mir-432 induces nrf2 stabilization by directly targeting keap1 · oncogenes and tumor suppressors...

Oncogenes and Tumor Suppressors

miR-432 Induces NRF2 Stabilization by DirectlyTargeting KEAP1Burak Akdemir1, Yasuaki Nakajima2, Johji Inazawa1,3, and Jun Inoue1,3

Abstract

NF-E2–related factor 2 (NRF2) is a master transcriptional regu-lator that integrates cellular stress responses and is negatively reg-ulated by Kelch-like ECH-associated protein 1 (KEAP1) at the post-translational level. In human cancers, aberrantly stabilizedNRF2, bythemutationof eitherNRF2orKEAP1or by the potential inhibitionof autophagy, plays a vital role in tumor growth and chemoresis-tance through the activation of target genes. MicroRNAs (miRNA)are endogenous small noncoding RNAs that can negatively regulategene expression by interfering with translation and/or stability oftarget transcripts. However, miRNA-mediated regulation of theNRF2–KEAP1 pathway under physiological conditions is poorlyunderstood. Here, miR-432-3p positively regulates NRF2 activitythrough the downregulation of KEAP1 by a direct-binding mech-

anism to the coding regionofKEAP1.OverexpressionofmiR-432-3presulted in a decreased sensitivity of esophageal squamous cellcarcinoma (ESCC) cells to chemotherapy drugs including cisplatin(CDDP).Conversely, the inhibitionofmiR-432-3p expressionby theCRISPR/Cas9 system resulted in an increased sensitivity of ESCCcells to CDDP. Furthermore, miR-432-3p was overexpressed inprimary ESCC tumors (55 of 84, 65.5%) and a negative correlationbetween the expression level of KEAP1 and miR-432-3p in primaryESCC tumors was observed.

Implications: These findings provide novel insights into themechanism of NRF2 stabilization in human cancers. Mol CancerRes; 15(11); 1570–8. �2017 AACR.

IntroductionNRF2 is a master transcriptional regulator of cytoprotection

against cellular damage from chemotherapy and oxidative stress(1–3). Under physiological conditions, NRF2 is ubiquitinated bythe cullin 3 (CUL3)-KEAP1 ubiquitin E3 ligase complex and isconstantly degraded in the proteasome, resulting in a low cellularconcentration of the NRF2 protein. Under conditions of cellularstress, KEAP1 is inactivated and NRF2 is stabilized in the nucleus,resulting in cell survival through the transcriptional activation ofNRF2 target genes by the direct binding of NRF2 at antioxidative-responsive elements (ARE) within the corresponding promoters(4, 5). In human cancers, aberrantly stabilization of NRF2, bymutation of either NRF2 or KEAP1, plays a vital role in tumorgrowth and chemoresistance through the activation of targetgenes (1, 6–9). Furthermore, excess accumulation (as aggregates)of the p62 protein, a substrate for protein degradation by autop-hagy, can also stabilize NRF2 by competitively interacting withKEAP1 in hepatocellular carcinoma (10–13). Thus, NRF2 has an

oncogenic function in cancer cells, and a high level of NRF2protein is associated with a poor prognosis (14–16).

MicroRNAs (miRNA) are endogenous small noncoding RNAsthat can negatively regulate gene expression by interfering withtranslation and/or stability of target transcripts by directly bindingto the 30-untranslated region (30-UTR) or coding region (17–20).Cancer-related miRNAs are divided into two groups, oncogenicmiRNAs (oncomiR) targeting tumor-suppressor (TS) genes andTS-miRNAs targeting oncogenes (21–24). In particular, the ther-apeutic replacement of TS-miRs or the administration of oncomiRinhibitors is expected to be effective for cancer therapy (25–30). Ina previous study, we identified candidate miRNAs that negativelyor positively regulate transcriptional activity of theNRF2 pathwayby miRNA library screening using ARE reporters (31). Further-more, we demonstrated that the in vivo administration ofmiR-507or miR-634, which can negatively regulate the NRF2 pathway bydirectly targeting NRF2, was therapeutically effective againstNRF2-stabilized cancers (28, 31). However, the physiologicalmiRNA(s)-mediated regulation of the NRF2–KEAP1 pathway isnot fully understood.

In the present study, we focused on miRNAs that positivelyregulate the NRF2 pathway based on our screening data (31). Wefound that among the candidate miRNAs, miR-432-3p can stabi-lize NRF2 protein by directly targeting the coding region ofKEAP1. Overexpression of miR-432-3p resulted in the decreasedsensitivity of cancer cells to chemotherapeutic drugs, includingcisplatin (CDDP) via the activation of NRF2. Conversely, theinhibition of miR-432-3p expression by CRISPR-Cas9 system-mediated gene editing resulted in an increased sensitivity of ESCCcells to CDDP. Importantly, miR-432-3p was highly expressed in55of 84 primary ESCC tumors (65.5%) comparedwith that in thecorresponding non-cancerous esophageal mucosa, and weshowed a negative correlation between the expression level ofKEAP1 and miR-432-3p in primary ESCC tumors. Thus, our

1Department of Molecular Cytogenetics, Medical Research Institute, TokyoMedical and Dental University, Tokyo, Japan. 2Department of Surgical Gastro-enterology, Graduate School, Tokyo Medical and Dental University, Tokyo,Japan. 3Bioresource Research Center, Tokyo Medical and Dental University,Tokyo, Japan.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Authors: Jun Inoue and Johji Inazawa, Medical Research Insti-tute, Tokyo Medical and Dental University. 1-5-45 Yushima, Bunkyo-ku, Tokyo113-8510, Japan. Phone: 81-3-5803-5821; Fax: 81-3-5803-0244; E-mail:[email protected] and [email protected]

doi: 10.1158/1541-7786.MCR-17-0232

�2017 American Association for Cancer Research.

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findings provide novel insights for the regulation of the NRF2pathway and for miR-432-3p as a target for overcomingchemoresistance.

Materials and MethodsCell culture and primary tumor samples

HeLa (cervical cancer cell line) and SH-SY5Y (neuroblastomacell line) were obtained from ATCC and cultured in DMEMcontaining 10% FBS. Cells were detected as Mycoplasma-free byPCR-based method and were cultured for no more than 20passages from the validated stocks. ESCC cell lines (KYSE170,KYSE770, and KYSE2270), which were kindly given to us byDr. Shimada Y (Toyama University; refs. 32–34), were culturedin RPMI-1640 medium containing 10% FBS. All cell lines weremaintained at 37�C with 5% CO2.

A total of 84 primary ESCC tumor samples and the correspond-ing noncancerous esophageal mucosa were obtained frompatients treated at the Tokyo Medical and Dental UniversityHospital from November 2007 to October 2012, frozen imme-diately in liquid nitrogen, and stored at�80�Cuntil the extractionof total RNA was extracted. The collection and analysis of patientsamples were approved by the Tokyo Medical and Dental Uni-versity Institutional Review Board (approval #2010-5-4), andwritten consent was obtained from all patients.

Antibodies and reagentsRabbit polyclonal anti-NRF2 (Santa Cruz Biotechnology), rab-

bit polyclonal anti-KEAP1 (Proteintech), mouse monoclonalanti-FLAG (Sigma) and mouse monoclonal anti–b-actin (Sigma)antibodies were used. Cisplatin (CDDP) was purchased fromWAKO, and 5-fluorouracil (5-FU) and actinomycin D (Act D)were obtained from Sigma.

Plasmid construction and transfectionPCRproducts including the coding region and30-UTRofKEAP1

was inserted into pCMV-3Tag-1A (Flag-tagged) expression vector(Stratagene). The generated constructs were verified by sequenc-ing. All site-specificmutations in the coding regionwere generatedusing the KODPlusMutagenesis Kit (Toyobo). The plasmidsweretransfected intoHeLa cells using Lipofectamine 2000 (Invitrogen)according to the manufacturer's instructions.

Gene editing using CRISPR/Cas9 systemCRISPR/Cas9-mediated knockout was performed using the

GeneArt CRISPR Nuclease Vector with OFP Reporter Kit (Invitro-gen) according to the manufacturer's instructions. Target-specificguide RNA within miR-432 gene locus was designed on CRISPRDESIGN (http://crispr.mit.edu/). Annealed oligonucleotides forguide RNA were inserted into CRISPR Nuclease vector and thenthe vector was transfected into KYSE2270 cells using Lipofecta-mine 2000 (Invitrogen) according to the manufacturer's instruc-tions. After 2 days of transfection, OFP-positive cells were col-lected by FACS and were seeded at a single cell per one well of the96-well plate and cultured for 3 weeks. One clone (named as"miR-432KOcells") was isolated as a survived clone. Gene editingwas checked by sequencing analysis. Furthermore, a subclone(named as "miR-432 KO subclone") was isolated by single-cellcloning frommiR-432 KO cells. The information for oligonucleo-tides used is provided in Supplementary Table S1.

Cell survival assayCell survival was assessed by the crystal violet staining assay.

The cells were washed in PBS and fixed with 0.2% crystal violet in10% formaldehyde in PBS for 3 minutes. The excess crystal violetsolution was discarded and after being completely air-dried, thestained cells were lysed with a 2% SDS solution by shakingthe plates for 1 hour. The optical density (OD) was measured at560 nm using a microplate reader. The percentage absorbance ofeach well was determined. The OD values of cells in the controlwells were arbitrarily set at 100% to determine the percentage ofviable cells.

Transfection of miRNAs and siRNAsmiRNAor siRNAwas transfected into cells using Lipofectamine

RNAiMAX (Invitrogen) according to the manufacturer's instruc-tions. PremiRmiRNAmimics formiR-125a (PM12561),miR-125b(PM10148), miR-297 (PM10176), miR-432-3p (PM10838),miR-507 (PM10509), and negative control (#1) and miRVanamiRNA mimics for miR-432-3p (MC10838) and negative control(#1),whichwere used for the functional assay ofmiR-432-3p, wereobtained from Thermo Scientific. siRNAs against NRF2 (siGEN-OME SMARTpool; M-003755-02-0005) and KEAP1 (siGENOMESMARTpool; M-012453-00-0005) were obtained from ThermoScientific.

Conventional luciferase assayReporter plasmids were constructed by inserting each fragment

of the genomic region including the candidate sites for binding ofmiR-432-3p into the pmirGLO dual-luciferase miRNA targetexpression vector (Promega). All site-specific mutations in thecoding region of KEAP1 were generated using the KOD PlusMutagenesis Kit (Toyobo). Firefly and Renilla luciferase activitieswere measured using the dual-luciferase reporter assay system(Promega). The relative luciferase activity was calculated bynormalizing the firefly luciferase activity with the correspondinginternal Renilla luciferase activity.

Western blottingWhole cell lysates were subjected to SDS-PAGE, and the pro-

teins were transferred to polyvinylidene difluoride (PVDF) mem-branes (GE Healthcare). After blocking with TBS containing0.05% Tween-20 and 5% nonfat dry milk for 1 hour, the mem-brane was reacted with primary antibodies overnight. The dilu-tions for the primary antibodies were 1/1,000 for rabbit anti-NRF2, 1/1,000 for rabbit anti-KEAP1, 1/2,000 for anti-FLAG, and1/4000 for mouse anti–b-actin. The membrane was washed andexposed to horseradish peroxidase (HRP)–conjugated anti-mouse or rabbit immunoglobulin G (IgG) antibodies (both at1/4,000) for 1 hour. The bound antibodies were visualized withHRP staining solution or with an ECL Western detection kitaccording to the manufacturer's instructions (Cell SignalingTechnology).

Quantitative reverse transcription-PCRTotal RNA was isolated using TRIsure reagent (Nippon Genet-

ics) according to standard procedures. Quantitative RT-PCR(qRT-PCR) was performed on an ABI PRISM 7500 sequencedetection system (Applied Biosystems) according to the manu-facturer's instructions. The expression levels of coding genes ormiRNAs were based on the amount of the target product relative

miR-Mediated Regulation of the NRF2–KEAP1 Pathway

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Figure 1.

Identification of KEAP1 as a direct target of miR-432-3p. A, Western blotting analysis of transfected HeLa cells. The cells were transfected with 20 nmol/Lof negative control-miRNA (miR-NC) or miR-432-3p. After 2 days, the cell lysates were subjected to SDS-PAGE and immunoreacted with the indicatedantibodies. B, Expression analysis by qRT-PCR in transfected HeLa cells. The cells were transfected as described in A and after 2 days total RNA wasextracted; bar, SD. C, Stability assay of KEAP1 mRNA in the miR-432-3p- or miR-NC–transfected HeLa cells. The cells were transfected as described inA, and after 2 days, the cells were treated with actinomycin D (ActD). Total RNA was extracted at the indicated times. The t1/2 indicates the half-lifeof KEAP1 mRNA. The error bars are not visualized due to too small; bar, SD. D, Western blotting analysis of exogenously transfected Flag-KEAP1 andendogenous KEAP1. (Continued on the following page.)

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to that ofGAPDH orRNU6B transcript, respectively, as the controlto normalize the initial input of total RNA. The information forthe primers and TaqMan probes used is provided in Supplemen-tary Table S1.

Strand-specific RT-PCR for RTL1 genecDNA was synthesized using a specific primer to paternally

expressed RTL1-sense (se) transcript (50�C, 30 minutes), RT-PCRwas performed using nested PCR primer sets, and then PCRproducts were electrophoresed on agarose gel. The informationfor the primers used is provided in Supplementary Table S1.

mRNA stability assayCells were incubated with Act (5 mg/mL) and total RNA was

extracted at subsequent time intervals (0, 2, or 4 hours). Tomeasure the stability of KEAP1 mRNA, qRT-PCR analysis wasperformed as described above.

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) assay was performed

using ChIP-IT Express Enzymatic (Active Motif) according to themanufacturer's instructions. KYSE2270WT andmiR-432 KO cellswere fixed with 1.0% formaldehyde for 10 minutes at roomtemperature. Cells were lysed and nuclei were pelleted by centri-fugation. Nuclei were resuspended and sheared using EnzymaticShearing Cocktail for 10minutes at 37�C. Sheared chromatin wasimmunoprecipitated with rabbit polyclonal anti-NRF2 antibody(Santa Cruz Biotechnology) or control rabbit IgG antibody (SantaCruz Biotechnology). The cross-links reversed overnight at 65�Cand deproteinated with 0.5 mg/mL proteinase K. DNAwas purifiedwith a NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel)and quantitative RT-PCR (qRT-PCR) was performed with theprimers are indicated in a previous study (35). The informationfor the primers used is provided in Supplementary Table S1.

Statistical analysisThe experiments were independently performed in triplicate.

All P values were calculated by the 2-tailed Student t test andconsidered significant at <0.05.

ResultsIdentification of KEAP1 as a direct target of miR-432-3p

On the basis of the data from our miRNA library screeningusing ARE reporter plasmids (31), we focused on four miRNAs,miR-297, miR-432-3p, miR-125a-5p, andmiR-125b-5p, which wereshown to most potently regulate the NRF2 pathway. Overexpres-

sion of these miRNAs resulted in increased expression of NRF2protein inHeLa and twoESCC cell lines (KYSE170 andKYSE770),whereas the overexpression of miR-507 induced a decrease inNRF2 protein as reported previously (Supplementary Fig. S1; 31).Among these miRNAs, we focused on miR-432-3p, because theoverexpression of this miRNA induced a decrease in KEAP1expression at both protein and mRNA levels and reduction ofKEAP1 mRNA stability in HeLa cells (Fig. 1A–C). Therefore, weexamined whether miR-432-3p could directly target KEAP1. Thelevel of exogenously expressed Flag-tagged KEAP1 protein, as wellas that of endogenous KEAP1 protein, was clearly decreased in themiR-432-3p–transfected cells (Fig. 1D). Furthermore, we foundthree candidate binding-sites for the seed sequence ofmiR-432-3pwithin the coding region of KEAP1 gene (Fig. 1E). To evaluatewhether miR-432-3p binds to these sites, synonymous mutationswere generated at each site of the Flag-KEAP1 protein (Fig. 1E).Although the level of exogenously expressed Flag-tagged wild-type KEAP1 (WT) was decreased in the miR-432-3p-transfectedcells, the miR-432-3p-induced decrease of wild-type KEAP1 wasrestored by a mutation at the R1 site (R1 Mut.) but not at the twoother sites of R2 (R2Mut.) and R3 (R3Mut; Fig. 1F). Furthermore,the luciferase activity for the R1 vector was significantly reducedcompared to that of the empty vector, and this reduction wascompletely restored by the mutation (Fig. 1G and H). Thesefindings suggest thatmiR-432-3p can downregulate KEAP1 expres-sion by directly targeting its coding region.

Effect ofmiR-432-3p overexpression on the sensitivity of cancercells to chemotherapy drugs

Next, we examined the effect of miR-432-3p overexpression onchemosensitivity in HeLa cells and the two ESCC cell lines(KYSE170 and KYSE770). As shown in Fig. 2A, when miR-432-3pwas transfected at a concentration of 20 or 2 nmol/L, the rate ofcell survival in response to CDDP treatment was markedlyincreased compared with that in the miR-NC-transfected cells.The increase in the rate of cell survival by miR-432-3p was alsoobserved in response to 5-FU treatment (Supplementary Fig.S2A). In particular, the siRNA-mediated inhibition of KEAP1expression induced an increase in the rate of cell survival inresponse to CDDP treatment and the inhibition of NRF2 expres-sion attenuated the miR-432-3p-induced resistance to CDDP inKYSE770 cells (Fig. 2B and C). However, nevertheless NRF2expression is knocked down with siRNA, overexpression ofmiR-432-3p partially increased the rate of cell survival in responseto CDDP treatment in KYSE770 cells, suggesting that miR-432-3pmay concurrently regulate the expressionof other genes, except for

(Continued.) HeLa cells were transfected with miR-432-3p or miR-NC, and after 24 hours, the cells were transfected with empty vector (vec.) orFlag-KEAP1 plasmids. After 24 hours, the cell lysates were subjected to SDS-PAGE and immunoreacted with the indicated antibodies. The anti-KEAP1antibody detects both Flag-KEAP1 (arrow) and endogenousKEAP1 (range).E,Three regions (R1, R2, and R3) containing candidate binding sites ofmiR-432-3pwithinthe coding region of the KEAP1 gene. Candidate-binding sites for the seed sequence of miR-432-3p within the coding region and corresponding mutantsequences are indicated in each column. F, Western blotting analysis of Flag-KEAP1 WT or its mutants. The cells were transfected as described in D and thecell lysates were subjected to SDS-PAGE and immunoreacted with the indicated antibodies. Images of Western blots are indicated in the top. The anti-KEAP1antibody detects both Flag-KEAP1 (arrow) and endogenous KEAP1 (range). The relative expression level of Flag-KEAP1 protein in miR-432-3p–transfectedcells to that in the miR-NC–transfected cells was indicated as a graph in the bottom; bar, SD. � , P < 0.05. G, Luciferase assay using reporter plasmids.HeLa cells were transfected with miR-NC or miR-432-3p, and after 24 hours, the cells were transfected with a reporter plasmid (empty vector, R1, R2, or R3).After 24 hours of transfection with reporter plasmids, firefly or Renilla luciferase activities were measured. The luciferase activity in the miR-432-3p–transfectedcells relative to that in the miR-NC–transfected cells is indicated on the vertical axis of the graph; bar, SD. � , P < 0.05. H, Luciferase assay using reporter plasmids.HeLa cells were transfected with miR-NC or miR-432-3p, and after 24 hours, the cells were transfected with a reporter plasmid (empty vector, R1 WT, or R1 Mut).After 24 hours of transfection with reporter plasmids, firefly or Renilla luciferase activities were measured. The luciferase activity is indicated as described inG; bar, SD; � , P < 0.05; �� , P < 0.01.






Figure 2.

Decrease in sensitivity to CDDP by the overexpression of miR-432-3p. A, Effect of miR-432-3p overexpression on cell survival in responseto CDDP treatment. The cells were transfected with 20 or 2 nmol/L of miR-NC or miR-432-3p. After 24 hours, the cells were treated with CDDP (15 mmol/Lin HeLa cells, 10 mmol/L in KYSE170 cells, and 60 mmol/L in KYSE770 cells) for 24 hours. Cell survival rates were measured by CV staining. An increasein NRF2 protein level was confirmed by western blotting as shown in the top. The relative rate of cell survival of CDDP-treated cells to that of the untreatedcells is indicated as a graph in the bottom; bar, SD. �� , P < 0.01; �� , P < 0.001. B, Effect of siRNA-mediated inhibition of KEAP1 on cell survival in responseto CDDP treatment. KYSE770 cells were transfected with 20 nmol/L of negative control-siRNA (control-siRNA) or KEAP1-siRNA. After 24 hours, the cellswere treated with 60 mmol/L of CDDP for 24 hours. Cell survival rates were measured by CV staining. Decrease in KEAP1 protein level and increase inNRF2 protein level were confirmed by Western blotting as shown in the top. The relative rate of cell survival of CDDP-treated cells to that of the untreatedcells is indicated as a graph in the bottom; bar, SD; � ,P <0.05.C,Effect of siRNA-mediated inhibition ofNRF2on cell survival in response toCDDP treatment. KYSE770cells were transfected with 20 nmol/L of negative control-miRNA (miR-NC) or miR-432-3p, and after 5 hours, the cells were transfected with control-siRNAor NRF2-siRNA. After 24 hours of transfection with siRNA, the cells were treated with 60 mmol/L of CDDP for 24 hours. Cell survival rates were measuredby CV staining. Increase or decrease in NRF2 protein level was confirmed by Western blotting as shown in the top. The relative rate of cell survivalof CDDP-treated cells to that of the untreated cells is indicated as a graph in the bottom; bar, SD; �� , P < 0.001.

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KEAP1, to affect sensitivity toCDDP (Fig. 2C).Wehave previouslydemonstrated that the overexpression ofmiR-634 could overcomeCDDP resistance by the direct targeting of NRF2 bymiR-634 (28).

We showed that co-transfection with miR-432-3p and miR-634attenuated themiR-634-mediated increase in sensitivity to CDDP(Supplementary Fig. S2B). These findings strongly suggest that the

Figure 3.

Increase in sensitivity to CDDP by the inhibition of miR-432-3p. A, The three edited sequences within the mir-432 locus in a subline of KYSE2270 cellsgenerated by the CRISPR/Cas9 system. B, Expression analysis of miR-432-3p in the miR-432-KO or WT cells by qRT-PCR. Bar, SD. C, Expression analysisof KEAP1 mRNA in the miR-432-KO or WT cells by qRT-PCR; bar, SD. D, Stability assay of KEAP1 mRNA in the miR-432-KO or WT cells. The cells weretreated with actinomycin D (ActD) and total RNA was extracted at the indicated times. The t1/2 indicates the half-life of KEAP1 mRNA; bar, SD. E, Westernblotting analysis of themiR-432-KO orWT cells. The cell lysates were subjected to SDS-PAGE and immunoreacted with the indicated antibodies. F, Luciferase assayusing reporter plasmids. The cells were transfected with reporter plasmids (empty vector or ARE-containing reporter vector) and internal control vector.After 48 hours of transfection, firefly or Renilla luciferase activities were measured. The relative luciferase activity is indicated on the vertical axis in graph;bar, SD; �� , P < 0.001. G, Expression analysis of NQO1mRNA in themiR-432-KO or WT cells by qRT-PCR. Bar, SD. H, ChIP-qPCR analysis for binding of NRF2 to theNQO1 promoter region in miR-432-KO or WT cells. qRT-PCR was performed on DNAs immunoprecipitated with rabbit polyclonal anti-NRF2 antibody orrabbit IgG antibody (negative control) using primers that flank the ARE region in the NQO1 promoter; bar, SD; �� , P < 0.001. I, Cell survival assay of miR-432-KOor WT cells in response to CDDP treatment. The cells were treated with 30 mmol/L of CDDP for 24 hours. Cell survival rates were measured by CV staining.The relative rate of cell survival of CDDP-treated cells to that of the untreated cells was indicated as a graph; bar, SD; �� , P < 0.01.






overexpression of miR-432-3p is involved in the decrease ofchemosensitivity through the upregulation of NRF2 protein incancer cells.

Effect of miR-432-3p inhibition on CDDP sensitivityNext, to investigate the effect of inhibition of miR-432-3p

expression on sensitivity to chemotherapy drugs, we establishedmiR-432 knock out (KO) cells in which the endogenous expres-sion level of miR-432-3p was inhibited by gene editing via theCRISPR/Cas9 system using a guide sequence designed within themiR-432 locus in KYSE2270 cells, which display high levels ofmiR-432-3p and lack mutations in the NRF2 and KEAP1 genes(Fig. 3A and Supplementary Fig. S3).We showed thatmiR-432KOcells was a mixture of several cells having different editing (either1bp-, 2bp- or 5bp-deletion) by sequencing analysis and that themature miR-432-3p expression was markedly inhibited in themiR-432 KO cells compared with that in the wild-type (WT) cells(Fig. 3A and B). Furthermore, the expression level or stability ofKEAP1 mRNA was increased in the miR-432-KO cells comparedwith that in the WT cells (Fig. 3C and D). By western blotting, wefound that the expression level of KEAP1 protein was increasedand that of NRF2 protein was decreased in the miR-432-KO cellscompared with those in the WT cells (Fig. 3E). In addition, theactivity of AREs was clearly reduced and the expression level ofNQO1 mRNA, a transcriptional target of NRF2, was decreased inthe miR-432-KO cells (Fig. 3F and G). Furthermore, ChIP-qPCRanalysis revealed the decreased enrichment of NRF2 at the pro-moter region of NQO1 gene including ARE in miR-432 KO cellscompared with that in WT cells (Fig. 3H). These results suggestthat the transcriptional activity ofNRF2was effectively reduced byinhibition of miR-432-3p expression in miR-432 KO cells. Thesensitivity of the miR-432-KO cells to CDDP was increased com-pared with that of the WT cells (Fig. 3I). Moreover, we isolated asubclone from miR-432-KO cells, which has only 1bp-deletionwithin miR-432 locus. This subclone exhibited the same pheno-types as miR-432 KO cells such as effective inhibition of miR-432-3p expression, upregulation of KEAP1, downregulation ofNRF2, and increased sensitivity to CDDP (Supplementary Fig.S4). On the other hand, miR-432-3p is harbored within animprinted RTL1 (retrotransposon like 1) genetic locus at14q32.2 and is functionally processed frommaternally expressedand non-coding RTL1-antisense (as) transcript, not from pater-nally expressed and coding RTL1-sense (se) transcript (36). Toexamine whether the loss-of-function mutation in RTL1-se bygene editing has an impact onphenotypes inmiR-432KOcells, wevalidated expression of RTL1-se by strand-specific RT-PCR. As aresult, we found that RTL1-se was not expressed in parentalKYSE2270 cells, miR-432 KO cells, and miR-432 KO subclone,suggesting that the phenotypes in miR-432 KO cells were actuallydue to the inhibition of miR-432-3p expression (SupplementaryFig. S5). Taken together, these findings strongly suggest that theinhibition ofmiR-432-3p expression is involved in the increase inCDDP sensitivity of ESCC cells.

Expression analysis of miR-432-3p in ESCC cell lines andprimary ESCC tumors

We first examined the expression level of miR-432-3p in 42ESCCcell lines. The expression level ofmiR-432-3pwas elevated in40 of 42 ESCC cell lines, compared with the normal esophagealtissues (95.2%, fold change > 2.0, Supplementary Fig. S3). Wenext examined the expression level of miR-432-3p by qRT-PCR in

84 paired samples from patients with ESCC. The expression levelof miR-432-3p was elevated in 55 of 84 primary ESCC tumors,compared with the corresponding non-cancerous esophagealmucosa (65.5%, fold change > 2.0, Fig. 4A). Furthermore, weshowed a negative correlation between KEAP1mRNA expressionand miR-432-3p expression in 58 primary ESCC cases, which areinformative for both KEAP1 mRNA expression and miR-432-3pexpression (Fig. 4B). There was no significant relationshipbetween the expression levels and the clinical implications andprognosis in those patients. These results suggest thatmiR-432-3pis frequently overexpressed in primary ESCC tumors and that thehigh expression ofmiR-432-3pmay be a potential mechanism fordownregulation of KEAP1 in ESCC.

DiscussionA key finding of our study is thatmiR-432-3p can downregulate

KEAP1 by directly binding to its coding region, and high expres-sion of miR-432-3p can contribute to decrease the sensitivity ofESCC cells to chemotherapy drugs probably due to the increase inNRF2 protein. It is well known that aberrantly stabilizedNRF2, bymutation of either NRF2 or KEAP1 or by the potential inhibitionof autophagy in hepatocellular carcinoma, plays a vital role ingrowth and development of resistance to chemotherapeutic

Figure 4.

Expression analysis of miR-432-3p in primary ESCC tumor samples.A, Relative expression level of miR-432-3p in 84 primary ESCC samplesrelative to that in corresponding non-cancerous tissues. Bar, SD.B, Negative correlation between miR-432-3p and KEAP1 mRNA expressionlevels in 58 primary tumor tissues.


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agents and radiotherapy in a variety of cancer (6–13). We havepreviously demonstrated that the down-regulation of expressionfor four miRNAs that negatively regulate NRF2, namely,miR-507,miR-634, miR-450a, and miR-129-5p, identified by our miRNAlibrary screening,was also involved in the stabilization ofNRF2 inESCC (31). In this study, we showed that the expression level ofmiR-432-3p was inversely correlated with KEAP1 mRNA level inprimary ESCC tumors. This suggests that the high expression ofmiR-432-3p and downregulation of miRNAs that negatively reg-ulate NRF2, as well as somatic mutations inNRF2 or KEAP1,maybe a possible mechanism of NRF2 stabilization, indicating thepresence of multiple pathways in NRF2 stabilization in cancer.Hence, determination of the genetic status of NRF2 and KEAP1and the expression status of NRF2-regulating miRNAs, includingmiR-432-3p in a large cohort of tumor samples, together withelucidating the mechanism of upregulation of miR-432-3p isneeded to understand the various mechanisms of NRF2 stabili-zation and to further develop a method for the classification ofpatients with NRF2-stabilized tumors for overcoming therapeuticresistance.

So far, it has been reported that three miRNAs, miR-200a,mir-141, and mir-125b-5p, target KEAP1 in MDA-MD-231 (breastcancer cell line), A2780 (ovarian cancer cell line), and primarymouse hepatocytes, respectively (37–39). However, among thesemiRNAs, miR-200a and miR-141, were not highly ranked ascandidates that positively regulate the NRF2 pathway in ourmiRNA library screening using HeLa cells (31). The miRNA-mediated regulation of KEAP1 expression may be dependent onthe cell-type or tissue-type. Furthermore, although we found thatthe transfection of miR-125b-5p and other candidate miRNAs,includingmiR-125a-5p andmiR-297 could also induce an increaseof NRF2 expression in HeLa cells and the two ESCC cell lines(KYSE170 and KYSE770), we did not observe a down-regulationof KEAP1 expression in these three cell lines transfected with themiRNAs. This suggests that these miRNAs may be involved inNRF2 stabilization through an unknown mechanism other thanthat involving KEAP1.

Administration of TS-miRNAs is expected to be useful strategyfor cancer therapy (25, 40). Indeed, clinical trials on the admin-istration of miR-34a, which can directly target several oncogenes,including BCL2, are currently on-going in hepatocellular carci-noma and hematopoietic tumors (41, 42). We recently demon-strated that in vivo therapeutic administration of miR-507 andmiR-634, which can negatively regulate NRF2, enhanced chemo-therapy-induced cytotoxicity against NRF2-stabilized cancers(28, 31). On the other hand, the therapeutic effect of anti-miRsin inhibiting the expressionof oncogenicmiRNAs such asmiR-10band miR-155 has also been demonstrated in xenograft tumors

in vivo (29, 30). Our in vitro results in this study suggest that theinhibition of endogenous miR-432-3p may overcome chemore-sistance through the upregulation of KEAP1 and downregulationof NRF2 in ESCC cells. Furthermore, we showed that gene editingby the CRISPR/Cas9 systemon the stem-loop structure of primarymiR-432-3p can effectively inhibit endogenousmiR-432-3p expres-sion probably due to the failure of miR-432-3p biogenesis. How-ever, transfection with commercially obtained anti-miR-432-3pwas not effective for the inhibition of endogenous miR-432-3pexpression (data not shown). Thus, the improvement of chemicalmodification and sequence-based design of anti–miR-432-3p tomore effectively inhibit the endogenousmiR-432-3p expression isrequired to achieve therapeutic inhibition of the NRF2-mediatedoncogenic pathway via theupregulationofKEAP1 inpatientswithNRF2-stabilized tumors.

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

Authors' ContributionsConception and design: B. Akdemir, J. Inazawa, J. InoueDevelopment of methodology: B. Akdemir, J. Inazawa, J. InoueAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Akdemir, Y. Nakajima, J. Inazawa, J. InoueAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B. Akdemir, J. Inazawa, J. InoueWriting, review, and/or revision of the manuscript: B. Akdemir, J. Inazawa,J. InoueAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Inazawa, J. InoueStudy supervision: J. Inazawa, J. Inoue

AcknowledgmentsThe authors thank Ayako Takahashi and Rumi Mori (Tokyo Medical and

Dental University, Japan) for their technical assistance.

Grant SupportThis work was supported in part by Grant-in-Aid for Scientific Research

(C:15K08301; to Jun Inoue), Grant-in-Aid for Scientific Research on InnovativeAreas "Conquering cancer through NEO-dimensional systems understandings"(15H05908; to Johji Inazawa) from JSPS and MEXT, a research program of theProject for Cancer Research and Therapeutic Evolution (P-CREATE), the Tailor-Made Medical Treatment with the BioBank Japan Project (BBJ) from the JapanAgency for Medical Research and Development (AMED), and the Joint Usage/Research Program of MRI, TMDU.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 27, 2017; revised June 16, 2017; accepted July 25, 2017;published OnlineFirst July 31, 2017.

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Akdemir et al.




2017;15:1570-1578. Published OnlineFirst July 31, 2017.Mol Cancer Res Burak Akdemir, Yasuaki Nakajima, Johji Inazawa, et al.

Induces NRF2 Stabilization by Directly Targeting KEAP1miR-432

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