Copine 1 predicts poor clinical outcomes by promoting M2 macrophage activation in ovarian cancer

Abstract

Objective: Copine 1 (CPNE1), a membrane-binding protein, influences the prognosis of various cancers. According to cBioPortal, CPNE1 amplification is a prevalent genetic mutation in ovarian cancer but with unknown oncogenic mechanism.

Methods: This study analysed the CPNE1 expression in ovarian cancer using online datasets, as validated by immunohistochemistry (IHC), quantitative polymerase chain reaction (qPCR) and western blotting. Concurrently, the prognostic value of CPNE1 was accessed. Cell Counting Kit-8, colony formation, transwells and xenograft experiments were performed to evaluate the functions of CPNE1 during ovarian cancer carcinogenesis. CPNE1 and its related genes were analysed by g:Profiler and Tumour Immune Estimation Resource. Furthermore, human monocytic THP-1 cells were co-cultured with ES2 cells to investigate the effect of CPNE1 on macrophage polarization.

Results: The results of bioinformatic analysis, IHC, qPCR and western blotting indicated a higher CPNE1 in ovarian cancer. CPNE1 overexpression demonstrated an association with a poor prognosis of ovarian cancer. Functionally, CPNE1 overexpression increased ES2 and SKOV3 cell proliferation, invasion and migration in vitro and promoted ovarian tumour xenograft growth in vivo, while CPNE1 knockdown led to opposite effects. Additionally, CPNE1 expression demonstrated an association with immune cell infiltration in ovarian cancer, especially macrophage. CPNE1 promoted protumour M2 macrophage polarization by upregulating cluster of differentiation 163 (CD163), CD206 and interleukin-10.

Conclusions: Our study revealed that CPNE1 mediated M2 macrophage polarization and provided a therapeutic target for ovarian cancer.

Graphical Abstract

Figure 1.

Upregulated CPNE1 expression in ovarian cancer. (A and B) The mRNA level of CPNE1 in ovarian cancer and normal tissues from GSE26712 (A) to GSE36668 (B). (C) Representative images of IHC staining with anti-CPNE1 antibody in ovarian cancer and normal tissues. (D) The IHC score of CPNE1 protein in ovarian cancer and normal tissues. (E) The expression of CPNE1 mRNA in IOSE and ovarian cancer cells as shown by PCR. (F) The expression of CPNE1 protein in IOSE and ovarian cancer cells as shown by western blotting. (G) The quantitative analysis of (F). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 2.

CPNE1 knockdown in ES2 and SKOV3 cells inhibits proliferation motility, invasion and migration. (A) Western blot analysis of CPNE1 knockdown efficacy in ES2 and SKOV3 cells. (B) The quantitative analysis of (A). (C–E) The effect of CPNE1 knockdown on cell viability in ES2 and SKOV3 cells was detected via CCK-8 assay (C) and colony formation (D, E). (F and G) The number of invasive cells (F) and migratory cells (G) in the control and CPNE1-knockdown groups. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 3.

CPNE1 promotes cell proliferation, invasion and migration of ES2 and SKOV3 cells. (A) CPNE1-overexpressing ES2 and SKOV3 cells were constructed and confirmed via western blot. (B) The quantitative analysis of (A). (C–E) The effect of CPNE1 overexpression on cell viability in ES2 and SKOV3 cells was detected via CCK-8 assay (C) and colony formation (D, E). (F and G) The number of invasive (F) and migratory (G) cells in the control group and CPNE1-overexpression group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 4.

CPNE1 overexpression promotes tumour growth in nude mice. (A) Photograph of CPNE1-overexpression ES2 xenografts (up) relative to the control (down). (B–D) The growth curves (B), final volume (C) and weight (D) of xenograft tumours in the CPNE1-overexpression and control groups. OE: CPNE1-overexpression. ^*P < 0.05.

Figure 5.

CPNE1 and its related genes regulate immunity in ovarian cancer. (A) The protein–protein interaction (PPI) network presented the proteins interacting with CPNE1. (B) Human Phenotype Ontology analysis of CPNE1 and its related genes. (C) Correlations between CPNE1 and multiple immune infiltrations. (D) Correlations between CPNE1 and B cell subtypes in ovarian cancer. (E) Correlations of CPNE1 and macrophage subtypes in ovarian cancer.

Figure 6.

CPNE1 promotes M2 polarization of TAMs by upregulating CD163 and CD206. (A) Experimental diagram of generating TAMs. (B) The morphology of THP-1 after co-culturing with PMA. (C) The mRNA level of macrophage marker CD68 in M0 macrophages compared with THP-1 cells, as shown by PCR. (D) The mRNA level of M2 macrophage markers (CD163, CD206 and IL-10) in the TAM_CPNE1-OE group compared with the TAM_NC group, as shown by PCR. (E) Immunofluorescence staining of CD206 in macrophages after co-culturing with different ES2 cells (ES2 CPNE1-overexpression or its corresponding empty vectors). ^**P < 0.01, ^***P < 0.001.