High expression of RAB38 promotes malignant progression of pancreatic cancer

Abstract

Ras‑Related Protein Rab‑38 (RAB38), which belongs to the RAB family, is involved in the biogenesis of lysosome‑related organelles and defense against certain microbial infections. However, the clinical significance and potential function of RAB38 in pancreatic adenocarcinoma remain unclear. In the present study, an immunohistochemical assay was performed to analyze the expression of RAB38 in pancreatic adenocarcinoma tumor specimens from 82 patients, and the clinicopathological characteristics and survival rate of these patients were further examined. To validate the role of RAB38 in tumors, the effect of RAB38 on tumor cell proliferation, migration and invasion was assessed by establishing RAB38 knockdown cell lines. Reverse transcription‑quantitative polymerase chain reaction and western blotting were used to examine the expression levels of proteins associated with the cancer cell behavior. In addition, the inhibitory effect of RAB38 silencing on pancreatic cancer was examined in mice. The immunohistochemistry results revealed that RAB38 was upregulated and positively correlated with the grade of progression in pancreatic adenocarcinoma patients. Further investigation indicated that RAB38 downregulation significantly suppressed the proliferation, migration and invasive capacity of pancreatic cancer cells, as well as decreased the expression levels of Ki67, proliferating cell nuclear antigen, and matrix metalloproteinases 2 and 9. RAB38 silencing also inhibited the development of pancreatic cancer in vivo. Taken together, a high level of RAB38 was significantly associated with the malignant phenotypes of pancreatic cancer, suggesting that RAB38 may serve as a novel biomarker and a potential therapeutic target for pancreatic cancer.

Keywords: RAB38; clinicopathological characteristics; pancreatic cancer; RNA interference; in vitro; in vivo.

Figure 1.

RAB38 expression is associated with the clinicopathological features of patients with pancreatic ductal adenocarcinoma. Representative immunohistochemical analysis images of RAB38 protein expression in (A) pancreatic ductal adenocarcinoma and (B) para-carcinoma tissues. (C) Association of OS and RFS rates with RAB38 expression in the 82 pancreatic cancer patients. Significantly shorter OS or RFS rates were observed in patients with high in comparison with low RAB38 expression (P<0.05). RAB38, Ras-related GTP-binding protein 38; OS, overall survival; RFS, relapse-free survival.

Figure 2.

Effects of shRNA targeting of RAB38 in pancreatic cancer cell lines by lentivirus-based RNA interference transfection. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot assay were used to examine RAB38 mRNA and protein expression, respectively, in PANC-1 and BxPC-3 cells transfected with RAB38 shRNA or negative control shRNA. Western blotting data were quantified by densitometric analysis with ImageJ software. Data are reported as the mean ± standard deviation (n=3). *P<0.05 vs. control group. shRNA, small hairpin RNA; RAB38, Ras-related GTP-binding protein 38.

Figure 3.

Effects of RAB38 silencing on the proliferation ability of PANC-1 and BxPC-3 cells. (A) Colony formation assay, indicating decreased colonies in the RAB38 shRNA-transfected group compared with the control group in vitro. (B) Cell viability in the RAB38 shRNA and control groups was analyzed using an MTT assay. Decreased OD value was detected in the RAB38 shRNA group as compared with the control group in vitro. The expression levels of the proliferation-associated proteins (C) Ki67 and (D) PCNA in PANC-1 and BxPC-3 cells were detected by western blot analysis. Data are reported as the mean ± standard deviation (n=3). *P<0.05 vs. control group. shRNA, small hairpin RNA; RAB38, Ras-related GTP-binding protein 38; OD, optical density; PCNA, proliferating cell nuclear antigen.

Figure 4.

Influence of RAB38 on the migration and invasion of PANC-1 and BxPC-3 cells. (A) Scratch migration assay, indicating significantly a larger scratch width in the shRNA group as compared with that in the control group at 48 h. Magnification, ×200. (B) Transwell invasion assays were performed to analyze PANC-1 and BxPC-3 cells transfected with RAB38 shRNA and control shRNA. Cells were examined by light microscopy, and the cell numbers were determined in five different fields-of-view for each condition in triplicate. Magnification, ×400. The expression levels of the metastasis-associated proteins (C) MMP2 and (D) MMP9 were detected by western blot analysis in the shRNA and control groups. Data are reported as the mean ± standard deviation (n=3). *P<0.05 vs. control group. shRNA, small hairpin RNA; RAB38, Ras-related GTP-binding protein 38; MMP, matrix metalloproteinase.

Figure 5.

Effects of RAB38 silencing on the growth of PANC-1 ×enograft tumors in vivo. (A) Tumor volume in the control and shRNA groups of mice. After 29 days of treatment, the tumors volume in the RAB38 shRNA group was significantly smaller compared with that in the control group. (B) Western blot analysis results of RAB38 expression in the two different groups. RAB38 expression was decreased in mice injected with RAB38 shRNA-transfected PCNA-1 cells to induce tumor xenograft. (C) Metastatic tumor volumes of mice in different groups. After 28 days of treatment, the tumor volume in the RAB38 shRNA group was smaller compared with that in the control group. *P<0.05 vs. control group. shRNA, small hairpin RNA; RAB38, Ras-related GTP-binding protein 38.