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. 2018 Jan 31;17(1):19.
doi: 10.1186/s12943-018-0771-7.

Circular RNA circ-ITCH inhibits bladder cancer progression by sponging miR-17/miR-224 and regulating p21, PTEN expression

Chengdi Yang  1 , Wenbo Yuan  1 , Xiao Yang  1 , Peng Li  1 , Jingzi Wang  1 , Jie Han  1 , Jun Tao  1 , Pengchao Li  1 , Haiwei Yang  2 , Qiang Lv  3 , Wei Zhang  1
Affiliations

Circular RNA circ-ITCH inhibits bladder cancer progression by sponging miR-17/miR-224 and regulating p21, PTEN expression

Chengdi Yang et al. Mol Cancer. .

Abstract

Background: Circ-ITCH is a circRNA generated from several exons of itchy E3 ubiquitin protein ligase (ITCH) and tumor suppressor served as a sponge for certain miRNAs targeting their parental transcripts of ITCH. However, the role of circ-ITCH in bladder cancer (BCa) was not reported. In the present study, we investigated the role of circ-ITCH in BCa.

Methods: Quantitative real-time PCR was used to detect the expression of circ-ITCH and survival analysis was adopted to explore the association between circ-ITCH expression and the prognosis of BCa. BCa cells were stably transfected with lentivirus approach and cell proliferation, migration, invasion, cell cycle and cell apoptosis, as well as tumorigenesis in nude mice were performed to assess the effect of circ-ITCH in BCa. Biotin-coupled probe pull down assay, Biotin-coupled miRNA capture, Fluorescence in situ hybridization and Luciferase reporter assay were conducted to confirm the relationship between the circ-ITCH and the microRNA.

Results: In the present study, we found that circ-ITCH, is down-regulated in BCa tissues and cell lines. BCa patients with low circ-ITCH expression had shortened survival. Enforced- expression of circ-ITCH inhibited cells proliferation, migration, invasion and metastasis both in vitro and in vivo. Mechanistically, we demonstrated that circ-ITCH up-regulates the expression of miR-17 and miR-224 target gene p21 and PTEN through 'sponging' miR-17 and miR-224, which suppressed the aggressive biological behaviors of BCa.

Conclusions: circ-ITCH acts as a tumor suppressor by a novel circ-ITCH/miR-17, miR-224/p21, PTEN axis, which may provide a potential biomarker and therapeutic target for the management of BCa.

Keywords: Bladder cancer; PTEN; circ-ITCH; miR-17; miR-224; p21.

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Conflict of interest statement

Ethics approval and consent to participate

The present study was approved by the Hospital’s Protection of Human Subjects Committee.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Cir-ITCH was significantly decreased in BCa and correlated with prognosis of BCa patients. a and b Relative expression level of cir-ITCH in BCa tissues (T) and adjacent normal tissues (N) (a: n = 72, b: n = 59) using qRT-PCR. cir-ITCH expression was significantly lower in bladder cancer tissues, compared with that in adjacent normal tissues (*P < 0.05, Student’s t-test). c Relative expression level of in bladder cancer cell lines and SV-HUC cell using qRT-PCR. d qRT-PCR confirmed the over-expression of cir-ITCH in BCa cell lines EJ and T24 after transfection (*P < 0.05, **P < 0.01, Student’s t-test). e Kaplan-Meier plotter analysis of the correlation of circ-ITCH expression level with overall survival of BCa patients
Fig. 2
Fig. 2
Over-expression of cir-ITCH inhibits bladder cancer cell growth, colony formation, migration and invasion in vitro. a and b Cir-ITCH inhibited cell proliferation as indicated by CCk-8 assays in EJ and T24 cells. Data are mean ± S.D. from triplicate experiments (*P < 0.05, **P < 0.01, Student’s t-test). c and d Transwell invasion assay was measured and the results were expressed as the number of invaded cells per field compared with respective control (magnification, × 100, *P < 0.05, Student’s t-test). e and f Wound healing assay showed that cir-ITCH resulted in a slower closing of scratch wounds (*P < 0.05, **P < 0.01, Student’s t-test). g and h Cir-ITCH inhibited the colony formation of EJ and T24 cells as demonstrated by colony formation assays (*P < 0.05, **P < 0.01, Student’s t-test)
Fig. 3
Fig. 3
Cir-ITCH induced cell cycle arrest and cell apoptosis in BCa cells. a Representative images of the cell cycle analysis using flow cytometry. b Cir-ITCH arrested the EJ and T24 cell cycle at the G1/S phase, while miR-17 and miR-224 turned the G1/S transition. c Representative images of Annexin V and 7-AAD staining apoptosis assay in EJ and T24 cells. d and e Apoptosis assay showed that increased the rate of apoptosis in EJ and T24 cells. Data are the means ± SEM of three experiments, (*P < 0.05, **P < 0.01, Student’s t-test)
Fig. 4
Fig. 4
Cir-ITCH acted as a sponge for miR-17 and miR-224. a Cir-ITCH in BCa cell lysis was pulled down and enriched with cir-ITCH specific probe and then detected by qRT-PCR(**P < 0.01, Student’s t-test). b miR-17 was pulled down and enriched with cir-ITCH specific probe and then detected by qRT-PCR(**P < 0.01, Student’s t-test). c miR-224 was pulled down and enriched with cir-ITCH specific probe and then detected by qRT-PCR(**P < 0.01, Student’s t-test). d and f Biotin-coupled miR-17/miR-224 captured a fold change of cir-ITCH in the complex as compared with biotin-coupled NC in biotin-coupled miRNA capture(*P < 0.05, **P < 0.01, Student’s t-test). e and g The product of d and f was detected using qRT-PCR, followed by agarose gel electrophoresis. h and i RNA FISH for cir-ITCH and miR-17/miR-224 was detected in BCa cells, miR-17 and miR-224 were co-localized with cir-ITCH in cytoplasm (magnification, × 400). Nuclei was stained blue (DAPI), cir-ITCH was stained red, miR-17/miR-224 was stained green
Fig. 5
Fig. 5
miR-17 and miR-224 played as oncogenes in BCa and eliminated the repression function of cir-ITCH. a and b miR-17 and miR-224 were up-regulated in BCa tissues as compared with adjacent normal tissues using qRT-PCR (n = 28, **P < 0.01, Student’s t-test). c and d A moderate negative correlation between the expression of cir-ITCH and miR-17/miR-224 was showed using Pearson correlation analysis. e and f CCK-8 assay showed that miR-17 and miR-224 promoted the vitality of EJ cell (*P < 0.05, **P < 0.01, Student’s t-test). g Cloning formation assay showed that miR-17 and miR-224 increased in cloning number of EJ cell (*P < 0.05, **P < 0.01, Student’s t-test). h miR-17 and miR-224 reversed the inhibitory effect of cir-ITCH on cell proliferation by CCK-8 assay (*P < 0.05, **P < 0.01, compared to circ-ITCH, Student’s t-test.)
Fig. 6
Fig. 6
Cir-ITCH modulated the expression of endogenous miR-17 and miR-224 targets p21 and PTEN. a Putative miR-17/miR-224 binding sequence in the 3′-UTR of p21 and PTEN mRNA. b and c cir-ITCH up-regulated the mRNA expression level of p21 and PTEN in BCa cells by qRT-PCR (*P < 0.05, **P < 0.01, Student’s t-test). c and d Circ-ITCH up-regulated the protein expression level of p21 and PTEN by western blot. e and f miR-17 and miR-224 decreased the protein expression level of p21 and PTEN, individually. g and h Dual luciferase reporter assays demonstrated that p21 and PTEN are direct target of miR-17(*P < 0.05, **P < 0.01, Student’s t-test). i and j Dual luciferase reporter assays demonstrated that p21 and PTEN are direct target of miR-224(*P < 0.05, Student’s t-test). k and l Western blot showed that mir-17 and miR-224 could partly decrease the protein expression level of p21 and PTEN which were promoted by cir-ITCH. m Mode pattern of the cir-ITCH-miR-17/miR-224-p21/PTEN regulatory network
Fig. 7
Fig. 7
Cir-ITCH suppressed tumor formation of xenograft in nude mice. a Representative image of the nude mice injected with T24 cells. b Representation picture of tumor formation of xenograft in nude mice (n = 5). c Summery of tumor volume of mice which were measured every week(*P < 0.05, **P < 0.01, Student’s t-test). d Weights of tumors in two groups were measured using electronic scales (*P < 0.05, Student’s t-test). e Proteins was extracted from the tumors and the protein expression of p21/PTEN was measured using western blot. f The expression of p21/PTEN was also measured using IHC in xenograft (Magnification, × 200)
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