A comprehensively prognostic and immunological analysis of actin-related protein 2/3 complex subunit 5 in pan-cancer and identification in hepatocellular carcinoma

Abstract

Background: Actin-related protein 2/3 complex subunit 5 (ARPC5) is one of the members of actin-related protein 2/3 complex and plays an important role in cell migration and invasion. However, little is known about the expression pattern, prognosis value, and biological function of ARPC5 in pan-cancer. Thus, we focus on ARPC5 as cut point to explore a novel prognostic and immunological biomarker for cancers.

Methods: The public databases, including TCGA, GTEx, and UCEC, were used to analyze ARPC5 expression in pan-cancer. The Human Protein Atlas website was applied to obtain the expression of ARPC5 in different tissues, cell lines, and single-cell types. Univariate Cox regression analysis and Kaplan-Meier analysis were used to explore the prognosis value of ARPC5 in various cancers. Spearman's correlation analysis was performed to investigate the association between ARPC5 expression and tumor microenvironment scores, immune cell infiltration, immune-related genes, TMB, MSI, RNA modification genes, DNA methyltransferases, and tumor stemness. Moreover, qPCR, Western blot, and immunohistochemistry were carried out to examine the differential expression of ARPC5 in HCC tissues and cell lines. CCK8, EdU, flow cytometry, wound-healing assays, and transwell assays were conducted to explore its role in tumor proliferation, apoptosis, migration, and invasion among HCC cells.

Results: ARPC5 expression was upregulated in most cancer types and significantly associated with worse prognosis in KIRC, KIRP, LGG, and LIHC. mRNA expression of ARPC5 showed low tissue and cell specificity in normal tissues, cell lines, and single-cell types. ARPC5 expression was positively correlated with the tumor microenvironment scores, immune infiltrating cells, immune checkpoint-related genes in most cancers. ARPC5 in STAD and BRCA was positively associated with TMB, MSI, and neoantigens. We also discovered that ARPC5 was correlated with the expression of m1A-related genes, m5C-related genes, m6A-related genes, and DNA methyltransferases. In experiment analyses, we found that ARPC5 was significantly highly expressed in HCC tissues and HCC cells. Functionally, silencing ARPC5 dramatically decreased proliferation, migration, and invasion ability of HCC cells.

Conclusions: ARPC5 expression affects the prognosis of multiple tumors and is closely correlated to tumor immune infiltration and immunotherapy. Furthermore, ARPC5 may function as an oncogene and promote tumor progression in HCC.

Keywords: ARPC5; biomarker; hepatocellular carcinoma; immune; pan-cancer; prognosis.

Figure 1

The expression levels of APRC5 in different cancers, normal tissues, and cells. (A) The differential expression of ARPC5 in pan-cancer tissues from TCGA datasets. (B) The differential expression of ARPC5 in pan-cancer tissues based on TCGA and GTEx datasets. (C) ARPC5 expression in paired cancer tissues and adjacent normal tissues from TCGA datasets. (D) The mRNA expression levels of ARPC5 in different normal tissues from HPA database. (E) The mRNA expression of ARPC5 in cancer cell lines from HPA database. (F) ARPC5 mRNA expression in different single cell types from HPA database. ns: no significance; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 2

Genetic alteration of ARPC5 in pan-cancer. (A) Mutation type and mutation frequency of ARPC5 obtained from the cBioPortal website. (B) The expression levels of ARPC5 in various CNV status of pan-cancer, CNV, copy number variations; *p < 0 . 0 5 ; * *p < 0.01; ****p < 0.0001.

Figure 3

Prognosis analyses of ARPC5 in pan-cancer based on univariate Cox regression method. (A) The correlation between ARPC5 expression and OS. (B) The correlation between ARPC5 expression and PFI. (C) The correlation between ARPC5 expression and DSS. OS: overall survival; PFI: progression-free interval; DSS: disease-specific survival.

Figure 4

ARPC5 expression significantly correlated with OS based on Kaplan–Meier analysis. (A) The correlation in ESCA. (B) The correlation in HNSC. (C) The correlation in KIRC. (D) The correlation in KIRP. (E) The correlation in LIHC. (F) The correlation in LGG. (G) The correlation in OV. (H) The correlation in SKCM. The optimal cutoff of ARPC5 expression were used to divide patients into high- and low-expression groups.

Figure 5

The correlation between ARPC5 expression and clinical stage, histologic grade, and tumor molecular subtypes in various cancers based on Spearman’s correlation analysis (the correlation with p < 0.05 were displayed). (A) The correlation between ARPC5 expression and clinical stage in pan-cancer. (B–C) The expression levels of ARPC5 in different clinical stages of KIRC (B) and KIRP (C). (D) The correlation between ARPC2 expression and histologic grade in pan-cancer. (E–H) The expression levels of ARPC5 in different histologic grades of KIRC (E), LGG (F), LIHC (G), and UCEC (H). (I–R) The correlation between ARPC5 expression and molecular subtypes in ACC (I), BRCA (J), LGG (K), HNSC (L), KIRP (M), OV (N), LUSC (O), PCPG (P), STAD (Q), UCEC (R). rho; rank coefficient of Spearman. Pv; p-value. NS, no significance.

Figure 6

The correlation between ARPC5 and immune infiltration cells in pan-cancer based on TIMER algorithm. (A) Heatmap displayed the correlation between ARPC5 expression and the proportions of B cell, CD4⁺ T cell, CD8⁺ T cell, neutrophil, macrophage, and DC cell. (B) The top five cancer types (including KIRC, LGG, PRAD, THCA, and THYM) with most significant correlation between ARPC5 and immune infiltration cells were displayed with scatterplots. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 7

The correlation between ARPC5 expression and immune subtypes in pan-cancer using TISIDB. The cancers with significant correlation were displayed. (A) In BLCA. (B) In BRCA. (C) In CESC. (D) In KICH. (E) In KIRC. (F) In LGG. (G) In LIHC. (H) In LUAD. (I) In PAAD. (J) In OV. (K) PCPG. (L) PRAD. (M) In READ. (N) In SARC. (O) In SKCM. (P) In STAD. (Q) In TGCT. (R) In THCA. (S) In UCS. (T) In UCEC. Pv; p-value. C1, wound healing; C2, IFN-gamma dominant; C3, inflammatory; C4, lymphocyte depleted; C5, immunologically quiet; C6, TGF-b dominant.

Figure 8

The relationship between ARPC5 and immune-checkmate inhibitors biomarkers in pan-cancer. (A) The heatmap showing the co-expression relationship between ARPC5 and 47 immune checkpoint–related genes. (B) Radar plot showing the relationship between ARPC5 and tumor mutation burden (TMB). (C) Radar plot showing the correlation of ARPC5 with microsatellite instability (MSI). (D) Radar plot showing the correlation of ARPC5 with neoantigens. The number in radar plot represents Spearman’s correlation coefficient. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 9

Correlation analysis between ARPC5 expression and RNA modification-related genes, DNA methyltransferases, and tumor stemness score in 33 cancer types. (A) Co-expression of ARPC5 with m1A-related genes. (B) Co-expression of ARPC5 with m5C-related genes. (C) Co-expression of ARPC5 with m6A-related genes. (D) Co-expression of ARPC5 with DNA methyltransferases. (E) The correlation between ARPC5 expression and Tumor Stemness score (DNAss). (F) The correlation between ARPC5 expression and Tumor Stemness score (RNAss). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 10

ARPC5 is upregulated in HCC cells and primary HCC tissues. (A) qPCR analysis of ARPC5 mRNA expression in four HCC cell lines (MHCC97-H, Huh7, HCC-LM3, and HepG2) and normal liver cell line (LO2). GAPDH was used as an internal control error bars represent M ± SEM (triplicate experiments). (B, C) The protein expression of ARPC5 was detected in four HCC cell lines and normal liver cell line with Western blot analysis. Error bars represent M ± SD of triplicate measurements. (D) The mRNA expression of ARPC5 in 40 pairs HCC tissues and adjacent para-carcinoma tissues was evaluated using qPCR. (E) Western blot analysis of ARPC5 protein expression in 10 paired HCC tissues and adjacent normal tissues. The number presented the relative protein expression levels of ARPC5. (F) Representative images of ARPC5 immunohistochemical staining analysis in the HCC tissue and adjacent normal liver tissue, original magnifications: ×40 and ×200. Scale bars, 50 μm. (G) Quantitative analysis of ARPC5 expression in HCC tissues based on mean optical density of immunohistochemical staining. Error bars represent the M ± SD of multiple tissues. (H) Kaplan–Meier curves showed that higher expression of ARPC5 was associated with poor DFS in HCC patients. *p < 0.05; **p < 0.01; ***p < 0.001. ns, no significance.

Figure 11

Silencing of ARPC5 inhibits cell proliferation and promotes cell apoptosis of HCC. (A) The knockdown efficiency of siRNA–ARPC5 was examined in HCC-LM3 and MHCC97-H cells with qPCR. (B) The knockdown efficiency of siRNA-ARPC5 was examined in HCC-LM3 and MHCC97-H cells with Western blot. The number presented as relative protein expression levels of ARPC5. (C–D) EdU assays for HCC-LM3 and MHCC 97-H were performed to evaluate cell proliferation ability after transfecting siRNA-ARPC5#1. Representative images (C) and the number of proliferative cells were calculated (D); original magnification, ×200. (E–F) Cellular growth curves were evaluated by CCK-8 assays in HCC-LM3 and MHCC97-H cells. (G–H) Flow cytometry was applied to test the apoptosis of HCC cells transfected with si-ARPC5 #1 in HCC-LM3 and MHCC 97-H cells. All data are presented as the M ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. ns, no significance.

Figure 12

Role of ARPC5 inhibition on migration, invasion, and epithelial–mesenchymal transition (EMT) of HCC cells. (A–D) Migration ability was assessed by scratch wound healing assay, representative images (A, B) were shown (original magnification, ×200; scale bars, 50 µm), and wound healing areas were calculated (C, D). (E, F) Transwell assay was applied to examine the invasion ability, representative images (F) were shown (original magnification, ×200; scale bars, 50 µm), and the histogram showed the number of invasion cells (E). (G) Western blot showed the changes of EMT proteins in HCC-LM3 and MHCC97-H cells transfected with si-ARPC5#1. *p < 0.05; **p < 0.01; ***p < 0.001.