CYP27B1 Downregulation: A New Molecular Mechanism Regulating EZH2 in Ovarian Cancer Tumorigenicity

Abstract

Background: Ovarian cancer has the highest mortality rate among gynecologic cancers, and most patients are diagnosed in advanced stages. Enhancer of zeste homolog 2 (EZH2) is a major tumor marker and an effective therapeutic target for ovarian cancer, but the underlying molecular mechanism remains unclear. The present study investigated the biological effects of EZH2 knockout in SKOV3 cells in vitro and in vivo and explored the molecular mechanism by integrated analysis of messenger RNA sequencing (mRNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data.

Methods: The CRISPR/Cas9 system was used to establish EZH2 knockout SKOV3 cells. Protein expression was evaluated by Western blotting. The effect of EZH2 on ovarian cancer was evaluated in vitro with MTT, wound healing, Transwell, and apoptosis assays and in vivo with a xenograft model. mRNA-seq and ChIP-seq were performed to explore the molecular mechanism underlying the biological function of EZH2. Immunohistochemical staining (IHC) of tissue arrays was used to analyze the correlations among EZH2 and CYP27B1 expressions and prognosis.

Results: We obtained three EZH2 knockout subclones. EZH2 knockout SKOV3 cells exhibited significantly suppressed proliferation, migration, and invasion and a significantly increased apoptosis rate. The subcutaneous tumor formation rate decreased from 100 to 0% in the EZH2 knockout group. Integrated analysis of the mRNA-seq and ChIP-seq data identified 1,455 significantly upregulated genes with matching downregulated trimethylation of histone H3 lysine 27 (H3K27me3) methylation binding sites in 1b11H cells compared to SKOV3 cells. The set of downregulated genes in EZH2 knockout cells was highly enriched in genes regulating the activation of steroid biosynthesis; the top-ranked hub gene was CYP27B1. The EZH2 and CYP27B1 expression levels showed a statistically significant inverse correlation, which was also associated with unfavorable prognosis. The in vitro experiment demonstrated that CYP27B1 can suppress the proliferation, migration, and invasion of ovarian cancer cells. Moreover, the levels of AKT and p-AKT were significantly increased, whereas STAT3 was downregulated, in 1b11H cells compared to SKOV3 cells. Moreover, STAT3 and AKT overexpression was observed in 1b11H siRNA for CYP27B1 (siCYP27B1) cells.

Conclusion: EZH2 plays an important role in promoting cell proliferation, migration, and invasion in ovarian cancer by regulating the core steroid biosynthesis gene via H3K27me3 methylation. Moreover, CYP27B1, the steroid biosynthesis hub gene, might be a novel therapeutic target for ovarian cancer.

Keywords: CYP27B1; ChIP-seq; EZH2; ovarian cancer; steroid biosynthesis.

FIGURE 1

(A) Clones with obviously shortened nucleic acid sequences were identified on agarose gels after PCR. (B) Changes in target fragments were identified by Sanger sequencing. (C) Western blot results. (D) Cell immunofluorescence assay results (green fluorescence represents EZH2 and blue fluorescence represents DAPI).

FIGURE 2

Changes in biological behaviors after knockout of EZH2. (A) Cell growth curve and doubling time. (B) Transwell migration and invasion assay results. (C) Wound healing assay results. (D) Flow cytometric analysis results. (E) In vivo experiment results. ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

Integrated analysis of RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data. (A) Heatmap of the differentially expressed genes identified by RNA-seq. (B) Heatmap of the ChIP-seq read distributions within peaks. (C) Average read signals across all transcription start sites (TSSs). (D) Functional prediction according to integrated analysis of RNA-seq and ChIP-seq data. (E) Pathway enrichment of matched downregulated genes. (F) Gene set enrichment analysis (GSEA) of matched downregulated genes. (G) Heatmap of the differentially expressed genes in steroid biosynthesis. (H) ChIP-seq read distributions at the TSS of CYP27B1.

FIGURE 4

Changes in biological behaviors and relative gene expression levels after silencing or overexpression of CYP27B1. (A) Western blot results. (B) Transwell migration and invasion assay results. (C,D) Cell growth curves. *p < 0.05; **p < 0.01.

FIGURE 5

Immunohistochemistry for enhancer of zeste homolog 2 (EZH2) and CYP27B1 and survival analysis of patients stratified by the EZH2 and CYP27B1 expression status. Ovarian cancer samples (N = 160). Ovarian cancer samples with weak and strong immunostaining scores for EZH2 and CYP27B1, respectively (A) Correlation between the expression levels of EZH2 and CYP27B1 in ovarian tissues (B) Overall (OS) and disease-free (DFS) survival curves for patients with ovarian cancer (N = 160) stratified according to EZH2 (C,D) or CYP27B1 (E,F) gene expression status (low or high). Stratification by the gene expression status was conducted according to the median value.