(Pro)renin receptor promotes colorectal cancer through the Wnt/beta-catenin signalling pathway despite constitutive pathway component mutations

Figures

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  Fig. 1. (P)RR is highly expressed in human CRC tissues. a Representative image of (P)RR staining by IHC in colorectal tissues, which contains both cancer and normal tissues. Yellow arrow: cancer tissue with messy and irregular gland structure. Black arrow: normal tissue with regular gland structure. b, c Representative IHC images of (P)RR staining in normal (b) and cancer (c) tissues with higher magnification. d Western blotting analysis of (P)RR expression levels in cancer and matched normal tissues (n = 5). Data are presented as mean ± SEM. e Representative IHC images of (P)RR staining in CRC tissues with different differentiated grades. f Comparison of percentages of patients showing strong, middle or weak (P)RR expression in groups with different CRC grades (n = 17, 30 and 13 in Grade 1, 2 and 3). *P < 0.05, ***P < 0.001

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  Fig. 2. (P)RR level in CRC is related to clinical characteristics and the activation of Wnt/β-catenin signalling. a Comparison of percentages of patients showing strong, middle or weak (P)RR expression in different stages (n = 20 in each stage). b Comparison of percentages of patients showing strong, middle or weak (P)RR expression in groups with and without recurrence and/or metastasis to other organ(s) (n = 23 without recurrence and metastasis; n = 37 with recurrence and/or metastasis). c Relevancy between (P)RR expression level and recurrence-free survival time of 20 patients in Stage II (n = 10) and III (n = 10) who had recurrence after radical operation (n = 16 with weak or middle (P)RR expression; n = 4 with strong (P)RR expression). d Comparison of percentages of patients showing strong, middle or weak (P)RR expression in groups with and without a 5-year survival (5-year follow-up after diagnosis of CRC was accomplished on 47 patients; 25 patients had survived over 5 years; 22 patients died within 5 years). e Representative IHC images of active β-catenin and c-Myc staining in CRC tissues with different (P)RR levels. Black arrow: staining of active β-catenin translocated into…

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  Fig. 3. (P)RR silencing attenuates the activation of Wnt/β-catenin signalling in CRC cells. a, b Representative western blotting images showing protein levels of components of the Wnt/β-catenin pathway in DLD-1 cells with mutated APC (a) and HCT116 cells with mutated β-catenin (b) after siRNA transfection. c, d Relative mRNA levels of components of the Wnt/β-catenin pathway in DLD-1 (c) and HCT116 (d) cells after siRNA transfection. Values were calculated from signal intensities obtained by real-time PCR (n = 3~6). Data are expressed as relative ratios to the scrambled siRNA group. e, f Wnt/β-catenin signalling activity of DLD-1 (e) and HCT116 (f) cells transfected with β-catenin-responsive TOP flash reporter and siRNA (n = 4). Data are expressed as relative ratios to vehicle (only transfected with the TOP flash reporter without siRNA) group. Cells transfected with the FOP flash reporter, of which the TCF/LEF binding sites were mutated, served as negative control. Numerical data are presented as mean ± SEM. N.S.: not significant, *P < 0.05, **P < 0.01, ***P < 0.001

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  Fig. 4. (P)RR silencing inhibits proliferation and induces apoptosis of CRC cells in vitro. a, b Cell number counting of DLD-1 (a) and HCT116 (b) cells during 72 h after siRNA transfection (n = 4). N.S.: not significant, **P < 0.01, ***P < 0.001 vs. scrambled siRNA group. c, d Proliferative activity of DLD-1 (c) and HCT116 (d) cells measured by WST-1 assay at 72 h after siRNA transfection (n = 6). Data are expressed as relative ratios to vehicle (without siRNA transfection) group. N.S.: no significant difference, *P < 0.05. e, f Flow cytometry detection of apoptosis and cell cycle distribution of DLD-1 (e) and HCT116 (f) cells at 72 h after siRNA transfection. Numerical data are presented as mean ± SEM

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  Fig. 5. (P)RR silencing attenuates growth and Wnt/β-catenin signalling of CRC xenografts in vivo. a Tumour formation of scrambled shRNA- and (P)RR shRNA-transfected DLD-1 cells at day 21 after subcutaneous transplantation into nude mice (n = 6). Successful (P)RR silencing was confirmed by western blotting before cell transplantation. b Growth patterns of tumour volume during 21 days. c Tumour weights at day 21. d Representative IHC images of (P)RR, active β-catenin and c-Myc staining in xenograft tumour tissues. Black arrow: staining of active β-catenin translocated into nuclei. Numerical data are presented as mean ± SEM. *P < 0.05 vs. scrambled shRNA group

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  Fig. 6. Induction of (P)RR overexpression enhances the Wnt/β-catenin signalling and proliferation of normal colon epithelial cells. a Representative western blotting images showing expression levels of components of the Wnt/β-catenin pathway in CCD841 CoN cells, a normal colon epithelial cell line, as well as DLD-1 and HCT116 cells. b Representative western blotting images showing expression levels of components of the Wnt/β-catenin pathway in CCD841 CoN cells, transfected with either plasmid vector containing (P)RR encoding gene ATP6ap2 ((P)RR vector) or empty vector. c Wnt/β-catenin signalling activity of CCD841 CoN cells transfected with β-catenin-responsive TOP flash reporter as well as (P)RR vector or empty vector (n = 4). Data are expressed as relative ratios to vehicle (only transfected with the TOP flash reporter) group. Cells transfected with the FOP flash reporter, of which the TCF/LEF binding sites were mutated, served as negative control. d Cell number counting of CCD841 CoN cells during 72 h after vector transfection (n = 3). e Proliferative activity of CCD841 CoN cells measured by WST-1 assay at 72 h after vector transfection. Numerical data are presented as mean ± SEM.…