Deregulated expression and subcellular localization of CPSF6, a circRNA-binding protein, promote malignant development of esophageal squamous cell carcinoma

Abstract

Objective: Cleavage and polyadenylation specific factor 6 (CPSF6) has been documented as an oncoprotein in different types of cancer. However, functions of CPSF6 have not been investigated yet in esophageal squamous cell carcinoma (ESCC). Here, we aimed to investigate the potential clinical values and biological functions of CPSF6 in ESCC.

Methods: For determining the expression level of CPSF6 in ESCC patients, we analyzed published data, performed quantitative real-time polymerase chain reaction (RT-qPCR) and immunohistochemistry assays. Kaplan-Meier curves and log-rank tests were used for survival analyses. GO and KEGG analyses were done for CPSF6-related genes. Cell proliferation, colony formation and xenograft assays were conducted to verify the effects of CPSF6 on ESCC. In addition, cell cycle and apoptosis assays were also performed to manifest the functions of CPSF6 and circCPSF6. RNA pulldown and radioimmunoprecipitation (RIP) assays were used for confirming the interaction between circCPSF6 (hsa_circ_0000417) and CPSF6 protein. The regulatory relationship between CPSF6 protein and circCPSF6 was determined by RT-qPCR.

Results: We found that CPSF6 was upregulated in ESCC tissues and overexpression of cytoplasmic CPSF6 was associated with poor prognosis. GO and KEGG analyses suggested that CPSF6 could mainly affect cell division in ESCC. Further experiments manifested that CPSF6 promoted cell proliferation and colony formationin vitro. Xenograft assay showed that knockdown of CPSF6 significantly decreased tumor growth rate in vivo. Subsequently, we verified that depletion of CPSF6 led to cell cycle arrest and apoptosis. Finally, we validated that CPSF6, as a circRNA-binding protein, interacted with and regulated its circular isoform circCPSF6 (hsa_circ_0000417), of which depletion also resulted in cell cycle arrest and cell apoptosis in ESCC.

Conclusions: These findings gave us insight that overexpression of cytoplasmic CPSF6 protein is associated with poor prognosis in ESCC and CPSF6 may function as an oncoprotein, at least in part, through regulating circCPSF6 expression.

Keywords: CPSF6; cell proliferation; circCPSF6; circRNA-binding protein; esophageal squamous cell carcinoma; prognosis.

Figure 1

CPSF6 is highly expressed in ESCC. (A) CPSF6 mRNA expression profile across all types of tumor and normal tissues in GEPIA 2. Each dot represents CPSF6 expression (TPM) of one sample. Statistically significant differences between tumor and normal samples are marked in red; (B) RT-qPCR analysis for relative CPSF6 mRNA expression of ESCC tumor tissues (n=29) and normal tissues (n=30) (P<0.001); (C) RT-qPCR analysis for relative CPSF6 mRNA expression in 2 immortalized esophageal epithelial cell lines and 10 ESCC cell lines; (D) Western blot assay for CPSF6 protein expression in 2 immortalized esophageal epithelial cell lines and 10 ESCC cell lines. Bar plot shows the relative CPSF6 protein normalized to β-actin. ESCC, esophageal squamous cell carcinoma; TPM, transcripts per million; RT-qPCR, quantitative real-time polymerase chain reaction

Figure 2

High mRNA level of CPSF6 is associated with poor prognosis in ESCC. (A) Relative CPSF6 mRNA expression (Log2 FPKM) of ESCC tumor samples (n=11) and normal samples (n=81) from the TCGA (P<0.001); (B) Relative CPSF6 mRNA expression (Log2 FPKM) of ESCC tumor samples (n=119) and normal samples (n=119) from the GSE53624 dataset (P<0.001); (C-F) Kaplan-Meier survival analyses for the correlation between CPSF6 mRNA expression and overall survival with all cases (C), stage I+II cases (D), stage III cases (E), and lymph node metastasis cases (F) using the data of GSE53624 dataset. ESCC, esophageal squamous cell carcinoma; TCGA, The Cancer Genome Atlas.

Figure 3

High cytoplasmic protein level of CPSF6 is detrimental to ESCC patient prognosis. (A) IHC staining of CPSF6 protein in ESCC and adjacent normal tissues; (B) Distribution of samples with different grades of total CPSF6 protein in ESCC (n=55) and adjacent normal tissues (n=49) (P=0.005); (C) Comparing total expression of CPSF6 protein in ESCC and adjacent normal tissues by IHC index [intensity × positive rate (%)] (P<0.001); (D) Kaplan-Meier survival analysis of cases according to the level of total expression of CPSF6 protein [HR=1.027 (95% CI: 0.637−1.965), P=0.934]; (E) Western blot assay for detecting the subcellular locations of CPSF6 protein in ESCC cell lines; (F) Distribution of samples with different grades of cytoplasmic CPSF6 protein in ESCC and adjacent normal tissues (P=0.001); (G) Comparing cytoplasmic expression of CPSF6 protein in ESCC and adjacent normal tissues by IHC index (P=0.051); (H) Kaplan-Meier survival analysis of cases according to the level of cytoplasmic expression of CPSF6 protein (P<0.001); (I) Distribution of samples with different grades of nucleoplasmic CPSF6 protein in ESCC and adjacent normal tissues (P=0.760); (J) Comparing nucleoplasmic expression of CPSF6 protein in ESCC and adjacent normal tissues by IHC index [HR=1.820 (95% CI: 0.964−3.439), P=0.062]; (K) Kaplan-Meier survival analysis of cases according to the level of nucleoplasmic expression of CPSF6 protein [HR=0.821 (95% CI: 0.427−1.576), P=0.554]. ESCC, esophageal squamous cell carcinoma; IHC, immunohistochemistry; HR, hazard ratio; 95% CI, 95% confidence interval.

Figure 4

Gene enrichment analyses of CPSF6-related genes. (A) Top 20 biological process terms enriched in GO analysis; (B) Top 10 pathways enriched in KEGG analysis.

Figure 5

CPSF6 plays oncogenic roles in vitro and in vivo. (A,B) RT-qPCR assay (A) and Western blot assay (B) for detecting the mRNA and protein level of CPSF6 in ESCC cell lines (KYSE410 and KYSE450) after knockdown or overexpression of CPSF6; (C) Proliferation curves of ESCC cell lines (KYSE410 and KYSE450) after knockdown or overexpression of CPSF6; (D) Colony formation assay of ESCC cell lines (KYSE410 and KYSE450) after knockdown or overexpression of CPSF6. The number of colonies is presented in bar plots; (E) Subcutaneous tumorigenesis assay was performed after knockdown of CPSF6 in KYSE450 cells. Tumor weight was measured on d 30 after subcutaneous injection. A box-plot is presented for tumor weight. ESCC, esophageal squamous cell carcinoma.

Figure 6

Knockdown of CPSF6 induces cell-cycle arrest and apoptosis of ESCC. (A,B) Cell cycle assay was performed after knockdown of CPSF6 in KYSE410 (A) and KYSE450 (B) cells, and the percentage of cells in each cell cycle phase is presented in bar plots; (C) Apoptosis assay was performed after knockdown of CPSF6 in ESCC cell lines (KYSE410 and KYSE45); (D) Bar plots are presented for the proportion of apoptotic cells; (E) Caspase 3 and cleaved caspase 3 were detected by Western blot after knockdown of CPSF6 in ESCC cell lines (KYSE410 and KYSE45). ESCC, esophageal squamous cell carcinoma

Figure 7

CPSF6 protein binds to and regulates its circular isoform circCPSF6 (hsa_circ_0000417). (A) A bar plot is presented for CPSF6 protein-binding RNA of CLIP-seq data from the ENCORI database; (B) Identification of circCPSF6 through RNase R treatment and Sanger sequencing of junction site; (C,D) Interaction between CPSF6 protein and circCPSF6 was detected by RIP assay (C) and RNA-protein pulldown assay (D); (E) Subcellular distribution of circCPSF6 in ESCC presented by a bar plot; (F,G) RT-qPCR assay for detecting the RNA level of CPSF6 and circCPSF6 after depletion of CPSF6 (F) and circCPSF6 (G), respectively; (H) Cell cycle assay was performed after knockdown of circCPSF6, and each cell cycle phase is presented by a bar plot; (I) Apoptosis assay was performed after knockdown of circCPSF6, and a bar plot is presented for the proportion of apoptotic cells. CLIP-seq, cross-linking immunoprecipitation sequencing; ESCC, esophageal squamous cell carcinoma.