circPRRC2A promotes angiogenesis and metastasis through epithelial-mesenchymal transition and upregulates TRPM3 in renal cell carcinoma

Background: Circular RNAs (circRNAs) have been identified as essential regulators in a plethora of cancers. Nonetheless, the mechanistic functions of circRNAs in Renal Cell Carcinoma (RCC) remain largely unknown. Methods: In this study, we aimed to identify novel circRNAs that regulate RCC epithelial-mesenchymal transition (EMT), and to subsequently determine their regulatory mechanisms and clinical significance. Results: circPRRC2A was identified by circRNA microarray and validated by qRT-PCR. The role of circPRRC2A in RCC metastasis was evaluated both in vitro and in vivo. We found that increased expression of circPRRC2A is positively associated with advanced clinical stage and worse survivorship in RCC patients. Mechanistically, our results indicate that circPRRC2A prevents the degradation of TRPM3, a tissue-specific oncogene, mRNA by sponging miR-514a-5p and miR-6776-5p. Moreover, circPRRC2A promotes tumor EMT and aggressiveness in patients with RCC. Conclusions: These findings infer the exciting possibility that circPRRC2A may be exploited as a therapeutic and prognostic target for RCC patients.


Microarray analysis
In this study, microarray-based profiling was performed from 3 lung metastatic tissue samples from 3 patients with renal cell carcinoma (RCC). For controls, matched 3 renal carcinoma samples from 3 patients with RCC were used. The sample preparation and microarray hybridization were performed based on the Arraystar's standard protocols. After being digested with Rnase R (Epicentre Technologies, Madison, USA) to remove linear RNAs, circular RNAs were amplified and transcribed into fluorescent circRNA utilizing Arraystar Super RNA Labeling Kit (Arraystar V2). Subsequently, the labeled circRNAs were hybridized onto the Arraystar Human circRNA Array V2 (8×15K, Arraystar). Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed with using the GeneSpring GX v12.1 software package (Agilent Technologies). CircRNAs showing fold changes ≥ 2 and P values＜0.05 were regarded as significantly differentially expressed.
For lentiviral transduction, virus-containing supernatant was collected 48 h after the cotransfection of packaging plasmids and the shRNA-or circRNAoverexpressing vector into HEK-293t cells, followed by its addition to the target cells. The stable cell lines were then infected by miRNAs and corresponding controls according to the manufacturer's instructions. 48 hours later, the infected cells were selected with 2 μg/ml of puromycin (Gibco, USA).

Biotin coupled miRNAs capture
The biotin-coupled miRNA pull-down assay was performed as described previously16,23. Briefly, the 3'-end biotinylated miRNA-RNA mimic or control biotin-RNA was transfected into RCC cells at final concentration of 25 nmol/L for 24 h. The biotin-coupled RNA complex was then pulled down by incubating cell lysates with streptavidin-coated magnetic beads (IBSBIO). miRNA and TRPM3 levels in bound fractions were evaluated by qRT-PCR.

Western blot assay
Total proteins from cells were extracted using RIPA lysis buffer (Thermo Fisher, USA) and protein concentrations were measured with BCA protein assay (Pierce, IL, USA). Protein bands were visualized by ECL (Thermo Fisher).
Western blot analyses were performed according to standard protocols as described previously. The antibodies used are listed in Supplementary Table 1d.

Actinomycin D and RNase R treatment
Transcription was interfered with the addition of 2 μg/ml Actinomycin D (Sigma-Aldrich, USA) at indicated time point. Total RNA (3 μg) was incubated for 15 min at 37°C with 3 U/μg of RNase R (Lucigen Technologies, WI, USA).
After treatment with Actinomycin D or RNase R, the RNA expression levels of circPRRC2A and linar-PRRC2A were analyzed by qRT-PCR.

Cell proliferation and colony formation assay
Cell proliferation was examined using the Cell Counting Kit (CCK)-8 assay Wound healing and cell invasion assay 5 × 10 5 cells were cultured in 6-well plate until confluence and then wounded with a 20 μl pipette tip. Migration photos were captured at 0, 24 h and 48 h after scratching. Transwell assays using Boyden chambers containing 24well Transwell plates (Sigma-Aldrich, CLS3460-48EA) with 8 mm pore size were used to evaluate the migration and invasiveness of cells. All experiments were performed in duplicate and repeated three times.

RNA fluorescence in situ hybridization (FISH)
The FISH assay was performed in ACHN/Caki-1 cells as previously described. Biotin-labelled probes specific to circPRRC2A and Dig-labelled miR-514a-5p and miR-6776-5p probes were used in the hybridization. The sequence is listed in Supplementary Table 1b, c. The signals of biotin-labelled probes were detected using Cy5-Streptavidin (Life Technologies). The signals of Dig-labelled miR-514a-5p and miR-6776-5p probes were detected using a tyramide-conjugated Alexa 488 fluorochrome TSA kit. Nuclei were counterstained with 4,6-diamidino-2-phenylindole. Images were acquired on a Leica confocal microscope (Leica Microsystems, Germany).

Immunofluorescence
HUVEC cells grown on the confocal dish (Corning) were rinsed three times with PBS and fixed with 4% paraformaldehyde for 15 min. After washing with PBS, cells were permeabilized with 0.1% TritonX-100 in PBS for 10 min on ice.
Cells were then washed three times with PBS, blocked with 5% BSA in TBST for 30 min at 37 °C and incubated for 1 h at 37°C with primary antibodies. After washing with TBST, cells were incubated with corresponding secondary antibody for 1 h at 37°C. HUVECs were then stained with DAPI for nucleus staining. The relative mean fluorescence densities were analyzed by Image J, and plotted using GraphPad Prism 5.0 software.

Luciferase reporter assay
The sequence of circPRRC2A was cloned downstream of Luci-reporter vector (IBSBIO). Mutations were performed in the binding sites. Luci-reporter vector was cotransfected with the predicted miRNAs or miR-NC into 293t or RCC cells by Lipofectamine-mediated gene transfer. The relative luciferase activity was normalized to Renilla luciferase activity 48 hours after transfection.

RIP assay was performed by using a Magna RIP RNA-Binding Protein
Immunoprecipitation Kit (Millipore) according to the manufacturer's instructions.

In vivo metastasis mice model
All procedures involving mouse were approved by the Institutional Animal