Overexpression of RAB31 in gastric cancer is associated with released exosomes and increased tumor cell invasion and metastasis

Abstract

Background: Gastric cancer (GC) is one of most common cancers worldwide. Several studies have suggested that Rab31 functions as a membrane vesicle transport regulator; however, the mechanism by which RAB31 regulates exosome secretion and promotes metastasis remains to be clarified.

Methods: We examined the expression of RAB31 protein and mRNA in GC tissue samples via immunohistochemistry and reverse transcription-polymerase chain reaction assays, respectively. We elucidated the function of RAB31 in GC cells by constructing a cell model and a pulmonary metastatic model of GC with overexpression of RAB31. Protein mass spectrometry was used to identify the exosomal protein.

Results: RAB31 expression increased at both the protein and mRNA levels with the development of GC. Cells overexpressing RAB31 showed an enhanced ability to migrate in both the in vitro cell model and the pulmonary metastatic model of GC. Exosome nanoparticle tracking analysis and electron microscopy revealed that the both the number and size of the exosomes secreted by GC cells were reduced when RAB31 expression was depleted. Injection of exosomes derived from RAB31 overexpressing cells promoted pulmonary metastasis in vivo. Analysis of the exosomal proteins revealed that PSMA1 was overexpressed in GC tissue in accordance with RAB31 expression. PSMA1 overexpression was highly associated with poor prognosis of GC patients.

Conclusion: Our findings revealed a key role for RAB31 in GC metastasis through regulation of exosome secretion.

Keywords: PSMA1; RAB31; exosome; gastric cancer; metastasis.

FIGURE 1

Overexpression of RAB31 predicted poorer prognosis in GC patients. (A) Representative images of immunohistochemistry staining of RAB31 expression normal and GC tissues in different stages of GC. (B) RT‐qPCR analysis of RAB31 mRNA expression levels in GC and para‐carcinoma tissues of 22 patients (p < 0.01). (C) Overall survival analysis of GC patients with low versus high RAB31 expression. Survival rate was calculated by Kaplan–Meier survival analysis (p < 0.001, log‐rank test). GC, gastric cancer; RT‐PCR, reverse transcription‐polymerase chain reaction.

FIGURE 2

Overexpression of RAB31 promoted metastasis of GC in vivo and in vitro. (A) The pulmonary metastasis model of gastric cancer was established by injecting GC cells into nude mice via the tail vein. Representative images of HE staining of the NC and RAB31 OE groups. (B) Representative images showing the invasion and migration of MGC803 cells after knockdown of RAB31 expression. (C) Representative images showing the invasion and migration of MKN45 cells after knockdown of RAB31 expression. (D) Western blot analysis of EMT‐related proteins after knockdown of RAB31 expression.

FIGURE 3

Depletion of RAB31 reduced the release of exosomes. (A and B) Representative electron microscopic images of exosomes in the normal control (NC) group (A), and the RAB31 depletion group (B). (C and D) Exosome nanoparticle tracking analysis of the NC group (C) and the RAB31 depletion group (D). Western blot analysis of exosome‐related proteins in the MGC803 cells and MKN45 cells between NC group and RAB31‐depleted group (E).

FIGURE 4

Overexpression of RAB31 enhanced metastasis of GC by promoting exosome secretion. (A) Pulmonary metastasis model of gastric cancer was established by injecting gastric cancer cells into nude mice via the tail vein. Representative images of HE staining of the NC, RAB31 OE, NC‐Exo (exosomes secreted by the control group), and OE‐Exo (exosomes secreted by RAB31 overexpressing cells) 20 groups. (B) Representative images of MGC803 and MKN45 cells in invasion and migration assays (left); data represent the mean ± standard deviation of three experiments (right) (p < 0.01). (C) Western blot analysis of EMT‐related proteins in the four groups of MGC803 cells described in (B).

FIGURE 5

Protein mass spectrometry to identify the differentially expressed exosomal proteins between control group and RAB31 depleted group. (A) Volcano plot showing the differentially expressed proteins. (B) Histogram showing the results of the KEGG analysis. (C) Heat map showing the differentially expressed proteins. (D and E) Ranking of the differentially expressed proteins by FC value (D) and p‐value (E).

FIGURE 6

RAB31 expression was associated with PSMA1, and PSMA1 overexpression was associated with a worse prognosis. (A) Representative images of IHC staining of RAB31 and PSMA1 in gastric tumor and non‐tumor tissues. (B) PSMA1 mRNA level was assessed by TCGA database. (C and D) Overall survival analysis of GC patients with low versus high PSMA1 expression. Survival rate was calculated by Kaplan–Meier survival analysis (p < 0.001, log‐rank test). (E) Overall survival analysis of our GC patients with low versus high PSMA1 expression.