OncoTargets and Therapy Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH CircHIPK3 Facilitates the G2/M Transition in Prostate Cancer Cells by Sponging miR-338-3p This article was published in the following Dove Press journal: OncoTargets and Therapy Fengchun Liu1 Background: Circular RNAs (circRNAs) play a crucial role in gene expression regulation. Yanru Fan 1 CircHIPK3 is a circRNA derived from Exon 2 of HIPK3 gene and its role in prostate cancer Liping Ou1 (PCa) is still unclear. fl Ting Li1 Methods: CCK8 assays, ow cytometry and colony formation assays were performed to assess Jiaxin Fan1 the effects of circHIPK3 in PCa cells. Bioinformatics analysis, RNA pull-down assay, RNA immunoprecipitation assay (RIP), and luciferase activity assay were performed to dissect the Limei Duan1 mechanism underlying circHIPK3-mediated G2/M transition in PCa cells. Jinxiao Yang1 1 Results: CircHIPK3 expression was upregulated in PCa cells and prostate cancer tissues. Chunli Luo Overexpression of circHIPK3 or circHIPK3 silencing altered PCa viability, proliferation and 2 Xiaohou Wu apoptosis in vitro. CircHIPK3 could sponge miR-338-3p and inhibit its activity, resulting in 1Department of Laboratory Diagnosis, increased expression of Cdc25B and Cdc2 in vitro. Chongqing Medical University, Yuzhong, Conclusion: CircHIPK3 promotes G2/M transition and induces PCa cell proliferation by Chongqing 408000, People’s Republic of China; 2Department of Urology, The First sponging miR-338-3p and increasing the expression of Cdc25B and Cdc2. CircHIPK3 may Affiliated Hospital of Chongqing Medical play an oncogenic role in PCa. University, Yuzhong, Chongqing 408000, Keywords: prostate cancer, circHIPK3, miR-338-3p, Cdc25B, G2/M transition People’s Republic of China Introduction Prostate cancer (PCa) is one of the most commonly diagnosed cancers among men worldwide.1 Once the tumor has migrated out of the gland, an incurable metastatic disease may inevitably occur.2–4 Circular RNA (circRNA) is an endogenous non-coding RNA.4 Unlike linear RNA terminated with a 5ʹ cap and a 3ʹ tail, circRNA forms a covalent closed-loop structure.4,5 At present, molecular purification methods combined with next- generation RNA sequencing have led to an in-depth understanding of circRNA.6 A mass of circRNAs are conserved throughout species with specific expression at 7,8 Correspondence: Xiaohou Wu the tissue/developmental stage, and are stable and resistant to RNaseR. Department of Urology, The First CircRNAs provide new insights into the pathogenesis of diseases.9 Affiliated Hospital of Chongqing Medical University, No. 1 Medical College Road, MicroRNAs (miRNAs) are endogenous ~23 nt RNAs that play important gene- ’ Yuzhong, Chongqing 408000, People s regulatory roles in animals by pairing to the mRNAs of protein-coding genes to Republic of China Email [email protected] direct their posttranscriptional repression.10 Gain-of-function and loss-of-function Chunli Luo experiments, in combination with target prediction analyses, have demonstrated that Department of Laboratory Diagnosis, microRNAs can affect different steps of the tumorigenesis and progression of Chongqing Medical University, No. 1 11,12 Medical College Road, Yuzhong, various cancers, including PCa. ’ Chongqing 408000, People s Republic of CircHIPK3 is derived from exon 2 of the HIPK3 gene. It has been shown that China 13 Email [email protected] circHIPK3 sponges miR-124 and suppresses miR-124 activity. In colorectal submit your manuscript | www.dovepress.com OncoTargets and Therapy 2020:13 4545–4558 4545 DovePress © 2020 Liu et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php – http://doi.org/10.2147/OTT.S242482 and incorporate the Creative Commons Attribution Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Liu et al Dovepress cancer, circHIPK3 may have considerable potential as (20–30 μg/mL) and rEGF (0.1–0.2 ng/mL) containing a prognostic biomarker, and support the notion that ther- 10% FBS and 1% PS. The cells were placed in an apeutic targeting of the c-Myb/circHIPK3/miR-7 axis may incubator at 37°C with 5% CO2. The cells were trans- be a promising treatment approach for patients.14 fected at approximately 60% confluence with synthesized CircHIPK3 inhibited the expression of heparanase small interfering RNAs (siRNAs) (100 nmol/L; 15 (HPSE) in bladder cancer by sponging miR-558. GenePharma, Shanghai, China) targeting circHIPK3 CircHIPK3 plays an important role in tumors and diabetic using EndoFectin™-Max reagent (GeneCopoeia Inc., 16 retinopathy by sponging miR-30a. Recently, it has been Rockville, MD, USA). reported that circHIPK3 was upregulated in PCa tissues and correlated with proliferation and invasion.17,18 Until now, little known about the potential role of circHIPK3 in Quantitative PCR (qPCR) cell cycle of prostate cancer cells, and its molecular Total RNA was extracted from cells and clinical samples mechanism still remains elusive. with TRIzol (Life Technologies, Carlsbad, CA, USA). The In this study, we reported that circHIPK3 is an onco- nuclear and cytoplasmic fractions were extracted with NE- gene that is upregulated in PCa. Importantly, we found that PER nuclear and cytoplasmic extraction kits (Thermo circHIPK3 knockdown markedly arrested the G2/M tran- Scientific, Waltham, MA, USA). cDNA was synthesized sition in PCa cells. Furthermore, circHIPK3 could sponge from total RNA using the PrimeScript RT Master Mix miR-338-3p to inhibit miR-338-3p activity, thereby lead- (Takara, Dalian, China), and qPCR was carried out using ing to increased Cdc25B and Cdc2 expression. Our find- TB Green Premix Ex Taq II (Takara). Stem-loop RT-PCR ings may reveal a novel mechanism of circHIPK3 in the was used to detect the amount of miRNA. The primers are development of PCa and provide a new perspective for the listed in Table S1. treatment of PCa. RNA Fluorescence in situ Hybridization Materials and Methods (FISH) Tissue Samples The Cy3-labelled circHIPK3 probe and FAM-labelled PCa tissues and benign prostatic hyperplasia (BPH) tissues miR-338-3p probe were designed and synthesized by were surgically removed from 45 patients with PCa and 25 GenePharma (Shanghai, China). The probe sequences are patients with BPH, respectively, and frozen in liquid nitro- listed in Table S1. FISH was carried out using the kit gen immediately. Clinical pathology data were collected (GenePharma). The images were gained on a ZEISS 800 from patients. Patients volunteered to participate in the laser scanning confocal microscope (ZEISS, Jena, study and signed written informed consent. This study Germany). was approved by the Ethics Committee of Chongqing Medical University (Approval No. 20176503) and was implemented following the Helsinki Declaration. CircHIPK3 Plasmids Construction To construct circHIPK3 over-expression plasmids, human circHIPK3 cDNA was synthesized by Cell Culture and Transfection TSINGKE (Nanjing, China) and cloned into pLCDH- Human prostate cell lines (LNCaP, DU145, PC3 and ciR vector (Geenseed Biotech Co, Guangzhou, China). 22RV1) and Human prostate epithelial cell line RWPE- The pLCDH-ciR vector contained a front circular frame 1 were obtained from the American Type Culture and a back circular frame. Collection (ATCC, Manassas, VA, USA). 22RV1, LNCaP and DU145 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS; Gibco, Cell Counting Kit-8 Proliferation Assay Grand Island, NY, USA) and 1% penicillin/streptomycin The proliferative capacity of LNCaP and DU145 cells was (PS; Sangon, Shanghai, China). PC3 cells were cultured evaluated by CCK8 (Dojindo Laboratories, Kumamoto, in F-12 medium containing 10% FBS and 1% PS. Japan) analysis. The results were read on an automatic RWPE-1 cells were cultured in Keratinocyte-SFM microplate reader (Synergy 4; BioTek, Winooski, VT, (K-sfm; Gibco) medium with Bovine Pituitary Extract USA) at 450 nm spectrophotometry. 4546 submit your manuscript | www.dovepress.com OncoTargets and Therapy 2020:13 DovePress Dovepress Liu et al Colony Formation Assay and resuspended in 0.5 mL TriReagent. Finally, coprecipi- PCa cells were seeded in 6-well plates at a density of 500 tated RNA was detected by qPCR. cells/well and cultured for 2 weeks. The colonies were fixed with methanol and stained with crystal violet. RNA Pull-Down Finally, colonies containing ≥50 cells were counted under Pull-down assay was performed following the manufac- ’ TM a microscope (Olympus, Toyko, Japan). turer s instructions of BersinBio RNA pull down kit. Biotinylated circHIPK3 probe was designed and synthe- sized by BersinBio. In brief, 107 cells were washed in cold Flow Cytometry Analysis PBS, lysed in 750 μL RIP buffer, and incubated with 2 μg Flow cytometry was applied to detect cell cycle and apop- biotinylated circHIPK3 probes, at room temperature for 2 tosis. PCa cells were stained with propidium iodide (PI) h. A total of 50 μL washed StreptavidinC1 magnetic beads (Beyotime, C1052, Shanghai, China). Cells were washed were added to each binding reaction and further incubated and resuspended in cold phosphate buffered saline (PBS) at room temperature for another hour. The beads were and incubated in ice-cold 70% ethanol for 4 h. The cells washed briefly with RIP buffer for five times. The eluted were then centrifuged at 1500 rpm for 10 min and resus- RNA in the pull-down materials were analyzed by qPCR. pended in propidium iodide (PI) master mix at a density of The circHIPK3 probes sequences are listed in Table S1. 5ⅹ105 cells/mL and incubated at 37°Cfor 30 min before analysis with flow cytometry as described.