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Full Terms&Conditions of access and use can be found athttps:/ homepage:https:/ RNA circHIPK3 modulates autophagy viaMIR124-3p-STAT3-PRKAA/AMPK signaling in STK11mutant lung cancerXiuyuan Chen,Rui Mao,Wenmei Su,Xia Yang,Qianqian Geng,ChunfangGuo,Zhuwen Wang,Jun Wang,Laura A.Kresty,David G.Beer,Andrew C.Chang&Guoan ChenTo cite this article:Xiuyuan Chen,Rui Mao,Wenmei Su,Xia Yang,Qianqian Geng,ChunfangGuo,Zhuwen Wang,Jun Wang,Laura A.Kresty,David G.Beer,Andrew C.Chang&Guoan Chen(2019):Circular RNA circHIPK3 modulates autophagy via MIR124-3p-STAT3-PRKAA/AMPKsignaling in STK11 mutant lung cancer,Autophagy,DOI:10.1080/15548627.2019.1634945To link to this article:https:/doi.org/10.1080/15548627.2019.1634945View supplementary material Accepted author version posted online:23Jun 2019.Published online:28 Jun 2019.Submit your article to this journal Article views:308View Crossmark dataRESEARCH PAPERCircular RNA circHIPK3 modulates autophagy via MIR124-3p-STAT3-PRKAA/AMPKsignaling in STK11 mutant lung cancerXiuyuan Chena,b,Rui Maoc,Wenmei Sud,Xia Yange,Qianqian Genge,Chunfang Guob,Zhuwen Wangb,Jun Wanga,Laura A.Krestyb,David G.Beerb,Andrew C.Changb,and Guoan ChenfaDepartment of Thoracic Surgery,Peking University Peoples Hospital,Beijing,China;bSection of Thoracic Surgery,Department of Surgery,RogelCancer Center,University of Michigan,Ann Arbor,USA;cCancer Center,Xinjiang Medical University,Urumqi,China;dDepartment of Oncology,Affiliated Hospital of Guangdong Medical University,Zhanjiang,China;eThe First Affiliated Hospital,Xian Jiaotong University,Xian,China;fSchoolof Medicine,Southern University of Science and Technology,Shenzhen,ChinaABSTRACTThe role of circular RNA in cancer is emerging.A newly reported circular RNA HIPK3(circHIPK3)is criticalin cell proliferation of various cancer types,although its role in non-small cell lung cancer(NSCLC),hasyet to be elucidated.Our results provided evidence that silencing of circHIPK3 significantly impaired cellproliferation,migration,invasion and induced macroautophagy/autophagy.Mechanistically,we uncov-ered that autophagy was induced upon loss of circHIPK3 via the MIR124-3p-STAT3-PRKAA/AMPKa axis inSTK11 mutant lung cancer cell lines(A549 and H838).STAT3 abrogation as well as transfection witha MIR124-3p mimic,recapitulated the induction of autophagy.We also demonstrated antagonisticregulation on autophagy between circHIPK3 and linear HIPK3(linHIPK3).We therefore propose that theratio between circHIPK3 and linHIPK3(C:L ratio)may reflect autophagy levels in cancer cells.We observedthat a high C:L ratio(0.49)was an indicator of poor survival,especially in advanced-stage NSCLCpatients.These results support that circHIPK3 is a key autophagy regulator in a subset of lung cancer andhas potential clinical use as a prognostic factor.The circular RNA HIPK3(circHIPK3)functions as anoncogene and autophagy regulator may potential use as a prognostic marker and therapeutic target inlung cancer.Abbreviations 3-MA:3-methyladenine;AMPK:AMP-activated protein kinase;ATG7:autophagy related7;Baf-A:bafilomycin A1;BECN1:beclin 1;circHIPK3:circular HIPK3;CQ:chloroquine;GAPDH:glyceralde-hyde-3-phosphate dehydrogenase;GFP:green fluorescent protein;HIPK3:homeodomain interactingprotein kinase 3;IL6R:interleukin 6 receptor;MAP1LC3B/LC3B:microtubule associated protein 1 lightchain 3 beta;NSCLC:non-small cell lung cancer;RFP:red fluorescent protein;RPS6KB1/S6K:ribosomalprotein S6 kinase B1;SQSTM1/p62:sequestosome 1;STAT3:signal transducer and activator of transcrip-tion 3;STK11:serine/threonine kinase 11ARTICLE HISTORYReceived 18 June 2018Revised 10 June 2019Accepted 19 June 2019KEYWORDSCell death;ceRNA;circRNA;HIPK3;prognosis;STK11IntroductionCircular RNAs are covalently-closed,single-stranded transcriptsderived from pre-mRNAs 1.Discovered in the 1970s 24,they were then regarded as a rare product arising from mRNAsplicing errors.Their abundance in human cells has recentlybeen clarified by RNA-seq analysis 57,and they appear tohave a significant role in tumorigenesis and development 8.Circular RNAs can be divided into 4 different categoriesbased on their origin 9,10.CircRNAs derived from exons arethe most widely studied,with other types including ciRNAsderived from introns,ElciRNAs derived from a combination ofexons and introns,as well as group I and II ribozymes.Multiplehypotheses have been proposed regarding the biogenesis ofcircRNAs,among which alternative splicing,reverse comple-mentary intronic sequence paring,or RNA binding proteinregulation being the most accepted ones 9,11.Understandingof circRNA function however remains incomplete but it is wellaccepted that circRNAs can act as microRNA sponges to reg-ulate gene expression and may have diagnostic or therapeuticpotential 12,13.Recent studies have focused on the relationship betweencircular RNA and cancer 8,1416.Among all cancers,NSCLC(non-small cell lung cancer)carries the greatest mor-tality worldwide with over 150,000 deaths estimated in 2018in the United States 17.Although circular RNAs areinvolved in the development of various cancers 13,16,18,yet their function in lung cancer remains to be characterized.Circular RNA circHIPK3 is a known oncogene derivedfrom chromosomal region 11p13 and originating fromthe second exon of HIPK3 16.It appears to regulate cellgrowth in liver,colon and cervical cancer cell lines by spong-ing multiple miRNAs 16.Clinically circHIPK3 has beenreported to be negatively correlated with bladder cancerCONTACT Guoan CSchool of Medicine,Southern University of Science and Technology,1088 Xueyuan Bivd,Nanshan District,Shenzhen,Guangdong 518055,P.R.China;Jun WDepartment of Thoracic Surgery,Peking University Peoples Hospital,Beijing100044,P.R.ChinaSupplementary material for this article can be accessed here.AUTOPHAGYhttps:/doi.org/10.1080/15548627.2019.1634945 2019 Informa UK Limited,trading as Taylor&Francis Groupgrade,invasion as well as lymph node metastasis 19.Thefunction and clinical potential of circHIPK3 in NSCLC isunknown.In this study,we investigated the function andmechanism of action of circHIPK3 in NSCLC cell lines andexamined its clinical significance in patients with primaryNSCLC.ResultsCircHIPK3 is abundantly expressed in a wide spectrumof lung cancer cell linesAlthough circHIPK3 was reported to play divergent roles indifferent cancer types and may have different biological func-tions 16,19,the role of circHIPK3 in lung cancer remainsunknown.We systematically investigated the expression ofcircHIPK3 among multiple lung cancer cell lines carrying differ-ent driver gene mutations(Table S1).Derived from the secondexon of HIPK3(Figure S1A),circHIPK3 was expressed in alltested cell lines(Figures 1A and S1B).It was prone to RNaseR digestion and was undetectable in genomic DNA(Figure S1C,D).Although circHIPK3 levels were relatively lower than thehousekeepingmRNAGAPDH,itwasasabundantasthepoly(A)isoform linear HIPK3(linHIPK3)(Figure 1A).Considering thatthe ratio between most circular RNAs and their linear counter-part is only one percent 7,we suspected that circHIPK3 ishighly functional in lung cancer cell lines given its prominentexpression in comparison with linHIPK3.To determine the cellular distribution of circHIPK3 andlinHIPK3,we performed RT-PCR using RNA samples collectedexclusively from either the cytoplasm or nuclear fractions of lungcancer cell line lysates.We found that circHIPK3 was distributedpredominantly in the cytoplasm,while linHIPK3 was more abun-dant in the nucleus in these cell lines(Figures 1B,C and S1E,F).To test whether circHIPK3 was functional in lung cancer,wegenerated small interfering RNAs(siRNAs)for gene abrogationexperiments.We utilized 3 siRNAs from the publication byZheng et al.16:si-circ-1 targeting circHIPK3,si-lin targetinglinHIPK3,and si-both targeting both isoforms(circHIPK3 andlinHIPK3)(Figure S2A).We generated an additional siRNA si-circ-2 to target circHIPK3 using CircInteractome Database 20.On average,si-circ-1,si-circ-2 and si-both treatment resulted inless than 5%of the residual circHIPK3 level seen with si-Ctrl;whilesi-linandsi-bothtreatment resulted in approximately80%reductionoflinHIPK3 expression(Figures 1D,EandS2B,C).Weobserved an approximately 2-fold increase of linHIPK3 in wholecelllysatesuponsilencingcircHIPK3inall3celllines(Figures1EandS2B).TheincreasedlinHIPK3arosemostlyfromthenucleusin H1299 cells(Figure 1G)indicating a possible feedbackmechanism in response to loss of circHIPK3.We also noticedthatuponlinHIPK3abrogation,circHIPK3wasdistributedmorein nuclear fractions(Figure 1F,G),although it remained steadilyexpressed in whole cell lysates.PreviousresearchrevealedthatHIPK3isafrequentmethylatedtarget in multiple diseases and disorders 21,22.We investigatedwhether circHIPK3 was regulated by epigenetic modification.InA549,by inhibiting deacetylation using TSA or demethylationA549H12990123linHIPK3Relative expressionCytoplasmNucleuscircHIPK3linHIPK3GAPDH0102030RNA expressionCT valueA549H838H1299HCC827PC-9H1650H1975HCC4006ABsi-Ctrlsi-circ-1si-lin0.00.51.01.52.02.5H1299 circHIPK3Relative expressionNucleusCytoplasm*FA549H129902468circHIPK3Relative expressionCytoplasmNucleus*Csi-Ctrlsi-circ-1si-lin0.00.51.01.52.02.5H1299 linHIPK3Relative expressionNucleusCytoplasm*Gsi-Ctrlsi-circ-1si-linsi-bothsi-circ-20.01.02.0H1299 linHIPK3Relative expression*Esi-Ctrlsi-circ-1si-linsi-bothsi-circ-20.00.51.0H1299 circHIPK3Relative expression*DFigure 1.TheexpressionandcellulardistributionofcircHIPK3.(A)RT-PCRanalysesoftheexpressionofcircHIPK,linHIPK3,andGAPDHcDNAinvariousNSCLCcelllines.Y-axisis the raw Ct value.(B,C)RT-PCR resultof the cellular distributionof circHIPK3 and linHIPK3 mRNA inA549 and H1299.(D,E)Cells were treated with si-Ctrl,si-circ-1,si-lin,si-both and si-circ-2 respectively for 48 h prior to collecting RNA.RT-PCR showed over 95%knockdown effect of circHIPK3 upon si-circ-1,si-both and si-circ-2 treatment,andover80%knockdown oflinHIPK3 uponsi-lin and si-both treatments.(F,G)RT-PCRresult indicated the change incellular distributionofcircHIPK3 and linHIPK3 upon si-circ-1and si-lin treatment,respectively.All data are presented as the means SD of at least 3 independent experiments.*P 0.05,*P 0.01.2X.CHEN ET AL.using 5-azacytidine(5-AZA),we observed a significant decreasein circHIPK3 but an increase in linHIPK3.Although the change isstatistically significant,the fold change is not profound ata 1.5-fold max.We consider this more of a general rather thanspecificeffectofepigeneticregulation(FigureS2D)inlungcancer.We did not find DNA amplification or loss in circHIPK3 regionby SNP array analysis(Figure S2E).Silencing of circHIPK3 inhibits cancer cell proliferation,migration and invasion in vitroTo investigate the function of circHIPK3 at the cellular level,cell proliferation assays were measured using water-solubletetrazolium 1(WST-1).We evaluated the loss of functionalimpact on cell viability by circHIPK3 and/or linHIPK3 inmultiple lung cancer cell lines including A549,H838,H1299,PC-9,H1975,H1650 and HCC827.Our analysis revealed thatloss of circHIPK3 conferred a time-dependent reduction incell viability(Figures 2AC and S3A).Exclusively silencing oflinHIPK3 minimally affected cell viability in most of the celllines,and even resulted in a minor increased viability in A549and H838 at 96 h(Figures 2AC and S3A,B).We also showedthat treatment with si-both(loss of both linHIPK3 andcircHIPK3)did not reverse the growth inhibition caused bycircHIPK3 abrogation alone.Corroborating these results,col-onyformationcapacitywasimpairedbysilencingofcircHIPK3(with or without linHIPK3)(Figure 2D,E).Onthe other hand,loss of linHIPK3 alone exhibited onlya minor effect(Figure 2D,E).These results indicated thatcircHIPK3 was the dominant isoform in modulating cell pro-liferation in lung cancer as compared to linHIPK3.In addition to cell proliferation,we also questionedwhether circHIPK3 modulates cell migration and invasion.We selected H1299 for Boyden chamber Matrigel invasionand migration assays since these cells were derived fromlymph node metastasis and have been reported to exhibithigh invasive potential 23.Consistent with cell viabilityassays,loss of circHIPK3 resulted in significant reduction incell invasion and migration capacity(Figures 2FH and S3C).Exclusive loss of linHIPK3 slightly decreased invasion capacityand had no influence on migration.Collectively,these cellfunction analyses demonstrate that circHIPK3,independent ofits linear counterpart,was an important modulator of lungcancer cell growth,invasion and migration.As the cell pro-liferation assays showed that A549,H838 and H1299 hada modest response to circHIPK3 abrogation,we thereforeselected these 3 cell lines for further mechanism-based studies.Abrogation of circHIPK3 induces autophagy in a subsetof lung cancer cell linesA role for HIPK3 protein as an autophagy regulator has beendemonstrated in a recent publication in Huntington disease24,25.As circHIPK3 is derived from the same pre-mRNA aslinHIPK3,we therefore investigated whether circHIPK3 affectedlung cancer cell viability by modulating this same mechanism.LC3B is a ubiquitin-like protein that has a non-lipidatedform,LC3B-I,and a lipidated form,LC3B-II.The accumulationof LC3B-II and the conversion from LC3B-I to LC3B-II canserve as sensitive marker for autophagy induction 26,27.Upon circHIPK3 knockdown,increased LC3B-II expression,upregulated LC3B-I/II conversion along with SQSTM1/p62degradation were observed in both A549 and H838,indicatingenhanced autophagosome synthesis and the onset of LC3B-mediated protein degradation(Figure 3A).In H1299,however,LC3B-II expression and LC3B-I/II conversion was suppressed,the degradation of SQSTM1 was also not observed(Figure 3A),suggesting the induction of autophagy via circHIPK3 abrogationappeared only in a subset of cell lines(further referred as autop-hagy-induced cell lines).TofurtherconfirmthatcircHIPK3silencingcaninduceautop-hagy,we measured the autophagic flux using the PremoAutophagy Tandem Sensor RFP-GFP-LC3B on both autophagy-induced cell lines(A549 and H838)and non-autophagy-inducedcell line(H1299).With RFP-GFP-LC3B assay,GFP and RFP areboth fluorescence-emitting markers in autophagosomes,while inautolysosomes,the GFP signal is lost due to the low pH environ-ment,allowing only RFP to fluoresce.This assay allows us toinspect the completion of autophagy flux that is deemed to bemoresensitiveandquantitativethanwesternblotmarkers26.InA549 and H838,we found that silencing circHIPK3 increased theformation of total autophagosomes and autolysosomes especiallyin H838 cells(Figures 3B,C,E,F and S4A,B).Loss of linHIPK3 incontrast,decreased the autophagic flux by 1050%in these 2 celllines(Figure 3B,C,E,F).Furthermore,si-both treatment thatsilenced both circHIPK3 and linHIPK3 reversed the increase inthe autophagic flux caused by c-circ-1 and c-circ-2 in both celllines(Figure3B,C,E,F).Theseresultssuggestedthattheregulationof autophagy by circHIPK3(si-circ-1)and