USP14 de-ubiquitinates vimentin and miR-320a modulates USP14 and vimentin to contribute to malignancy in gastric cancer cells

Abstract

Vimentin plays important roles in the epithelial-to-mesenchymal transition (EMT). In this study, we found that vimentin was highly expressed in human gastric cancer (GC) tissues and cell lines and significantly promoted cell growth, migration and invasion. Ubiquitin-specific protease 14 (USP14) interacted with the vimentin protein, which led to its de-ubiquitination. miR-320a was found to bind to the 3'UTR of both vimentin and USP14 transcripts and downregulate the expression of both proteins. The downregulation of miR-320a upregulates vimentin expression by directly binding to the 3'UTR of vimentin to derepress expression and indirectly by augmenting USP14 to increase vimentin stability in GC cells. Taken together, these results provide new insight into malignancy in gastric cancers.

Keywords: USP14; gastric cancer; miR-320a; vimentin.

Figure 1. Vimentin promotes the aggressiveness of gastric cancer cells

(A and B) The mRNA levels of vimentin in GC tissues (n = 19) and cell lines were analyzed by RT-qPCR. (C) Protein expression levels of vimentin in GC cell lines and the control cell line. (D and E) MTT assays performed in GES-1 cells with over-expressed vimentin and in BGC-823 cells with the inhibition of vimentin. (F) Colony formation assays examined the proliferation rates of GES-1 and BGC-823 transfected with flag-vimentin and shR-vimentin. (G and H) Transwell assays showed the migration and invasion in GES-1 cells with over-expressed vimentin and in BGC-823 cells with inhibited vimentin. The data are presented as the mean ± S.D. n = 3,*P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group.

Figure 2. USP14 can influence the expression of vimentin by affecting its ubiquitination

(A) Western blot detecting the ubiquitination of vimentin in GC cells and GES-1 cell lines. (B and C) Exogenous and endogenous Co-IP experiments exploring the interactions between USP14 and vimentin in MGC-803 cells. (D and E) Western blot analysis of the impact of USP14 on vimentin expression 48h after transfection, with and without the treatment of MG132 in BGC-823. (F and G) Western blot detecting the ubiquitination of vimentin in BGC-823 and MGC-803 cells after transfection with HA-USP14, siUSP14 and the respective controls. (H–J) The impact of HA-mut-USP14 and USP14 inhibitor (IU1) on vimentin protein levels and its ubiquitin levels as examined by western blotting.

Figure 3. USP14 increases the malignant behavior of gastric cancer cells

(A) An MTT assay tested the viabilities of BGC-823 and MGC-803 cells transfected with HA-USP14 and siUSP14. (B) Colony formation, (C) migration and (D) invasion of BGC-823 and MGC-803 cells were affected by transfection with HA-USP14 and siUSP14. (E–H) MTT, colony formation, migration and invasion assays were performed to investigate the effects of shR-vimentin on USP14's promotion of gastric malignant behaviors. (I–L) The biological functions of mut-USP14 and WT-USP14 in GC cells, as determined by MTT, colony formation, migration and invasion assays. The data are presented as the mean ± S.D. n = 3, *P < 0.05, **P < 0.01 compared with the control group.

Figure 4. USP14 and vimentin are suppressed by miR-320a

(A) The vimentin and USP14 3′UTR and mutant 3′UTR containing a miR-320a-binding sites are shown. (B and C) BGC-823 cells were transfected with EGFP reporter vectors containing either wild-type or mutant USP14 3′UTR and wild-type or mutant vimentin 3′UTR. The relative EGFP intensity was determined after transfection. (D and E) mRNA and protein expression levels of USP14 and vimentin were measured by RT-qPCR and western blotting after transfection with pri-miR-320a and ASO-miR-320a. (F) MTT, (G) colony formation, (H) migration and (I) invasion of BGC-823 and MGC-803 cells assays were affected by transfection with pri-miR-320a and ASO-miR-320a. The data are presented as the mean ± S.D. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group.

Figure 5. miR-320a functions as a tumor suppressor in GC cells via USP14 and vimentin

(A) The mRNA levels of USP14 in GC tissues were analyzed by RT-qPCR. (B) The positive correlation of USP14 and vimentin in GC tissues. (C) The mRNA levels of miR-320a in GC tissues analyzed by RT-qPCR. (D and E) Pearson's correlation analysis between miR-320a/USP14 and miR-320a/vimentin levels in GC tissues (n = 19). Overexpression of USP14 and vimentin rescues the effect of miR-320a on cell viability (F and G), colony formation (H and I), migration (J and K) and invasion (L and M) in BGC-823 and MGC-803 cells, respectively. The data are presented as the mean ± S.D. n = 3, *P < 0.05, **P < 0.01 compared with the control group. (N) miR-320a not only suppresses vimentin directly but also binds to USP14 to inhibit vimentin indirectly in GC.