Downregulated USP3 mRNA functions as a competitive endogenous RNA of SMAD4 by sponging miR-224 and promotes metastasis in colorectal cancer

Abstract

Increasing evidence shows that competitive endogenous RNAs (ceRNAs) can affect the expression of other transcripts by sequestering common microRNAs (miRNAs), and participate in tumourigenesis. As a potent tumour suppressor in colorectal cancer (CRC), SMAD4 is regulated by many miRNAs. However, the regulation of SMAD4 by ceRNAs has never been examined. In the present study, we found that USP3 modulated SMAD4 expression in a miRNA dependent, and protein-coding gene independent manner. USP3 and SMAD4 were directly targeted by miR-224, and overexpression of the USP3 3'UTR could inhibit metastasis caused by the loss of USP3. The correlation of USP3, SMAD4 and miR-224 expression was further verified in CRC specimens. Additionally, the loss of USP3 was associated with distal metastasis and a poor prognosis. Altogether, our study demonstrates USP3 as a bona fide SMAD4 ceRNA. The results from this study may provide new insights into the prevention and treatment of CRC.

Figure 1

USP3 interacted with SMAD4 at the post-transcriptional level. (a,b) Protein and mRNA expression of USP3 and SMAD4 were examined by western blot and qRT-PCR in CRC cells. (c,e) USP3 and SMAD4 protein and (d,f) mRNA levels were decreased after knockdown of either USP3 or SMAD4 by transfection of siRNA pools in LoVo and HCT116 cells, respectively. (g,h) Overexpression of the USP3 or SMAD4 CDS did not affect the other’s expression at both protein and mRNA levels. All values are represented as the mean ± SD (n = 3). Western blot images are cropped; full-length blots are included in Supplementary Figures S7 to S10. The densitometric measurement was performed with each western blot analysis; relative protein expression was normalized to β-actin. *P < 0.05.

Figure 2

Crosstalk between USP3 and SMAD4 was 3′UTR and miRNA dependent. (a,b) USP3 3′UTR or SMAD4 3′UTR mutually modulated protein expression of the other when endogenous USP3 or SMAD4 expression was first reduced by siRNA transfection. (c,d) Deletion of USP3 or SMAD4 attenuated luciferase activity of the PGL3-USP3 3′UTR or PGL3-SMAD4 3′UTR reporters; (e,f) overexpression of USP3 3′UTR or SMAD4 3′UTR increased the luciferase activity of the corresponding reporter plasmids. (g,h) USP3 and SMAD4 expression was elevated when Dicer depletion was combined with USP3 or SMAD4 knockdown, compared with treatment of si-USP3 or si-SMAD4 alone. All values are represented as the mean ± SD (n = 3). Western blot images are cropped; full-length blots are included in Supplementary Figure S11 to S14. Quantitative protein expression data were normalized to β-actin. *P < 0.05.

Figure 3

miR-224 modulated expression of USP3 and SMAD4. (a) miR-224 mimics attenuated protein expression of USP3 and SMAD4 and (b) miR-224 inhibitor enhanced USP3 and SMAD4 expression. Western blots are cropped; full-length blots are included in Supplementary Figures S15 and S16. The results were quantified by ImageJ software, and the relative protein ratio was normalized to β-actin. (c) Expression of USP3 and SMAD4 mRNA were reduced by miR-224 mimics in LoVo and HCT116 cells. (d,e) Left upper panel: Schematic diagram of miR-224-binding sites in the SMAD4 or the USP3 3′UTRs from microRNA.org. Left lower panel: Construction of USP3 or SMAD4 luciferase reporter plasmids containing wild-type miR-224 MREs (WT) or mutant sequences (MUT). Right panel: Luciferase reporters harbouring wild-type or mutant miR-224-binding sites of the USP3/SMAD4 3′UTR were co-transfected with miR-224 mimics/mi-NC. Relative luciferase activity is presented as the mean ± SD (n = 3). (f,g) miR-224 expression was determined by qRT-PCR after USP3/SMAD4 silencing or USP3/SMAD4 CDS or 3′UTR overexpression in CRC cells. *P < 0.05.

Figure 4

Reduced USP3 expression promoted migration and invasion of CRC cells in vitro and in vivo. (a) Wound-healing assays were performed to detect CRC cell migration after transfection with si-USP3 or co-transfection with si-USP3 and USP3 3′UTR. Photographs were obtained 0 and 24 h after scratching. (b,c) Transwell assays with or without Matrigel coatings were used to evaluate migration and invasion by LoVo and HCT116 cells after the indicated transfection treatment. The migratory or invasive cells were stained with crystal violet, and counted under a microscope in five random fields at 100 × magnification. Quantification data are shown as the mean ± SD (n = 3). *P < 0.05. (d) After transduction of the sh-USP3 lentivirus, mRNA expression of USP3 and SMAD4 were both reduced in LoVo cells compared with the group transduced with sh-NC lentivirus. (e) Representative photographs of lungs stained with picric acid, which were harvested from NOD/SCID mice eight weeks after injection with stably transduced sh-NC or sh-USP3 LoVo cells (2 × 10⁶ cells per injection) via tail veins (n = 8); arrows indicate metastatic tumours. (f) Representative images of haematoxylin and eosin-stained sections in sh-NC and sh-USP3 lung metastatic foci. Scale bars, 100 μm. (g) The number of lung metastatic nodules was counted under a microscope. The results are presented as the mean ± SD (n = 8), *P < 0.05.

Figure 5

Expression and correlation of USP3, SMAD4 and miR-224 in primary CRC samples (a–c) USP3, SMAD4 and miR-224 expression in 40 paired primary CRC and adjacent non-cancer tissues were evaluated by qRT-PCR. (d) Comparison of the USP3 expression levels between “SMAD4 low” and “SMAD4 high” subgroups that were classified according to the median SMAD4 expression levels. (e–g) Correlation analyses of USP3 mRNA, SMAD4 mRNA and miR-224 were conducted in these CRC specimens.

Figure 6

Kaplan-Meier curves for the disease-free survival and overall survival of the 40 CRC patients. High USP3 mRNA expression was notably correlated with favourable DFS (a) and OS (b) in the CRC patients.

Figure 7

Analyses of USP3 mRNA expression in the CRC samples from the GEO database. (a) USP3 mRNA was decreased in CRCs compared with their adjacent noncancerous tissues (GEO datasets: GSE9348, GSE46200 and GSE4107). (b) Reduced expression of USP3 mRNA was associated with an advanced tumour stage in the primary CRC tissues (GEO datasets: GSE39582, GSE14333 and GSE33193). (c) A schematic model illustrates how a dysregulated USP3-miR-224-SMAD4 axis facilitates CRC metastasis during cancer development.