Journal: Clinical and Translational Medicine

Article Title: LncRNA CARMN m6A demethylation by ALKBH5 inhibits mutant p53 ‐driven tumour progression through miR‐5683/FGF2

doi: 10.1002/ctm2.1777

Figure Lengend Snippet: Downregulation of LncRNA CARMN is associated with high methylation levels in colorectal cancer with mutant p53 . (A) A volcano plot displayed the significantly expressed LncRNAs between TP53 mutant and wild‐type patients in CRC. (B) The plot of the survival curve showed the low expression of CARMN led to a poor prognosis in advanced colorectal patients with mutant p53 and wild‐type p53 . (C) The correlation between the demethylase LncRNA CARMN and mutant p53R273H was analysed and visualised using corrplot package with colon cancer data retrieved from the TCGA database. (D, E) The qPCR assays were employed to assess CARMN RNA levels in SW480 and HT29 cells transfected with human empty non‐p53 construct vector with GFP (NC), sh‐p53R273H or OE‐p53R273H, respectively. (F) Relative expression of LncRNA CARMN in the normal colorectal cancer cells (HIEC‐6, FHC) and other different colorectal cancer cell lines (HCT116, SW480, SW620). The scale bar in 10 µM, * p < 0.05 and ** p < 0.01 as indicated. (G, M) m6A dot blot assays of SW480 cells with knockdown or overexpression of p53 ‐R273H (G) or ALKBH5 (M) were designed to measure the m6A level from total RNA diluted with 200, 400 and 800 ng, methylene blue (MB) staining worked as a loading control. (H, I) The mRNA and protein expression of ALKBH5 in colon cancer patients with mutant p53 . (J) The relative protein level of ALKBH5 in SW480 cells transfected with sh‐ p53R273H or OE‐ p53R273H . (K) The mRNA expression levels of the demethylase ALKBH5 were compared between mutant and wild‐type p53 using R Studio with data sourced from the TCGA database. (L) The correlation between the demethylase ALKBH5 and mutant p53R273H was analysed and visualised using corrplot package with colon cancer data retrieved from the TCGA database. (P) Human colorectal tissue samples containing mutant p53 were utilised to investigate the correlation between CARMN and ALKBH5 through the RIP assay, employing the ALKBH5 antibody. (N) The location of CARMN was imaged by confocal microscopy in SW480 transfected with GFP‐ CARMN . (O) Combined immunofluorescence obtained from DAPI (blue) and RNA‐FISH analysis of LncRNA CARMN (green), and ALKBH5 (red) in SW480 cells. (Q) The expression of CARMN in the subcellular fractions of colorectal cancer with mutant p53 was detected by RT‐PCR. U6 and actin were utilised as nuclear and cytoplasmic markers, respectively.

Article Snippet: Human colorectal cancer cell lines (HIEC‐6, FHC, HCT116, SW480, SW620, HT29) were authenticated by the International Cell Line Authentication Committee and obtained from ATCC.

Techniques: Methylation, Mutagenesis, Expressing, Transfection, Construct, Plasmid Preparation, Dot Blot, Knockdown, Over Expression, Staining, Control, Confocal Microscopy, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction

Journal: Clinical and Translational Medicine

Article Title: LncRNA CARMN m6A demethylation by ALKBH5 inhibits mutant p53 ‐driven tumour progression through miR‐5683/FGF2

doi: 10.1002/ctm2.1777

Figure Lengend Snippet: FGF2 is correlated with miR‐5683 and has a promotional effect on tumour progression. (A) Upregulation (red) and downregulation (blue) genes were displayed on the volcano plot with mutant p53 of colorectal cancer. (B) Protein–protein interaction (PPI) networks between the De‐mRNAs were constructed by the online tool STRING. (C) The PPI network was simplified by Cytoscape, which was used to calculate the degree value of De‐mRNAs in PPI networks. (D) The core genes in this PPI network. (E) The Venn plot was used to obtain core genes that combined with miR‐5683 . (F) The expression levels of these selected genes in (E) were compared in SW480 cells transfected with control mimics (mimics NC), and miR‐5683 mimics, revealing differential expression. (G) The survival plot revealed higher expression of FGF2 resulted in lower survival. (H) The box diagram displayed the expression of FGF2 in TP53 mutant and wild‐type patients. (I) The expression of FGF2 was measured by RT‐PCR in HCT116 −/− cells transfected with OE‐ p53R273H . (J) The location of FGF2 was observed by confocal microscopy in SW480 cells transfected with GFP‐ FGF2 . (K–M, P, Q) Transwell migration assays (K) and CCK8 assays (L, M, P, Q) were used to detect the effect on SW480 cells transfected with shRNA‐ FGF2 or OE‐ FGF2 . (N, R) Expression of ULK1, P62, and LC3II/I were obtained by Western blotting in SW480 and SW620 with FGF2 overexpressed or knockdown. (O, S) Overexpression of FGF2 suppressed autophagy in CRC cells.

Article Snippet: Human colorectal cancer cell lines (HIEC‐6, FHC, HCT116, SW480, SW620, HT29) were authenticated by the International Cell Line Authentication Committee and obtained from ATCC.

Techniques: Mutagenesis, Construct, Expressing, Transfection, Control, Quantitative Proteomics, Reverse Transcription Polymerase Chain Reaction, Confocal Microscopy, Migration, shRNA, Western Blot, Knockdown, Over Expression

Journal: Clinical and Translational Medicine

Article Title: LncRNA CARMN m6A demethylation by ALKBH5 inhibits mutant p53 ‐driven tumour progression through miR‐5683/FGF2

doi: 10.1002/ctm2.1777

Figure Lengend Snippet: Mutant p53 inhibits the transcription of ALKBH5 to promote the malignant proliferation of colorectal cancer with mutant p53 . (A) The live SW480 cells transfected with GFP‐ p53R273H and mCherry‐ ALKBH5 were imaged by confocal microscopy. (B) Co‐immunoprecipitation (Co‐IP) analysis of the interaction between ALKBH5 and mutant p53 in SW480 cells. (C) Three potential mutant‐ p53 binding sites on the ALKBH5 promoter were shown in the diagram. (D–I) A specific binding site was demonstrated by ChIP‐qPCR analysis. DNA fragments were precipitated using either a p53‐specific antibody or an IgG antibody. The input was employed as internal positive controls, whereas IgG served as an internal negative control. (J, K) ChIP‐qPCR assays further confirmed the direct binding of mutant p53 to the promoters of ALKBH5, PHLPP2 and p21 in SW480 cells with mutant p53 knockdown (J), as well as in HCT116 p53 −/− cells with mutant p53 overexpression (K). (L) ChIP‐qPCR analysis conducted on human tissue confirmed three sites of mutant p53 binding to the ALKBH5 promoter, with IgG served as an internal negative control. (M) The CUT&Tag assay confirmed that mutant p53 directly bound to ALKBH5 promoter region. Compared to the untreated control group, ALKBH5 promoter signal was significantly decreased in sw480 cells with knockdown of mutant p53R273H. The red box indicated the binding site of mutant p53 on ALKBH5 promoter. Characteristic signal peaks in this region were shown for both the control group (upper) and knockdown p53R273H (lower). (N) Profile plots and heatmaps were displayed the mutant p53 signal across the gene bodies or transcription start sites (TSS) of ALKBH5 RT&Tag‐enriched transcripts based on varying levels of p53 CUT&Tag signal over their gene bodies. The heatmaps were arranged in descending order of CUT&Tag signal strength. (O) The relative protein level of ALKBH5 in SW480 cells transfected with OE‐ ALKBH5 or si‐ ALKBH5 . (P–T) The CCK8 assays (P, Q), the EdU assays (R) and colony formation assays (T) were obtained to measure the effect on SW480 cells transfected with OE‐ ALKBH5 or si‐ ALKBH5 . (S) The SW480 cells were treated with overexpression of ALKBH5 or knockdown of ALKBH5 to induce apoptosis. The cells were stained with Annexin V‐iFluor 488 and Propidium Iodide. Apoptosis cells can be observed in the bottom right quadrant. (U, V) The transwell migration assays (U) and wound‐healing assays (V) were applied to compare the cell proliferation or migration ability in SW480 cells transfected with OE‐ ALKBH5 or si‐ ALKBH5 .

Article Snippet: Human colorectal cancer cell lines (HIEC‐6, FHC, HCT116, SW480, SW620, HT29) were authenticated by the International Cell Line Authentication Committee and obtained from ATCC.

Techniques: Mutagenesis, Transfection, Confocal Microscopy, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, ChIP-qPCR, Negative Control, Knockdown, Over Expression, Control, Staining, Migration

Journal: Clinical and Translational Medicine

Article Title: LncRNA CARMN m6A demethylation by ALKBH5 inhibits mutant p53 ‐driven tumour progression through miR‐5683/FGF2

doi: 10.1002/ctm2.1777

Figure Lengend Snippet: MiR‐5683 combines with LncRNA CARMN , and downregulation of it enhances the proliferation capability and tumour growth of colorectal cancer with mutant p53 . (A) A volcano plot displayed the DE‐miRNAs between TP53 mutant and wild‐type patients in colorectal cancer. (B) The Venn diagram was drawn to take an intersection for miRNAs by the bioinformatics tool Venn. (C) The differentially expressed miRNAs identified in (B) were detected in SW480 cells with either knockdown or overexpressed of CARMN. (D) The association between miR‐5683 and CARMN was determined by the luciferase activities in SW480 cells cotransfected with WT‐ CARMN or MUT‐ CARMN and miR‐5683 mimics. (E, F) The expression of miR‐5683 was measured by RT‐PCR in HCT116 −/− cells transfected with OE‐ p53R273H (E) and SW480 cells transfected with shRNA‐ p53 (F). (G, H) The expression of miR‐5683 was measured by RT‐PCR in SW480 and SW620 cells transfected with miR‐5683 mimics (G) or miR‐5683 inhibitors (H). (I–K, M) The proliferation and migration abilities of SW480 cells transfected with miR‐5683 mimics or miR‐5683 inhibitors were tested by CCK8 (I, J), colony formation assays (K), and transwell migration assays (M). (L) Dot plots of Annexin V and PE after transfected with miR‐5683 mimics and miR‐5683 inhibitors in SW480 cells. (N, O) The apoptosis‐related and autophagy‐related proteins were detected in SW620 cells by Western blotting. (P) The SW480 cells cotransfected with GFP‐mRFP‐ LC3 and miR‐5683 mimics or GFP‐mRFP‐ LC3 and miR‐5683 inhibitors were observed by confocal microscopy.

Article Snippet: Human colorectal cancer cell lines (HIEC‐6, FHC, HCT116, SW480, SW620, HT29) were authenticated by the International Cell Line Authentication Committee and obtained from ATCC.

Techniques: Mutagenesis, Knockdown, Luciferase, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, shRNA, Migration, Western Blot, Confocal Microscopy