese population consisting of 4293 cases and 7176 controls. d Validation in European populations from GECCO, with a combined sample size of 17,789 cases and 19,951 controls. P -values were calculated by an unconditional logistical regression analysis with an additive model adjusting for gender, age, smoking, and drinking status. P < 0.05 was considered statistically significant (indicated by the red line). e Epigenetic tracks obtained from the Cistrome database show the enrichment of enhancer markers (H3K4me1 and H3K27ac peaks) in the rs3094296 region. f eRNAQTL analysis demonstrates the correlation between the rs3094296 genotype and the expression of ENSR00000155786 in the TCGA CRC samples and our own CRC tissues. Data were shown as the median (minimum to maximum). g Relative reporter gene activity of the vectors containing the rs3094296-T or rs3094296-C allele in HCT116 cell. h The rs3094296-T allele resides within the HOXA5 binding motif. i The effect of HOXA5 knockdown on the relative luciferase activity of vectors containing the rs3094296-T or rs3094296-C allele in HCT116 cell. Data were presented as the median (minimum to maximum) from three repeated experiments, each has three technical replicates. P -values were calculated by a two-sided Student’s t -test. j Scatter plots show the correlations between HOXA5 and ENSR00000155786 expression in our CRC tumor tissues and stratified by SNP rs3094296 genotype. All P -values and correlation coefficients were calculated by Spearman’s correlation analysis. k Histogram displays the effect of HOXA5 knockdown on the expression of ENSR00000155786 in SW480 and HCT116 cells. Data were presented as the mean ± SD from three repeated experiments with three technical replicates. All P -values were calculated using a two-sided Student’s t -test. * P < 0.05, *** P < 0.001. CRC colorectal cancer, FDR false discovery rate, GECCO Genetics and Epidemiology of Colorectal Cancer Consortium, LD linkage disequilibrium, FPKM fragments per kilobase of exon model per million mapped fragments, RPKM reads per kilobase per million mapped reads, HOXA5 homeobox A5, SD standard deviation

Article Snippet: The normal human intestinal epithelial cells HIEC-6 and CRC cell lines HCT116, HT15, and SW480 were obtained from the China Center for Type Culture Collection (Wuhan, China) in September 2021.

Techniques: Expressing, Variant Assay, Biomarker Discovery, Activity Assay, Binding Assay, Knockdown, Luciferase, Standard Deviation

Journal: Military Medical Research

Article Title: Genome-wide enhancer RNA profiling adds molecular links between genetic variation and human cancers

doi: 10.1186/s40779-024-00539-2

Figure Lengend Snippet: eRNA ENSR00000155786 inhibits colorectal cancer (CRC) cell proliferation by activating SENP7 expression. a Scatter plots show the correlations between ENSR00000155786 and SENP7 expression, as well as the correlations between HOXA5 and SENP7 expression in our CRC tumor tissues both of them, are stratified by SNP rs3094296 genotype. All P -values and correlation coefficients were calculated by Spearman’s correlation analysis. b eQTL analyses demonstrate the correlation between the rs3094296 genotype and the expression of SENP7 in the TCGA CRC samples and our own CRC tissues. Data were shown as the median (minimum to maximum). ChIRP assay was performed using two different sets of antisense probes (“even group” and “odd group”) that detected ENSR00000155786 or control probes (anti-LacZ) in CRC cell lines. The enrichment of genomic DNA in the ChIRP and input samples was measured by qPCR ( c ) and agarose gel electrophoresis ( d ). Data were shown as the mean ± SD. P -values were calculated by a two-sided Student’s t- test. The histogram displays the effect of ENSR00000155786 knockdown on the expression of SENP7 ( e ) and the effect of HOXA5 knockdown on the expression of SENP7 ( f ) in SW480 and HCT116 cells. Data were presented as the mean ± SD from three repeated experiments with three technical replicates. All P -values were calculated using a two-sided Student’s t -test. g Western blotting analysis shows that the knockdown of HOXA5 and ENSR00000155786 reduced the protein expression level of SENP7 in SW480 and HCT116 cells. h ENSR00000155786 and SENP7 are significantly decreased in tumor tissues compared with normal tissues from our own CRC tissues. P -values were calculated by a paired two-sided Student’s t -test. i Cell proliferation assay with knockdown of ENSR00000155786 and SENP7 in SW480 and HCT116 cells. Results were shown as the means ± SEM from three experiments, each with six replicates. P -values were calculated from a two-sided Student’s t -test by comparing with controls in 96 h. j SENP7 is essential for cell growth with higher CERE scores in COLO205 CRC cell lines from the data of genome-wide CRISPR/Cas9-based loss-of-function screen. Higher CERE scores demonstrate an elevated dependency of cell viability on given genes. k The graph shows the mechanism that eRNAQTL rs3094296 contributes to a decreased risk of CRC by playing allele-specific effect to transcribe eRNA ENSR00000155786 , which further exerts a transcriptional activator promoting target gene SENP7 expression, then these two genes display a synergistic effect to inhibit tumor cells proliferation. * P < 0.05, ** P < 0.01, *** P < 0.001. FPKM fragments per kilobase of exon model per million mapped fragments, RPKM reads per kilobase per million mapped reads, HOXA5 homeobox A5, SENP7 sentrin-specific protease 7, ChIRP chromatin isolation by RNA purification, CERES CRISPR enrichment-based screening, SEM standard error of the mean

Article Snippet: The normal human intestinal epithelial cells HIEC-6 and CRC cell lines HCT116, HT15, and SW480 were obtained from the China Center for Type Culture Collection (Wuhan, China) in September 2021.

Techniques: Expressing, Control, Agarose Gel Electrophoresis, Knockdown, Western Blot, Proliferation Assay, Genome Wide, CRISPR, Isolation, Purification