RETRACTED ARTICLE: Hsa_circ_0001944 promotes the growth and metastasis in bladder cancer cells by acting as a competitive endogenous RNA for miR-548

We used 4-week-old BALB/c nude mice weighing 15–20 g (SLARC, Shanghai, China) in this study. The Ethics Committee of Shanghai University of Medicine and Health Sciences approved all animal experiments.

In total, we enrolled 90 pairs of BC tissues and adjacent normal tissues from BC patients who were treated at Huashan Hospital, Fudan University School of Medicine between January 2007 and January 2013. No patients received preoperative local or systemic treatment prior to specimen collection. We acquired informed consent from every patient or his/her relative(s). The Board and Ethics Committee of Huashan Hospital and Fudan University School of Medicine approved this project. We directly deposited collected tissues in liquid nitrogen for further usage.

We extracted total RNA from paired BC and adjacent noncancerous tissues with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). We subjected nearly 3 μg of total RNA from each sample to the VAHTS Total RNA-seq (H/M/R) Library Prep Kit from Illumina (Vazyme Biotech Co., Ltd., Nanjing, China) to erase ribosomal RNA, but retain other RNA classes such as non-coding RNAs and mRNAs. We treated the RNAs with 40 U of RNase R (Epicenter) at 37 °C for 3 h, followed by TRIzol purification. We prepared RNA-seq libraries by the KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland) and subjected them to deep sequencing through Illumina HiSeq 4000 at Aksomics, Inc. (Shanghai, China). For miRNA and mRNA analyses, T24 cells transfected with siRNA against hsa_circ_0001944 or negative control (NC) vector were used for high-throughput RNA-Seq of miRNAs as previously mentioned.

We purchased SV-HUC-1 cells and the BC cell lines 5637, UM-UC-3, T24, and RT-4 from Type Culture Collection in Chinese Academy of Sciences (Shanghai, China) and cultured them in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO₂.

We transfected small interfering RNAs (siRNAs; si-hsa_circ_0001944 and si-circRNA), miR-548 mimics, miR-548 inhibitors, PROK2 overexpression vector, and their NCs into cultured T24 or UM-UC-3 cells via Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the standard process. To further verify the effect of hsa_circ_0001944 using in vivo experiments, lentiviral-stabilized hsa_circ_0001944-silenced (sh-circ0001944) T24 cells were constructed.

We identified circRNA/miRNA target genes with CircularRNAInteractome. We predicted the interactive relationship between miR-548 and PROK2 using TargetScanHuman.

We made specific probes against hsa_circ_0001944 (Dig-5′-GATACTTTATGAGGAGACTAAGGTGTCAGTATG-3′-Dig) with help from Geneseed Biotech (Guangzhou, China). We explored signals using Cy3-conjugated anti-digoxin and FITC-conjugated anti-biotin antibodies (Jackson ImmunoResearch Inc., West Grove, PA, USA). We counterstained nuclei with 4,6-diamidino-2-phenylindole (DAPI). Afterwards, we acquired images with a Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany).

We isolated total RNA from tumor tissues or cells with TRIzol reagent (Invitrogen) following the standard protocol. We examined RNA samples for purity and concentration spectrophotometrically by detecting absorbance at 260 nm, 280 nm, and 230 nm with a NanoDrop ND-1000 (Thermo Fisher Scientific, Wilmington, DE, USA). In particular, we deemed OD260/OD280 ratios ranging between 1.8–2.1 and OD260/OD230 ratios > 1.8 as acceptable.

We reverse transcribed total RNA before RT-qPCR detection. We obtained primers specific for hsa_circ_0001944, miR-548, and PROK2 from GenePharma (Shanghai, China). We performed RT-qPCR with AB7300 thermo-recycler (Applied Biosystems, Carlsbad, CA, USA) with primers and the TaqMan Universal PCR Master Mix. We employed GAPDH as reference gene for circRNAs and mRNAs. We used U6 as an internal control for miRNA expression levels. We quantified gene expression via the 2^−ΔΔCt method. The primers utilized to assay hsa_circ_0001944 expression included 5′-CTCTTTGACATCATAATAAAATACT-3′ forward, and 5′-GGCTGAGGCAGGAGAATAGCTTGGG-3′ reverse. The miR-548 primers were 5′-ATTGGAACGATACAGAGAAGATT-3′ forward and 5′-GGAACGCTTCACGAATTTG-3′ reverse. The PROK2 primers were 5′-GGGGATCCATGAGGAGCCTGTGCTGCGCCCCA-3′ forward, and 5′-GGGAATTCCTTTTGGGCTAAACAAATAAATCG-3′ reverse. The U6 primers were 5′-CTCGCTTCGGCAGCACA-3′ forward, and 5′-AACGCTTCACGAATTTGCGT-3′ reverse. The GAPDH primers were 5′-GCACCGTCAAGGCTGAGAAC-3′ forward, and 5′-GGATCTCGCTCCTGGAAGATG-3′ reverse.

We inserted binding sites for hsa_circ_0001944 and the PROK2 3′-UTR, termed hsa_circ_0001944-WT, hsa_circ_0001944-Mut, PROK2–3′UTR-WT, and PROK2–3′UTR-Mut into HindIII and KpnI sites of pGL3 promoter vector (Realgene, Nanjing, China) of the dual-luciferase reporter assay. We first plated cells into 24-well plates, and transfected 80 ng of plasmid, 50 nM of miR-548 mimics, 5 ng of the Renilla luciferase vector pRL-SV40, and NC reagents into cells with lipofectamine 2000 (Invitrogen). We collected cells and measured them 2 d after transfection with Dual-Luciferase Assay (Promega, Madison, WI, USA), following standard instructions. We independently repeated all experiments three times.

We used the Cell Counting Kit-8 (CCK-8) assay to detect cell proliferation. We seeded transfected cells into 96-well plates at a density of 5000 cells/well in triplicate. We measured cell viability through the CCK-8 system (Gibco) at 0, 1, 2, and 3 d after seeding, following standard procedures.

For colony formation assays, we seeded transfected cells into six-well plates at a density of 2000 cells/well and maintained them in DMEM containing 10% FBS for 10 d. We imaged and counted the resultant colonies after fixing and staining them.

We used the EdU assay kit (RiboBio, Guangzhou, China) to investigate cell proliferation and DNA synthesis. We seeded 10,000 T24 or UMUC3 treated cells into 96-well plates overnight. The second day, we added EdU solution (25 μM) to the plate and incubated the cells for 1 d. We then used 4% formalin to fix the cells at room temperature for 2 h. We utilized 0.5% TritonX-100 to permeabilize the cells for 10 min, and added Apollo reaction solution (200 μL) to stain EdU and DAPI (200 μL) to stain nuclei for 30 min. Lastly, a Nikon microscope (Nikon, Tokyo, Japan) was used to measure cell proliferation and DNA synthesis, which were reflected by blue and red signals, respectively.

We suspended BC cells in 200 μL of serum-free medium and inserted them into upper chamber of Transwell plates with 8-μm pores (Corning Costar, Corning, NY, USA). We also placed 600 μL medium containing 20% FBS in lower chamber as chemoattractant. After incubation for 1 d, we fixed cells in the filter with methanol, stained them with 0.1% crystal violet solution, and then counted them in three random fields of view (200×).

For wound healing assays, we seeded BC cells in 6-well plates. We created a linear scratch wound with a 20-μL pipette tip in the confluent monolayer of cells. After 2 d of incubation in medium without FBS, we observed and photographed wound closure under microscope. We conducted experiments in triplicate and repeated them three times.

We fixed cells in 70% ethanol overnight at 4 °C, resuspended them in staining solution (Beyotime, Shanghai, China), and then incubated them for 30 min at 4 °C. We measured stained cells by flow cytometry (Beckman Coulter, Franklin Lakes, NJ, USA).

For the xenograft assays, we subcutaneously injected 1 × 10⁶ modified (hsa_circ_0001944-silenced) or NC T24 cells into the right side of each male nude mouse. We calculated tumor volumes (length × width² × 0.5) at the indicated timepoints and excised tumors 4 weeks after the injection.

For metastasis analysis, we transfected 2 × 10⁵ NC or hsa_circ_0001944-silenced T24 cells with luciferase expression vectors, and injected the cells intravenously into the tails of mice. After 30 d, T24 cell metastases were analyzed by bioluminescence imaging following an intravenous injection of luciferin (150 mg luciferin/kg body weight) into the tails.

We fixed tumor tissue samples in 10% formalin and embedded them in paraffin. We stained sections (5-μm thick) with Ki67 to explore proliferation. We examined sections with an Axiophot light microscope and imaged them with digital camera.

We assessed differences among groups via paired/unpaired t-tests (two-tailed). We used Pearson’s correlation test to obtain associations between groups. Data are presented as mean ± SEM. We considered P-values < 0.05 as significant. We performed all statistical analyses with GraphPad Prism (GraphPad Inc., San Diego, CA, USA).

To reveal correlations between circRNA expression and BC progression, two BC samples were used for circRNA high-throughput sequencing. The results show that 2113 circRNAs were upregulated and 1551 circRNAs were downregulated in BC tissues compared with adjacent normal tissues (Fig. 1a). However, only 12 circRNAs were significantly upregulated and 78 were significantly downregulated (Fig. 1b). Among the upregulated circRNAs, hsa_circ_0001944 expression was significantly increased in BC tissues (supplementary materials. S1). FISH assays demonstrated that hsa_circ_0001944 expression increased in BC tissues compared with adjacent normal tissues. The results also showed that hsa_circ_0001944 was localized predominately to cytoplasm (Fig. 1d). RT-qPCR detection of 90 patient samples suggested hsa_circ_0001944 expression increased in BC tissues compared with adjacent normal tissues (Fig. 1e). To further understand prognostic value of hsa_circ_0001944 expression, we analyzed correlations with patient characteristics. This illustrated that high hsa_circ_0001944 expression was correlated with increased tumor size, higher stage, lymph node metastasis, and higher pathological T stage (Table. 1). Also, the correlation analysis demonstrated that high hsa_circ_0001944 expression was associated with poorer overall survival compared with patients with low hsa_circ_0001944 expression (Fig. 1f). hsa_circ_0001944 is derived from circularizing exons from gene TCONS_l2_00030860, which located at chr5:73069679–73,076,570. TCONS_l2_00030860 consists of 45,161 bp and spliced mature circRNA is 1096 bp (Fig. 1g). In this regard, these findings suggest that hsa_circ_0001944 functions in BC progression.

Rt-qPCR experiments showed that hsa_circ_0001944 expression in BC cell lines was increased compared with the normal cell line SV-HUC-1 (Fig. 2a). T24 and UMUC3 cells had the highest hsa_circ_0001944 levels, so they were selected for further experiments. hsa_circ_0001944 expression was significantly decreased in both UMUC3 and T24 cells after transfecting a siRNA against hsa_circ_0001944 (Fig. 2b). Cell cycle distribution analysis demonstrated that the S-phase proportion was significantly decreased, while the G2/M-phase proportion was increased after hsa_circ_0001944 depletion (Fig. 2c), suggesting a cell cycle arrest at G2/M phase. CCK8 (Fig. 2d and e), colony formation assays (Fig. 2f and g), and DNA synthesis determination by the EdU assay (Fig. 2h and i) showed that silencing hsa_circ_0001944 suppressed cell proliferation in UMUC-3 and T24 cells. The xenograft results verified that hsa_circ_0001944 knockdown suppressed T24 tumor growth (both weight and volume) compared with the NC group (Fig. 3a-c). Immunohistochemical detection of Ki67 demonstrated that hsa_circ_0001944 silencing suppressed Ki67 expression in tumor tissues (Fig. 3d), which verified that hsa_circ_0001944 knockdown suppressed tumor growth. RT-qPCR analysis showed that silencing hsa_circ_000194 decreased hsa_circ_000194 and PROK2 expression in tumor tissues, but promoted miR-548 expression (Fig. 3e-g).

Transwell migration (Fig. 4a and b) and wound healing (Fig. 4c and d) assays to detect for invasive ability showed that silencing hsa_circ_0001944 decreased migration and invasion of both T24 and UMUC-3 cells. The metastasis ability of T24 cells was also decreased after hsa_circ_0001944 silencing using live imaging analysis of metastasis model mice (Fig. 4e). Together, these data suggest that knocking down hsa_circ_0001944 suppressed the invasion ability of BC cells both in vivo and in vitro. Immunohistochemical detection of Ki67 demonstrated that hsa_circ_0001944 silencing suppressed Ki67 expression in metastatic lung tissue (Fig. 3f), which verified that hsa_circ_0001944 knockdown suppressed tumor growth. RT-qPCR analysis showed that silencing hsa_circ_000194 decreased hsa_circ_000194 and PROK2 expression in metastatic lung tissue, but promoted miR-548 expression (Fig. 3g-i).

Increasingly, studies have confirmed that circRNAs, including microRNA (miRNA/miR) response elements, interact with miRNAs as competitive endogenous RNAs (ceRNAs) to regulate target mRNA expression [14, 15]. Therefore, we selected T24 cells with or without hsa_circ_0001944 silencing for high-throughput sequencing. The results showed that hsa_circ_0001944 depletion resulted in a series of upregulated miRNAs (supplementary materials. S2). Combined with the biological analysis that showed miR-548 was a hsa_circ_0001944 target, bioinformatic analyses also illustrated that miR-548 was a downstream hsa_circ_0001944 target (Fig. 5a). To further verify the relationship between hsa_circ_0001944 and miR-548, we prepared wild-type (WT) or mutated (MUT) hsa_circ_0001944 sequences that included the miR-548 binding sequence into a luciferase reporter vector (Fig. 5b). We then transfected this reporter vector into 293 T cells combined with a miR-548 mimic or not. Luciferase reporter analysis suggested that miR-548 inhibited luciferase activity in WT-transfected cells, yet not in MUT-transfected cells (Fig. 5c), suggesting that miR-548 was a hsa_circ_0001944 target.

Bioinformatic analyses illustrated that PROK2 was a miR-548 downstream target. To further validate the correlation between miR-548 and PROK2, we constructed WT or MUT 3′UTR-PROK2 sequences that included that miR-548 binding sequence into a luciferase reporter vector (Fig. 5d). We then transfected this reporter vector into 293 T cells combined with a miR-548 mimic or not. Luciferase reporter analysis demonstrated that miR-548 inhibited luciferase activity in WT-transfected cells, yet not in MUT-transfected cells (Fig. 5e), suggesting that PROK2 was a miR-548 target.

To identify the regulatory relationships between hsa_circ_0001944, miR-548, and PROK2, both T24 and UMUC3 cells were transfected with si-hsa_circ_0001944, miR-548 inhibitor or PROK2 overexpression vector, either singly or in combination. RT-qPCR detection showed that hsa_circ_0001944 expression decreased significantly after transfection with the siRNA against hsa_circ_0001944. miR-548 downregulation or PROK2 overexpression could not reverse the hsa_circ_0001944 expression in UMUC3 or T24 cells (Fig. 5f and h). hsa_circ_0001944 downregulation increased miR-548 expression in UMUC3 and T24 cells. Treating cells with the miR-548 inhibitor suppressed miR-548 expression, but PROK2 overexpression could not reverse miR-548 expression (Fig. 5j and g). We also found that silencing hsa_circ_0001944 decreased PROK2 expression in both T24 and UMUC3 cells, while treatment with the miR-548 inhibitor partially recovered PROK2 expression. Transfecting the PROK2 overexpression vector increased PROK2 expression (Fig. 5i and k). Together, these data suggest that miR-548 and PROK2 are downstream targets of hsa_circ_0001944 and that PROK2 is a miR-548 downstream target.

Colony formation (Fig. 6a-c) and EdU (Fig. 6d-f) assays showed that silencing hsa_circ_0001944 suppressed the proliferation of both UMUC3 and T24 cells. Further silencing miR-548 or overexpressing PROK2 rescued proliferation ability of UMUC3 and T24 cells. Transwell assays to measure cell invasion (Fig. 6g-i) also found that silencing miR-548 or overexpressing PROK2 rescued invasion ability of both T24 and UMUC3 cells.

Colony formation (Fig. 7a-c) and EdU (Fig. 7d-f) assays showed that overexpressing miR-548 suppressed proliferation of T24 and UMUC3 cells. Overexpressing PROK2 rescued the proliferation ability of T24 and UMUC3 cells because miR-548 could not interact with exogenous PROK2, which lacked a 3′UTR after transcription. Transwell assays to measure cell invasion (Fig. 7g-i) also found that overexpressing PROK2 rescued the invasion ability of both T24 and UMUC3 cells that overexpressed miR-548.