Dysregulation of p53-RBM25-mediated circAMOTL1L biogenesis contributes to prostate cancer progression through the circAMOTL1L-miR-193a-5p-Pcdha pathway

p53, circRNAs and miRNAs are important components of the regulatory network that activates the EMT program in cancer metastasis. In prostate cancer (PCa), however, it has not been investigated whether and how p53 regulates EMT by circRNAs and miRNAs. Here we show that a Amotl1-derived circRNA, termed circAMOTL1L, is downregulated in human PCa, and that decreased circAMOTL1L facilitates PCa cell migration and invasion through downregulating E-cadherin and upregulating vimentin, thus leading to EMT and PCa progression. Mechanistically, we demonstrate that circAMOTL1L serves as a sponge for binding miR-193a-5p in PCa cells, relieving miR-193a-5p repression of Pcdha gene cluster (a subset of the cadherin superfamily members). Accordingly, dysregulation of the circAMOTL1L-miR-193a-5p-Pcdha8 regulatory pathway mediated by circAMOTL1L downregulation contributes to PCa growth in vivo. Further, we show that RBM25 binds directly to circAMOTL1L and induces its biogenesis, whereas p53 regulates EMT via direct activation of RBM25 gene. These findings have linked p53/RBM25-mediated circAMOTL1L-miR-193a-5p-Pcdha regulatory axis to EMT in metastatic progression of PCa. Targeting this newly identified regulatory axis provides a potential therapeutic strategy for aggressive PCa.


All cells were transfected using Lipofectamine 2000 (Invitrogen/Life Technologies)
according to the manufacturer's protocol. Small interfering RNAs (siRNAs) targeting the indicated gene sequences (Supplementary Table 2) were designed and synthesized by GenePharma (Shanghai, PR China). Expression plasmids for circAMOTL1L, p53 and RBM25 are described below (vector construction).

Cell migration, invasion, and wound-healing assays
Approximately 3 × 10 4 PC3 or DU145 cells per well were plated in the upper chambers of 24-well Transwell plates (#3422; Corning) overnight and transfected with circ-pcDNA3.1-circAMOTL1L (pcDNA-circAMOTL1L) or circ-pcDNA3.1 plasmids. After 6 h, the medium was replaced with complete medium (10% fetal bovine serum, FBS), and cells were cultured for an additional 18 h. Cells remaining on the upper filter were removed; those that passed through the Transwell filter were stained using hematoxylin-eosin (HE). Images were taken from six random optical fields (200×) per filter, and cell numbers were quantified using a Leica microscope (DM6000B; Leica Microsystems, Heerbrugg, Switzerland) and digitized using LAS V.4.4 software (Leica).
To study cell invasion, Transwell membranes were coated with matrigel (Sigma-Aldrich, St. Louis, MO, USA) prior to plating cells. The matrigel served as a basement membrane barrier that cells would have to destroy before invading the lower chamber.
After a 24-hour treatment, HE staining and cell counting were performed as above. For the wound-healing assay, PC3 or DU145 cells were grown on 12-well plates and transfected with pcDNA-circAMOTL1L or circ-pcDNA3.1 plasmids. Confluent cell layers were scraped using a 200-μL pipette tip, washed twice with PBS, and cultured in media containing 10% serum for 24 h. Images were acquired using an Olympus microscope (CKX41; Olympus Corporation, Tokyo, Japan) equipped with a camera (EOS 600D; Canon Inc., Tokyo, Japan) immediately (0 h) and at 24 h after wounding.

Plasmid and lentivirus expression vector constructs
In order to overexpress a circular RNA with a seamless connection between its 5' and 3' ends without redundant nucleotides, we constructed a circ-pcDNA3.1 vector with a reverse repeat sequence combined with 5' donor splice sequences and 3' acceptor splice sequences (Supplementary Table 2). Between the donor splice site and acceptor splice site, we inserted a EcoNI enzyme site with sequences of 5'-CCTCAG ∨ CTAGG-3' and a PmlI enzyme site with sequences of 5'-CAC ∨ GTG-3'. The full length sequence of circAMOTL1L was amplified from cDNA and inserted to EcoNI (NEB) and PmlI (NEB)-digested circ-pcDNA3.1 with one-step cloning (C112-02; Vazyme Biotech Co., Ltd.). Sequences of WT or mutant circAMOTL1L or the 3' untranslated region (UTR) sequences of Pcdha8 containing WT or mutated miR-193a-5p binding site were inserted into the pmir-GLO Dual-Luciferase miRNA Target Expression Vector (Promega Corp., Madison, WI, USA). All plasmids were sequenced for confirmation. All oligos used as primers for plasmid construction and probe preparation, siRNA, and biotinylated-oligos are listed in Supplementary Table 2.

Xenograft animal model
All animal studies were approved by the Institutional Animal Care and Use Committee   Table 2) and the internal control U6, according to the manufacturer's protocol. For large mRNA, cDNA was synthesized using a M-MLV First Strand Kit (Life Technologies) with random hexamer primers. mRNAs or circRNAs were subjected to qRT-PCR using the Platinum SYBR Green qPCR Super Mix UDG Kit (Invitrogen) and the ABI 7500 FAST system (Life Technologies). Relative transcript expression levels were normalized to GAPDH and calculated using the 2 −ΔΔCt formula as previously described (2). Supplementary Table 2 summarizes the primer sequences.

Western blot analysis
Western blotting was carried out using a standard protocol as previously described (2,3). The total protein from tumor tissues were extracted by homogenization, and cultured cells were lysated with lysis buffer (1% Triton X-100, 150 mM NaCl, 10

Morphometry and histology
Human PCa and BPH tissues were fixed in 10% neutral buffered formalin solution and then processed for routine embedding in paraffin. Ten consecutive 5-μm-thick sections were prepared for hematoxylin and eosin staining. PCa cross-section images were acquired using a Leica digital pathology scanner (Aperio CS2, Switzerland) and digitized with Image-Scope-Rev-v12.1.0.5029 (Leica).

Fluorescence In situ hybridization
In situ hybridization was performed as described previously (3). In brief, cells were fixed in 4% paraformaldehyde for 5 min at room temperature and subsequently washed with PBS. Paraffin-embedded cross-sections (5-μm-thick) from clinical tissues were deparaffinized and rehydrated for in situ hybridization according to the miRCURY LNA TM microRNA ISH Optimization Kit manual (Exiqon, Vedbaek, Denmark).
Hybridization with fluorescence-labeled circAMOTL1L or miR-193a-5p probes (Supplementary Table 2) in hybridization buffer (Exiqon) was performed during a 1-h incubation at 55°C in a thermoblock (Labnet, Edison, NJ, USA). After stringent washing with SSC buffer, samples were treated with DAPI (157574, MB biomedical) for nuclear counterstaining. Images were captured using a confocal microscope (DM6000 CFS; Leica) and processed using LAS AF software. Further images were acquired using a Leica DM6000B microscope and digitized using LAS V.4.4 software.

Immunofluorescence staining
Immunofluorescence staining was performed as described previously (2)(3)(4). In brief, five-micrometer paraffin-embedded cross-sections of tissues or cultured PC3 cells were subjected to immunofluorescence staining. Sections were deparaffinized with xylene, rehydrated, and pre-incubated with 10% normal goat serum (710027, KPL, USA) followed by incubation with the following primary antibodies: anti-E-cadherin Biomedical) was used for nuclear counterstaining. Images were captured using a confocal microscope (DM6000 CFS; Leica) and processed using LAS AF software.

Biotinylated-oligo pulldown for RNAs or circAMOTL1L-binding proteins
To detect interactions between circAMOTL1L and miRNAs, a biotin-oligo pulldown was performed as previously described, with some modifications (5). Briefly, log-phase cells were cross-linked with 1% glutaraldehyde or formaldehyde in PBS for 10 min at room temperature; the cross-linking was then quenched with 0.125 M glycine for 5 min. The

Northern blotting
Northern blot analysis of circRNAs or linear RNAs was performed as described previously (2). PCR primers including T7 promoter sequences were designed to target the linear transcript of AMOTL1, or the circAMOTL1L transcript exclusively (AMOTL1: 603 bp; circAMOTL1L: 421 bp). Supplementary Table 2

RNA immunoprecipitation (RIP) assays
PC3 cells were co-transfected with pcDNA3.1-RBM25 and pcDNA3.1-circAMOTL1L for 48 h, and then cells were used to conduct RIP experiments using a RBM25 antibody Technologies) reader 24 h after transfection. The specific activity was expressed as the relative activity ratio of firefly to Renilla luciferase. All promoter constructs were evaluated in a minimum of three separate wells per experiment.

Chromatin immunoprecipitation (ChIP) assay
The chromatin immunoprecipitation (ChIP) assay was performed as described previously (2). In brief, PC3 cells were treated with 1% formaldehyde for 10 min to cross-link proteins with DNA. The cross-linked chromatin was then prepared and sonicated to an average size of 400-600 bp. The samples were diluted 10-fold and then precleared with protein A-agarose/salmon sperm DNA for 30 min at 4 °C. The DNA fragments were immunoprecipitated overnight at 4 °C with anti-p53, or anti-IgG (as negative control) antibodies. After cross-linking reversal, p53 occupancy on the RBM25 promoter was examined. Results were determined by qRT-PCR. ChIP primer sequences were summarized in Supplementary Table 2.

Deletion of a target gene by CRISPR/Cas9 technology
For the CRISPR/Cas9 assay, sgRNA design and cloning into retroviral construct were carried out as described previously (2,6). In brief, sgRNAs were constructed by annealing each pair of oligos (Supplementary Table 2) and ligating them to BsaIlinearized SaCas9 (Plasmid#61591, Addgene). The vectors were confirmed by sanger sequence. PC3 cells were seeded in 6-well plates at a density of 2 × 10 5 cells per well.
After 24 h, the cells were transiently transfected with 2 μg CRISPR/Cas9 plasmid using Lipofectamine 2000 (Life Technologies). Genomic DNA and total RNA were extracted after 72 h of transfection using DNA Extraction Kit (Omiga) or QIAzol Lysis Reagent (79306). PCR was conducted to amplify the targeting region by using DNA template.
The expression of circAMOTL1L was detected by qRT-PCR.

p53 knockout and RNA-Seq analysis
The p53 gene in PC3 cells was knocked out (p53-KO) by CRISPR/Cas9 technique as described previously (7). In brief, PC3 cells were transfected with constructs expressing Cas9-D10A (Nickase) and control sgRNAs or sgRNAs targeting p53 exon 3 (sc-437281 for control; sc-416469-NIC for targeting p53). After 48 h of transfection, cells were suspended, diluted and re-seeded to make sure single clone formation. GFP positive clones were picked up, and the expression of p53 in each single clone was evaluated by Western blotting with p53 (sc-126) antibody. The stable clones were seeded again in 10-cm plates at 3-5 days after culture, when the cells reached 50% confluency, the cells were washed with ice-cold PBS twice and RNA extraction was To further analyze the differentially expressed transcripts in a more reliable interval, the following filter strategies were applied: 1) the average of FPKM in either sample group > 0.1; 2) the fold change between Ctl group and p53 group >2; 3) p value between Ctl group and p53 group <0.01. RNA-seq data have been deposited into NCBI Gene Expression Omnibus (GEO) with accession number GSE113180.