Comprehensive analysis of prognostic value, immune implication and biological function of CPNE1 in clear cell renal cell carcinoma

Abstract

Background: Elevated expression of Copine-1 (CPNE1) has been proved in various cancers; however, the underlying mechanisms by which it affects clear cell renal cell carcinoma (ccRCC) are unclear. Methods: In this study, we applied multiple bioinformatic databases to analyze the expression and clinical significance of CPNE1 in ccRCC. Co-expression analysis and functional enrichment analysis were investigated by LinkedOmics, cBioPortal and Metascape. The relationships between CPNE1 and tumor immunology were explored using ESTIMATE and CIBERSORT method. In vitro experiments, CCK-8, wound healing, transwell assays and western blotting were conducted to investigate the effects of gain- or loss-of-function of CPNE1 in ccRCC cells. Results: The expression of CPNE1 was notably elevated in ccRCC tissues and cells, and significantly correlated with grade, invasion range, stage and distant metastasis. Kaplan-Meier and Cox regression analysis displayed that CPNE1 expression was an independent prognostic factor for ccRCC patients. Functional enrichment analysis revealed that CPNE1 and its co-expressed genes mainly regulated cancer-related and immune-related pathways. Immune correlation analysis showed that CPNE1 expression was significantly related to immune and estimate scores. CPNE1 expression was positively related to higher infiltrations of immune cells, such as CD8⁺ T cells, plasma cells and regulatory T cells, exhibited lower infiltrations of neutrophils. Meanwhile, elevated expression of CPNE1 was characterized by high immune infiltration levels, increased expression levels of CD8⁺ T cell exhaustion markers (CTLA4, PDCD1 and LAG3) and worse response to immunotherapy. In vitro functional studies demonstrated that CPNE1 promoted proliferation, migration and invasion of ccRCC cells through EGFR/STAT3 pathway. Conclusion: CPNE1 is a reliable clinical predictor for the prognosis of ccRCC and promotes proliferation and migration by activating EGFR/STAT3 signaling. Moreover, CPNE1 significantly correlates with immune infiltration in ccRCC.

Keywords: CPNE1; EGFR/STAT3; immune infiltration; prognosis; renal cell carcinoma.

FIGURE 1

Pan-cancer analysis. (A) CPNE1 was aberrantly expressed among multiple cancer types. (B) High expression of CPNE1 predicted worse prognosis in HNSC, KIRC, and MESO. (C) High expression of CPNE1 was associated with advanced stage in BRCA, COAD, KIRC, KIRP, UCEC. (D) High CPNE1 expression was correlated with high grade in CESC, KIRC, LGG, LIHC, UCEC. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (Supplementary Table S3 showed a complete list of the TCGA cancer-type abbreviations).

FIGURE 2

CPNE1 mRNA expression, diagnostic and prognostic value in ccRCC. (A) CPNE1 was significantly overexpressed in tumors compared with the adjacent normal tissue in TCGA-KIRC dataset (p < 0.001). (B) CPNE1 was significantly overexpressed in tumors compared with the adjacent normal tissue in GSE40435 (p < 0.001). (C) ROC curve showed that the prediction of the AUC of the 1-year, 3-year and 5-year OS were 0.649, 0.634 and 0.684 respectively. (D) Kaplan–Meier plot demonstrated a correlation between poor prognosis and the high CPNE1 expression in TCGA-KIRC dataset (p < 0.001). (E) Kaplan–Meier plot demonstrated a correlation between poor prognosis and the high CPNE1 expression in E-MTA-1980 dataset (p = 0.04).

FIGURE 3

CPNE1 was correlated with various clinicopathological parameters in ccRCC. High CPNE1 expression was positively correlated with (A) tumor grade, (B) invasion range, (C) stage and (D) distant metastasis. CPNE1 was not associated with (E) age, (F) gender and (G) lymph node metastasis in TCGA-KIRC dataset. (H) High CPNE1 expression was associated with advanced tumor grade in E-MTAB-1980 dataset. (I) Heatmap showed the association of CPNE1 and clinicopathological features. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4

Independent prognostic analysis. (A) Univariate analysis showed that CPNE1, age, grade and stage were related to OS (p < 0.01). (B) Multivariate analysis showed that CPNE1, age, grade and stage were strong independent prognostic factors. (C) Nomogram represented the multivariate model. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5

Co-expression genes of CPNE1 and miRNA-target enrichment analysis. (A) Heatmaps showed top 50 genes positively correlated with CPNE1. (B) Heatmaps showed top 50 genes negatively correlated with CPNE1. (C) Volcano plot showed the CPNE1 highly correlated genes. Positive correlations are displayed in red and negative correlations in green color. The top 3 genes co-expressed with CPNE1 were verified in the cBioPortal database. (D) CTNNBL1 was positively correlated with CPNE1 (R = 0.62, p < 0.001). (E) BCL2L12 was positively correlated with CPNE1 (R = 0.54, p < 0.001). (F) LRBA was negatively correlated with CPNE1 (R = −0.52, p < 0.001). (G) miRNA-target enrichment of CPNE1 based on LinkedOmics. R, Pearson correlation coefficient.

FIGURE 6

Function enrichment analysis of co-expressed genes with CPNE1 by Metascape. (A) GO annotation showed that CPNE1 was related to the GO term “cellular process”, “metabolic process” and “regulation of biological process.” (B) KEGG pathway showed that CPNE1 was related to “metabolism of RNA”, “Cell Cycle” and “ncRNA processing”.

FIGURE 7

Tumor immunology analysis. (A) CPNE1 was positively correlated with immune and estimate scores. (B) CPNE1 high- and low-expression groups displayed different fraction of immune cell types. The x-axis represents the types of immune cell. The y-axis represents the proportion of each immune cell type. (C) CPNE1 was significantly related to the proportions of multiple immune cell types. (D) Heatmap showed CPNE1 was associated with multiple immune checkpoint members. Heatmap colors: correlation (cor) coefficient. CPNE1 high -expression groups displayed higher (E) TIDE score, (F)T cell dysfunction score, and (G) T cell exclusion score. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 8

Construction of CPNE1 knockdown cell lines and cell proliferation assays. (A) The mRNA and protein expression level of CPNE1 in ccRCC cell lines was generally higher than that in renal epithelial cell. (B) Successful knockdown by the siRNA was confirmed by RT-PCR and WB in 786-O cells. (C) Successful knockdown by the siRNA was confirmed by RT-PCR and WB in OS-RC-2 cells. (D) Knockdown of CPNE1 inhibited 786-O cells proliferation. (E) Knockdown of CPNE1 inhibited OS-RC-2 cells proliferation. **, p < 0.01; ***, p < 0.001. NC: Negative control. Si: ccRCC cells transfected with CPNE1-small interfering RNA.

FIGURE 9

Biological functions of CPNE1 knockdown in vitro. (A) Knockdown of CPNE1 decreased migration of 786-O cells (scale bar: 50 μm). (B) Knockdown of CPNE1 decreased migration of OS-RC-2 cells (scale bar: 50 μm). (C) CPNE1 knockdown significantly inhibited 786-O and OS-RC-2 cells invasion (scale bar: 200 μm). (D) EGFR and STAT3 phosphorylation were inhibited by the knockdown of CPNE1 in 786-O cells. (E) EGFR and STAT3 phosphorylation were inhibited by the knockdown of CPNE1 in OS-RC-2 cells. NC: Negative control. Si: ccRCC cells transfected with CPNE1-small interfering RNA. **, p < 0.01; ***, p < 0.001.

FIGURE 10

Biological functions of CPNE1 overexpression in vitro. (A) The successful construction of CPNE1 overexpression (ov) plasmid was validated via RT-PCR and WB in A-498 cells. (B) Overexpression of CPNE1 promoted proliferation in A-498 by CCK8 assays. (C) Scratch-wound healing assays demonstrated that migration abilities of A-498 cells were enhanced by overexpression of CPNE1 (scale bar: 50 μm). (D) Invasive capacities of A-498 cells were enhanced by CPNE1 overexpression (scale bar: 200 μm). (E) EGFR and STAT3 phosphorylation were activated by the overexpression of CPNE1 in A-498 cells. NC: negative control. ***, p < 0.001.