CircDUSP16 promotes the tumorigenesis and invasion of gastric cancer by sponging miR-145-5p

Abstract

Background: Circular RNAs (circRNAs) as a novel subgroup of non-coding RNAs act a critical role in the pathogenesis of gastric cancer (GC). However, the underlying mechanisms by which hsa_circ_0003855 (circDUSP16) contributes to GC are still undocumented.

Materials: The differentially expressed circRNAs were identified by GEO database. The association of circDUSP16 or miR-145-5p expression with clinicopathological features and prognosis in GC patients was analyzed by FISH and TCGA-seq data set. Loss- and gain-of-function experiments as well as a xenograft tumor model were performed to assess the role of circDUSP16 in GC cells. circDUSP16-specific binding with miR-145-5p was confirmed by bioinformatic analysis, luciferase reporter, and RNA immunoprecipitation assays.

Results: The expression levels of circDUSP16 were markedly increased in GC tissue samples and acted as an independent prognostic factor of poor survival in patients with GC. Knockdown of circDUSP16 repressed the cell viability, colony formation, and invasive potential in vitro and in vivo, but ectopic expression of circDUSP16 reversed these effects. Moreover, circDUSP16 possessed a co-localization with miR-145-5p in the cytoplasm, and acted as a sponge of miR-145-5p, which attenuated circDUSP16-induced tumor-promoting effects and IVNS1ABP expression in GC cells. MiR-145-5p had a negative correlation with circDUSP16 expression and its low expression was associated with poor survival in GC patients.

Conclusions: CircDUSP16 facilitates the tumorigenesis and invasion of GC cells by sponging miR-145-5p, and may provide a novel therapeutic target for GC.

Keywords: Gastric cancer; Growth; Invasion; circDUSP16; miR-145-5p.

Fig. 1

Identification of a novel circDUSP16 in GC cells. a GSE78092 analysis of the differentially expressed circRNAs between GC and adjacent normal tissues. b The genomic loci of the DUSP16 gene and circDUSP16. Arrows represent divergent primers that bind to the genomic region of circDUSP16. c qRT-PCR analysis of circDUSP16 and DUSP16 expression after treatment with RNase R in SGC-7901 and MKN-45 cells. d qRT-PCR analysis of the half-life of circDUSP16 and DUSP16 after treatment with Actinomycin D in SGC-7901 and MKN-45 cells. e, f qRT-PCR and FISH analysis of the cellular localization of circDUSP16 in GC cells and tissue cells. Data are the means ± SEM of 3 experiments. **P < 0.01

Fig. 2

Upregulation of circDUSP16 was associated with poor survival in GC patients. a qRT–PCR analysis of the expressed levels of circDUSP16 in eight paired GC tissues. b, c FISH analysis of the expression levels of circDUSP16 in 40 paired GC tissues. d The cut-off value of circDUSP16 divided the GC patients into high expression (n = 12) and low expression groups (n = 28). e, f Kaplan–Meier analysis of the association of high or low circDUSP16 expression with overall survival in GC patients or those in early stage

Fig. 3

Knockdown of circDUSP16 inhibited cell proliferation, colony formation, and invasion in GC cells. a qRT–PCR analysis of the expression levels of circDUSP16 in different GC cell lines. b qRT–PCR analysis of the transfection efficiency of sh-circDUSP16 in MKN-28 and MKN-45 cell lines. c MTT, d colony formation, and e transwell analysis of the cell viability, colony number, and cell invasion after the transfection with sh-circDUSP16 in MKN-28 and MKN-45 cell lines. f Western blot analysis of the protein expression of PCNA and MMP2 after the transfection with sh-circDUSP16 in MKN-28 and MKN-45 cell lines. Data are the means ± SEM of 3 experiments. *P < 0.05, **P < 0.01

Fig. 4

Overexpression of circDUSP16 facilitated cell proliferation, colony formation, and invasion in GC cells. a qRT–PCR analysis of the transfection efficiency of circDUSP16 plasmid in BGC-823 and SGC-7901 cell lines. b MTT, c colony formation, and d transwell analysis of the cell viability, colony number, and cell invasion after the transfection with circDUSP16 plasmid in BGC-823 and SGC-7901 cell lines. e Western blot analysis of the protein expression of PCNA and MMP2 after the transfection with circDUSP16 plasmid in BGC-823 and SGC-7901 cell lines. Data are the means ± SEM of 3 experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

CircDUSP16 acted as a sponge of miR-145-5p in GC cells. a Luciferase activity of WT circDUSP16 3′UTR after the transfection with miR-134-5p, miR-145-5p, miR-556-5p, miR-1224-3p, and miR-130b-5p in HEK293T cells. b Schematic representation of the binding sites of miR-145-5p with circDUSP16. c Luciferase activity of WT or Mut circDUSP16 3′UTR after the transfection with miR-145-5p mimic or miR-NC in BGC-823 and SGC-7901 cell lines. d FISH analysis of the co-localization of circDUSP16 with miR-145-5p in BGC-823 cells. e TCGA analysis of the expression of miR-145-5p in paired (n = 32) and unpaired GC tissue samples (n = 368). f ROC curve analysis of the cut-off value of miR-145-5p in GC patients and Kaplan–Meier analysis of the association of high or low miR-145-5p expression with overall survival in GC patients. g Pearson correlation analysis of the correlation of circDUSP16 expression with miR-145-5p in GC tissues. h qRT–PCR analysis of the expression levels of miR-145-5p after the transfection with circDUSP16 plasmid in BGC-823 and SGC-7901 cell lines. i RIP analysis of the enrichment of circDUSP16 and miR-145-5p pulled down from the Ago2 protein in BGC-823 and SGC-7901 cell lines. Data are the means ± SEM of 3 experiments. *P < 0.05, **P < 0.01

Fig. 6

MiR-145-5p reversed the tumor-promoting effects of circDUSP16 in GC cells. a MTT and b transwell analysis of the effects of co-transfection with circDUSP16 and miR-145-5p mimic on cell proliferation and invasive potential in BGC-823 and SGC-7901 cell lines. c Schematic representation of the binding sites between miR-145-5p and WT or Mut IVNS1ABP 3′ UTR. d Luciferase activity of WT or Mut IVNS1ABP 3′ UTR after co-transfection with miR-145-5p mimic and WT or Mut IVNS1ABP 3′ UTR reporter in BGC-823 and SGC-7901 cell lines. e qRT-PCR and Western blot analysis of IVNS1ABP expression levels after co-transfection with circDUSP16 and miR-145-5p mimic in BGC-823 and SGC-7901 cells. Data shown are the mean ± SEM of three experiments. *P < 0.05; **P < 0.01

Fig. 7

Knockdown of circDUSP16 inhibited in vivo tumor growth. a Representative photographs of MKN-45 xenograft tumors after treatment with sh-circDUSP16 or sh-NC. b Growth curve analysis of tumor proliferation activity after treatment with sh-circDUSP16 or sh-NC. c, d Comparison of the tumor volumes and weights between the sh-circDUSP16 and sh-NC groups. e qRT-PCR analysis of circDUSP16 and miR-145-5p expression levels after treatment with sh-circDUSP16 or sh-NC. f Pearson correlation analysis of the relationship between circDUSP16 and miR-145-5p expression in sh-circDUSP16-treated tumor tissues. **P < 0.01