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Molecular Cell Biology

The Circular RNA circPRKCI Promotes TumorGrowth in Lung AdenocarcinomaMantang Qiu1,2,Wenjia Xia1, Rui Chen3,4, Siwei Wang1,3, Youtao Xu1, Zhifei Ma1,3,Weizhang Xu1,3, Erbao Zhang5, Jie Wang1,6, Tian Fang7, Jingwen Hu1,3,Gaochao Dong1,6, Rong Yin1, Jun Wang2, and Lin Xu1

Abstract

Somatic copy number variations (CNV) may drive cancerprogression through both coding and noncoding transcripts.However, noncoding transcripts resulting from CNV are largelyunknown, especially for circular RNAs. By integrating bioin-formatics analyses of alerted circRNAs and focal CNV in lungadenocarcinoma, we identify a proto-oncogenic circular RNA(circPRKCI) from the 3q26.2 amplicon, one of the mostfrequent genomic aberrations in multiple cancers. circPRKCIwas overexpressed in lung adenocarcinoma tissues, in part dueto amplification of the 3q26.2 locus, and promoted prolifer-ation and tumorigenesis of lung adenocarcinoma. circPRKCI

functioned as a sponge for both miR-545 and miR-589and abrogated their suppression of the protumorigenic tran-scription factor E2F7. Intratumor injection of cholesterol-conjugated siRNA specifically targeting circPRKCI inhibitedtumor growth in a patient-derived lung adenocarcinoma xeno-graft model. In summary, circPRKCI is crucial for tumorigenesisand may serve as a potential therapeutic target in patients withlung adenocarcinoma.

Significance: These findings reveal high expression of thecircular RNA circPRKCI drives lung adenocarcinoma tumori-genesis. Cancer Res; 78(11); 2839–51. �2018 AACR.

IntroductionCopy number variation (CNV) is a form of genomic structural

variation leading to gains and losses of DNA segments. SomaticCNVs are extremely common due to the genomic instability andplay a significant role in tumorigenesis inmany cancers, includingcolorectal, gastric, and lung cancers (1–4). During cancer devel-

opment, proliferation-related genes are often amplified, in com-parison with frequent loss of apoptosis effector genes. A largenumber of cancer-driving CNV loci that encode proteins havebeen successfully identified using high-throughput genomesequencing technologies (5, 6). Taking epithelial cancers as anexample, integrated cancer genomic analysis and transgenic ani-malmodel have confirmed somewell-known amplicons inducedproto-oncogenic proteins likeMYC (7) and PIK3CA (8), as well asdeletions induced tumor suppressor–like RB1 (9) and PTEN (10).

Oncogenic proteins are not the only entities involved intumorigenesis. Noncoding RNAs are also critical in cancer devel-opment, such as long noncoding RNA (lncRNA) PVT1 in the 8q24"genomic desert" region, lncRNA FAL in the 1q21 amplificationregion (11), recurrently deleted lncRNA-PRAL on chromosome17q13.1 (12), as well as the oncogenic miR-569 on the 3q26.2amplicon (13). These findings indicated that noncoding RNAs, asthe majority of transcriptome, may represent a large number ofunexplored targets of genomic aberrations. Therefore, furtherexploration of the hidden noncoding transcripts within recurrentCNV loci in cancers is warranted.

Circular RNAs (circRNA), a naturally occurring family of non-coding RNAs, are involved in multiple biological processes(14, 15), including cancers (16, 17). One of the earliest charac-terized circRNAs was the sex-determining region of ChrY (Sry) inmice (18), and the well-known "miRNA sponging" function hasbeen demonstrated for an antisense transcript to cerebellar degen-eration-related protein 1 (CDR1as/ciRS-7; ref. 19), which con-tains 70 binding sites for miR-7 and suppresses miR-7 activity.Several cancer-derived circRNAs include circHIPK3 in multiplecancers (16), the hepatocellular carcinoma suppressor circMTO1(20), the colon cancer progression promotor circCCDC66 (21),and gastric cancer highly expressed circPVT1 in the 8q24 ampli-fication locus (22), which is highly expressed in gastric cancer.

1Department of Thoracic Surgery, Jiangsu Cancer Hospital, Jiangsu Institute ofCancer Research, Nanjing Medical University Affiliated Cancer Hospital, JiangsuKey Laboratory of Molecular and Translational Cancer Research, Nanjing, China.2Department of Thoracic Surgery, Peking University People's Hospital, Beijing,China. 3The Fourth Clinical College of NanjingMedical University, Nanjing, China.4Department of Cardiothoracic Surgery, Taixing People's Hospital, TheAffiliatedTaixing Hospital of Yangzhou University, Taixing, China. 5Department of Epi-demiology and Biostatistics, Jiangsu Key Lab of Cancer Biomarkers, Preventionand Treatment, Collaborative Innovation Center for Cancer Personalized Med-icine, School of Public Health, Nanjing Medical University, Nanjing, China.6Department of Scientific Research, NanjingMedical University AffiliatedCancerHospital, Cancer Institute of Jiangsu Province, Nanjing, China. 7Department ofComparative Medicine, Jingling Hospital, Nanjing University School of Medicine,Nanjing, China.

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

M. Qiu, W. Xia, R. Chen, and S. Wang contributed equally to this article.

Corresponding Authors: Lin Xu, Department of Thoracic Surgery, NanjingMedical University Affiliated Cancer Hospital, Cancer Institute of Jiangsu Prov-ince, 42 Baiziting, Nanjing 210009, China. Phone: 8625-8328-4700; Fax: 8625-8364-1062; E-mail: [email protected]; Rong Yin, [email protected]; andJunWang, Department of Thoracic Surgery, PekingUniversity People's Hospital,11 South Xizhimen St., Beijing 100044, China. Phone: 8610-8832-5952; Fax: 8610-6834-9763; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-2808

�2018 American Association for Cancer Research.

CancerResearch

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However, CNV-associated circRNAs in lung cancer have beenreported rarely.

Lung adenocarcinoma is currently themost commonhistologictype of lung cancer (23). In the current study, we identified 107differently expressed circRNAs in lung adenocarcinoma by usingmicroarray, followed by an integrated analysis of published lungadenocarcinoma–specific CNV data derived from The CancerGenome Atlas (TCGA; ref. 24). We further characterized a circularRNA, termed as circPRKCI, produced from the PRKCI gene at3q26.2 amplicon. Subsequent biotin-coupled miRNA pulldownexperiment revealed circPRKCI could function as a sponge forboth miR-545 and miR-589, thus increasing expression levels ofE2F7 and promoting tumorigenesis of lung adenocarcinoma.

Materials and MethodsPatients and tissue samples

All primary lung adenocarcinoma tissues and adjacent nontu-mor tissues were collected from patients who had undergonesurgery at the Department of Thoracic Surgery, Nanjing MedicalUniversity Affiliated Cancer Hospital (Nanjing, China). All tumorsand paired nontumor tissues were confirmed by experiencedpathologists. Written informed consent was obtained from allpatients. Collection of human tissue samples wad conducted inaccordance with the International Ethical Guidelines for Biomed-ical Research Involving Human Subjects. This study was approvedby the Ethics Committee of the Nanjing Medical University Affil-iated Cancer Hospital and was performed in accordance with theprovisions of the Ethics Committee ofNanjingMedical University.This study was approved by the Nanjing Medical University.

Cell cultureAll cell lines [A549, NCI-H1975, NCI-H1703, NCI-H226,

NCI-H46, PC9, NCI-H1299, SPC-A1, HCC827, and human bron-chial epithelial cell (HBE)] were purchased from Shanghai Insti-tutes for Biological Science, (Shanghai, China). NCI-H1975,A549, NCI-H1703, NCI-H226, NCI-H46, PC9, HCC827, andNCI-H1299 cellswere cultured inRPMI1640medium(KeyGene);SPC-A1 and HBE cells were cultured in DMEM medium(KeyGene), supplemented with 10 % FBS with 100 U/mL pen-icillin and 100 mg/mL streptomycin included. All cell lines weregrown in humidified air at 37 �Cwith 5%CO2. Cell cultures wereoccasionally tested for mycoplasma (last tested 2016). Authen-tication of cells was verified by short tandem repeat DNAprofilingwithin 6 months of use for the current study. The cells used inexperiments were within 10 passages from thawing.

circRNA microarrayFive lung adenocarcinoma tissues and paired nontumor tissues

were used formicroarray analysis. Themicroarray experimentwasperformed by Kangcheng Bio-tech Inc. The microarray data weresubmitted to the Gene Expression Omnibus, and the data can beaccessed by the accession number GSE101586.

Tissue microarray and chromogenic in situ hybridizationTissue microarray (TMA) was constructed as described previ-

ously (25). Eighty-nine pairs of lung cancer tissues and adjacentnontumor tissues were used to construct the TMA. RNA chromo-genic in situ hybridization (CISH) was performed to detectcircPRKCI expression in TMA using digoxigenin-labeled probe(50-GTATGCGAATTTGTTTTTCCAAAATAACATATCCCAATCA-30).Briefly, after dewaxing and rehydration, the samples were digested

with proteinase K, fixed in 4% paraformaldehyde, and hybridizedwith the digoxin-labeled probe overnight at 55�C. The sampleswere then incubated overnight at 4�C with an anti-digoxin mAb(Roche Applied Science). The sections were stained with nitro bluetetrazolium/5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP)in the dark, mounted, and observed.

RNA extraction, gDNA extraction, and qRT-PCR analysisRNA extraction and qRT-PCR were performed as described

previously (26). Genomic DNA (gDNA) was extracted fromtissues or cultured cells according to the PureLink Genomic DNAMini Kit protocol (Thermo Fisher Scientific, K182001). GAPDH,ACTB, and snRNA U6 were used as internal controls. All primersequences are listed in Supplementary Table S1.

Nucleic acid electrophoresisThe cDNA and gDNAPCRproducts were investigated using 4%

agarose gel electrophoresis with TBE running buffer. DNA wasseparated by electrophoresis at 110 V for 30minutes. The DNAmarker used was DL600 (KeyGen, Nanjing). The bands wereexamined by UV irradiation.

RNA isolation of nuclear and cytoplasmic fractionsThe subcellular localization of circPRKCI was detected using

the PARIS Kit according to the manufacturer's protocol (Ambion,Life Technologies).

siRNA and plasmid construction and cell transfectionThe siRNAs were provided by Life Technologies. The miRNA

mimics and primers were provided by RiboBio. The full-lengthcDNA of human circPRKCI was synthesized by Invitrogen andcloned into the expression vector pCDNA3.1 (ClontechLaboratories, Inc.). The final construct was verified by sequencing.Plasmid vectors for transfection were prepared using DNA Mid-iprep Kits (E.Z.N.A Endo-Free PlasmidMini Kit II) and transfectedinto lung adenocarcinoma cells using Lipofectamine 3000 (Invi-trogen). The siRNAs andmiRNAmimicwere transfected into lungadenocarcinoma cells using RNAiMAX (Invitrogen) according tothe manufacturer's instructions. All siRNA sequences used arelisted in Supplementary Table S1.

Cell proliferation, cell cycle, and apoptosis assaysCell proliferation was examined using a CCK-8 Kit (Roche

Applied Science), EdU assay (RiboBio), and Real timexCELLigence analysis system (RTCA) following the research pro-tocol afforded by the manufacturer (Roche Applied Science andACEA Biosciences; ref. 27). Colony formation assays were per-formed to monitor lung adenocarcinoma cell cloning capability.Lung adenocarcinoma cells were transfected with si-circPRKCI ornegative control (NC) and analyzed on a flow cytometer(FACScan; BD Biosciences) equipped with CellQuest software(BD Biosciences). Gefitinib (SML1657, Sigma) was dissolved inDMSO and used for in vitro studies at concentrations not exceed-ing 0.1% DMSO.

RNA immunoprecipitationThe EZMagna RIP Kit (Millipore) was used following the

manufacturer's protocol. Lung adenocarcinoma cells were lysedin complete RNA immunoprecipitation (RIP) lysis buffer, andthe cell extract was incubated with magnetic beads conjugatedwith anti-Argonaute 2 (AGO2) or control anti-IgG antibody

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(Millipore) for 6 hours at 4�C. The beads were washed andincubated with Proteinase K to remove proteins. Finally, purifiedRNA was subjected to qRT-PCR analysis.

Biotin-coupled miRNA captureThe biotin-coupled miRNA pull-down assay was performed as

described previously by Zheng and colleagues (16). Briefly, the 30

end biotinylated miR-RNA mimic or control biotin-RNA(RiboBio) was transfected into SPC-A1 cells at a final concentra-tion of 20 nmol/L for 1day. The biotin-coupled RNA complexwaspulled downby incubating the cell lysatewith streptavidin-coatedmagnetic beads (Ambion, Life Technologies). The abundance ofcircPRKCI andE2F7 inbound fractionswas evaluatedbyqRT-PCRanalysis.

Luciferase reporter assaysThe E2F7-binding sites of miRNAwere predicted by TargetScan

(http://www.targetscan.org/vert_71/). The different fragmentsequenceswere synthesized and then inserted into the pGL3-basicvector (Promega). All vectors were verified by sequencing, andluciferase activity was assessed using the Dual Luciferase Assay Kit(Promega) according to the manufacturer's instructions.

In vivo tumor growth assaysFemale BALB/c nude mice (4 weeks old) were maintained

under specific pathogen-free conditions andmanipulated accord-ing to protocols approved by the Nanjing Medical ExperimentalAnimal Care Commission. NC and si-circPRKCI transfectedSPC-A1 cells were harvested. For the tumor formation assay,1–2 � 106 cells were subcutaneously injected into a single flankof each mouse. Tumor growth was examined every week, andtumor volume was calculated using the following equation: V ¼0.5 � D � d2 (V, volume; D, longitudinal diameter; d, transversediameter).

Animal care, tumor engraftment, and PDTX maintenanceAnimal experiments were conducted in accordance with the

Institute for Laboratory Animal Research Guide for the Care andUse of Laboratory Animals and followed protocols approved bythe Animal Committee of Nanjing Origin Biosciences. BALB/cmale nude mice, ages 4 to 6 weeks and weighing 20 to 25 g, werepurchased from the Beijing Vital River Laboratory Animal Tech-nology Co., Ltd. All animals were fed an autoclaved laboratoryrodent diet.

Primary lung adenocarcinoma samples were cut into approx-imately 1-mm3 fragments in 0.1 mL 50% Matrigel BasementMembrane Matrix (BD Biosciences) and directly implanted intothe subcutaneous space (n ¼ 5 for each tumor sample). Patient-derived tumor xenografts (PDTX)were harvested anddivided intothree portions for the generation of the second in vivo passagexenograft tumors, protein and DNA/RNA extraction, and histo-pathologic examination.Micewith palpable tumors (�200mm3)were randomly divided into two experimental groups with intra-tumoral injection twice weekly for two weeks: 1.5 mg/kg controlsiRNA and si-circPRKCI. At the end of the experiment, tumorswere weighed and processed for knockdown activity by qRT-PCRand further histologic analysis.

Western blot analysisWestern blot analyses were performed according to standard

protocols as described previously (26). Anti-ACTB, anti-E2F7, andanti-Hedgehog acyltransferase (HHAT) were purchased from

Abcam. Anti-CDKN1A, anti-CCND1, anti-KI67, and anti-SOX2were purchased from Cell Signaling Technology.

Data AvailabilityMicroarray data have been submitted to GEOdatabase and can

be accessed with the ID: GSE101586.

Statistical analysisDifferences between groups were assessed by a paired two-

tailed t test. One-way ANOVA or the nonparametric Kruskal–Wallis test was applied to assess the relationship between cir-cPRKCI expression and other characteristics. The strength of theassociation between continuous variables was tested with theSpearman correlation. Multivariate Cox regression was used toidentify factors associated with survival of lung adenocarcinoma.All statistical analyses were performed using SPSS 20 software(Abbott Laboratories).

ResultsExpression profiles and integrated screening of circRNAs inlung adenocarcinoma

We performed microarrays to characterize the expression pro-files of circRNAs and mRNAs in paired lung adenocarcinomatissues and adjacent nontumor tissues from 5 patients with lungadenocarcinoma.A total of 107circRNAs (P<0.05and fold change> 1.5) and 1,691 mRNAs (P < 0.05 and fold change > 2.0) weredifferentially expressed between the lung adenocarcinoma tumortissues and paired adjacent normal tissues. Among the 107 differ-entially expressed circRNAs (Supplementary Table S2), 28 wereupregulated and 79 were downregulated in lung adenocarcinomatissues compared with nontumor tissues (Fig. 1A). These circRNAsand their host genes are located at diverse genomic regions.Notably, Circos plot showed that the expression levels of circRNAsdid not correlatewith themRNA levels of their host genes (Fig. 1B).

To further screen candidate circRNAs, we extracted the pre-viously published lung adenocarcinoma–associated somaticCNV data from TCGA (24) and found the 50 most frequentlyrecurrent variation regions (both P and residual P < 0.05),which included 20 amplified (689 genes) and 30 deleted (2160genes) regions (Supplementary Table S3). By intersecting theamplified regions with host genes of upregulated circRNAswithin these loci, and the deleted ones with downregulatedcircRNAs, respectively, we identified two amplified host genes(PRKCI and P4HB) and three deleted host genes (RABL2B,FLI1, and TM4SF19; Fig. 1C). Because PRKCI has been previ-ously demonstrated as an important stemness maintainer oflung cancer (28, 29) in 3q26.2 amplicon, which is one of themost frequent genomic aberrations in lung cancer (30), wetherefore hypothesized that PRKCI gene generated circRNA(Hsa_circ_0067934, termed as circPRKCI below) might alsoplay an important role in lung adenocarcinoma and eventuallychose circPRKCI for further investigation.

Characterization of circPRKCI in lung adenocarcinomacircPRKCI is back-spliced of two exons (exons 15 and 16) of

PRKCI gene (chr3: 170013698–170015181), located at 3q26.2amplicon (Fig. 1D). We firstly verified its existence in manycircRNA databases. According to the circBase database, circPRKCIis detected in many types of cancer cell lines, including K562 andA549 (http://www.circbase.org/cgi-bin/singlerecord.cgi?id¼hsa_

circPRKCI Promotes Lung Adenocarcinoma Tumorigenesis

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http://www.targetscan.org/vert_71/

http://www.circbase.org/cgi-bin/singlerecord.cgi?id=hsa_circ_0067934



Figure 1.

circRNA expression profile in lung adenocarcinoma and characterization of circPRKCI. A, Heatmap of the differentially expressed circRNAs in five pairs ofhuman lung adenocarcinoma tissues and matched nontumor tissues. Red, upregulated circRNAs in lung adenocarcinoma; green, downregulated circRNAs inlung adenocarcinoma.B,Circos plots showing the differentially expressed circRNAs and their host genes. Outer, host genes; inner, differentially expressed circRNAs.The values represent the log (fold change) of cancer versus normal. Red, upregulated. C, Screening strategy of candidate circRNAs. Amp, amplified genes;Del, deleted genes. D, circPRKCI is back-spliced by exons 15 and 16 of PRKCI. E, The divergent primers detected circular RNAs in cDNA but not in gDNA. GAPDHwas used as a control for a linear RNA transcript. F, qRT-PCR analysis of circPRKCI and PRKCI mRNA after treatment with RNase R in lung adenocarcinoma cells.CircPRKCI was resistant to RNase R treatment. � , P < 0.05, ��P, < 0.01. Error bars, SEM.

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circ_0067934). The circNet database also supported the exist-ence of circPRKCI (http://circnet.mbc.nctu.edu.tw/ by searchingcircPRKCI).

To further characterize circPRKCI, we designed two sets ofprimers: the divergent primers were used to amplify the circulartranscripts and the convergent primers were used to detect thelinear transcripts. The two sets of primers were then used toamplify the circular and linear transcripts of PRKCI in both cDNAand gDNA. PCR results indicated that the circular form wasamplified using the divergent primers in cDNA but not gDNA.Convergent primers amplified in both cDNA and gDNA. GAPDHwas used as a linear RNA control (Fig. 1E). RNase R is a 30 to 50

exoribonuclease that degrades linear RNA but does not act oncircular RNA. Aswas expected, the linear transcripts of PRKCIweredegradedbyRNase R,whereas the circular transcripts of circPRKCIwere resistant to RNase R treatment (Fig. 1F). Indeed, these dataconfirmed the existence of circPRKCI.

Correlation between circPRKCI expression and clinicalcharacteristics of lung adenocarcinoma

The expression of circPRKCIwas detected in 48 pairs of primarylung adenocarcinoma and adjacent nontumor tissues using qRT-PCR. circPRKCI was highly upregulated in lung adenocarcinoma,with an average fold of 6.89 (P < 0.01; Fig. 2A). We next evaluatedthe association between circPRKCI and clinical and pathologicparameters (Supplementary Table S4). Patients with larger tumorsize exhibited higher expression of circPRKCI (P¼ 0.001; Fig. 2B).Patients with tumor–node–metastasis (TNM) stage II–III exhib-ited higher circPRKCI expression than patients with TNM stage I(P ¼ 0.009; Fig. 2C). Taken together, circPRKCI is upregulated inlung adenocarcinoma tissues and positively correlated withtumor size and TNM stage.

CircPRKCI expression was then detected in lung cancer tissuesby CISH using TMA of 89 pairs of lung adenocarcinomaand adjacent nontumor tissues. There was also a positive corre-lation between circPRKCI expression and T stage and TNM stage(Fig. 2D; Supplementary Table S5). Kaplan–Meier survival curvesshowed that patients with higher levels of circPRKCI had a shorteroverall survival [HR ¼ 1.977; 95% confidence interval (CI),1.153–3.579; P¼ 0.037; Fig. 2E]. Multivariate analyses indicatedthat high circPRKCI level is an independent poor prognosisfactor for patients with patients with lung adenocarcinoma(HR ¼ 2.664; 95% CI, 1.327–5.347; P ¼ 0.006; Fig. 2F; Supple-mentary Table S6).

PRKCI amplification is correlated with the upregulation ofcircPRKCI in lung adenocarcinoma

To determine whether PRKCI gene amplification increasescircPRKCI expression, we first examined the PRKCI copy numberand circPRKCI expression in lung adenocarcinoma cell lines.Compared with the internal control, CEP3, we observed ampli-fication of the PRKCI gene in A549, SCPA1, and H1299 cells(Fig. 2G). Accordingly, the expression of circPRKCI in the threecell lines was significantly higher than that in other cell lines(Fig. 2H). Next, we examined PRKCI copy number variation andcircPRKCI expression in 60 pairs of lung adenocarcinoma tumortissues. The PRKCI copy number was amplified in tumor tissuescompared with paired nontumor tissues and positively correlatedwith circPRKCI expression (R2 ¼ 0.4366, P < 0.01; Fig. 2I). Theseresults suggested that high expression of circPRKCI is at leastpartially due to PRKCI gene amplification.

CircPRKCI promotes the proliferation and migration of lungadenocarcinoma cell lines in vitro

To investigate the biological function of circPRKCI, wedesigned two siRNAs: One is circPRKCI siRNA (si-circPRKCI),which specifically targets the back-splice junction site ofcircPRKCI, and the other is PRKCI siRNA (si-PRKCI), whichtargets the exon 18 of the linear transcript of PRKCI only(Fig. 3A). For ectopic overexpression of circPRKCI, exon 15and exon 16 of PRKCI were cloned into expression vectors,together with upstream and downstream flanking introns topromote the formation of circPRKCI as in a previous study(Fig. 3A; ref. 31).

Compared with the negative control siRNA, the expressionof circPRKCI was only downregulated by si-circPRKCI but wasnot affected by si-PRKCI (Fig. 3B). Also, the expressionvector markedly increased the expression of circPRKCI comparedwith the empty vector (Fig. 3C). By using Cell Counting Kit-8(CCK-8), colony formation, and 5-ethynyl20deoxyuridine (EdU)proliferation assays, we determined that knockdown of circPRKCIgreatly impaired the proliferation ability of SPC-A1 cells, whereasectopic expression of circPRKCI promoted cell proliferation(Fig. 3D–F). We further evaluated whether circPRKCI affectsapoptosis or cell-cycle progression of SPC-A1 cells. After treatmentwith si-circPRKCI, lung adenocarcinoma cells were arrested atG1 phase (Fig. 3G), but circPRKCI knockdown did not affectapoptosis. Terminal dexynucleotidyl transferase–mediated dUTPnick end labeling (TUNEL) experiment also confirmed thatknockdown of circPRKCI did not affect apoptosis in lung adeno-carcinoma cells. The Matrigel assay showed that si-circPRKCItreatment markedly impaired the invasion capacity of SPC-A1cells (Fig. 3H). All of the above experiments were also repeated inA549 cells, and similar results were achieved (SupplementaryFig. S1A–S1L). These in vitro experiments suggested thatcircPRKCI promotes the proliferation and migration of lungadenocarcinoma cells.

circPRKCI serves as a sponge for both miR-545 and miR-589To explore themolecular mechanisms of circPRKCI-promoting

lung adenocarcinoma tumorigenesis, we first determined thesubcellular localization of circPRKCI in lung adenocarcinomacell lines using the nuclear mass separation assay (Fig. 4A) andFISH analysis (Fig. 4B). We found more than 90% of circPRKCIwas present in the cytoplasm.

Given that many circRNAs can function as miRNA sponges inthe cytoplasm (32), we determined whether circPRKCI mayalso bind to miRNAs as a sponge and regulate targets via thecompetitive endogenous RNA (ceRNA) mechanism. We there-fore analyzed the sequence of circPRKCI using the miRandaalgorithm and identified five miRNA-binding sites with rela-tively high scores (miR-545, miR-589, miR-600, miR-144, andmiR-670; Supplementary Fig. S2A). It is well known thatmiRNAs usually silence gene expression by combining withthe AGO2 protein and form the RNA-induced silencing com-plex (RISC). In the context of ceRNA mechanism, it might be aprevalent phenomenon that AGO2 could bind with bothcircRNAs and miRNAs based on previous studies (16, 33,34). We therefore conducted an RIP assay to pull down RNAtranscripts that bind to AGO2 in SPC-A1 and A549 cells.Indeed, endogenous circPRKCI was efficiently pulled down byanti-Ago2 (Fig. 4C). To further detect whether circPRKCI couldsponge miRNAs, we performed a miRNA pull-down assay using






http://circnet.mbc.nctu.edu.tw/


biotin-coupled miRNA mimics (miR-545, miR-589, miR-600,miR-144, and miR-670). Interestingly, circPRKCI was onlyefficiently enriched by miR-589 and miR-545, but not by theother three miRNAs (Fig. 4D). In addition, RIP assay revealedthat miR-545 and miR-589 were efficiently pulled down by theanti-AGO2 antibody but not by the nonspecific anti-IgG anti-body (Fig. 4E). Furthermore, silencing of circPRKCI did not

affect the expression of miR-545 or miR-589, and transfectionof miR-545 and miR-589 mimics did not affect the expressionof circPRKCI (Fig. 4F and G), which indicated circPRKCI func-tions as an miRNA sponge without affecting the expression ofsponged miRNAs.

Because there are few reports about miR-545 and miR-589,we next investigated the biological functions of miR-545 and

Figure 2.

circPRKCI is frequently upregulated in lung adenocarcinoma due to gene amplification of PRKCI. A, circPRKCI expression was upregulated in 48 pairednormal and cancerous lung adenocarcinoma tissues by qRT-PCR. B and C, circPRKCI was upregulated in patients with T 2-3 and TNM stage II–III. D, The expressionof circPRKCI was analyzed by in situ hybridization on lung adenocarcinoma tissue. CircPRKCI was upregulated in lung adenocarcinoma tumor tissuescompared with normal tissues and circPRKCI positively correlated with larger tumors and a higher TNM stage. E, Kaplan–Meier analysis of the correlationbetween circPRKCI expression and overall survival. Patients with high levels of circPRKCI had a significantly shorter overall survival. F, Multivariate Coxregression showed high circPRKCI expression was an independent prognostic factor for poor survival. G and H, circPRKCI was upregulated in lung adenocarcinomacell lines after normalizing to HBE. PRKCI gene was amplified in A549, SCPA1, and H299 cells. The expression of circPRKCI was dramatically higher in thesethree cell lines than in other cell lines. I, Positive correlation between circPRKCI expression and PRKCI genomic DNA content in lung adenocarcinoma tissues.� , P < 0.05; �� , P < 0.01. Error bars, SEM.

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miR-589. We found that they significantly inhibit the prolifera-tion of lung adenocarcinoma cells and induce a G1 phase arrest(Supplementary Fig. S2B–S2E). These results suggest thatcircPRKCI functions according to the ceRNA mechanism andserves as a sponge for miR-545 and miR-589, both of whichsuppressed the tumorigenesis of lung adenocarcinoma cells.

Both miR-545 and miR-589 decrease E2F7 expression and itsdownstream signaling

To identify the potential target genes of miR-545 andmiR-589,we filtered 958 (total contextþþ score < �0.17) and 781 (totalcontextþþ score < �0.18) genes, respectively, by using theTargetScan prediction program. According to the ceRNA theory,circPRKCI expression is positively correlated with its target genes.Next, we filtered genes that positively correlated with circPRKCI

expression in our microarray data. By plotting a Venn diagramusing the 3 gene sets, we identified 7 candidate target genes (E2F7,CENPA, DNAJC3, FRK, LRRC17, MON2, and RMI2; Fig. 4H). Tofurther verify the downstream targets of circPRKCI, mRNAlevels of 7 candidate target genes were detected after silencingcircPRKCI, and we found only E2F7 was downregulated (Fig. 4I).In addition, literature review suggests E2F7 is closely involved intumorigenesis; we therefore reasoned that E2F7 may be a down-stream target of circPRKCI.

To verify whether E2F7 was the direct target of miR-545 andmiR-589, we first performed the miRNA biotin pull-downassay. We found that both miR-545 and miR-589 could sig-nificantly enrich the 30UTR of E2F7 mRNA (Fig. 4J). Aftertransfection of either miR-545 or miR-589 mimics, both themRNA and protein levels of E2F7 were significantly decreased

Figure 3.

circPRKCI promotes the malignant progression of lung adenocarcinoma cells. A, Schematic illustration showing siRNAs and circPRKCI expression vectors.Si-circPRKCI targets the back-splice junction of circPRKCI. B and C, qRT-PCR analysis of circPRKCI and PRKCI RNA expression after treatment with two siRNAs andcircPRKCI expression vectors.D–F,CircPRKCI promotes the proliferation of SPC-A1 cells shownby the colony formation (D), EdU (E), and CCK8 (F) assays.G, SPC-A1cells transfected with si-circPRKCI are arrested at G1 phase.H, circPRKCI promotes themetastasis of SPC-A1 cells, as evidenced byMatrigel assays. � , P < 0.05; �� , P <0.01. N.S, nonsignificant. Error bars, SEM.






Figure 4.

circPRKCI serves as a sponge for miR-545 and miR-589 to increase E2F7 in lung adenocarcinoma. The majority of circPRKCI was present in the cytoplasmaccording to the nuclear mass separation assay (A) and FISH (B). C, RIP assay using an antibody against Ago2. D, Pulldown assay using biotin-coupled miR-545,miR-589, miR-600, miR-144, and miR-670. E, The Ago2 RIP also showed that Ago2 significantly enriched miR-545 and miR-589. F and G, Silencing ofcircPRKCI did not affect the expression of miR-545 or miR-589. miR-545 and miR-589 did not affect the expression of circPRKCI. H and I, Schematic drawingof the screening procedure of candidate genes. E2F7 was the most significantly downregulated gene among all candidate genes after the inhibition of circPRKCI.J, Biotin pulldown assay demonstrates that the miR-589- and miR-545–captured fractions distinctly enrich E2F7. K, After transfection with miR-545 andmiR-589, E2F7 expression significantly decreased. L, Dual luciferase reporter assays showed that miR-545 and miR-589 directly bind to the 3'-UTR of E2F7 andinhibit luciferase activity. M, miR-545 and miR-589 upregulated CDKN1A and downregulated CCND1 and E2F7. N, miR-545 and miR-589 are negativelycorrelated with E2F7 in lung adenocarcinoma tissues according to TCGA database. � , P < 0.05, �� , P < 0.01. N.S, nonsignificant. Error bars, SEM.

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(Fig. 4K and M). Furthermore, we cloned the wild-typeand mutant (predicted miR-545 and miR-589–binding siteswere mutated) 30-UTR of E2F7 mRNA and performed a dualluciferase reporter assay. Compared with the control RNAgroup, both miR-545 and miR-589 mimics efficiently inhibitedluciferase activity of wild-type group but not mutant one.However, after mutating the binding sites, the inhibitoryeffect was abolished (Fig. 4L). These results suggested that bothmiR-545 and miR-589 bind to the 30-UTR of E2F7 and directlydownregulate E2F7 expression.

E2F7 was previously identified as a negative regulator ofCDKN1A transcription and subsequently upregulated down-stream CCND1 so as to induce cell-cycle arrest (35, 36). Weconfirmed that both miR-545 and miR-589 mimics upregulatedCDKN1A while downregulating CCND1 as compared with con-trol RNA (Fig. 4M). In addition, by analyzing the RNA sequencingand miRNA microarray data derived from TCGA, we foundexpression levels of both miR-545 and miR-589 are negativelycorrelated with E2F7 (Fig. 4N). It has been reported that E2F7promotes cancer cell proliferation (35). In support of this finding,silencing of E2F7 indeed inhibited lung adenocarcinoma cellproliferation and induced G1 phase arrest (Supplementary Fig.S3A–S3D). Taken together, these data indicated that both miR-545 and miR-589 could directly downregulate E2F7 and therebyregulate its downstream signaling.

CircPRKCI promotes cell proliferation via the circPRKCI-miR-545/589-E2F7 axis

To validate whether circPRKCI promotes cell proliferationvia the circPRKCI-miR-545/589-E2F7 axis, we first confirmedthat silencing circPRKCI decreased the protein levels of E2F7,whereas overexpressing circPRKCI increased the protein levelsof E2F7. The protein levels of downstream CDKN1A andCCND1 were also altered accordingly (Fig. 5A). Second, in theexpression cohort of 48 patients with lung adenocarcinoma,circPRKCI expression was positively correlated with mRNAlevels of E2F7 (R2 ¼ 0.49, P < 0.01; Fig. 5B). Then, we designedrescue experiments using miRNA-545 and miR-589 mimics(Fig. 5C). At protein level, miR-545 and miR-589 partiallyreversed the effects of circPRKCI on E2F7, CDKN1A, andCCND1 in SPC-A1 cell (Fig. 5D). More importantly, as revealedby colony formation, CCK-8, and EdU assays, both the miR-545 and miR-589 mimics could partially rescue the prolifera-tion-promoting effect induced by circPRKCI and the combina-tion of two miRNA mimics showed a stronger rescue effect (Fig.5E; Supplementary Fig. S4A–S4C).

To further determine whether circPRKCI exerts the prolifera-tion-promoting effect dependent on the miR-545/589–bindingsites, we constructed circPRKCI vectors withmutated binding sitesof miR-545 (circPRKCI Mut1), miR-589 (circPRKCI Mut2), andboth binding sites (circPRKCI Mut1þ2), respectively. Colony

Figure 5.

circPRKCI promotes proliferation by binding to miR-545 and miR-589. A, Knockdown of circPRKCI increased CDKN1A expression but decreased CCND1 andE2F7 expression, whereas overexpression of circPRKCI produced the opposite results. B, circPRKCI was significantly positively correlated with E2F7 in theexpression cohort of 48 patients with lung adenocarcinoma. C, Schematic drawing of the study design for the rescue experiments. D, miR-545 and miR-589partially reversed the effects of circPRKCI on E2F7, CDKN1A, and CCND1. E, miR-545 and miR-589 partially abolished the effects of circPRKCI on cell growth,as revealed by colony formation assay. F, Mutation of miR-545 and miR-589–binding sites abolishes the proliferation-promoting effect of circPRKCI.Mut1, miR-545–binding site mutation; Mut2, miR-589–binding site mutation; Mut1þ2, mutation of both miR-545- and miR-589–binding sites. ��, P < 0.01.N.S, nonsignificant. Error bars, SEM.






formation andEdUassay showed thatmutationof themiR-545ormiR-589–binding site could partially reverse the cell proliferationinduced by circPRKCI, and mutation of both miR-545 and miR-589–binding sites completely abolished the enforced cell prolif-eration induced by circPRKCI (Fig. 5F; Supplementary Fig. S5A–S5C). Taken together, we demonstrated that circPRKCI promoteslung adenocarcinoma cell proliferation via the circPRKCI–miR-545/589–E2F7 axis.

circPRKCI does not function through the PRKCI/SOX2signaling pathway

PRKCI and SOX2 are coamplified in 3q26. Previous work (28)has demonstrated PRKCI could regulate SOX2-mediated HHATexpression, a key component of Hedgehog pathway. PCR in lungadenocarcinoma tumor tissues showed circPRKCI expressionwas not correlated with PRKCI (R2 ¼ 0.0026, P ¼ 0.72) or SOX2(R2 ¼ 0.049, P¼ 0.170; Supplementary Fig. S6A and S6B). SOX2and HHAT expression were not altered at the protein level aftersilence or overexpression of circPRKCI (Supplementary Fig. S6Cand S6D). To seewhether SOX2 has synergistic effect to circPRKCIor not, we silenced circPRKCI and SOX2 alone or both in lungadenocarcinoma cells and performed colony formation and Eduassay. The results showed that silencing circPRKCI or SOX2 aloneinhibited cell proliferation, while silencing both somehowenforced the inhibitory effect (Supplementary Fig. S6E–S6H).These lines of evidence suggest circPRKCI might not functionthrough PRKCI/SOX2 signaling pathway.

circPRKCI promotes lung adenocarcinoma tumorigenesisin vivo and acts as a potential therapeutic target

To investigate the biological function of circPRKCI in vivo, weestablished a xenograft tumor model in nude mice. SPC-A1 cellstransfected with si-circPRKCI or a control siRNA were subcuta-neously injected into nude mice. The tumors derived from cellstransfected with si-circPRKCI had a smaller size and lower weightcompared with those derived from cells transfected with thecontrol siRNA (Fig. 6A). IHC revealed that tumor tissues collectedfrom the si-circPRKCI group had fewer E2F7 andCCND1-positivecells but more CDKN1A-positive cells when compared withcontrol group (Fig. 6B). In addition, it is well known that PDTXsmaintained better cell differentiation ability, morphology, andarchitecture of the original patient tumors compared with thecell line–derived xenografts; therefore, PDTX is considered as atranslationalmodel for cancer research (37).We then developed aPDTX model from a female patient with lung adenocarcinomaand evaluated the therapeutic potential of circPRKCI by intratu-moral injection of cholesterol-conjugated si-circPRKCI and acontrol siRNA (4 times and twice a week). As a result, treatmentof si-circPRKCI significantly inhibited growth of PDTX in vivo(Fig. 6C), suggesting that circPRKCI could serve as a promisingtherapeutic target of lung adenocarcinoma.

Because EGFR tyrosine kinase inhibitors (EGFR-TKI) have beenwidely used in patients with lung adenocarcinoma with EGFRmutations, to find whether circPRKCI could influence the thera-peutic effects of EGFR-TKI, we performed proliferation assay inHCC827 cells (with EGFR E746-A750 deletion, which is sensitiveto EGFR-TKI) by using RTCA system. As was expected, eitherEGFR-TKI (gefitinib) alone or silencing circPRKCI alone showedcytotoxic effect in HCC827 cells. Combining gefitinib with silenc-ing circPRKCI showed remarkably stronger inhibitory effect com-pared with gefitinib or si-circPRKCI alone (Fig. 6D), which

suggested that combined inhibitionof EGFRand circPRKCImighthave potential synergistic inhibition effect.

DiscussionRecent high-throughput sequencing studies havedemonstrated

that the human genome is actively transcribed, and noncodingtranscripts are abundant in the human transcriptome. circRNAsare a type of noncoding RNAs that have recently attracted greatresearch interest. The majority of circRNAs are generated by exoncircularization from precursor RNAs; thus, the expression ofcircRNAs would be affected by genetic alterations, like transloca-tion and CNV (22). Guarnerio and colleagues have proved thatchromosomal translocations give rise to fusion circRNAs andthe fusion circRNA generated by MLL/AF9 translocation is onco-genic and promotes leukemia progression (38). However, thefunction of deregulated circRNAs caused by CNVs remains largelyunknown.

In the current study, we filtered several deregulated circRNAsthat are localized in recurrent CNV locus in lung adenocarcinomaby integrated analysis of microarray and TCGA data. Amongthem, we characterized that circPRKCI is a highly upregulatedcircRNAs in lung adenocarcinoma due to the amplification of3q26.2 locus.During the period of investigating circPRKCI in lungadenocarcinoma, we interestingly found circPRKCI was alsoupregulated in esophageal squamous cell carcinoma (39). How-ever, the molecular mechanism of circPRKCI, as well as thecorrelation between increased circPRKCI expression and 3q26amplification, remains unclear at that moment. circPRKCI isderived from exons 15 and 16 of its host gene PRKCI, which isa known proto-oncogenic gene in lung cancer that could drive astem-like phenotype by competing with SOX2 (28). It has beenproven that amplification of the 3q26.2 locus leads to increasedPRKCI expression (40), and our data also demonstrated thatPRKCI gene amplification was positively correlated with cir-cPRKCI expression. In addition to lung cancer, accumulatingevidences have revealed the amplification of 3q26.2 is frequentlyidentified in multiple cancers, such as esophageal cancer (41),ovarian cancer (42), thyroid cancer (43), and testicular germ celltumor (44). At this region, many proto-oncogenic genes havebeen identified, including not only coding genes like PRKCI,SEC62 (45), and MDS1/EVI1 (46), but also some noncodinggens like miR-551b-3p (42) and miR-569 (13). These data indi-cated the noncoding transcripts at the 3q26.2 amplicon also playan important role in cancer development.On theother hand, geneamplification leads to overexpression of both linear and circulartranscripts. Considering that many oncogenes generate circulartranscripts, like circFOXO3 (14), it is plausible to hypothesize thatcircular transcripts of those oncogenes may also play a role in thepathogenesis of cancer independent of their linear transcripts.

The ceRNA hypothesis suggests that RNA transcripts, includingmRNAs, lncRNAs, pseudogenes, and circRNAs, could communi-cate with each other via miRNAs. They usually bind to miRNAsand then regulate expression of RNA transcripts harboring thesamemiRNA-binding sites, constructing a complex posttranscrip-tional regulatory network (47). circRNAs were first observeddecades ago; however, their functional roles in biological pro-cesses have not beenwell characterized until recently (48). Owingto its biological stability, many researchers have shown thatcircRNAs are perfect miRNA sponges. ciRS-7 is the first circRNAthat is confirmed as a miRNA sponge, which contains more than

Qiu et al.





70 binding sites of miR-7, and is involved in the development ofbrain (19). They demonstrated "ciRS-7 serves as a binding plat-form for Ago2 andmiR-7" and "the widespread Ago occupancy iscaused by miR-7-directed association of AGO2 proteins to theprevalent and conserved miR-7 target sites in the ciRS-7 RNA."Thereafter, many other studies have also validated that circRNA

could bind with AGO2 and "sponge"miRNAs (16, 33, 34). In thecurrent study, we also found Ago2 could pull both miR-545/589and circPRKCI. Taking the literatures and our data together, itmight be a broad phenomenon that both circRNAs and miRNAscould bind with AGO2, despite the underlying mechanisms needfurther investigation.

Figure 6.

circPRKCI promotes lung adenocarcinoma tumorigenesis in vivo and is a potential therapeutic target. A, Xenograft tumor models show that tumorsgrown from circPRKCI knockdown cells were smaller than those grown from control cells. B, IHC staining of CCND1, CDKN1A, E2F7, and Ki67. C, PDTX modelshowed that intratumoral injection of siRNA targeting circPRKCI significantly inhibited tumor growth. D, Silence of circPRKCI significantly increasedtherapeutic effect of gefitinib in EGFR-mutant HCC827 cells. �� , P < 0.01. Error bars, SEM.






A growing number of studies have shown that several circRNAsthat can sponge miRNAs have been reported in various types ofcancer. For an instance, circHIPK3 is upregulated inmultiple solidcancers and binds to miR-124 so as to inhibit cancer growth (16).Subsequent studies found that circHIPK3 could also bind tomiR-558 (49) andmiR-30a-3p (50). Other circRNAs with similarceRNA functions have also been reported, such as circMTO1binding to miR-9 (20), as well as circCCDC66 binding to miR-33b and miR-93 (21). It suggests that one circRNA could bind tomore than one miRNA, and consistent with these findings, ourdata showed circPRKCI binds to both miR-545 and miR-589.Considering the relative low abundance of circRNAs and variousmiRNA-binding sites within mRNA and circRNA transcripts,spongingmultiplemiRNAs by a single circRNA could be a generalmechanism in cells.

Finally, we also evaluated the clinical and translational rele-vance of circPRKCI. Results of RT-PCR and CISH both showedcircPRKCI is highly expressed in lung adenocarcinoma tissues,and further analyses indicated high expression of circPRKCIcorrelates with higher tumor grade and poor prognosis of lung

adenocarcinoma. These lines of evidence demonstrate thatcircPRKCI is a potential biomarker for lung adenocarcinoma.Considering the translational significance of circPRKCI, in thePDTXmodel, intratumor injection of siRNA inhibited lung cancergrowth in vivo. Moreover, in EGFR-mutant HCC827 cells, silenceof circPRKCI greatly increased the inhibitory effect of gefitinib.These data indicate that circPRKCI may be a potential therapeutictarget for lung adenocarcinoma.

In summary, as shown in Fig. 7, we identified a CNV-associatedcircular RNA, circPRKCI, which is overexpressed in lung adeno-carcinoma tissues, at least in part due to the amplification of3q26.2 locus, promotes proliferation and tumorigenesis of lungadenocarcinoma. circPRKCI binds to bothmiR-545 andmiR-589and subsequently inhibits their suppressing capability on E2F7.Our study provides a novel potential target for patients with lungadenocarcinoma in the sight of circular RNAs.

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

Authors' ContributionsConception and design: M. Qiu, W. Xia, R. Yin, Jun Wang, L. XuDevelopment of methodology: M. Qiu, W. Xia, R. Chen, S. Wang, J. HuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Wang, Y. Xu, Z. Ma, T. Fang, J. HuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Qiu, W. Xia, S. Wang, Y. Xu, W. Xu, E. Zhang,R. Yin, Jun Wang, L. XuWriting, review, and/or revision of the manuscript: M. Qiu, S. Wang, Z. Ma,R. Yin, Jun Wang, L. XuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Jie Wang, J. Hu, G. Dong, L. XuStudy supervision: R. Yin, Jun Wang, L. Xu

AcknowledgmentsThis work was supported by the National Natural Science Foundation of

China (81372321, 81472200, 81572261, and 81702256), Innovation Capa-bility Development Project of Jiangsu Province (BM2015004), Project ofJiangsu Provincial Medical Talent (ZDRCA2016033), and the Key Project ofCutting-edgeClinical Technologyof JiangsuProvince (BE2016797).M.Qiuwassupported in part by the Postdoctoral Fellowship of Peking-Tsinghua Center forLife Sciences. We greatly appreciate Dr. Mark K. Ferguson's (Department ofSurgery and the Cancer Research Center, the University of Chicago Medicine &Biological Sciences) kind help for manuscript language editing.

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 September 13, 2017; revised February 13, 2018; acceptedMarch 23,2018; published first March 27, 2018.

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2018;78:2839-2851. Published OnlineFirst March 27, 2018.Cancer Res Mantang Qiu, Wenjia Xia, Rui Chen, et al. AdenocarcinomaThe Circular RNA circPRKCI Promotes Tumor Growth in Lung

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