VPS53 Suppresses Malignant Properties in Colorectal Cancer by Inducing the Autophagy Signaling Pathway

Abstract

Background: Many studies found that VPS53, one of the subunits of the golgi-associated retrograde protein (GARP) complexes, was aberrantly expressed in human diseases.

Aim: This study investigated the functions and molecular mechanisms of VPS53 in colorectal cancer (CRC).

Methods: Expression and correlation of Beclin 1 and VPS53 were analyzed by RT-qPCR and Pearson's correlation in CRC tissues, and VPS53 expression was also determined in CRC cells. The changes of proliferation, migration, invasion, apoptosis, and autophagy of CRC cells were examined by a succession of functional experiments including CCK-8, flow cytometry, transwell assay, and electron microscopy. The levels of autophagy related proteins were evaluated by Western blotting analysis.

Results: RT-qPCR results found that VPS53 was downregulated in CRC tissues and cells, and Beclin 1 expression was also decreased in CRC tissues. There was a positive correlation between VPS53 and Beclin 1. Functional results showed that overexpression of VPS53 could suppress proliferation, migration, and invasion, and accelerate apoptosis and autophagy of CRC cells. Also, VPS53 could upregulate Beclin 1 and LC3BII, suggesting the inductive effect of VPS53 on CRC cell autophagy. Furthermore, it was found that the autophagy inhibitor (Inhb) could attenuate the inhibition of VPS53 on CRC progression.

Conclusion: VPS53 repressed CRC progression by regulating the autophagy signaling pathway, suggesting that VPS53 might be a promising therapeutic target for CRC.

Keywords: VPS53; autophagy; cell invasion; cell migration; colorectal cancer.

Figure 1

Downregulated VPS53 was positively correlated with Beclin 1 in CRC. (A) RT-qPCR results of expression of Beclin 1 in adjacent normal tissues (n=15) and CRC tissues (n=15), **P < 0.01. (B) The level of VPS53 as also examined by RT-qPCR analysis in adjacent normal tissues (n=15) and CRC tissues (n=15), **P < 0.01. (C) Western blotting analysis of VPS53 in adjacent normal and CRC tissues. (D) The correlation analysis of Beclin 1 and VPS53 in CRC tissues (R²=0.4719, p=0.0047). (E) The level of VPS53 as determined by RT-qPCR assay in normal colon FHC cells and human CRC cells (SW620, HT29, SW480, HCT116, and LoVo), **P < 0.01.

Figure 2

Overexpression of VPS53 suppressed proliferation, migration, and invasion, and facilitated apoptosis and autophagy of CRC cells. SW620 and LoVo cells were transfected with the pcDNA-VPS53 plasmid and NC, respectively. (A) VPS53 expression was determined by Western blotting analysis and the VPS53 level was normalized to GAPDH, ***P < 0.001. (B) CCK-8 assay investigated the effects of VPS53 overexpression on SW620 and LoVo cells, **P < 0.01. (C) Apoptotic analysis of SW620 and LoVo cells was evaluated by flow cytometry, and then the percentage of apoptosis was calculated, ***P < 0.001. (D and E) Transwell assays were used, and the results show the number of migrated and invaded cells per field, magnification, ×200; **P < 0.01. (F) Western blotting analysis displays expressions of Beclin 1 and LC3B, and the ratio of LC3BII/I and Beclin 1 level were counted based on the grayscale, *P < 0.05, **P < 0.01, ***P < 0.001. (G) Electron microscopy shows the number of autophagosomes in each group. The red arrow denotes autophagosomes and the green arrow denotes the lysosomes.

Figure 3

VPS53 inhibited CRC cell carcinogenesis by regulating autophagy. SW620 and LoVo cells were treated with the VPS53-overexpressed plasmid and the autophagy inhibitor (Inhb). (A) CCK-8 assay indicates the influence of Inhb on the VPS53-inhibited proliferation of SW620 and LoVo cells, **P < 0.01 vs NC group; ^#P < 0.05, ^##P < 0.01 vs VPS53 group. (B) The role of Inhb on the VPS53-induced apoptosis analyzed by flow cytometry, and the apoptosis percentage is shown, **P < 0.01, ***P < 0.001 vs NC group; ^#P < 0.05 vs VPS53 group. (C and D) VPS53 plasmid and Inhb-treated SW620 and LoVo cells were subjected to the migration and invasion assays, and the migrated and invaded cells were counted, **P < 0.01, ***P < 0.001 vs NC group; ^#P < 0.05, ^##P < 0.01 vs VPS53 group. (E) Western blot analysis results show expressions of LC3B and Beclin 1, and the quantitative results were analyzed, **P < 0.01, ***P < 0.001 vs NC group; ^#P < 0.05, ^##P < 0.01 vs VPS53 group. (F) Ultrastructure of autophagic bodies observed by an electron microscope. The red arrow denotes autophagosomes and the green arrow denotes the lysosomes.