RAB42 is a Potential Biomarker that Correlates With Immune Infiltration in Hepatocellular Carcinoma

Abstract

Backgrounds: Hepatocellular carcinoma (HCC) is a malignant cancer with high mortality. Previous studies have reported that RAB42 is associated with prognosis and progression in glioma. However, the role of RAB42 in HCC is still unknown. Therefore, we aimed to elucidate the value of RAB42 in the predicting prognosis of HCC, and its relationship with immune cells infiltration. Methods: UALCAN, HCCDB, and MethSurv databases were used to examine the expression and methylation levels of RAB42 in HCC and normal samples. cBioPortal and MethSurv were used to identify genetic alterations and DNA methylation of RAB42, and their effect on prognosis. The correlations between RAB42 and the immune cells and cancer-associated fibroblasts infiltration were analyzed by TIMER, TISIDB, and GEPIA database. The LinkedOmics database was used to analyze the enriched pathways associated with genes co-expressed with RAB42. EdU assay was used to evaluate the proliferation ability of liver cancer cells, and transwell assay was used to detect the invasion and migration ability of liver cancer cells. Results: The expression levels of RAB42 were increased in HCC tissues than that in normal tissues. Highly expressed RAB42 was significantly correlated with several clinical parameters of HCC patients. Moreover, increased RAB42 expression clearly predicted poor prognosis in HCC. Furthermore, multivariate Cox regression analysis showed that RAB42 was an independent prognostic factor in HCC. The RAB42 genetic alteration rate was 5%. RAB42 DNA methylation in HCC tissues was lower than that in normal tissues. Among the 7 DNA methylation CpG sites, two were related to the prognosis of HCC. The results of gene set enrichment analysis (GSEA) showed that RAB42 was associated with various immune cells and cancer-associated fibroblasts infiltration in HCC. Meanwhile, we found RAB42 methylation was strongly correlated with immune infiltration levels, immunomodulators, and chemokines. Experiments in vitro indicated that knockdown of RAB42 inhibited the proliferation, invasion, and migration of liver cancer cells. Conclusions: Our study highlights the clinical importance of RAB42 in HCC and explores the effect of RAB42 on immune infiltration in the tumor microenvironment, and RAB42 may act as a pro-oncogene that promotes HCC progression.

Keywords: CAFs; HCC; RAB42; biomarker; immune infiltration.

FIGURE 1

Workflow of this study.

FIGURE 2

The expression level of RAB42 in HCC and its relationship with individual clinical parameters. (A) The table and (B) histogram listed eight HCC cohorts in the HCCDB database, and RAB42 was significantly highly expressed compared to the adjacent liver tissues in four of these datasets, namely HCCDB12 and HCCDB13, HCCDB16, HCCDB18. (C) Analysis using the UALCAN database also showed that RAB42 had higher expression in HCC compared to normal tissues. The expression of RAB42 significantly increased with advancing stage (D), increasing T stage (E) and histologic grade. (F) *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance; HCC, hepatocellular carcinoma.

FIGURE 3

The survival analysis and the prognostic value of RAB42 in HCC. (A – D) The OS (A), DSS (B), PFS (C), and RFS (D) survival curves comparing patients with high (red) and low (black) RAB42 expression in HCC were plotted using the Kaplan–Meier plotter database at the threshold of the p-value of <0.05. (E) Univariate Cox regression analyses of OS-related factors in HCC from TCGA dataset. (F) Multivariate Cox regression analyses of OS-related factors in HCC from TCGA dataset. HCC, hepatocellular carcinoma; OS, overall survival; DSS, disease specific survival; PFS, progress free survival; RFS, relapse free survival.

FIGURE 4

Genetic alteration of RAB42 in HCC. (A) OncoPrint summarized the genetic alteration in RAB42 based on LIHC (TCGA, Firehose Legacy, 379 samples) from cBioPortal database. (B) The histogram showed the gene alteration frequency of RAB42 from TCGA. (C) Kaplan-Meier analysis showed genetic alterations in RAB42 were related to shorter overall survival (p = 0.0153) in HCC patients. (D) Kaplan-Meier analysis showed genetic alterations in RAB42 were related to shorter disease free survival (p = 0.0312) in HCC patients.

FIGURE 5

DNA methylation expression level of RAB42 in HCC. (A) Promoter methylation level of RAB42 in HCC and normal tissues were analyzed with TCGA data by using UALCAN database. (B) Correlation between RAB42 mRNA expression and DNA methylation level was analyzed with TCGA data by the cBioPortal database (Spearman’s correlation r = −0.49, p-value = 3.40E−24). (C) The heat map showed the RAB42 DNA methylation at CpG sites by using the MethSurv database. (D) HCC patients with lower RAB42 methylation of cg03757398 CpG sites had a worse overall survival than those with higher RAB42 methylation (HR = 0.671, p = 0.022). (E) HCC patients with lower RAB42 methylation of cg04896949 CpG sites had a worse overall survival than those with higher RAB42 methylation (HR = 0.675, p = 0.048).

FIGURE 6

RAB42 co-expressed genes and KEGG, GO enrichment analysis in HCC patients. (A) Volcano plots showed the differentially co-expressed genes of RAB42 in HCC by the LinkedOmics database. (B,C) The heatmap showed the top 50 positively (B) and negatively (C) differentially correlated genes of RAB42 in HCC by the LinkedOmics database. (D) KEGG enrichment analysis revealed diseases and signaling pathways related to RAB42 co-related genes. (E – G) RAB42 co-expressed genes were annotated by using GO analysis. KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; BP, Biological Process; CC, Cellular Component; MF, Molecular Function.

FIGURE 7

Relationship between RAB42 expression and immune cells infiltration in HCC. (A) Correlation analysis based on TIMER database showed RAB42 expression was positively correlated with tumor infiltrating lymphocytes in HCC tissues, including B cells, CD8⁺ T cells, CD4⁺ T cells, macrophages, neutrophils, and dendritic cells. Heat map showed the correlation between the RAB42 expression and various types of immune cells (B), immunoinhibitors (C), immunostimulators (D), chemokines (E), receptors (F) in HCC from the TISIDB database. The GEPIA database (G) and the TISIDB database (H) were used to evaluate the relationship between RAB42 expression and major immune checkpoint molecules in HCC, including PDCD1, CTLA-4, HAVCR2, LAG3, TNFRSF18 and TIGIT.

FIGURE 8

Correlation between RAB42 expression and CAFs in HCC. (A) The association between RAB42 expression and the infiltration level of CAFs was investigated by using various algorithms in HCC from the TCGA. (B) RAB42 expression was positively correlated with several markers of CAFs (ACTA2, FAP, VIM, S100A4, PDGFRB) and ECM-related genes (ELN, FLNA, COL1A1, COL1A2) based on GEPIA database. CAFs, cancer-associated fibroblasts; ECM, extracellular matrix.

FIGURE 9

Association between the methylation level of RAB42 with immune cells infiltration in HCC. (A) Heatmap showed the correlation of RAB42 methylation status with immune-infiltrating lymphocytes in HCC. RAB42 methylation status was negatively associated with gdT helper cells, Activated CD4⁺ T, T follicular helper cells, and Type 2 T helper cells in HCC from the TISIDB database. RAB42 methylation status was negatively correlated with immune checkpoint molecules (TNFRSF18, CD86, ICOS, CD80) (B), immunoinhibitors (PDCD1, CTLA4, LAG3, TIGIT) (C), chemokines (XCL1, CCL20, CXCL5, CCL8) (D) and receptors (CXCR4, CXCR3, CCR10, CCR6) (E) in HCC from the TISIDB database.

FIGURE 10

Knockdown of RAB42 inhibited the proliferation, invasion, and migration of liver cancer cells. (A) qRT-PCR assay was performed to compare the mRNA expression level of RAB42 in different liver cancer cell lines, including SMMC7721, Huh7, MHCCLM3, MHCC97L, Hep3B, HepG2 and normal hepatic cell line, LO2. n = 3. (B) qRT-PCR assay was performed to detect the silencing efficiency of RAB42 by three different siRNA in SMMC772 cells and Hep3B cells. Data were presented as the mean ± SD, n = 3, *p < 0.05, **p < 0.01. ns, no significance; siRNA, small interfering RNA; NC, negative control. (C , D) Representative images of the EdU assay of SMMC772 cells transfected with siRAB42-3 and quantitative measurement of the proportion of EdU-positive SMMC7721 cells. Silencing RAB42 expression decreased the proliferation of SMMC7721 cells. Data were presented as the mean ± SD, n = 3. ***p < 0.001. (E , F) Representative images of the EdU assay of Hep3B cells transfected with siRAB42-2 and quantitative measurement of the proportion of EdU-positive Hep3B cells. Silencing RAB42 expression decreased the proliferation of Hep3B cells. Data were presented as the mean ± SD, n = 3. **p < 0.01. (G , H) Transwell assays showed the invasion and migration of SMMC7721 cells (G) and Hep3B cells (H) transfected with or without siRAB42. Silencing RAB42 expression inhibited the invasion and migration of SMMC7721 and Hep3B cells. Data were presented as the mean ± SD, n = 3. **p < 0.01, ***p < 0.001.