Fig 1: A candidate enhancer variant rs73613962 is screened and validated to be significantly associated with HCC risk.a Flowchart shows the candidate single-nucleotide polymorphisms (SNPs) in enhancers selected from the discovery stage and validated in two stages of replication. HRC Haplotype Reference Consortium, MAF minor allele frequency, HWE Hardy-Weinberg equilibrium, eQTL expression quantitative trait loci, TF transcription factor. b Regional association plot shows the association results (-log10 P) of all the SNPs in the region 100 kb upstream and 100 kb downstream of PRMT7 rs73613962 in Discovery stage (n = 1161 cases, n = 1353 controls). The association of each SNP with HCC risk was evaluated through logistic regression under an additive model adjusting for gender and age. As the index SNP, rs73613962 is shown in purple, and the r2 values of the remaining SNPs are indicated by color. The genes within the region are annotated and shown as arrows. c The meta forest plot shows the association results of rs73613962 in the five independent populations of the discovery stage, replication stage 1, and replication stages 2a, 2b, and 2c. The association of rs73613962 with HCC risk in each population was calculated through logistic regression under an additive model adjusting for gender and age. The MAFs in cases and controls are shown for each population. The ORs and 95% CIs were calculated by considering the major allele as a reference. The center of each square and the horizontal line show the OR and the corresponding 95% CI, respectively. The pooled OR was obtained using the fixed-effects model and is represented by a hollow diamond, where its center indicates the OR and its ends correspond to the 95% CI.
Fig 2: PRMT7 regulates DNA damage response. (A,B) Levels of DNA damage-associated proteins using western blotting. U2OS cells were treated with 10 µM SGC8158 (A) or transfected with PRMT7 siRNA (B) for 2 days, followed by treatment with 10 µM etoposide for indicated times. Cell lysates were subjected to immunoblotting analysis with the indicated antibodies. (C–F) Analysis of ?H2AX levels during DNA repair process. After suppressing PRMT7 in U2OS cells by treating with 10 µM SGC8158 (C,E) or the siRNA (D,F) for 2 days, the cells were treated with 10 µM etoposide for 2 h, and then further incubated with fresh medium without etoposide for an additional 4 h. ?H2AX levels were measured using western blotting (C,D) or immunostaining (?H2AX; green, PRMT7; red, DAPI; blue) (E,F). The fluorescence intensity of ?H2AX was quantified by image analysis software (630× magnification). Data are presented as means ± SD of six independent experiments. *** p < 0.001. DAPI: 4',6-diamidino-2-phenylindole; SD: standard deviation.
Fig 3: PRMT7 regulates DNA repair processes. Schematic of the DR-GFP and Ej5-GFP reporter assays. (A) DR-GFP reporter system was used to monitor HR process. Modified GFP gene (SceGFP) contains an I-SceI site in-frame. Expression of I-SceI causes DSBs at internal SceGFP sites and a non-functional GFP fragment gene (iGFP) is used to repair the DSB by HR, generating a functional GFP gene. In addition, the Ej5-GFP reporter system was used to monitor the total NHEJ process. The GFP gene is separated from its promoter by a puromycin resistance gene (puro) flanked by two I-SceI sites. Transient expression of I-SceI endonuclease causes the excision of the puro and induces the repair of the DSB by NHEJ, thus creating a functional GFP gene. (B,C) U2OS stable cell lines expressing DR-GFP or Ej5-GFP reporter constructs were co-treated (or transfected) with PRMT7 inhibitor (or siRNA) and I-SceI plasmid. After 3 days, the efficiency of HR or NHEJ was determined by counting the number of GFP-positive cells using flow cytometry. BRCA2 siRNA and Ku80 siRNA were used as positive controls to validate HR and NHEJ reporter systems, respectively. The percentage of GFP-positive cells from three independent experiments is indicated as the means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001. (D) The expression levels of HR or NHEJ-related proteins (BRCA2, Ku80, and 53BP1) in the presence of SGC8158 were determined using western blotting under the same conditions. HR: homologous recombination; DSB: double-strand break; NHEJ: non-homologous end-joining; SD: standard deviation.
Fig 4: PRMT7 inhibition induces cell cycle arrest and cellular senescence via p21 accumulation. (A) After treatment of A549 cells with 10 µM SGC8158 for 3 days, the proportion of cells in each phase of the cell cycle was measured by FACS analysis. Data are presented as means ± SD of three independent experiments. * p < 0.05 and ** p < 0.01. (B) The expression profiles of cell cycle-related proteins. After treatment of A549 cells with 10 µM SGC8158 or PRMT7 siRNA transfection for 3 days, the levels of cell cycle-related proteins were analyzed by western blotting. (C,D) Representative images illustrating SA-ß-gal-positive cells (100× magnification). Percentage of SA-ß-gal-positive cells was shown as means ± SD from three independent experiments. * p < 0.05 and ** p < 0.01. (E) p21 mRNA levels were evaluated by quantitative RT-PCR and presented as means ± SD (n = 3). (F,G) The cells were treated with 10 µM SGC8158 or transfected with PRMT7 siRNA for 3 days, and then treated with 100 µg/mL CHX for the indicated times. Band intensities were quantitated using image processing software. Error bars indicate the SD of three independent replicates. FACS: fluorescent-activated cell sorting; SD: standard deviation; CHX: cycloheximide.
Fig 5: PRMT7 inhibition causes growth inhibition in various human cancer cells. (A) Structure of SGC8158 (B) A549 cells were treated with 10 µM SGC8158 or transfected with PRMT7 siRNA for 3 days. Whole-cell lysates were subjected to immunoprecipitation using Hsp70 antibody, followed by immunoblotting with MMA antibodies. (C) The growth inhibition of SGC8158 in various human cancer cells was determined using an MTT assay. Data are presented as floating bar plots from three independent experiments. (D) A549 cells were treated with 10 µM SGC8158 or transfected with PRMT7 siRNA as described. After five days, the colonies were stained using crystal violet (10× magnification) and the absorbance was measured at 540 nm. Data are presented as means ± SD (n = 3). ** p < 0.01 and *** p < 0.001. MMA: monomethylarginine; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SD: standard deviation.
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