PRR11 promotes ccRCC tumorigenesis by regulating E2F1 stability

Abstract

Proline rich 11 (PRR11), a novel tumor-related gene, has been identified in different tumors. However, the relevant biological functions of PRR11 in human clear cell renal cell carcinoma (ccRCC) have not been studied. In this study, we first identified PRR11 as a biomarker of ccRCC and predictor of poor prognosis by bioinformatics. Then, we showed that PRR11 silencing substantially reduced ccRCC cell proliferation and migration in vitro and in vivo. Importantly, we found that PRR11 induced the degradation of the E2F1 protein through its interaction with E2F1, and PRR11 reduced the stability of the E2F1 protein in ccRCC cells, thereby affecting cell cycle progression. Further results indicated that the downregulation of E2F1 expression partially reversed the changes in ccRCC cell biology caused by PRR11 deletion. In addition, we showed that PRR11 was a target gene of c-Myc. The transcription factor c-Myc may have promoted the expression of PRR11 in ccRCC cells by binding to the PRR11 promoter region, thereby accelerating the progression of ccRCC. In summary, we found that PRR11 served as an oncogene in ccRCC, and PRR11 reduced the protein stability of E2F1 and could be activated by c-Myc.

Keywords: Cancer; Cell Biology; Cell cycle; Nephrology.

Figure 1. PRR11 is an independent factor of ccRCC.

(A and B) The expression level of PRR11 in ccRCC cell lines (ACHN, 796-P, 786-O, and Caki-1 cells) and a human renal tubular epithelial cell line (HK2; n = 3). (C) PRR11 staining score of ccRCC tissues and adjacent tissues based on a tissue microarray (n = 89 ccRCC tissues and n = 89 adjacent tissues). (D) Correlation between the PRR11 staining score and the ccRCC pathological grade based on a tissue microarray (n = 32 for grade I, n = 31 for grade II, n = 25 for grade III, and n = 1 for grade IV). (E) Representative pattern of PRR11 protein expression in ccRCC tissues and adjacent tissues using tissue microarray sections. (F) Survival analysis of PRR11 expression based on a ccRCC tissue microarray. Scale bar: 400 μm (left) and 100 μm (right). The data are shown as mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons test (A), 2-tailed t test (C), and 1-way ANOVA with Tukey’s multiple comparisons test (D) analyses were performed. *P < 0.05, **P < 0.01, ***P < 0.001. PRR11, proline rich 11; ccRCC, clear cell renal cell carcinoma.

Figure 2. PRR11 knockdown inhibited ccRCC cell proliferation.

(A–D) MTT assay was used to evaluate the proliferation and viability of PRR11 knockdown and PRR11-overexpressing ccRCC cells (n = 3). (E–H) A clonogenic assay was used to estimate the colony formation ability of PRR11 knockdown and PRR11-overexpressing ccRCC cells, and the clone numbers were statistically analyzed (n = 3). The data are shown as mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons test (A, C, and F) and 2-tailed t test (B, D, and H) analyses were performed. *P < 0.05, **P < 0.01, ***P < 0.001. PRR11, proline rich 11; ccRCC, clear cell renal cell carcinoma.

Figure 3. PRR11 knockdown induced the accumulation of ccRCC cells in the S phase.

(A–D) Flow cytometry analysis of the effect of PRR11 knockdown on ccRCC cell cycle progression (n = 3). (E) GSEA was used to show the correlation between increased PRR11 expression and cell cycle progression. Nominal P calculated by the permutation test and FDR-corrected q values were obtained by the multiple hypothesis test in GSEA. (F and G) Expression of cycle-related proteins in PRR11 knockdown or PRR11-overexpressing ccRCC cells via Western blotting (n = 3). See complete unedited blots in the supplemental material. One-way ANOVA with Dunnett’s multiple comparisons test analyses were performed. **P < 0.01, ***P < 0.001. PRR11, proline rich 11; ccRCC, clear cell renal cell carcinoma; GSEA, Gene set enrichment analysis; NES, normalized enrichment score.

Figure 4. The silencing of PRR11 expression inhibited the migration and decreased the EMT of ccRCC cells.

(A–D) Transwell migration assays were used to evaluate the migration of PRR11 knockdown and PRR11-overexpressing ccRCC cells, and the relative number of migrated cells was statistically analyzed (n = 3). Scale bar: 150 μm. (E–L) Wound healing assays were used to evaluate the migration of PRR11 knockdown and PRR11-overexpressing ccRCC cells, and the relative number of migrated cells was statistically analyzed (n = 3). Scale bar: 150 μm. (M and N) Western blotting analysis of EMT-related protein expression in PRR11-silenced or PRR11-overexpressing ccRCC cells (n = 3). The data are shown as mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons test (B, F, and J) and 2-tailed t test (D, H, and L) analyses were performed. NS, *P < 0.05, **P < 0.01, ***P < 0.001. PRR11, proline rich 11; EMT, epithelial-mesenchymal transition; ccRCC, clear cell renal cell carcinoma.

Figure 5. Reduction in PRR11 expression attenuated the in vivo proliferation and migration of ccRCC cells.

(A and B) The knockdown effect of LV-shPRR11 in ACHN cells was verified at the mRNA and protein levels (n = 3). (C) LV-NC and LV-shPRR11 cells were subcutaneously injected into nude mice to establish xenograft models. After 7 weeks, the xenografts were removed and photographed (n = 8). (D and E) The tumor volumes and weights were analyzed and evaluated (n = 8). (F) H&E staining was used to determine the degree of tumor malignancy, and IHC staining was used to evaluate the expression of PRR11 and Ki67 (n =8). Scale bar: 200 μm. (G and H) Quantification of the percentage of Ki67-positive cells and the PRR11 staining score (n = 8). (I–K) We established a lung metastasis model, detected the fluorescence intensity of the lung tumors, and performed H&E staining (n = 6). Scale bar: 100 μm. The data are shown as mean ± SD. Two-tailed t test analyses were performed. *P < 0.05, **P < 0.01, ***P < 0.001. PRR11, proline rich 11; ccRCC, clear cell renal cell carcinoma; LV-NC, lentiviral control shRNA.

Figure 6. c-Myc promoted the expression of PRR11 in ccRCC cells by binding to the PRR11 promoter.

(A and B) The mRNA expression levels of PRR11 and c-Myc in ccRCC tissues were analyzed (n = 11). (C and D) The effects of changes in c-Myc expression on PRR11 expression were analyzed at the mRNA and protein levels (n = 3). (E) The binding site of c-Myc was obtained from the JASPAR database. (F) Schematic of the PRR11 promoter region. The box (P1-P4) indicates the sequence region covered by the ChIP-qPCR primers. The red area represents the core binding element. (G) ChIP-qPCR assay of the enrichment of c-Myc in the PRR11 promoter region (n = 3). (H) Luciferase activity verified that c-Myc promoted the transcription of PRR11 in 293T cells (n = 4). The upregulation of c-Myc expression was detected by Western blotting (n = 3). (I) A luciferase assay was performed on the WT and mutant promoters of PRR11 in ACHN cells (n = 4). The data are shown as mean ± SD. Two-tailed t test (A–C, left; and G), 1-way ANOVA with Dunnett’s multiple comparisons test (C, right; and H), and 2-way ANOVA with Tukey’s multiple comparisons test (I) analyses were performed. NS, *P < 0.05, **P < 0.01, ***P < 0.001. PRR11, proline rich 11; ccRCC, clear cell renal cell carcinoma.

Figure 7. PRR11 reduced the stability of the E2F1 protein by interacting with E2F1.

(A) Western blotting analysis of ACHN cells and Caki-1 cells subjected to double thymidine blocking (n = 3). (B) Co-IP assay of exogenous E2F1 protein and PRR11 protein was performed in 293T cells (n = 3). (C) Co-IP assay of endogenous E2F1 protein and PRR11 protein was performed in ACHN cells (n = 3). (D) The colocalization of PRR11 and E2F1 in ACHN cells was analyzed by observing fluorescence signals by confocal immunofluorescence microscopy. Scale bar: 15 μm. (E) The effect of PRR11 on E2F1 protein levels in ACHN cells was analyzed by Western blotting (n = 3). (F–H) The effect of PRR11 on E2F1 protein degradation was investigated with the exogenous and endogenous expression of the proteins by an CHX assay (n = 3). (I–K) Quantification of E2F1 protein degradation rate (n = 3). Two-tailed t test (I) and 1-way ANOVA with Dunnett’s multiple comparisons test (J and K) analyses were performed. *P < 0.05, **P < 0.01, ***P < 0.001. PRR11, proline rich 11; Co-IP, coimmunoprecipitation; CHX, cycloheximide.

Figure 8. E2F1 played an important role in the PRR11-mediated regulation of the proliferation and migration of ccRCC cells.

(A–D) Transwell and clonogenic assays were used to analyze the effect of E2F1 knockdown on the migration and colony formation of ACHN cells transfected with shPRR11 (n = 3). Scale bar: 150 μm. (E and F) E2F1 knockdown significantly reversed the effect on the cell cycle progression of shPRR11-transfected ACHN cells (n = 3). (G) MTT assay confirmed that E2F1 knockdown significantly enhanced the proliferation of ACHN cells transfected with shPRR11 (n = 3). (H–J) The expression of PRR11, E2F1, cell cycle–related genes, and migration-related genes in ccRCC cells was determined by Western blotting. The data are shown as mean ± SD. Two-way ANOVA with Tukey’s multiple comparisons test analyses were performed (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. ccRCC, clear cell renal cell carcinoma; PRR11, proline rich 11.