Fig 1: RASAL1 is downregulated in colon cancer samples and cell lines. a Expression of RASAL1 in 27 pairs of colon cancer samples was compared with that in adjacent non-tumor tissues; determination via quantitative RT-qPCR. b Quantitative RT-qPCR and c Western blot were performed to detect the expression of RASAL1 in 5 colon cancer cell lines (LoVo, CaCo2, HCT-116, SW1116 and SW480) and a normal colonic cell line (NCM460). *P < 0.05
Fig 2: RASAL1 inhibited the activity of SCD1 3′-UTR through LXRα and SREBP1c. a, b SCD1 mRNA and protein levels were detected by RT-qPCR and western blot at 48 h after transfection with pcDNA3.1 or SREBP1c, or treated with 1 μM T0901317, β-actin was used as loading control. c Luciferase assay of HCT-116 cells co-transfected with the luci-SCD1 3′-UTR reporter with SREBP1c or treated with 1 μM T0901317. After 48 h, firefly luciferase values were normalized to β-gal luciferase activity. d Schematic illustration of point mutation of the binding site of RASAL1 and SCD1 promoter. e, f The effect of RASAL1 on luciferase intensity controlled by the wild type (WT) or mutant (Mut) 3′-UTR of SCD1 was determined using the luciferase assay. Each bar represents the mean ± SEM. The results were reproduced in three independent experiments. *P < 0.05
Fig 3: Targeted hydroxymethylation of four different aberrantly methylated genes by dCas9-TET3CD fusion protein in human kidney cells. a Schematic representing hypermethylated RASAL1 promoter region (upper panel) and reactivated RASAL1 expression through induction of RASAL1 promoter hydroxymethylation by dCas9-TET3CD fusion protein in complex with a sgRNA binding to its target region (lower panel). b Schematic of domain structure of the dCas9-TET3CD (upper panel) and dCas9-TETCDi (lower panel) fusion protein. c–e Locations for RASAL1/EYA1/LRFN2-sgRNAs are indicated by thick lines with corresponding PAM in magenta within the human RASAL1/EYA1/LRFN2 gene locus, respectively. Human fibrotic TK188 fibroblasts were transduced with lentivirus expressing demethylation constructs guided by RASAL1-sgRNAs 1–10, EYA1-sgRNA 1–6, LRFN2-sgRNA 1–8, or by LacZ control sgRNA. Results were normalized to reference gene GAPDH. f, g MeDIP and hMeDIP analysis of TK188 cells were transduced with dCas9-TET3CD-RASAL1-sgRNA3. The results were calculated relative to the input. h Bisulfite sequencing summary of promoter methylation status of the RASAL1 gene in TK188 cells transduced with demethylation constructs guided by RASAL1-sgRNA3, by LacZ control sgRNA or DNA treated with M.SssI serving as positive control. Each data point represents the mean of three independent transduction experiments with error bars indicating the standard error of the mean for six or more bisulfite sequencing results. i Locations for KL-sgRNAs are indicated by thick lines with corresponding PAM in magenta within the human KL gene locus. Three days TGFß1-treated HK2 cells were transduced with lentivirus expressing demethylation constructs guided by KL-sgRNAs 1–8 or by LacZ control sgRNA. j, k MeDIP and hMeDIP analysis of HK2 cells were transduction with dCas9-TET3CD-KL-sgRNA2. The results were calculated relative to the input. All data are presented as mean value; error bars represent S.D.; n = 3 independent biological replicates, n.s. not significant; *p < 0.05, **p < 0.01, ***p < 0.001
Fig 4: Targeted Rasal1 promoter demethylation by dCas9/dHFCas9-TET3CD-Rasal1-sgRNA fusion protein ameliorates kidney fibrosis. a Schematic showing the parenchymal injection of lentiviral particles containing dCas9/dHFCas9-TET3CD fusion protein into UUO-challenged kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Rasal1 mRNA expression was significantly induced in UUO-challenged kidneys transduced with dCas9/dHFCas9-TET3CD-Rasal1-sgRNA, but not in UUO-challenged kidneys transduced with control dCas9/dHFCas9-TET3CD-LacZ-sgRNA. There is no significant difference between dCas9-TET3CD and dHFCas9-TET3CD constructs. Results were normalized to reference gene Gapdh (expression is presented as mean value; error bars represent S.D., n = 5 in each group, # not significant, ***p < 0.001). d Western blots showing restored RASAL1 protein expression in UUO-challenged kidneys which were transduced with lentivirus expressing dCas9/dHFCas9-TET3CD-Rasal1-sgRNA. The membranes were restriped and re-probed with a-TUBULIN antibody to serve as equal loading control. e, f UUO-challenged kidneys which were transduced with lentivirus expressing dCas9/dHFCas9-TET3CD-Rasal1-sgRNA show significantly reduced Rasal1 promoter methylation by MeDIP-qPCR assay (e) and increased hydroxymethylation by hMeDIP-qPCR assay (f). There is no significant difference between dCas9-TET3CD and dHFCas9-TET3CD constructs. The results were calculated relative to input. The data are presented as mean value, error bars represent S.D., n = 5; # not significant, ***p < 0.001. g Kidney sections from UUO- and sham-operated mice which were transduced with lentivirus expressing dCas9/dHFCas9-TET3CD-Rasal1-sgRNA or dCas9/dHFCas9-TET3CD-LacZ-sgRNA were stained for Masson’s trichrome (MTS) (representative light microscopy images are shown in the top row), Collagen-1 or a-SMA (representative confocal images are shown in the middle and bottom row, respectively) (Scale bars: 25 µm or 50 µm). h–j Quantification of the percentage of total interstitial fibrosis and immunostained positive cells in each group is depicted (data are presented as mean value, error bars represent S.E.M., n = 5 in each group, # not significant, ***p < 0.001, ****p < 0.0001). Both dCas9-TET3CD and dHFCas9-TET3CD lentivirus transduced UUO-operated kidneys show significantly decreased interstitial fibrosis level and a significantly decreased number of a-SMA- and Collagen-1-positive cells. HFCas9-TET3CD shows significantly better efficacies when compared to dCas9-TET3CD
Fig 5: dCas9-TET3CD and dHFCas9-TET3CD fusion proteins induce targeted Rasal1/ Kl promoter demethylation in mouse kidney cells and dHFCas9-TET3CD largely reduced off-target effects. a Locations for Rasal1-sgRNAs are indicated by thick lines with corresponding PAM in magenta within the mouse Rasal1 gene locus. 10 days TGFß1-treated mKF were transduced with dCas9-TET3CD-Rasal1-sgRNAs1-8 or by LacZ control sgRNA. b Locations for Klotho-sgRNAs are indicated by thick lines with corresponding PAM in magenta within the mouse Klotho gene locus. Three days TGFß1-treated MCT were transduced with dCas9-TET3CD-Klotho-sgRNAs1-6 or by LacZ control sgRNA. c MeDIP and hMeDIP analysis of TGFß1-treated mKF were transduced with dCas9-TET3CD-Rasal1-sgRNA4. d Bisulfite sequencing summary of promoter methylation status of the Rasal1 gene in TGFß1-treated cells transduced with dCas9-TET3CD-Rasal1-sgRNA4 or by LacZ control sgRNA. e MeDIP and hMeDIP analysis of TGFß1-treated MCT cells transduced with dCas9-TET3CD-Kl1-sgRNA2 or with LacZ control sgRNA. f Bisulfite sequencing summary of promoter methylation status of the Klotho gene in TGFß1-treated MCT cells transduced with dCas9-TET3CD-sgRNA2 or by LacZ control sgRNA. g Schematic of domain structure of the dHFCas9-TET3CD fusion protein. h TGFß1-treated mKFs were transduced with dCas9/dHFCas9-TET3CD-Rasal1-sgRNA4 or LacZ control sgRNA (left panel). TGFß1-treated MCT cells were transduced with dCas9/dHFCas9-TET3CD-Klotho-sgRNA2 or LacZ control sgRNA (right panel). i MeDIP-qPCR analysis of TGFß1-treated mKFs transduced with dCas9/dHFCas9-TET3CD-Rasal1-sgRNA4 or LacZ control sgRNA (left panel) and TGFß1-treated MCT cells transduced with dCas9/dHFCas9-TET3CD-Klotho-sgRNA2 or LacZ control sgRNA (right panel). j Venn diagram summarizes the common off-targets identified by ChIP-seq analysis between dCas9-TET3CD and dHFCas9-TET3CD transduced mKF (left panel). Tracks indicate the binding regions and the enrichment of dCas9/dHFCas9-TET3CD-Rasal1/Klotho/LacZ sgRNA protein-RNA complexes in mKF cells as visualized in the IGV browser (right panel). Genomic coordinates are shown below the tracks (build mm9). All data are presented as mean value; error bars represent S.D, n = 3 independent biological replicates, n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001. qRT-PCR results were normalized to reference gene Gapdh. MeDIP and hMeDIP results were calculated relative to input. For bisulfate sequencing each data point represents the mean of three independent biological replicates with error bars indicating the standard error of the mean for six or more bisulfite sequencing results
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