PRMT5 promotes colorectal cancer growth by interaction with MCM7

Abstract

Protein arginine methyltransferase 5 (PRMT5) is a type of methyltransferase enzyme that can catalyse arginine methylation of histones and non-histone proteins. Accumulating evidence indicates that PRMT5 promotes cancer development and progression. However, its function in colorectal cancer (CRC) is poorly understood. In this study, we revealed the oncogenic roles of PRMT5 in CRC. We found that PRMT5 promoted CRC cell proliferation, migration and invasion in vitro and in vivo. We identified minichromosome maintenance-7 (MCM7) as the direct PRMT5-binding partner. A co-immunoprecipitation (co-IP) assay indicated that PRMT5 physically interacted with MCM7 and that the direct binding domain was located between residues 1-248 in MCM7. In addition, our results from analysis of 99 CRC tissues and 77 adjacent non-cancerous tissues indicated that the PRMT5 and MCM7 expression levels were significantly higher in CRC tissues than in control tissues, which was further confirmed by bioinformatic analysis using TCGA and GEO datasets. We also found that MCM7 promoted CRC cell proliferation, migration and invasion in vitro. Furthermore, we observed that increased PRMT5 expression predicted unfavourable patient survival in CRC patients and in the subgroup of patients with a tumour size of ≤5 cm. These data suggested that PRMT5 and MCM7 might be novel potential targets for the treatment of CRC.

Keywords: MCM7; PRMT5; colorectal cancer.

FIGURE 1

PRMT5 promotes the proliferation, migration and invasion of CRC cells in vitro and in vivo. A, Western blot analysis of PRMT5 levels in RKO and HT29 cells stably transfected with the control or PRMT5 expression vector (left panel; RKO‐NC/RKO‐PRMT5 and HT29‐NC/HT29‐PRMT5, respectively) and in HCT8 and HCT116 cells transfected with the control siRNA or either of two PRMT5‐specific siRNAs (right panel; HCT8‐NC/HCT8‐siPRMT5 and HCT116‐NC/HCT116‐siPRMT5, respectively). GAPDH was used as the loading control. B, CCK‐8 cell proliferation assay of the indicated cells at the indicated time points. All experiments were performed in triplicate, and the results are presented as the mean ± SD values. **P < .01; ***P < .001. C, Images (upper) and quantification (bottom) of migrated and invaded cells in the migration and invasion assays. Scale bar: 100 μm. All data are presented as the mean ± SD of three independent experiments. **P < .01; ***P < .001. D‐E, (D) RKO‐NC/RKO‐PRMT5 and (E) HT29‐NC/HT29‐PRMT5 cells were transfected with scramble siRNA or siPRMT5 for 48 hours prior to western blot analysis and migration and invasion assays. Upper panel: PRMT5 protein levels were verified by western blotting. GAPDH was used as the loading control. Middle panel: representative photographs of the migrated and invaded cells in the migration and invasion assays. Scale bar: 100 μm. Bottom panel: the bars show the mean ± SD of experiments performed in triplicate. *P < .05; **P < .01; ***P < .001. F, Representative bioluminescence images (left), gross images of excised lungs (middle) and images of H&E‐stained lung and brain sections (right) from each group of mice injected with HT29‐NC and HT29‐PRMT5 cells via the tail vein. Scale bars: 1 mm or 100 μm

FIGURE 2

PRMT5 directly interacts with MCM7, and MCM7 promotes proliferation, migration and invasion of CRC cells in vitro. A, Co‐IP analysis of the interaction between FLAG‐PRMT5 and MCM7 in FLAG‐tagged RKO‐NC/RKO‐PRMT5 and HT29‐NC/HT29‐PRMT5 cells with an anti‐FLAG antibody. B‐C, Co‐IP analysis of the interaction between PRMT5 and MCM7 in HEK293T cells transiently transfected with full‐length Myc‐PRMT5 and/or FLAG‐MCM7 vector with an (B) anti‐Myc or (C) anti‐FLAG antibody. D, Co‐immunoprecipitation of PRMT5 and MCM7 in HEK293T cells transiently transfected with vectors expressing full‐length Myc‐PRMT5 and various FLAG‐MCM7 truncations with an anti‐Myc antibody. E, western blot analysis verifying the efficiency of MCM7 knockdown in HCT8, HCT116 and RKO cells transfected with the scramble control siRNA or either of two siRNAs targeting MCM7 for 48 h. GAPDH was used as the loading control. F, Flow cytometric analysis of the S‐phase proportions of the indicated cells transfected with the scramble control siRNA or siMCM7. The data are presented as the mean ± SD of experiments performed in triplicate. *P < .05; **P < .01; ***P < .001. G, CCK‐8 cell proliferation assay of the indicated cells transfected with the scramble control siRNA or siMCM7 at the indicated time points. All experiments were performed in triplicate, and the results are presented as the mean ± SD values. **P < .01; ***P < .001. H, Images (upper) and quantification (bottom) of the migrated and invaded cells among the indicated cells transfected with the scramble control siRNA or siMCM7, as assessed by migration and invasion assays. Scale bar: 100 μm. All data are presented as the mean ± SD of three independent experiments. **P < .01; ***P < .001

FIGURE 3

PRMT5 and MCM7 expression levels are elevated in clinical samples, and high expression of PRMT5 is associated with poor prognosis in CRC. A‐B, Representative images (left) and quantification (right) of IHC staining for (A) PRMT5 and (B) MCM7 proteins in 99 CRC tissues and 77 adjacent non‐cancerous tissues. Scale bar: 50 μm. The data are presented as the mean ± SD values. ***P < .001. C, Dot plot showing the correlation between PRMT5 and MCM7 protein expression in all CRC tissues (R = .420, P < .001). D, Kaplan‐Meier analysis of overall survival comparing CRC patients (N = 99) stratified according to the PRMT5 expression level (high PRMT5 expression vs. low PRMT5 expression, P = .026). E, Kaplan‐Meier analysis of overall survival comparing CRC patients with a tumour size of ≤5 cm (N = 47) stratified according to the PRMT5 expression level (high PRMT5 expression vs. low PRMT5 expression, P = .021)

FIGURE 4

PRMT5 and MCM7 mRNA expression is upregulated in CRC samples in TCGA and GEO datasets. A, Bioinformatic analysis of PRMT5 mRNA expression in CRC and non‐cancerous tissues in the TCGA, GSE21510, GSE24514, GSE22598, GSE31737, GSE89076, GSE20842, GSE44861 and GSE33113 datasets. B, Bioinformatic analysis of MCM7 mRNA expression in CRC and non‐cancerous tissues in the TCGA, GSE21510, GSE24514, GSE22598, GSE31737, GSE60331, GSE89287, GSE23878 and GSE50421 datasets. The data are presented as the mean ± SD values. *P < .05; **P < .01; ***P < .001