BMB Reports 2020; 53(2): 82-87  https://doi.org/10.5483/BMBRep.2020.53.2.059
Circular RNA hsa_circ_0075828 Promotes Bladder Cancer Cell Proliferation Through Activation of CREB1
Chengle Zhuang1, Xinbo Huang1, Jing Yu2,* & Yaoting Gui1,*
1Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen-Peking University-the Hong Kong University of Science and Technology Medical Center, Shenzhen 518000, 2Department of Laboratory Medicine, Peking University Shenzhen Hospital, Shenzhen 518000, PR China
Correspondence to: Yaoting Gui, Tel: +86-755-839233; Fax: +86-755-839233; E-mail: guiyaoting2007@aliyun.com; Jing Yu, Tel: +86-755-839233; Fax: +86-755-839233; E-mail: jing_yu2004@aliyun.com
Received: February 25, 2019; Revised: March 27, 2019; Accepted: May 3, 2019; Published online: February 29, 2020.
© Korean Society for Biochemistry and Molecular Biology. All rights reserved.

cc This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Circular RNAs (circRNAs), one kind of non-coding RNA, have been reported as critical regulators for modulating gene expression in cancer. In this study, microarray analysis was used to screen circRNA expression profiles of bladder cancer (BC) 5637 cells, T24 cells and normal control SV-HUC-1 cells. The data from the microarray showed that hsa_circ_0075828 (named circCASC15) was most highly expressed in 5637 and T24 cells. circCASC15 was highly expressed in BC tissues and cells. Overexpression of circCASC15 was closely associated with BC tumor stage and promoted cell proliferation significantly in vitro and in vivo. Mechanistically, circCASC15 could act as miR-1224-5p sponge to activate the expression of CREB1 to promote cell proliferation in BC. In short, circCASC15 promotes cell proliferation in BC, which might be a new molecular target for BC diagnosis and therapy.
Keywords: Bladder cancer, circCASC15, Circular RNA, CREB1, miR-1224-5p
INTRODUCTION

Bladder cancer (BC) is one of the most common malignancies of the urinary system, with high morbidity and mortality rates worldwide (1). Surgery, radiation and chemotherapy are effective therapies for a subset of BC patients (2). However, the overall therapeutic efficacy and the 5-year survival rate are still low (3). In addition, the molecular mechanisms of BC pathogenesis are poorly understood (4). Thus, discovering novel molecular biomarkers and further investigation of the mechanisms of BC progression are important.

Circular RNAs (circRNAs), a class of non-coding RNAs, are closed covalent loops with neither a polyadenylate tail nor 5’ to 3’ polarity (5). More than 30,000 circRNAs have been identified, and they are stable, conserved and abundant in mammalian cells (6). Various circRNAs have been suggested to be involved in the occurrence of numerous tumors, including gastric cancer, hepatocellular carcinoma, prostate cancer, and BC (7). Numerous studies have reported that miRNA response elements (MREs) exist in circRNAs, which function as competing endogenous RNAs (ceRNAs) to sponge miRNAs or transcription regulators to regulate the expression of target genes (8). This hypothesis was first verified by solid evidence that there are 70 miR-7 binding sites in circRNA CiRS-7 that act as miR-7 sponges to regulate miR-7 target mRNA expression (8). Recently, circular RNA YAP1 has been shown to directly interact with miR-367-5p to modulate p27 expression and inhibit proliferation and invasion of gastric cancer (9). A study reported that circFBLIM1 promoted hepatocellular carcinoma through sponging miR-346 (10). Circular RNA cTFRC sponged miR-107 to increase TFRC expression to promote BC progression (11). Circular RNA circPRMT5 acted as a miR-30c sponge to induce epithelial-mesenchymal transition (EMT) in BC (12). Circular RNA circHIPK3 bound to miR-558 to suppress heparanase expression in BC (13). In brief, the circRNA/miRNA/mRNA axis may be extensively involved in the progression of cancer, including BC, and the underlying mechanisms of circRNAs are still not clear.

In this study, we mainly focus on a circRNA derived from CASC15 gene, hsa_circ_0075828, termed circCASC15, which was screened from microarray analysis. The expression of circCASC15 was increased significantly in BC tissues and notably associated with the tumor stage of BC. Additionally, the high abundance of circCASC15 promoted proliferation of BC through acting as a ceRNA for miR-1224-5p. It then activated oncogenic cAMP responsive element binding protein 1 (CREB1) expression in BC. Collectively, circCASC15 may be a novel potential target for the treatment of BC.

RESULTS

Various studies have shown that many circRNAs in the cytoplasm play oncogenic or tumor-suppressive roles in distinct human cancers through functioning as miRNA sponges (18). CircRNA circ-ITCH sponged miR-17/miR-224 to increase the expression of p21 and PTEN to suppress BC progression (19). CircRNA circCEP128 functioned as a ceRNA for miR-145-5p and promoted progression of BC through increasing the expression of SOX11 (20). Several studies have reported the expression characteristics of circRNAs in BC tissues (13, 21). However, functional experiments are performed in BC cell lines and no studies have revealed the profile of circRNA expression using a microarray assay in BC cells. Thus, circRNA microarray analysis was carried out in SV-HUC-1, BC 5637 and T24 cells. The circRNA expression level in 5637 and T24 cells was compared with control SV-HUC-1. We found that circCASC15, was the most increased in 5637 and T24 cells, and it was also a upregulated significantly circRNA in a previous study using High-Throughout Sequencing (21). Highly expressed circCASC15 was closely related to BC tumor stage. Upregulation of circCASC15 promoted cell proliferation in BC in vitro and in vivo. This is the first study to illustrate that circCASC15 is clinically and functionally oncogenic in BC.

To investigate the underlying mechanism of circCASC15 in BC, we firstly illustrated the subcellular location of circCASC15 in BC cells through separating subcellular fractionation. circCASC15 was mainly localized in the cytoplasm, which was the same as miRNAs. We found that circCASC15 could bind to miR-1224-5p directly through bioinformatics analysis, RIP and dual-luciferase assays. Many studies have reported that miRNAs regulated gene expression through target mRNA degradation and inhibition of translation (22). In this study, circCASC15 could function as a miRNA sponges for miR-1224-5p and regulate the expression of the downstream gene CREB1. Many studies have shown that CREB1 played oncogenic roles to promote cell proliferation in different kinds of cancers including BC (23-27). Thus, we suspected that circCASC15 promoted cell proliferation through activating downstream CREB1 expression. Our results demonstrated that downregulation of circCASC15 could inhibit the mRNA and protein expression levels of CREB1 in BC 5637 and T24 cells. A rescue assay was performed, and the results of the CCK-8 and EdU assays showed that overexpression of CREB1 could abrogate the effects of downregulation of circCASC15 on cell growth.

In conclusion, the expression level of circCASC15 was positively correlated with tumor stage in BC patients, and one of the main mechanisms of circCASC15 was to act as a miR-1224-5p sponge to activate the oncogenic CREB1 expression to promote cell proliferation in BC. circCASC15 might be a novel clinical and therapeutic factor in BC.

MATERIALS AND METHODS

Patient tissue specimens

Sixty-seven pairs of BC tissues and matched para-carcinoma tissues were acquired from patients who underwent radical cystectomy after being diagnosed with urothelial carcinomas of the bladder. This study was approved by the Institutional Ethical Committee Board of Peking University Shenzhen Hospital, and all patients signed informed consent before tissues collection. The 2004 World Health Organization Consensus Classification and Staging Systme for BC was used to classify specimens.

Cell lines and cell culture

Human BC (5637, T24, J82, UM-UC-3 and SW780) cell lines and the SV-HUC-1 cell line were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Complete growth medium of these six cell lines was chosen according to ATCC suggestions. Cells were cultured at 37°C supplied with 5% CO2 in an incubator.

RNA extraction and quantitative real time-PCR (qRT-PCR)

TRIzol reagent (Invitrogen, Grand Island, NY, USA) was used to isolate total RNA from tissues or cell lines according to the manufacturer’s instructions. Three U/mg RNase R was utilized to eliminate linear RNA at 37°C for 20 min. The complementary DNA (cDNA) was synthesized from total RNA using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara., Dalian, China) or a commerical miRNA reverse transcription PCR kit (RiboBio, Guangzhou, China). The SYBR Premix Ex TaqTM kit (Takara, Dalian, China) was used for the quantitative real-time PCR (qRT-PCR) analysis on the Roche lightcycler 480 Real-Time PCR System. Levels of GAPDH or U6 were used as an endogenous control to normalize the differences of circRNA and miRNA. The comparative 2−DDCT method was used to analyze the expression levels. All assays were carried out at least three times. The primer sequences were described in Table S3.

Additional methods

Cell transfection, microarray analysis, vector construction, cell proliferation assays, subcellular fractionation location, RNA-binding protein immunoprecipitation (RIP) assay, dual-luciferase reporter assay, Western blot analysis and the xenograft tumor model are described in the Supplementary Materials and Methods.

Statistical analysis

All statistical analyses were carried out using SPSS computer software version 20.0 (SPSS Inc., Chicago, IL, USA). All data are indicated as the mean ± standard error (SD). The data regarding circCASC15 expression level in BC tissues were analyzed using a paired-sample t-test. A Chi-square test was utilized to compare the difference in clinicopathological characteristics in BC patients. Student’s t-test, was used to analyze the group difference. A P-value < 0.05 (two-sided) was chosen for statistical significance.

Supplemental Materials
BMB-53-082_Supple.pdf
ACKNOWLEDGEMENTS

This work was supported by grants from the Guangdong Key Laboratory of Male Reproductive Medicine and Genetics (2017B030314074), the Shenzhen Project of Science and Technology (JCYJ20170413100245260) and the ‘San-ming’ Project of Medicine in Shenzhen (SZSM201612066).

CONFLICTS OF INTEREST

The authors have no conflicting interests.

Figures
Fig. 1. The expression characteristics of circCASC15, its correlation with prognosis and location in BC are presented. (A) The schematic diagram represents the genomic location and splicing pattern of circCASC15. Direct sequencing was used to verify the splice junction. (B) The expression pattern of circCASC15 in 67 pairs of BC tissues was shown. (C, D) circCASC15 was significantly upregulated in BC tissues and cells. (E, F) High circCASC15 expression indicated poor overall and disease free survival of BC patients. (G) circCASC15 was resistent to RNase R. (H, I) circCASC15 was mainly located in the cytoplasm of BC 5637 and T24 cells. *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig. 2. circCASC15 promotes BC cell proliferation significantly in vitro and in vivo. (A, B) The expression level of circCASC15 was decreased or increased significantly after knockdown or overexpression of circCASC15 in 5637 and T24 cells, respectively. (C, D) Cell proliferation was inhibited or promoted significantly after knockdown or overexpression of circCASC15 in 5637 and T24 cells using the CCK-8 assay, respectively. (E-H) Cell growth was suppressed or activated remarkably after knockdown or overexpression of circCASC15 in 5637 and T24 cells using the EdU assay, respectively (bar in EdU image represents 50 µm). (I, J) Representative images of the tumor in nude mice were shown. (K, L) Compared with the LV-sh-NC group, the tumor volume curve and tumor weight were significantly inhibited in the LV-sh-circ group. *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig. 3. circCASC15 directly binds to miR-1224-5p that targets downstream CREB1 in BC. (A) Bioinformatics analysis showed five possible miRNAs targeting circCASC15. (B, C) RIP assay demonstrated that circCASC15 bound to miR-1224-5p directly. (D, E) The mRNA expression level of CREB1 was decreased or increased significantly after overexpression or inhibition of miR-1224-5p in 5637 and T24 cells, respectively. (F, G) The CREB1 protein expression level was inhibited remarkably after transfection of miR-1224-5p mimics in 5637 and T24 cells. (H, I) The CREB1 protein expression level was increased notably after transfection of miR-1224-5p inhibitors in 5637 and T24 cells. *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig. 4. CREB1 was the functional target of circCASC15. (A, B) The CREB1 mRNA expression level was inhibited significantly after knockdown of circCASC15 in 5637 and T24 cells. However, overexpression of CREB1 could significantly abrogate the inhibitory effects of knockdown of circCASC15 on CREB1 mRNA expression level in 5637 and T24 cells. (C, D) The CREB1 protein expression level was suppressed remarkably after downregulation of circCASC15 in 5637 and T24 cells. But overexpression of CREB1 could notably counteract the inhibitory effects of knockdown of circCASC15 on CREB1 protein expression level in 5637 and T24 cells. (E-H) Cell proliferation was inhibited significantly after inhibition of circCASC15 in BC 5637 and T24 cells using CCK-8 and EdU assays. However, overexpression of CREB1 could markedly abolish the suppressive effects of knockdown of circCASC15 on cell growth (bar in EdU image represents 50 µm). *P < 0.05, **P < 0.01 and ***P < 0.001.
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Funding Information
  • Guangdong Key Laboratory of Male Reproductive Medicine and Genetics
     
      2017B030314074
  • Shenzhen Project of Science and Technology
     
      JCYJ20170413100245260
  • Sanming Project of Medicine in Shenzhen
      10.13039/501100012151
      SZSM201612066

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