RAB31 Targeted by MiR-30c-2-3p Regulates the GLI1 Signaling Pathway, Affecting Gastric Cancer Cell Proliferation and Apoptosis

Abstract

Background: Gastric cancer (GC), one of the most common cancers worldwide, is highly malignant and fatal. Ras-related protein in brain 31 (RAB31), a member of the RAB family of oncogenes, participates in the process of carcinogenesis and cancer development; however, its role in GC progression is unknown. Methods: In our study, 90 pairs of tissue microarrays were used to measure the levels of RAB31 protein by immunochemistry, and 22 pairs of fresh tissue were used to measure the levels of RAB31 mRNA by quantitative PCR. We also investigated the effects of RAB31 on tumor growth both in vitro and in vivo. Results: RAB31 was overexpressed in GC tissues, and its overexpression predicted poor survival in patients. In a nude mouse model, depletion of RAB31 inhibited tumor growth. In vitro, silencing of RAB31 suppressed cell viability, promoted cell cycle arrest, enhanced apoptosis, and affected the expression of cell cycle and apoptotic proteins; these effects were mediated by glioma-associated oncogene homolog 1 (GLI1). Co-immunoprecipitation and immunofluorescence assays confirmed that RAB31 interacted with GLI1. In addition, luciferase reporter assays and Western blotting showed that microRNA-30c-2-3p modulated the RAB31/GLI1 pathway by targeting the 3'-untranslated region of RAB31. Conclusions: Collectively, these data show that RAB31 is regulated by microRNA-30c-2-3p, and functions as an oncogene in GC tumorigenesis and development by interacting with GLI1. Therefore, targeting the miR-30c-2-3p/RAB31/GLI1 axis may be a therapeutic intervention for gastric cancer.

Keywords: GLI1; RAB31; gastric cancer; miR-30c-2-3p; prognosis; proliferation.

Figure 1

RAB31 is overexpressed in GC tissues and is positively correlated with poor prognosis. (A–D). Representative box plots of RAB31 expression from the Oncomine database (P < 0.001). (E) Expression level of RAB31 in cancer and para-carcinoma tissues of 22 patients as determined by qPCR (P < 0.01). (F) The receiver operating characteristic of specificity and sensitivity was created to assess potential predictive values. (G) The different expression levels of RAB31 in different stages of GC are shown by the violin plot, P < 0.05. (H) Representative IHC images of RAB31 staining in gastric tumor and non-tumor tissues (scale bar, 100 μm). (I) Overall survival analysis of GC patients with low versus high RAB31 expression. Survival rate was calculated by Kaplan–Meier survival analysis (n = 90; P < 0.001, log-rank test). (J) Survival curve was created using the GEPIA tool (P < 0.05).

Figure 2

Depletion of RAB31 inhibits GC cell proliferation, blocks cell cycle progression, and promotes apoptosis in vitro. (A) Expression of RAB31 protein in six GC cell lines. (B) Western blot analysis of RAB31 in AGS and MKN45 cell lines after transfection of RAB31 siRNA; GAPDH was used as the control. (C,D) Viability of AGS and MKN45 cells after depleting RAB31 expression, as determined by the CCK8 assay. (E) Images from the colony formation assays in AGS and MKN45 cells are shown on the left; the average number of colonies formed in three experiments is shown by histogram on the right (P < 0.01). (F,G) Cell cycle and apoptosis were assessed by flow cytometry in AGS and MKN45 cell lines after knockdown of RAB31 expression; representative images are shown on the left and the results of triplicate assays are shown on the right. Results are shown as the mean ± SD (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001).

Figure 3

Depletion of RAB31 suppressed tumor growth in vivo and biological data predicted RAB31 function via interaction with GLI1. (A) Xenograft tumors were excised from the mice. (B,C) Measurements of xenograft tumor volumes and weights. (D) IHC staining of RAB31 and Ki-67 in tumor specimens from mouse tumor tissues (Scale bar, 100 μm). (E) GSEA analysis of the TCGA dataset showed the association between RAB31 expression and apoptosis signaling pathways. The enrichment score (ES, green line) equals the degree to which the gene set is overrepresented. (G) Genes involved in apoptosis are listed according to rank metric score. (H) The relationship between RAB31 and GLI1 was determined using the GEPIA tool. (I) Fresh tissues from 22 pairs of patients were analyzed for a correlation between RAB31 and GLI1 mRNA levels by qPCR. (J) Association of RAB31 expression with GLI1 expression in 90 primary human GC specimens; the representative images are shown on the left (scale bar, 100 μm). The columns show the significant results on the right (^**P < 0.01, ^***P < 0.001).

Figure 4

RAB31 promotes GC progression through activation of the GLI1 pathway in vivo. (A) Western blotting was performed to evaluate the overexpression of RAB31 after transfection of plasmids into MKN45 cells. (B–F) AGS and MKN45 cells with enhanced RAB31 expression and vector control were transfected simultaneously with GLI1 siRNA and siNC, respectively. (B,C) The viability of AGS and MKN45 cells was determined by the CCK8 assay. (D) Representative images of colony formation assays are shown on the left; the average colony formation from three experiments is shown by the histogram on the right (P < 0.01). (E,F) Cell cycle and apoptosis were assessed by flow cytometry. Representative images are on the left and the results of triplicate assays are shown on the right. Results are shown as the mean ± SD (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001).

Figure 5

RAB31 mediated its downstream proteins via GLI1. (A,B) Expression of GLI1, cyclin D1, c-myc, BAX, and Bcl-2 were detected by Western blotting in AGS and MKN45 cells with RAB31 expression silenced. (C,D) Western blotting was conducted to detect the expression of GLI1, cyclin D1, c-myc, BAX, and Bcl-2 in AGS and MKN45 cells with simultaneous RAB31 plasmid and GLI1 siRNA co-transfection. (E,F) Co-IP assay was utilized to investigate the interaction between RAB31 and GLI1 expression. The assay was conducted using a GLI1 antibody to pulldown the RAB31 protein or a RAB31 antibody to pulldown the GLI1 protein. (G) Co-localization of the GLI1 and RAB31 proteins was evaluated by immunofluorescence. (H,I) Immunofluorescence was conducted to measure the fluorescence intensity of RAB31 and GLI1 in AGS and MKN45 cells transfected with the RAB31 plasmid and vector controls.

Figure 6

miR-30c-2-3p has low expression in GC and inhibits GC cell proliferation, blocks the cell cycle, and promotes apoptosis in vitro. (A) The results obtained on the Hh signaling pathway via analysis using Targetscan, miRWalk 2.0, miRDB databases, and KEGG pathway analysis are shown by the histogram, according to the P-value. (B,C) The heat plot showing miRNA expression in GC and the histogram showing miR-30c-2-3p expression were created using the YM500v2 tool. (D) Kaplan–Meier curve for overall survival was created using clinical information from the TCGA databank in GC patients with high and low miR-30c-2-3p expression levels (P < 0.05). (E) qPCR analysis of miR-30c-2-3p in MKN45 cells after transfection of mimics or the inhibitor. (F,G) Viability of AGS and MKN45 cells after upregulation or downregulation of miR-30-2-3p expression was determined by the CCK8 assay. (H) Significant images of colony formation assays using AGS and MKN45 cells are shown on the left, and the average colony formation from three experiments is shown by histogram on the right (P < 0.01). (I,J) Cell cycle and apoptosis were assessed by flow cytometry in AGS and MKN45 cell lines transfected with miR-30c-2-3p mimics and the inhibitor. Representative images are shown on the left and the results of triplicate assays are on the right. Results are shown as the mean ± SD (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001).

Figure 7

miR-30c-2-3p is involved in the RAB31/GLI1 pathway and regulates GC cell growth and apoptosis via binding to the 3′-UTR of RAB31. (A) Luciferase reporter assay was performed using MKN45 cells to determine if miR-30c-2-3p binds to the 3′-UTR of RAB31. (B,C) RAB31, GLI1, cyclin D1, BAX, and Bcl-2 expression was detected by Western blotting in AGS and MKN45 cells after transfection with miR-30c-2-3p mimics or the inhibitor; GAPDH was used as the control. (D,E) Cell viability was assessed in AGS and MKN45 cells transfected with the miR-30c-2-3p inhibitor and RAB31 siRNA. (F,G) Western blotting was conducted to investigate the levels of RAB31, GLI1, and its downstream proteins in AGS and MKN45 cells co-transfected with the miR-30c-2-3p inhibitor.