noncoding effects of circular rna ccdc66 promote colon ... · novel oncogenic function of circrna...

Molecular and Cellular Pathobiology

Noncoding Effects of Circular RNA CCDC66Promote Colon Cancer Growth and MetastasisKuei-Yang Hsiao1, Ya-Chi Lin2, Sachin Kumar Gupta3, Ning Chang1,Laising Yen3, H. Sunny Sun2, and Shaw-Jenq Tsai1

Abstract

Circular RNA (circRNA) is a class of noncoding RNA whosefunctions remain mostly unknown. Recent studies indicate circ-RNAmay be involved in disease pathogenesis, but direct evidenceis scarce. Here, we characterize the functional role of a novelcircRNA, circCCDC66, in colorectal cancer. RNA-Seq data frommatched normal and tumor colon tissue samples identifiednumerous circRNAs specifically elevated in cancer cells, severalof which were verified by quantitative RT-PCR. CircCCDC66expression was elevated in polyps and colon cancer and wasassociated with poor prognosis. Gain-of-function and loss-of-

function studies in colorectal cancer cell lines demonstrated thatcircCCDC66 controlled multiple pathological processes, includ-ing cell proliferation, migration, invasion, and anchorage-inde-pendent growth. In-depth characterization revealed thatcircCCDC66 exerts its function via regulation of a subset ofoncogenes, and knockdown of circCCDC66 inhibited tumorgrowth and cancer invasion in xenograft and orthotopic mousemodels, respectively. Taken together, these findings highlight anovel oncogenic function of circRNA in cancer progression andmetastasis. Cancer Res; 77(9); 2339–50. �2017 AACR.

IntroductionColorectal cancer was the third most common cancer with

approximately 1.4 million new cases in 2012 (1) and was rankedas the second leading cause of cancer-related death in the UnitedStates (2). Despite intensive investigations and therapeuticimprovements, over 50%of patients with colon cancer ultimatelydie from this disease, which highlights the necessity to unravelother unknown mechanisms contributing to colon cancermalignancy.

Circular RNA (circRNA), a class of RNAmolecules with circularconfiguration formed by either typical spliceosome-mediated orlariat-typed splicing between an upstream splice acceptor and adownstream splice donor, was recently found to be widely spreadin eukaryotes (3, 4). Circular RNA arises from exonic, intronic,and intergenic regions (3). The exonic circRNAs (exclusivelyhaving exon sequences) typically reside in the cytoplasm whileexon intronic circRNAs (intron-retaining circRNAs) remain innuclei (5). The exon-containing circRNAs are the end productsof splicing and are the most studied groups to date.

The biogenesis of circRNAs involves several distinct mechan-isms. An early studyproposes that the accumulationof circRNAs iscaused by the retardation of cell proliferation because circRNAsare passively diluted in proliferative cells (6). Others propose thatthe biogenesis of circRNAs is facilitated by an RNA bindingprotein like Quaking (7). The formation of exonic circRNAs isfacilitated by complementary sequences in the flanking introns(8–10) and by proteins modulating the proper interactionbetween upstreamand downstream introns (7, 11–13).However,the roles of these proteins in circRNA dysregulation in cancer havenot been fully characterized.

In addition to thebiogenesis of circRNA, genome-wide analysesrevealed that circRNAs are differentially expressed in variouscancerous tissues/cell lines. It has been reported that cancerouscell lines tend to express a more diverse pattern of circRNAscompared with noncancer cell lines (14). Bachmayr-Heyda andcolleagues reported that the overall levels of circRNAs wereglobally downregulated in colorectal and ovarian cancers andwere negatively correlated with the disease status and prolifera-tion (6). In adherent to Bachmayr-Heyda's study, two recentstudies also demonstrated that circITCH and circFOXO3 aredownregulated in esophageal squamous cell carcinoma and inabreast cancer cell line, respectively (15, 16). In contrast, the levelsof several circRNAswere elevated during the process of epithelial–mesenchymal transition (7) and in cultured primary endothelialcells (17). These studies demonstrate that the expression ofcircRNA is tightly regulated under distinct circumstances, andthat investigation into circRNA is still in its infancy, and yet theroles of circRNA in cancer are not fully characterized.

Despite the negative correlation between the overall circRNAexpression levels and normal/cancer status, the biological orpathological functions of individual circRNA in aparticular cancerremain largely unknown. CircRNAs are suggested to serve asmicroRNA (miRNA) sponges, regulators of splicing, and as tran-scription regulators (5, 13, 18, 19); nonetheless, the experimentaldata supporting these hypotheses are scarce. Herein, we aimed to

1Department of Physiology, College of Medicine, National Cheng Kung Univer-sity, Tainan, Taiwan. 2Institute of Molecular Medicine, College of Medicine,National Cheng Kung University, Tainan, Taiwan. 3Department of Pathologyand Immunology, Baylor College of Medicine, Houston, Texas.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Shaw-Jenq Tsai, National Cheng Kung University, 1University Road, Tainan 70101, Taiwan. Phone: 886-6-2353535, ext. 5426; Fax:886-6-2362780; E-mail: [email protected]; and H. Sunny Sun, Instituteof Molecular Medicine, College of Medicine, National Cheng Kung University, 1University Road, Tainan 70101, Taiwan. Phone: 886-6-2353535, ext. 3645; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-16-1883

�2017 American Association for Cancer Research.

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identify circRNAs that are clinically relevant to colorectal cancerand to investigate the roles of these circRNAs in colorectal cancerpathogenesis.

Materials and MethodsPatient enrollment and tissues collection

This study was approved by Institutional Reviewing Board ofNational Cheng Kung University Hospital and informed con-sent was acquired from each patient. Tissues were collectedfrom patients with colorectal cancer undergone surgery at theDepartment of Surgery at the National Cheng Kung UniversityHospital, and the nontumorous tissues were collected at the site10 cm away from the affected region. The stages and classifi-cation of collected tumors were histologically verified bypathologists.

RNA sequencingTotal RNA isolated from 12 paired tumor/adjacent nontu-

mor specimens was subjected to ribosomal RNA (rRNA)removal using the RiboMinus technology (Invitrogen). TherRNA-depleted RNA sample was quantified by the Agilent2100 Bioanalyzer, and used to construct cDNA template bythe SOLiD Total RNA-Seq Kit according to the manufacturer'sinstructions. In brief, the RNA was fragmented using RNase IIIhydrolysis followed by ligation with strand-specific adaptersand reverse transcribed to generate cDNA. Fragments of cDNAbetween 150 and 250 nucleotides were subsequently isolatedby electrophoresis in 6% urea-TBE acrylamide gel. The gel-isolated cDNA was amplified with 15 cycles of PCR to produceenough templates for the SOLiD EZ Bead system to generatetemplate bead library for ligation-based sequencing on theSOLiD Sequencer System. The sequencing raw data ran throughthe SOLiD Accuracy Enhancement Tool (Life Technologies) forcolor space correction (20). The color space reads were alignedto the human reference genome (hg19, http://genome.ucsc.edu) using the mapping algorithm implemented in the RNA-Seq pipeline of LifeScope Genomic Analysis Software (LifeTechnologies). The mapped reads were reported in BAM filesand used in the following bioinformatic analysis developed inhouse for circular RNA identification.

Bioinformatic identification of circRNAsThe steps for identification of circRNAs were illustrated

in Fig. 1A. Briefly, the first 25 bp and last 25 bp were extractedfrom the BAM files of hard-clipped reads (with H in CIGARstring) and unmapped reads and converted to fastq format,followed by second round of mapping to human genome(hg19) using CLC Genomics Workbench (QIAGEN). The pairsof half-reads mapped to the same genes were used for furtheranalyses. To identify the backsplice junctions in the full-lengthreads, the exon positions for the pairs of half-reads having thesame gene ID with reversed order in the genomic coordinatewere used to preassemble reference sequences as backsplicedexons. Full-length read sequences matched to the preassembledreference sequences, either one alignment in the preassembledexons or two alignments in a single exon (Supplementary Fig.S1), using BLAST2 (parameters: bl2seq -F F -U T -m T -T F -q -2-W 28 -G 0 -p blastn -D 1) were considered as the circRNAcandidates. The rest of full-reads either with zero or multiple(>2) matches were realigned to full length of mRNA, and

considered as the circRNA candidates if the matched positionshave reversed order.

Analysis of miRNA targeting sitesThemiRNA binding sites in circRNAs were analyzed bymiRan-

da (Version 3.3a, parameter: -strict) using the miRNA list frommiRBase 21 (downloaded in February 2015; refs. 21, 22), whilemiRTarBase (Release 6.0; ref. 23) was used to identify the miRNAtargeting sites in 30-UTR for genes.

Gene set enrichment analysisThe Gene Set Enrichment Analysis was performed according to

a previous study (24). The colorectal cancer RNA-Seq data wereused as expression profile, and the differentially expressed geneswith miRNA sites identified in circCCDC66 (designatedcircCCDC66-regulated genes) were inputted as the gene set forenrichment analysis. Similarly, the gene set of differentialexpressed genes without miRNA sites identified in circCCDC66was used as non–circCCDC66-regulated ones.

RNA interference knockdownSpecific knockdown of circCCDC66 was achieved using two

siRNA oligonucleotides designed and synthesized by Dharmaconto target the backsplice junction (siJCT1 and siJCT2, see sequencesin Fig. 4A and Supplementary Table S1). HCT116 or HT-29 cellswere transfected with desired siRNA oligonucleotides at a finalconcentration of 50 nmol/L, using Lipofectamine 2000 (LifeTechnologies) reagent, according to the manufacturer's instruc-tions. Expression of both linear and circular CCDC66 transcriptswas quantified using RT-qPCR.

Molecular cloning of circRNA for overexpression andvalidation

To validate the backsplice junction, a set of primers spanningthe splice site was design for each circRNA candidate (Supple-mentary Table S2). Amplicon with expected size was cloned topCRII (Invitrogen) and sequenced. pCIRC2, the circular RNAexpression plasmid, was developed by the Yen lab (detail to bepresented elsewhere). For overexpression of circCCDC66_10_8,we first used pCIRC2 vector to include part of the intronsequences from CiRS-7 for efficient circularization of tran-script. The sequence of exons 8 to 10 was amplified usingPCR and cloned to pCIRC2 through MfeI and XmaI sites,followed by reconstitution of the intron/exon junction using30 SS F/R and 50 SS F/R primers (see Supplementary Table S3for primer sequences). The full length of CCDC66 exons 8–10and the flanking intronic sequence were verified using Sangersequencing.

Xenograft and orthotopic models of colorectal cancer inmice

NOD/SCID mice (NOD.CB17-Prkdcscid/NcrCrl) were pur-chased from the Animal Center at the College of Medicine,National Cheng Kung University, and approval for the experi-ments was obtained from the institutional animal care and usecommittee. HCT116 cells (1� 106 cells) transfected with controloligonucleotides or oligonucleotides against circCCDC66 wereresuspended inmatrigel (BD, 354230; cells inmedium:matrigel¼1:1) and injected either subcutaneously into the dorsal flank orinto the surface of cecum of 8 weeks old mice. After 4 weeks, themice were sacrificed, xenografted tumors, cecum, colon, and liver

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were collected for inspection using hematoxylin and eosin stain-ing and immunohistochemical staining.

Statistical analysesThe data were expressed as means � standard deviation of the

mean in the bar charts and were analyzed by either Student t test(two samples) or one-way analysis of variance (ANOVA) usingGraphPad Prism 5.0 (GraphPad Software, Inc.). The categoricaldata were analyzed by the c2 test. Results with P values less than0.05 were considered statistically significant. The receiver oper-ating characteristic (ROC) analysis was applied to evaluate thepower of expression of circCCDC66 as a prognostic marker of

colorectal cancer, and the area under curve (AUC) was calculatedin Prism 5.0.

ResultsDifferential expression of circRNAs in colorectal cancer tissues

To identify the expression of circular RNAs in clinical samplesfrom patients with colorectal cancer, we applied an in-housedeveloped pipeline to consider that backspliced exons typicallycaused the problems to the reads aligner—typically unmapped orpartially mapped (clipped reads), thus we focused on thoseunmapped reads and reads with hard clipping (which indicates

Figure 1.

Expression of circRNAs in colorectal cancer. A, The flowchart delineates the steps used to extract the backsplice events from the RNA-Seq data. B, Distribution ofexonic circRNAs based on their location in gene body.C,RNA fromHCT116 andHT-29were treatedwithout orwith RNaseR (�/þ) prior to reverse transcription.�RT,reverse transcriptionwithout reverse transcriptase. NC, negative control for PCR.D,Results of RT-qPCR for circCCDC66_10_8, circCCNB1_7_6 and circCDK13_2_2 innontumor (N)/tumor (T) paired specimens from colorectal cancer patients (n ¼ 48). E, Representative images of PCR products from denoted circRNAs. 18S,18S rRNAas internal control. The bottom image shows the PCR amplicon of linear transcript of exons 6 to 11 of CCDC66 and exon-skipped transcript (not detected). F,RT-PCR results for circCCDC66 from indicated cell lines. G, Levels of small nucleolar RNA (RNU6B, as positive control for nuclear fraction), rRNA (positivecontrol for cytoplasmic fraction), and circRNAs fromnuclear and cytoplasmic fractions.H,Results of RT-qPCR for linear transcript of CCDC66 in nontumor (N)/tumor(T) paired specimens from colorectal cancer patients (n ¼ 48). I, Levels of circular and linear transcripts of CCDC66 from individual patients. J, Ratio ofcircular to linear CCDC66 transcript in HCT116 treated with normoxia or hypoxia. K, RNA stability of circular (exon 10_8), linear transcripts (exon 4/5, exon16/17) of CCDC66, and 18S rRNA. � , P < 0.05.

CircCCDC66 Promotes Colon Cancer Progression

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a fragment of sequence wasmissing in the reference sequence). Tofacilitate the mapping of those reads and identify the potentialbacksplice events, thefirst and last 25bpof the readswere extractedand remapped to the human genome (Fig. 1A). Pairs of half-readswere grouped "exonic" if they fall in the exon regions,while the restwere grouped "intergenic/intronic." Among the 1,645 exonic half-reads, the half-reads with reversed orders within the same genewere further manually curated and collapsed to 74 circular RNAswith canonical splice signals (Supplementary Table S4). Themajority (71.62%) of the 74 circRNA candidates sat within thecoding region, while 21.62% covered the 50-UTR and partial CDS,5.40% covered the 30-UTR and partial CDS, and 1.35% spannedfrom the beginning to the end of transcript (Fig. 1B).

As a proof-of-concept, several circRNAswere selected for furtherinvestigation. They were chosen because their parental genes areinvolved in cell-cycle regulation (CCNB1 and CDK13), signaltransduction (PLCE1 and USP3), epigenetic modulation(KDM1A and KMT2E), or transcriptional regulation (FOXK2 andBMP2K). In addition, we selected the circRNA derived fromCCDC66 because there is no known function associated with itsparental gene. The presence of these candidate circRNAs was firstvalidated with RT-PCR using outward primer pairs in two coloncancer cell lines, HT-29 and HCT116, and in a set of clinicalcolorectal cancer specimens. Circular RNAs derived from thegenes BMP2K, KMT2E, and USP3 showed multiple backspliceevents, while the rest of circular RNAs yielded a single amplicon(Fig. 1C and Supplementary Fig. S2A). Each selected circRNAcandidate was verified by omitting reverse transcriptase to excludethe potential DNA contamination and treated with RNase R priorto reverse transcription to confirm the lack of free 50- and 30-ends(Fig. 1C, top) and the levels of linear transcript were used todemonstrate the efficacy of RNase R treatment (Fig. 1C, bottom).The backsplice junctions were further confirmed by Sangersequencing of the excised bands (Supplementary Fig. S3). Next,differential expression of the circRNA candidates was quantifiedin 48 pairs of colorectal tumors and corresponding adjacentnontumor tissues. Among the investigated circRNA candidates,circCCDC66_10_8, circCCNB1_7_6, and circCDK13_2_2 weresignificantly elevated in the colorectal tumor tissues (Fig. 1D andE) while circFOXK2_3_2, circKDM1A_4_2, circKDM1A_9_2 andcircPLCE1_2_2 showed no significant difference (SupplementaryFig. S2B). Note that we used a set of inward primers to amplifyexons 6 to 11 of CCDC66 and did not detect the exon-skippedproduct (Fig. 1E, bottom), which indicates circCCDC66 is not aproduct of exon skipping.

To explore the novel functions of circular RNA, we chosecircCCDC66 (circCCDC66_10_8) for further investigation as itsparental transcript possesses no known biological function. RT-PCR results from multiple cell lines demonstrated thatcircCCDC66 were expressed in all tested colorectal (Caco-2,HCT116, HT-29, LS123), breast (MCF-7, MDA-MB-231, MDA-MB-468), pancreatic (BxPC-3, MIA PaCa-2), and cervical (HeLa)cancer cell lines except the cell line derived from normal colonepithelial cells (CCD 841 CoN), suggesting that the elevated levelof circCCDC66 may be general to tumorigenesis of these cancers(Fig. 1F). Cellular localization analysis revealed that circRNAsmainly resided in the cytoplasm (RNU6B as positive control fornuclear fraction; 18S rRNA for cytoplasmic fraction), a phenom-enon that is consistent with the property of exonic circRNAs (Fig.1G). Next, we quantified the level of the linear transcript ofCCDC66 by RT-qPCR to determine if the elevated level of

circCCDC66 is due to the higher levels of parental transcripts.Surprisingly, the level of linear CCDC66 was lower in cancer cellscompared with adjacent normal tissues (Fig. 1H). To answerwhether the low level of linear transcript is due to being processedto generate circular RNA,we analyzed the correlation of linear andcircular CCDC66 levels of each patient. The results showed thatthere is no negative correlation between the levels of circular andlinear transcripts (Fig. 1I), which suggests that the low levels oflinear CCDC66 in cancer is not caused by a high converting rate togenerate circCCDC66. Intriguingly, we found that the level oflinear CCDC66 transcript was dramatically decreased in cancercells treated with hypoxia while the level of circular CCDC66transcript remained unchanged (Supplementary Fig. S4), togethercaused the fraction of circular CCDC66 enriched (Fig. 1J). Thenature of high stability (Fig. 1K) of circular RNA might partlycontribute to this differential response to hypoxia between linearand circular transcripts. These data imply that the elevated/accu-mulated levels of circCCDC66 could be the consequence ofrecurring intratumoral hypoxic stress, which is further enhancedby the stable nature of circular RNA.

Wenext addressed the potential clinicopathologic implicationsof elevated circCCDC66 during the development of colon cancer.We found that the level of circCCDC66 was elevated in theprecancerous polyp tissues and became higher in the tumorous

Figure 2.

Expression of circCCDC66 as a prognostic marker. A, Levels of circCCDC66 innontumor (N; n ¼ 76), polyps (P; n ¼ 22), and tumor (T; n ¼ 131) tissuesfrom colorectal cancer patients. B, Levels of circCCDC66 in colorectal cancerpatients grouped by AJCC staging. C, Kaplan–Meier survival curves forpatients with low and high expression levels of circCCDC66. D, Similar to C, forlinear CCDC66 transcript. E, ROC curves of circCCDC66. � , P < 0.05.

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tissues (Fig. 2A). The level of circCCDC66 was increased in allstages of colorectal cancer tissues compared with their pairednoncancerous counterparts (Fig. 2B). Of note, patients with ahigher level of circCCDC66have a poor prognosis as evidenced bylower overall survival rates (Fig. 2C). This phenomenon is specificto circCCDC66 as the level of linear CCDC66 mRNA was notassociated with any prognostic markers (Fig. 2D, linear CCDC66transcript). To better demonstrate the circCCDC66maybeused asa diagnostic marker for colorectal cancer, the area under receiveroperating characteristic curve (AUC) using the expression levels ofcircCCDC66 was calculated. The AUC of circCCDC66 indicatedthat 88% of randomly chosen colorectal cancer patient will havehigher levels of circCCDC66 (ranked higher) than the randomlychosen normal subjects (Fig. 2E; ref. 25), suggesting the expres-

sion level of circCCDC66 is a good predictive biomarker forcolorectal cancer detection and prognosis.

CircCCDC66 promotes cancer cell proliferation, migration,and metastasis

To investigate the role of circCCDC66 in colorectal cancer, wedesigned two siRNA oligonucleotides to target the unique back-splice junction. The backsplice junction-specific siRNA (siJCT1and siJCT2) successfully knocked down circCCDC66 but had noeffect on the level of linear CCDC66mRNA (Fig. 3A). The specificknockdowns led to a significantly reduced cell number in bothHCT116 andHT-29 (Fig. 3B andC), whichwasmediated throughsuppression of cell proliferation (Fig. 3D), but not through theinduction of apoptosis (Fig. 3E). In addition to cell proliferation,

Figure 3.

CircCCDC66 is required for cell proliferation, migration, and invasion.A, The illustration shows the sequence around the backsplice junction and the siRNA targetingthe junction (siJCT1/J1 and siJCT2/J2; top). Results of RT-qPCR for circular and linear transcripts of CCDC66 in HCT116 treated with siRNA (siC1 and siC2,control oligonucleotides with scramble sequence; siJ1 and siJ2, targeting the backsplice junction). B, Representative images and quantification results ofcrystal violet staining of HCT116 treated with siRNA targeting backsplice junction for 48 hours. C, Similar to B, performed in HT-29 cells. D, Results of BrdUincorporation assay quantified by flow cytometry from HCT116 transfected with control siRNA oligonucleotide (siC1 þ siC2, denoted as siCON) or siRNA targetingbacksplice junction (siJ1 þ siJ2, denoted as siJCT) for 48 hours. E, Results of 7-AAD/Annexin V staining assay quantified by flow cytometry from HCT116transfected with control siRNA oligonucleotide (siCON) or siRNA targeting backsplice junction (siJCT). F, Representative images and quantification results ofmigration assays for HCT116 andHT-29 cells transfectedwith siCONor siJCT oligonucleotides.G, Invasion assays for HCT116 andHT-29 cells transfectedwith siCONorsiJCT oligonucleotides. � , P < 0.05.






our data demonstrated that knockdown of circCCDC66 signifi-cantly reduced cell migration as assayed by the number of cellsmigrating through the Transwell membrane and also reducedinvasion as assayed by the number of cells traveling through thematrigel in both HCT116 and HT-29 cells (Fig. 3F and G).

To further characterize the role of circCCDC66, we usedpCIRC2, a circRNA expression vector, to express circRNA withexons 8 to 10 of CCDC66 (Fig. 4A). The plasmid was firsttransfected to 3T3L1 cells, a mouse cell line without detectablecircCCDC66, to demonstrate the circularization of designedconstruct (Fig. 4A and B). The specificity of pCIRC2 was furtherconfirmed by mutating splice signals (mutated splice acceptor/mSA and mutated splice donor/mSD), which abolished theexpression of circCCDC66 (Fig. 4A and B). Transfection of

pCIRC2-CCDC66 e8-10 led to the expression of circCCDC66as confirmed by RT-PCR and Sanger sequencing of the back-spliced RNA junction (Fig. 4B). Next, pCIRC2-CCDC66 e8-10was transfected to HCT116 cells and the expression level ofcircCCDC66 was quantified (Fig. 4C and D). Overexpression ofcircCCDC66 significantly increased the number of cell coloniesin the soft agar as compared with the circular mCherry RNA thatwas used as a control (Fig. 4E, upper). In addition, the BrdUincorporation assay showed an increased number of BrdU-positive cells overexpressing circCCDC66 (Fig. 4E, bottom),suggesting that circCCDC66 stimulates DNA synthesis. Impor-tantly, the overexpression of circCCDC66 had no effect on thelevels of the endogenous parental linear CCDC66 transcript,which was measured using two independent sets of inward

Figure 4.

Expressionof circCCDC66promotes cell proliferation.A,Primer locationCCDC66gene (top), circCCDC66 (middle), andpCIRC2 expression vector.—, inwardprimersfor linear transcript; *, outward primers for circular transcript. B, Representative images for RT-PCR results from 3T3L1 cells without transfection (no tfx),with transfection of pCIRC2-mCherry (CON) or pCIRC2-CCDC66 exons 8–10. 18S, 18S rRNA. C, Representative images for RT-PCR results from HCT116 cellstransfected with pCIRC2-mCherry (CON), pCIRC2-CCDC66 with native splicing signals (wild-type, WT), or mutated splice signals as indicated in Fig. 5A (middle).D, Results of RT-qPCR for circular and linear transcripts of CCDC66 in HCT116 with/without overexpression of circCCDC66. E, Representative images andquantification results of soft agar assay (upper) and BrdU incorporation assay (middle and bottom) for HCT116 overexpressing circCCDC66 (exons 8–10).BrdU-positive cells were assessed using immunofluorescent staining (green, BrdU; blue, DNA counterstaining with Hoechst 33258) and flow cytometry.� , P < 0.05.

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primers designed to amplify the linear transcripts (Fig. 4C andD, center and right).

CircCCDC66 may serve as a miRNA spongeTo explore the molecular function of circCCDC66, we per-

formed amiRNA target sequence analysis using CCDC66 exons 8to 10. The analysis revealed that this region was potentiallytargeted by 99 miRNAs (Fig. 5A and Supplementary Table S5).Unlike miR-7, which has multiple binding sites in CDR1as (19),most of miRNAs targeting circCCDC66 have single binding siteexcept miR-670 and miR-4326 (Supplementary Table S5). Thedistribution of potential miRNA binding sites was illustrated andsummarized as cumulative counts at positions where miRNAsbind (Fig. 5B). This prompted us to hypothesize that circCCDC66may function as a miRNA sponge to protect a specific group ofoncogenes from attacks by miRNAs in colorectal cancer. Amongthese 99 miRNAs, 62 are expressed in colorectal cancer tissuesaccording to three independent genome-wide studies—GSE63119, GSE46622, and GSE43793 (Fig. 5A, left; refs. 26–28). Of special note, there were many miRNAs (high-numbered)registered after these three genome-wide studies were conducted;thus, the colorectal cancer–expressed miRNAs may be under-estimated in these three datasets. To test the miRNA spongehypothesis, we filtered oncogenes and tumor suppressor geneswith significant changes in colon cancer (fold change > 1.5, FDR <0.4, and P < 0.05) and inspected whether these genes could bepredicted as targets of miRNAs that have matching sequences incircCCDC66 (Fig. 5A, right). The results revealed that 37.5% ofoncogenes have the targeting sites of those colorectal cancer–expressed miRNAs (circCCDC66.CRC.miRNAþ), while only23.5% of the tumor suppressor genes contain such sites (Fig.5C and Supplementary Fig. S5A, top). This phenomenon ofpreferential regulation on oncogenes is not restricted to ourRNA-Seq expression profile because a similar result was observedwhen using an independent dataset of colorectal cancer fromGene Expression Omnibus (GSE32323; Fig. 5C and Supplemen-tary Fig. S5A, bottom). In contrast, when applying the sameanalyses using 5000 randomly selected miRNAs and the afore-mentioned colorectal cancer expression profiles, no differencewas found between oncogenes and tumor suppressor genes,suggesting that randomly selected miRNAs have no regulatorypreference on oncogenes (Fig. 5D). To test whether the predictedcircCCDC66-regulated genes are enriched in the tumor transcrip-tome, gene set enrichment analysis (GSEA) was performed andthe results indicated that predicted circCCDC66-regulated genes(by using colorectal cancer expressing miRNAs) were enriched inthe tumor transcriptome (Fig. 5E, left), while non–circCCDC66-regulated genes showedno enrichment (Fig. 5E, right). Consistentresults were observed when using circCCDC66-regulated genes(predicted by using the full set of miRNAs; SupplementaryFig. S5B).

The circCCDC66-miRNAs regulatory network is a critical com-ponent for oncogene activation. Using this regulatory axis ofcircCCDC66–miRNAs–oncogenes, we revealed that manycircCCDC66-regulated oncogenes are upregulated in the colorec-tal cancer tumor specimens (Fig. 6A). As a proof of concept, wemanipulated the levels of circCCDC66 using overexpression andknockdown approaches and investigated the expression levels ofseveral candidate oncogenes that are known to play pivotal rolesin colorectal cancer. The levels of DNMT3B, EZH2, MYC, andYAP1, which are the targets of the predicted miRNAs, were found

to be significantly upregulated by circCCDC66 overexpression(Fig. 6B) or downregulated by knockdown (Supplementary Fig.S6A). In contrast, EFNA4 and FGF9, which are not targeted by thepredicted miRNAs, were not affected (Fig. 6B and SupplementaryFig. S6A). Coherently, we found that the MYC protein level wasincreased by the overexpression of circCCDC66 in HCT116 whiledecreased by circCCDC66 depletion (Fig. 6C and SupplementaryFig. S6B). To further consolidate the role of circCCDC66 as amiRNA sponge, we developed a novel in vitro transcriptioncombined with in vivo circularization protocol to testcircCCDC66's miRNA sponge effect (Fig. 6D, top). The in vitrotranscribed biotinylated linear RNA was efficiently circularized incells (Fig. 6D, top), and was used to pull down potential miRNAs.As a proof of concept, miR-33b, miR-93, and miR-185, threemiRNAs predicted to be sponged by circCCDC66, were tested.Results demonstrated that significant levels of predicted miRNAswere pulled down by biotinylated circCCDC66 (Fig. 6E, top). Incontrast, the levels of unrelated miRNAs, miR-22, miR-148a, andmiR-152 were not enriched (Fig. 6E, bottom). In addition, wecloned the 30-UTR of human MYC gene to the downstream ofrenilla luciferase (Fig. 6F). The MYC 30-UTR activity was signifi-cantly increased by the overexpression of circCCDC66 (Fig. 6G)and suppressedby circCCDC66knockdown(Fig. 6H). In contrast,the nontargeted FGF9 30-UTR reporter activity was not affected bythe overexpression (Fig. 6G) or knockdown of circCCDC66(Supplementary Fig. S6C), further consolidating the hypothesisthat circCCDC66 regulates gene expression as miRNA sponge. Toinvestigate whether this protective effect of circCCDC66 on MYC30-UTR activity ismediated through sponging outmiR-33b and/ormiR-93, inhibitors of two miRNAs were administered to the cellswith circCCDC66 knockdown. Knockdown of circCCDC66released miR-33b and miR-93, which then targeted the 30-UTRof MYC but not FGF9 transcript and caused the reduction ofluciferase activity. Administration of inhibitors of miR-33b ormiR-93 relieved the suppressive effect of circCCDC66 knock-down on MYC 30-UTR activity but had no effect on FGF9 30-UTR activity (Fig. 6H and Supplementary Fig. S6C). Lastly,levels of MYC transcripts were quantified by RT-qPCR to testwhether a similar phenomenon can be observed in endogenousmRNA. As shown in Fig. 6I, knockdown of circCCDC66 (releaseof miR-33b and miR-93) inhibited MYC mRNA expressionwhile administration of miR-33b and miR-93 inhibitors abol-ished circCCDC66 knockdown effect. Taken together, thesedata support the idea that circCCDC66 functions as miRNAsponge to protect the MYC mRNA from the attack of miRNA-33b and miR-93.

CircCCDC66 promotes cancer cell proliferation, migration,and metastasis

To investigate the potential clinical relevance of circCCDC66 invivo, HCT116 cells with or without transient circCCDC66 knock-down were first injected subcutaneously into dorsal flanks ofNOD/SCID mice and allowed to proliferate for 4 weeks. Thegrowth of tumors derived fromHCT116with circCCDC66 knock-downwas significantly inhibited (Fig. 7A andB). Interestingly, theexpressionof circCCDC66 in xenograftedHCT116 tumor cells hasreturned to the pre-knockdown level as examined at the end ofweek 4 (Supplementary Fig. S7), whereas the level of MYCremained low (Fig. 7C), suggesting that suppression ofcircCCDC66 at early stage may have great impact on tumorgrowth. Furthermore, HCT116 cells with or without transient






circCCDC66 knockdown were inoculated orthotopically in thececum of NOD/SCIDmice and allowed to proliferate for 4 weeks(Fig. 7D, top). The tumors formed by the HCT116 cells with theknockdown control aggressively disrupted the lining of smoothmuscles and intercalated with the muscle cells (Fig. 7D, bottomleft). In contrast, tumors derived from the cells with thecircCCDC66 knockdown typically retained a distinct boundaryalong the edge of the smoothmuscle layer (Fig. 7D, bottom right).The ability of HCT116 cells to form nodules in the liver, presum-ably due to themetastasis of the tumor from the cecum to the liver,was significantly reduced with circCCDC66 knockdown (Fig. 7E).Immunohistochemical staining using an antibody specific forpan-keratin showed that the metastasized cells were indeed epi-thelial cells but not hepatocytes (Fig. 7F).

DiscussionCircRNAs are a group of noncoding RNAs with largely

unknown biological functions. In this study, we found that theexpression of several circRNAs is enriched in colorectal tumorspecimens. Among them, circCCDC66, a circRNA derived from agene with unknown molecular functions, was upregulated in allstages of colon cancer and negatively correlatedwith prognosis. Invitro and in vivo studies showed that circCCDC66 accumulated

under hypoxia and possesses oncogenic capability in terms ofpromoting cell proliferation, migration, and metastasis. Bioin-formatic and genome-wide analyses revealed that circCCDC66may preferentially protect a group of oncogenes from beingattacked by a panel of miRNAs and these circCCDC66-protectedgenes are positively correlated with colon cancer status. To ourknowledge, this is the first report that thoroughly investigates theexpression, regulation, function, and clinical implication of cir-cRNA, circCCDC66, in human cancer.

The expression of circRNAs in human diseases is an emergingresearch topic. One pioneer study showed that the molecularsignatures of circRNA among different types of cancer cell lines arehighly diverse compared with normal cell lines (14). A few earlierstudies reported that circRNAs are downregulated in cancer cells(29–31) while more and more recent studies demonstrated anenrichment of certain circRNAs in several types of human cancer(7, 32–35). To investigate the expression and functionof circRNAsin colon cancer pathogenesis, we performed RNA-Seq analysis of12 pairs of colorectal cancer and adjacent nontumor samples.Seventy-four novel exon-containing circRNAs were identified. Byusing RT-qPCR quantification, we showed that the expression of apanel of novel circRNAs including circCCDC66, circCCNB1, andcircCDK13 was elevated in colorectal cancer cell lines as well as inclinical specimens. RNase R treatment, sequencing verification,

Figure 5.

CircCCDC66 regulates tumor transcriptome as a miRNA sponge. A, Schematic flowchart shows the pipelines for miRNA analyses. B, Cumulative counts of miRNAtargeting sites in the region of CCDC66 exons 8–10. C and D, Differential expressed oncogenes and tumor suppressor genes (TS) identified in our RNA-Seq result(colorectal cancer RNA-Seq) or GSE32323 dataset were inspected whether having targeting sites of colorectal cancer–expressing miRNAs identified fromcircCCDC66 (circCCDC66.CRC.miRNA�/þ) or targeting sites of random miRNA set (rand.miRNA�/þ). The percentages shown in the result of random set were theaverages from 5,000 times of permutation, and the numbers of genes were calculated accordingly. E, The result of Gene Set Enrichment Analysis from thecircCCDC66-regulated genes in the tumor/nontumor transcriptomes (left) or from non–circCCDC66-regulated genes (right).

Hsiao et al.





and cellular localization analysis confirmed that they are bona fideexonic circRNAs. Our finding is consistent with the resultsobserved in TGFb-treated human mammary epithelial cells (7)and in hypoxia-treated human umbilical venous endothelial cells(17), which suggest that circRNAs are regulated by complicatedmechanisms in disease- andmicroenvironment-specificmanners.

Although the biogenesis and expression of circRNAs have beenintensively investigated in the past couple years, the functions ofcircRNAs in human cancers are not well understood. In esoph-ageal squamous cell carcinoma, circITCH protects its parental

transcript, ITCH, which functions as a tumor suppressor byinhibiting theWnt/b-catenin pathway-mediated cell proliferation(15). In addition, a recent study discovered that circFOXO3worksas a miRNA sponge to protect Foxo3 mRNA from miRNA attack(16). These two studies exemplify the tumor suppressive functionof circRNAs and how the loss of these circRNAs contributes totumor progression. In contrast, our current study reported thatcircCCDC66 possesses oncogenic capacity through protectingmultiple oncogenes from being attacked by a group of miRNAs.Thus, overexpression of circCCDC66 potentiates multiple tumor

Figure 6.

CircCCDC66 functions as miRNA sponge for a subset of oncogenes.A, Expression profile of circCCDC66-regulated oncogenes using the colorectal cancer RNA-Seqdata. The expression levels were expressed as z-scores in the indicated color scale. B, Results of RT-qPCR for circCCDC66-regulated oncogenes DNMT3B, EZH2,MYC, and YAP1 and non–circCCDC66-regulated genes EFNA4 and FGF9 in HCT116 with/without circCCDC66 overexpression. C, Representation images forimmunoblotting of MYC in HCT116 with or without circCCDC66 overexpression.D, The schematic diagram shows the strategy of in vitro transcription combined within vivo circularization of circCCDC66 in cells (TSS, transcription start site of T7 promoter). Results of RT-qPCR for circCCDC66 and its linear counterpart fromHCT116 transfected with indicated amount of in vitro transcribed biotinylated CCDC66 exons 8–10. E, Results of biotinylated RNA pulldown assay for indicatedmiRNAs from HCT116 transfected with/without biotinylated CCDC66 exon 8–10. F, The illustration shows the construct of MYC 30-UTR reporter and thealignment between MYC 30-UTR and indicated miRNAs. G, MYC (left) and FGF9 (right) 30-UTR reporter activity from HCT116 with/without circCCDC66overexpression.H and I,MYC 30-UTR reporter activity (H) andmRNA levels (I) fromHCT116 transfectedwith control siRNA oligonucleotide (siCON) or siRNA againstcircCCDC66 (siJCT) in the presence or absence of miRNA inhibitors to miR-33b and miR-93. � , P < 0.05.






characteristics, including proliferation, migration, and invasion.These functions greatly contribute to cancermetastasis andmalig-nancy, which supports the observation that high levels ofcircCCDC66 are associated with poor prognosis of colorectalcancer patients. It is worth noting that the relationship of highexpression levels and poor prognosis was not seen in linearCCDC66 mRNA, suggesting that it is likely mediated by thenoncoding function of circCCDC66. Indeed, gene set enrichmentanalysis revealed that most circCCDC66-protected genes areupregulated in cancer but not in normal colon cells. In vivo study

using orthotopic injection of circCCDC66 knockdown coloncancer cells showed that knockdown of circCCDC66 impairstumor nodule establishment in the liver. Taken together, ourstudy provides multiple lines of evidence to illustrate a novelmechanism of circRNA in cancer progression.

Our finding that circCCDC66 functions as an oncogenic cir-cRNA is different from the reported tumor-suppressive circRNAsin severalways (Fig. 7G). First, the expressionof linearCCDC66 intumor specimens is not correlated with circCCDC66, implyingthat the regulation ofCCDC66 transcription and the processing of

Figure 7.

CircCCDC66 is required for tumorigenesis in vivo. A, Growth curves of xenografted tumors derived from HCT116 with or without circCCDC66 knockdown (n¼ 8 foreach group). B, Tumors harvested after 4 weeks of inoculation (top), tumor volumes and weight (bottom). C, The representative images for MYC IHC (left) and theresults of scoring by two individuals (right) from tumors derived from HCT116 transfected with control oligonucleotides (siCON) or oligonucleotides againstcircCCDC66 (siJCT). D, The developed tumors from HCT116 transfected with control oligonucleotides (siCON) or oligonucleotides against circCCDC66 (siJCT) inthe mice cecum. The gross outlines of tumors are indicated in yellow (top). The representative images for hematoxylin and eosin staining for tumorsderived from HCT116 transfected with control oligonucleotides (siCON) or oligonucleotides against circCCDC66 (siJCT; bottom). T, tumor. Yellow arrowheads,tumor masses in the layers of smooth muscle; yellow dashed lines, the intact boundary between tumor mass and layers of smooth muscle. E, Grossview of tumor nodules established in liver (left) and quantification of numbers of tumor nodules in livers (right; n ¼ 4 for each group). Yellow arrowheads,tumor nodules in the liver. F, The microscopic and IHC images of liver metastasis (arrowheads, metastatic liver nodules). The number of metastatic liver nodulesis shown on the right. Scale bar, 200 mm. H&E, hematoxylin and eosin. G, Working model. �, P < 0.05.

Hsiao et al.





the circular form are two independent events. In contrast, positivecorrelationwas observed between circular and linear transcripts inthe cases of circITCH and circFOXO3 (15, 16). Second, theexpression of circCCDC66 does not affect the level of the lineartranscript in either overexpression or circCCDC66-specific knock-down experiments. This is different from the previously identifiedfunction of intron–exonic circRNA, which promotes transcriptionof parental genes (5). The lack of exon-skipped transcript (exons7–11) suggests that it is unlikely amodulator of splicing event or aproduct of exon skipping (13, 36). One may expect that theexpression of circCCDC66 would protect the parental lineartranscript frommiRNA-mediatedmRNAcleavage.However, thereis growing evidence that miRNAs targeting the coding region andthe 30-UTR have distinct effects onmRNA levels. In contrast to themiRNA binding sites in the 30-UTR, miRNA-mediated cleavage inCDS is typically hindered by translation machinery (37, 38).Nevertheless, the lack of association with the active translationmachinery may also make circRNA a better miRNA sponge ascompared with its linear transcript. Third, unlike ciRS-7, whichhas an extremely high density of targeting sites for a singlemiRNA,circCCDC66 suppresses multiple miRNAs (Supplementary TableS5) (18). Fourth, circCCDC66 impacts the expression of multiplegenes simultaneously while circITCH and circFOXO3 exert theirinfluence on the single mRNA target. Intriguingly, many of thegenes that harbor the same miRNA binding sites found incircCCDC66 are oncogenes, while only a few are tumor suppres-sor genes, implicating that circCCDC66-associated regulatorynetwork benefits the tumor development, and thus is preservedin the tumor cells (Fig. 1F). Similarly, during the preparation ofthis article, a report by Zheng and colleagues showed that cir-cHIPK3 sponges multiple miRNAs and promotes cell prolifera-tion in vitro (39), suggesting that the regulation of multiplemiRNAs by circRNA could be a general mechanism in cells.However, whether circHIPK3 is also elevated in the tumor tissues,preferentially regulates oncogenes, or promotes tumorigenesis invivo like circCCDC66 remains unsolved and warrants furtherinvestigation. Lastly, it is worth noting that the transient knock-down of circCCDC66 had a great suppressive effect on the tumorgrowth in vivo as evidenced by the return of circCCDC66 but notMYC expression in tumor cells. We reason that this might be dueto the complicated gene–gene and gene–microenvironment inter-actions during the early stage of tumor development. However,detailedmechanisms remain unknown. It would be interesting toinvestigate whether circCCDC66 exerts such effects by targetingmultiple oncogenes or epigenetic regulators and how to translatesuch findings to clinical application.

In summary, we identified a group of novel circRNAs expressedin colorectal cancer and, among these, circCCDC66 negatively

correlated with treatment outcomes. CircCCDC66 serves as asponge to protect multiple oncogenes from being attacked bymiRNAs. Our finding that one circRNA contains various bindingsites for different miRNAs reveals the complex role of circRNA incancer malignancy and highlights the importance of investigatingthe complicated circRNA–miRNA regulatory gene network incancer progression and treatment efficacy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K.-Y. Hsiao, H.S. Sun, S.-J. TsaiDevelopment of methodology: K.-Y. Hsiao, S.K. Gupta, L. Yen, H.S. SunAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.-Y. Hsiao, Y.-C. Lin, N. ChangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.-Y. Hsiao, Y.-C. Lin, N. ChangWriting, review, and/or revision of the manuscript: K.-Y. Hsiao, Y.-C. Lin,S.K. Gupta, N. Chang, L. Yen, S.-J. TsaiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y.-C. LinStudy supervision: H.S. Sun

AcknowledgmentsWe thank all the staffs in the NCKU Bioinformatics Center for assisting

the analyses of RNA-Seq data, Miss Yen-Yu Lai for GEO dataset processing,Miss Yi-Cheng Tang for animal handling, and Miss Bi-Hua Chao and Su-Chen Hung for processing clinical samples. The authors also want to showgreat appreciation to Dr. Trees-Juen Chuang (Division of Physical andComputational Genomics, Genomics Research Center, Academia Sinica)for his comments on the circRNA analyses and to Dr. Chu-An Wang(Institute of Basic Medical Sciences, College of Medicine, National ChengKung University) for kindly providing the RNA lysates from various breastand pancreatic cancer cell lines.

Grant SupportThis work was supported by the Ministry of Science and Technology of

Taiwan (MOST 104-2320-B-006-036-MY3; S.J. Tsai), the National HealthResearch Institute (NHRI-EX102-10244BI; S.J. Tsai), Top University Grant ofNational Cheng Kung University (grant # D103-35A17; S.J. Tsai), CancerPrevention Research Institute of Texas training grant (RP160283; S.K. Gupta),Duncan Cancer Center Pilot Grant, Cancer Prevention Research Institute ofTexas HIHRRA (RP160795), and National Institutes of Health (R01EB013584;L. Yen).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 10, 2016; revised August 18, 2016; accepted February 22, 2017;published OnlineFirst March 1, 2017.

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2017;77:2339-2350. Published OnlineFirst March 1, 2017.Cancer Res Kuei-Yang Hsiao, Ya-Chi Lin, Sachin Kumar Gupta, et al. Growth and MetastasisNoncoding Effects of Circular RNA CCDC66 Promote Colon Cancer

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