Ring finger protein 2 promotes colorectal cancer progression by suppressing early growth response 1

Abstract

Ring finger protein 2 (RNF2) is an important component of polycomb repressive complex 1. RNF2 is upregulated in many kinds of tumors, and elevated RNF2 expression is associated with a poor prognosis in certain cancers. To assess the function of RNF2 in colorectal cancer, we examined RNF2 protein levels in 313 paired colorectal cancer tissues and adjacent normal tissues. We then analyzed the association of RNF2 expression with the patients' clinicopathologic features and prognoses. RNF2 expression was upregulated in colorectal cancer tissues and was associated with the tumor differentiation status, tumor stage and prognosis. In colorectal cancer cell lines, downregulation of RNF2 inhibited cell proliferation and induced apoptosis. Gene microarray analysis revealed that early growth response 1 (EGR1) was upregulated in RNF2-knockdown cells. Knocking down EGR1 partially reversed the inhibition of cell proliferation and the induction of apoptosis in RNF2-knockdown cells. RNF2 was enriched at the EGR1 promoter, where it mono-ubiquitinated histone H2A, thereby inhibiting EGR1 expression. These results indicate that RNF2 is oncogenic in colorectal cancer and may promote disease progression by inhibiting EGR1 expression. RNF2 is thus a potential prognostic marker and therapeutic target in colorectal cancer.

Keywords: EGR1; RNF2; apoptosis; cell proliferation; colorectal cancer.

Figure 1

RNF2 expression was upregulated in CRC tumor tissues and was associated with patients’ prognoses. (A) Representative immunohistochemistry results depicting positive and negative RNF2 staining in clinical CRC tumor tissues and adjacent normal tissues. The pictures on the right are the magnified view of the yellow boxes on the left. Scale bar: 200 μm. (B) H-scores for RNF2 expression in 313 CRC tumor tissues and adjacent normal tissues. ***p<0.001 versus adjacent normal tissue. (C) Overall survival of RNF2-positive or -negative CRC patients. (D) Local relapse-free survival of RNF2-positive or -negative CRC patients. (E) Distant metastasis-free survival of RNF2-positive or -negative CRC patients.

Figure 2

The downregulation of RNF2 inhibited cell proliferation and increased apoptosis in CRC cells. (A) Western blot analysis showing the knockdown efficiency of RNF2 in HCT116 cells that had been infected with shRNA-expressing lentiviruses for 48 hours. (B) MTT assay showing the proliferation of RNF2-knockdown and control HCT116 cells. (C) Apoptosis analysis of RNF2-knockdown and control HCT116 cells. (D) MTT assay showing the proliferation of RNF2-knockdown and control WiDr cells. (E) Apoptosis analysis of RNF2-knockdown and control WiDr cells. (F) Western blot analysis showing the knockdown efficiency of RNF2 and the cleavage of PARP in RNF2-knockdown and control WiDr cells. (G) qRT-PCR analysis showing the mRNA levels of IL-6 and IL-8 in RNF2-knockdown and control HCT116 cells. (H) ELISA assay showing the soluble IL-6 and IL-8 levels in culture media. Three independent experiments were performed and analyzed for B-E. Data represent the mean ± standard deviation. *p<0.05 versus shCon group.

Figure 3

The downregulation of RNF2 induced EGR1 expression. (A) Heat map of some of the most differentially expressed genes in shRNF2-treated HCT116 cells, determined through microarray analyses (red: upregulated; green: downregulated). (B) RT-PCR analysis showing the increased EGR1 mRNA levels in RNF2-knockdown HCT116 cells. (C) Western blot showing the increased EGR1 protein levels in RNF2-knockdown HCT116 cells.

Figure 4

Knocking down EGR1 partially reversed the inhibition of cell proliferation and the increase in apoptosis in RNF2-knockdown cells. (A) Western blot showing the knockdown efficiency of RNF2 and EGR1 and the cleavage of PARP in RNF2/EGR1 double-knockdown, RNF2-knockdown and control HCT116 cells. (B) MTT assay showing the proliferation of RNF2/EGR1 double-knockdown, RNF2-knockdown and control HCT116 cells. (C) Apoptosis analysis of RNF2/EGR1 double-knockdown, RNF2-knockdown and control HCT116 cells. Three independent experiments were performed and analyzed for B and C. Data represent the mean ± standard deviation. *p<0.05 versus shCon, Δp<0.05 versus shRNF2.

Figure 5

The downregulation of RNF2 reduced both RNF2 enrichment and H2A mono-ubiquitination at the EGR1 promoter. (A) The positions of the three PCR primer sets used in the ChIP assay. (B) Quantitative PCR analysis after a ChIP assay using antibodies against RNF2 and H2A K119Ub. Three independent experiments were performed and analyzed. Data represent the mean ± standard deviation. *p<0.05, **p<0.01 and ***p<0.001 versus IgG. (C) Quantitative PCR analysis using primer set 2 after a ChIP assay to show the enrichment of RNF2 and the mono-ubiquitination of H2A at the EGR1 promotor in RNF2-knockdown and control HCT116 cells. Three independent experiments were performed and analyzed. Data represent the mean ± standard deviation. *p<0.05, **p<0.01 and ***p<0.001 versus shCon.