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miR-432 Induces NRF2 Stabilization by Directly Targeting KEAP1 1

Burak Akdemir1, Yasuaki Nakajima

3, Johji Inazawa

1,2, and Jun Inoue

1,2 2

3

Author’s affiliations: 1Department of Molecular Cytogenetics, Medical Research Institute, Tokyo 4

Medical and Dental University, Tokyo, Japan; 2Bioresource Research Center, Tokyo Medical and 5

Dental University, Tokyo, Japan; 3Department of Surgical Gastroenterology, Graduate School, Tokyo 6

Medical and Dental University, Tokyo, Japan. 7

8

Correspondence: Jun Inoue and Johji Inazawa, Department of Molecular Cytogenetics, Medical Research 9

Institute, Tokyo Medical and Dental University. 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan; e-10

mail: [email protected] (Jun Inoue), [email protected] (Johji Inazawa); TEL: +81-3-5803-11

5820/5821, FAX: +81-3-5803-0244. 12

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Conflicts of interest: The authors declare no conflicts of interest. 14

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Running title: miR-mediated Regulation of the NRF2-KEAP1 Pathway 16

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Keywords: NRF2-KEAP1 pathway, Chemosensitivity, microRNA, Gene editing, Esophageal 18

squamous cell carcinoma (ESCC) 19

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on October 11, 2020. © 2017 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 31, 2017; DOI: 10.1158/1541-7786.MCR-17-0232

mailto:[email protected]

http://mcr.aacrjournals.org/

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Financial support: This work was supported in part by Grant-in-Aid for Scientific Research 1

(C:15K08301, Jun Inoue), Grant-in-Aid for Scientific Research on Innovative Areas “Conquering 2

cancer through NEO-dimensional systems understandings” (15H05908, Johji Inazawa) from JSPS 3

and MEXT, a research program of the Project for Cancer Research and Therapeutic Evolution (P-4

CREATE), the Tailor-Made Medical Treatment with the BioBank Japan Project (BBJ) from the Japan 5

Agency for Medical Research and Development (AMED), and the Joint Usage/Research Program of 6

MRI, TMDU. 7

8

Notes: abstract; 207 words, text; 4,531 words, Figures; 4, Supplemental figures; 5, Supplemental 9

table; 1. 10




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ABSTRACT 1

NF-E2-related factor 2 (NRF2) is a master transcriptional regulator that integrates cellular stress 2

responses and is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1) at the post-3

translational level. In human cancers, aberrantly stabilized NRF2, by the mutation of either NRF2 or 4

KEAP1 or by the potential inhibition of autophagy, plays a vital role in tumor growth and 5

chemoresistance through the activation of target genes. MicroRNAs (miRNAs) are endogenous 6

small noncoding RNAs that can negatively regulate gene expression by interfering with translation 7

and/or stability of target transcripts. However, miRNA-mediated regulation of the NRF2-KEAP1 8

pathway under physiological conditions is poorly understood. Here, miR-432-3p positively regulates 9

NRF2 activity through the down-regulation of KEAP1 by a direct binding mechanism to the coding 10

region of KEAP1. Overexpression of miR-432-3p resulted in a decreased sensitivity of esophageal 11

squamous cell carcinoma (ESCC) cells to chemotherapy drugs including cisplatin (CDDP). 12

Conversely, the inhibition of miR-432-3p expression by the CRISPR/Cas9 system resulted in an 13

increased sensitivity of ESCC cells to CDDP. Furthermore, miR-432-3p was overexpressed in 14

primary ESCC tumors (55 of 84, 65.5%) and a negative correlation between the expression level of 15

KEAP1 and miR-432-3p in primary ESCC tumors was observed. 16

17

Implications: These findings provide novel insights into the mechanism of NRF2 stabilization in 18

human cancers. 19

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INTRODUCTION 1

NRF2 is a master transcriptional regulator of cytoprotection against cellular damage from 2

chemotherapy and oxidative stress (1-3). Under physiological conditions, NRF2 is ubiquitinated by 3

the cullin 3 (CUL3)-KEAP1 ubiquitin E3 ligase complex and is constantly degraded in the 4

proteasome, resulting in a low cellular concentration of the NRF2 protein. Under conditions of 5

cellular stress, KEAP1 is inactivated and NRF2 is stabilized in the nucleus, resulting in cell survival 6

through the transcriptional activation of NRF2 target genes by the direct binding of NRF2 at 7

antioxidative responsive elements (AREs) within the corresponding promoters (4,5). In human 8

cancers, aberrantly stabilization of NRF2, by mutation of either NRF2 or KEAP1, plays a vital role in 9

tumor growth and chemoresistance through the activation of target genes (1,6-9). Furthermore, 10

excess accumulation (as aggregates) of the p62 protein, a substrate for protein degradation by 11

autophagy, can also stabilize NRF2 by competitively interacting with KEAP1 in hepatocellular 12

carcinoma (10-13). Thus, NRF2 has an oncogenic function in cancer cells, and a high level of NRF2 13

protein is associated with a poor prognosis (14-16). 14

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that can negatively regulate 15

gene expression by interfering with translation and/or stability of target transcripts by directly binding 16

to the 3′-untranslated region (3′-UTR) or coding region (17-20). Cancer-related miRNAs are divided 17

into two groups, oncogenic miRNAs (oncomiRs) targeting tumor suppressor (TS) genes and TS-18

miRNAs targeting oncogenes (21-24). In particular, the therapeutic replacement of TS-miRs or the 19

administration of oncomiR inhibitors is expected to be effective for cancer therapy (25-30). In a 20

previous study, we identified candidate miRNAs that negatively or positively regulate transcriptional 21

activity of the NRF2 pathway by miRNA library screening using ARE reporters (31). Furthermore, we 22




5

demonstrated that the in vivo administration of miR-507 or miR-634, which can negatively regulate 1

the NRF2 pathway by directly targeting NRF2, was therapeutically effective against NRF2-stabilized 2

cancers (28,31). However, the physiological miRNA(s)-mediated regulation of the NRF2-KEAP1 3

pathway is not fully understood. 4

In the present study, we focused on miRNAs that positively regulate the NRF2 pathway 5

based on our screening data (31). We found that among the candidate miRNAs, miR-432-3p can 6

stabilize NRF2 protein by directly targeting the coding region of KEAP1. Overexpression of miR-432-7

3p resulted in the decreased sensitivity of cancer cells to chemotherapeutic drugs including cisplatin 8

(CDDP) via the activation of NRF2. Conversely, the inhibition of miR-432-3p expression by CRISPR-9

Cas9 system-mediated gene editing resulted in an increased sensitivity of ESCC cells to CDDP. 10

Importantly, miR-432-3p was highly expressed in 55 of 84 primary ESCC tumors (65.5%) compared 11

with that in the corresponding non-cancerous esophageal mucosa, and we showed a negative 12

correlation between the expression level of KEAP1 and miR-432-3p in primary ESCC tumors. Thus, 13

our findings provide novel insights for the regulation of the NRF2 pathway and for miR-432-3p as a 14

target for overcoming chemoresistance. 15

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MATERIALS AND METHODS 1

Cell culture and primary tumor samples 2

HeLa (cervical cancer cell line) and SH-SY5Y (neuroblastoma cell line) were obtained from ATCC 3

and cultured in Dulbecco's Modified Eagle’s Medium containing 10% FBS. Cells were detected as 4

mycoplasma-free by PCR-based method and were cultured for no more than 20 passages from the 5

validated stocks. ESCC cell lines (KYSE170, KYSE770, and KYSE2270), which were kindly given to 6

us by Dr. Shimada Y (Toyama University) (32-34), were cultured in RPMI1640 medium containing 7

10% FBS. All cell lines were maintained at 37°C with 5% CO2. 8

A total of 84 primary ESCC tumor samples and the corresponding noncancerous esophageal 9

mucosa were obtained from patients treated at the Tokyo Medical and Dental University Hospital 10

from November 2007 to October 2012, frozen immediately in liquid nitrogen, and stored at −80°C 11

until the extraction of total RNA was extracted. The collection and analysis of patient samples were 12

approved by the Tokyo Medical and Dental University Institutional Review Board (approval #2010-5-13

4), and written consent was obtained from all patients. 14

15

Antibodies and reagents 16

Rabbit polyclonal anti-NRF2 (Santa Cruz Biotechnology), rabbit polyclonal anti-KEAP1 (Proteintech), 17

mouse monoclonal anti-FLAG (Sigma) and mouse monoclonal anti-β-actin (Sigma) antibodies were 18

used. Cisplatin (CDDP) was purchased from WAKO, and 5-fluorouracil (5-FU) and actinomycin D 19

(Act D) were obtained from Sigma. 20

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Plasmid construction and transfection 1

PCR products including the coding region and 3′-UTR of KEAP1 was inserted into pCMV-3Tag-1A 2

(Flag-tagged) expression vector (Stratagene). The generated constructs were verified by sequencing. 3

All site-specific mutations in the coding region were generated using the KOD Plus Mutagenesis Kit 4

(Toyobo). The plasmids were transfected into HeLa cells using Lipofectamine 2000 (Invitrogen) 5

according to the manufacturer’s instructions. 6

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Gene editing using CRISPR/Cas9 system 8

CRISPR/Cas9-mediated knockout was performed using the GeneArt® CRISPR Nuclease Vector 9

with OFP Reporter Kit (Invitrogen) according to the manufacturer’s instructions. Target-specific guide 10

RNA within miR-432 gene locus was designed on CRISPR DESIGN (http://crispr.mit.edu/). 11

Annealed oligonucleotides for guide RNA were inserted into CRISPR Nuclease vector and then the 12

vector was transfected into KYSE2270 cells using Lipofectamine 2000 (Invitrogen) according to the 13

manufacturer’s instructions. After 2 days of transfection, OFP-positive cells were collected by FACS 14

and was seeded at a single cell per one well of the 96-well plate and cultured for 3 weeks. One clone 15

(named as “miR-432 KO cells”) was isolated as a survived clone. Gene editing was checked by 16

sequencing analysis. Furthermore, a subclone (named as “miR-432 KO subclone”) was isolated by 17

single-cell cloning from miR-432 KO cells. The information for oligonucleotides used is provided in 18

Supplementary Table 1. 19

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http://crispr.mit.edu/


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Cell survival assay 1

Cell survival was assessed by the crystal violet staining assay. The cells were washed in PBS and 2

fixed with 0.2% crystal violet in 10% formaldehyde in PBS for 3 minutes. The excess crystal violet 3

solution was discarded and after being completely air-dried, the stained cells were lysed with a 2% 4

SDS solution by shaking the plates for 1 hour. The optical density (OD) was measured at 560 nm 5

using a microplate reader. The percentage absorbance of each well was determined. The OD values 6

of cells in the control wells were arbitrarily set at 100% to determine the percentage of viable cells. 7

8

Transfection of miRNAs and siRNAs 9

miRNA or siRNA was transfected into cells using Lipofectamine RNAiMAX (Invitrogen) according to 10

the manufacturer's instructions. PremiR miRNA mimics for miR-125a (PM12561), miR-125b 11

(PM10148), miR-297 (PM10176), miR-432-3p (PM10838), miR-507 (PM10509), and negative 12

control (#1) and miRVana miRNA mimics for miR-432-3p (MC10838) and negative control (#1), 13

which were used for the functional assay of miR-432-3p, were obtained from Thermo Scientific. 14

siRNAs against NRF2 (siGENOME SMARTpool; M-003755-02-0005) and KEAP1 (siGENOME 15

SMARTpool; M-012453-00-0005) were obtained from Thermo Scientific. 16

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Conventional luciferase assay 18

Reporter plasmids were constructed by inserting each fragment of the genomic region including the 19

candidate sites for binding of miR-432-3p into the pmirGLO dual-luciferase miRNA target expression 20

vector (Promega). All site-specific mutations in the coding region of KEAP1 were generated using 21

the KOD Plus Mutagenesis Kit (Toyobo). Firefly and Renilla luciferase activities were measured 22




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using the dual-luciferase reporter assay system (Promega). The relative luciferase activity was 1

calculated by normalizing the firefly luciferase activity with the corresponding internal Renilla 2

luciferase activity. 3

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Western blotting 5

Whole cell lysates were subjected to SDS-PAGE, and the proteins were transferred to polyvinylidene 6

difluoride (PVDF) membranes (GE Healthcare). After blocking with TBS containing 0.05% Tween-20 7

and 5% nonfat dry milk for 1 hour, the membrane was reacted with primary antibodies overnight. 8

The dilutions for the primary antibodies were 1/1000 for rabbit anti-NRF2, 1/1000 for rabbit anti-9

KEAP1, 1/2000 for anti-FLAG, and 1/4000 for mouse anti-β-actin. The membrane was washed and 10

exposed to horseradish peroxidase (HRP)-conjugated anti-mouse or rabbit immunoglobulin G (IgG) 11

antibodies (both at 1/4,000) for 1 hour. The bound antibodies were visualized with HRP staining 12

solution or with an ECL Western detection kit according to the manufacturer's instructions (Cell 13

Signaling Technology). 14

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Quantitative reverse transcription-PCR (qRT-PCR) 16

Total RNA was isolated using TRIsure reagent (Nippon Genetics) according to standard procedures. 17

Quantitative RT-PCR (qRT-PCR) was performed on an ABI PRISM 7500 sequence detection 18

system (Applied Biosystems) according to the manufacturer's instructions. The expression levels of 19

coding genes or miRNAs were based on the amount of the target product relative to that of GAPDH 20

or RNU6B transcript, respectively, as the control to normalize the initial input of total RNA. The 21

information for the primers and TaqMan probes used is provided in Supplementary Table 1. 22




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1

Strand-specific RT-PCR for RTL1 gene 2

cDNA was synthesized using a specific primer to paternally expressed RTL1-sense (se) transcript 3

(50°C, 30min), RT-PCR was performed using nested PCR primer sets, and then PCR products were 4

electrophoresed on agarose gel. The information for the primers used is provided in Supplementary 5

Table 1. 6

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mRNA stability assay 8

Cells were incubated with Act (5 μg/ml) and total RNA was extracted at subsequent time intervals (0, 9

2, or 4 h). To measure the stability of KEAP1 mRNA, qRT–PCR analysis was performed as 10

described above. 11

12

Chromatin Immunoprecipitation (ChIP) Assay 13

ChIP assay was performed using ChIP-IT® Express Enzymatic (Active Motif) according to the 14

manufacturer's instructions. KYSE2270 WT and miR-432 KO cells were fixed with 1.0% 15

formaldehyde for 10 min at room temperature. Cells were lysed and nuclei were pelleted by 16

centrifugation. Nuclei were resuspended and sheared using Enzymatic Shearing Cocktail for 10 min 17

at 37°C. Sheared chromatin was immunoprecipitated with rabbit polyclonal anti-NRF2 antibody 18

(Santa Cruz Biotechnology) or control rabbit IgG antibody (Santa Cruz Biotechnology). The cross-19

links reversed overnight at 65 °C and deproteinated with 0.5 µg/μl proteinase K. DNA was purified 20

with a NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel) and quantitative RT-PCR (qRT-21



https://www.activemotif.com/catalog/details/53009/chip-it-express-enzymatic


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PCR) was performed with the primers are indicated in a previous study (42). The information for the 1

primers used is provided in Supplementary Table 1. 2

3

Statistics 4

The experiments were independently performed in triplicate. All P values were calculated by 2-tailed 5

Student t test and considered significant at < 0.05. 6

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RESULTS 1

Identification of KEAP1 as a direct target of miR-432-3p 2

Based on the data from our miRNA library screening using ARE reporter plasmids (31), we focused 3

on four miRNAs, miR-297, miR-432-3p, miR-125a-5p, and miR-125b-5p, which were shown to most 4

potently regulate the NRF2 pathway. Overexpression of these miRNAs resulted in increased 5

expression of NRF2 protein in HeLa and two ESCC cell lines (KYSE170 and KYSE770), whereas 6

the overexpression of miR-507 induced a decrease in NRF2 protein as reported previously 7

(Supplementary Fig. S1) (31). Among these miRNAs, we focused on miR-432-3p, because the 8

overexpression of this miRNA induced a decrease in KEAP1 expression at both protein and mRNA 9

levels and reduction of KEAP1 mRNA stability in HeLa cells (Fig. 1A-C). Therefore, we examined 10

whether miR-432-3p could directly target KEAP1. The level of exogenously expressed Flag-tagged 11

KEAP1 protein, as well as that of endogenous KEAP1 protein, was clearly decreased in the miR-12

432-3p-transfected cells (Fig. 1D). Furthermore, we found three candidate binding-sites for the seed 13

sequence of miR-432-3p within the coding region of KEAP1 gene (Fig. 1E). To evaluate whether 14

miR-432-3p binds to these sites, synonymous mutations were generated at each site of the Flag-15

KEAP1 protein (Fig. 1E). While the level of exogenously expressed Flag-tagged wild-type KEAP1 16

(WT) was decreased in the miR-432-3p-transfected cells, the miR-432-3p-induced decrease of wild-17

type KEAP1 was restored by a mutation at the R1 site (R1 Mut.) but not at the two other sites of R2 18

(R2 Mut.) and R3 (R3 Mut.) (Fig. 1F). Furthermore, the luciferase activity for the R1 vector was 19

significantly reduced compared to that of the empty vector, and this reduction was completely 20

restored by the mutation (Fig. 1G and 1H). These findings suggest that miR-432-3p can down-21

regulate KEAP1 expression by directly targeting its coding region. 22




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Effect of miR-432-3p overexpression on the sensitivity of cancer cells to chemotherapy drugs 1

Next, we examined the effect of miR-432-3p overexpression on chemosensitivity in HeLa cells and 2

the two ESCC cell lines (KYSE170 and KYSE770). As shown in Fig. 2A, when miR-432-3p was 3

transfected at a concentration of 20 nM or 2 nM, the rate of cell survival in response to CDDP 4

treatment was markedly increased compared with that in the miR-NC-transfected cells. The increase 5

in the rate of cell survival by miR-432-3p was also observed in response to 5-FU treatment 6

(Supplementary Fig. S2A). In particular, the siRNA-mediated inhibition of KEAP1 expression 7

induced an increase in the rate of cell survival in response to CDDP treatment and the inhibition of 8

NRF2 expression attenuated the miR-432-3p-induced resistance to CDDP in KYSE770 cells (Fig. 9

2B and 2C). However, nevertheless NRF2 expression is knocked down with siRNA, overexpression 10

of miR-432-3p partially increased the rate of cell survival in response to CDDP treatment in 11

KYSE770 cells, suggesting that miR-432-3p may concurrently regulate the expression of other 12

genes, except for KEAP1, to affect sensitivity to CDDP (Fig. 2C). We have previously demonstrated 13

that the overexpression of miR-634 could overcome CDDP resistance by the direct targeting of 14

NRF2 by miR-634 (28). We showed that co-transfection with miR-432-3p and miR-634 attenuated 15

the miR-634-mediated increase in sensitivity to CDDP (Supplementary Fig. S2B). These findings 16

strongly suggest that the overexpression of miR-432-3p is involved in the decrease of 17

chemosensitivity through the up-regulation of NRF2 protein in cancer cells. 18

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Effect of miR-432-3p inhibition on CDDP sensitivity 1

Next, to investigate the effect of inhibition of miR-432-3p expression on sensitivity to chemotherapy 2

drugs, we established miR-432 knock out (KO) cells in which the endogenous expression level of 3

miR-432-3p was inhibited by gene editing via the CRISPR/Cas9 system using a guide sequence 4

designed within the miR-432 locus in KYSE2270 cells, which display high levels of miR-432-3p and 5

lack mutations in the NRF2 and KEAP1 genes (Fig. 3A and Supplementary Fig. S3). We showed 6

that miR-432 KO cells was a mixture of several cells having different editing (either 1bp-, 2bp- or 7

5bp-deletion) by sequencing analysis and that the mature miR-432-3p expression was markedly 8

inhibited in the miR-432 KO cells compared with that in the wild-type (WT) cells (Fig. 3A and 3B). 9

Furthermore, the expression level or stability of KEAP1 mRNA was increased in the miR-432-KO 10

cells compared with that in the WT cells (Fig. 3C and 3D). By western blotting, we found that the 11

expression level of KEAP1 protein was increased and that of NRF2 protein was decreased in the 12

miR-432-KO cells compared with those in the WT cells (Fig. 3E). In addition, the activity of AREs 13

was clearly reduced and the expression level of NQO1 mRNA, a transcriptional target of NRF2, was 14

decreased in the miR-432-KO cells (Fig. 3F and 3G). Furthermore, ChIP-qPCR analysis revealed 15

the decreased enrichment of NRF2 at the promoter region of NQO1 gene including ARE in miR-432 16

KO cells compared with that in WT cells (Fig. 3H). These results suggest that the transcriptional 17

activity of NRF2 was effectively reduced by inhibition of miR-432-3p expression in miR-432 KO cells. 18

The sensitivity of the miR-432-KO cells to CDDP was increased compared with that of the WT cells 19

(Fig. 3I). Moreover, we isolated a subclone from miR-432-KO cells, which has only 1bp-deletion 20

within miR-432 locus. This subclone exhibited the same phenotypes as miR-432 KO cells such as 21

effective inhibition of miR-432-3p expression, up-regulation of KEAP1, down-regulation of NRF2, 22




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and increased sensitivity to CDDP (Supplementary Fig. S4). On the other hand, miR-432-3p is 1

harbored within an imprinted RTL1 (retrotransposon like 1) genetic locus at 14q32.2 and is 2

functionally processed from maternally expressed and non-coding RTL1-antisense (as) transcript, 3

not from paternally expressed and coding RTL1-sense (se) transcript (41). To examine whether the 4

loss-of-function mutation in RTL1-se by gene editing has an impact on phenotypes in miR-432 KO 5

cells, we validated expression of RTL1-se by strand-specific RT-PCR. As a result, we found that 6

RTL1-se was not expressed in parental KYSE2270 cells, miR-432 KO cells, and miR-432 KO 7

subclone, suggesting that the phenotypes in miR-432 KO cells were actually due to the inhibition of 8

miR-432-3p expression (Supplementary Fig. S5). Taken together, these findings strongly suggest 9

that the inhibition of miR-432-3p expression is involved in the increase in CDDP sensitivity of ESCC 10

cells. 11

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Expression analysis of miR-432-3p in ESCC cell lines and primary ESCC tumors 13

We first examined the expression level of miR-432-3p in 42 ESCC cell lines. The expression level of 14

miR-432-3p was elevated in 40 of 42 ESCC cell lines, compared with the normal esophageal tissues 15

(95.2%, fold change > 2.0, Supplementary Fig. S3). We next examined the expression level of miR-16

432-3p by qRT-PCR in 84 paired samples from patients with ESCC. The expression level of miR-17

432-3p was elevated in 55 of 84 primary ESCC tumors, compared with the corresponding non-18

cancerous esophageal mucosa (65.5%, fold change > 2.0, Fig. 4A). Furthermore, we showed a 19

negative correlation between KEAP1 mRNA expression and miR-432-3p expression in 58 primary 20

ESCC cases, which are informative for both KEAP1 mRNA expression and miR-432-3p expression 21

(Fig. 4B). There was no significant relationship between the expression levels and the clinical 22




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implications and prognosis in those patients. These results suggest that miR-432-3p is frequently 1

overexpressed in primary ESCC tumors and that the high expression of miR-432-3p may be a 2

potential mechanism for down-regulation of KEAP1 in ESCC. 3

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DISCUSSION 1

A key finding of our study is that miR-432-3p can down-regulate KEAP1 by directly binding to its 2

coding region, and high expression of miR-432-3p can contribute to decrease the sensitivity of 3

ESCC cells to chemotherapy drugs probably due to the increase in NRF2 protein. It is well known 4

that aberrantly stabilized NRF2, by mutation of either NRF2 or KEAP1 or by the potential inhibition of 5

autophagy in hepatocellular carcinoma, plays a vital role in growth and development of resistance to 6

chemotherapeutic agents and radiotherapy in a variety of cancer (6-13). We have previously 7

demonstrated that the down-regulation of expression for four miRNAs that negatively regulate NRF2, 8

namely, miR-507, miR-634, miR-450a, and miR-129-5p, identified by our miRNA library screening, 9

was also involved in the stabilization of NRF2 in ESCC (31). In this study, we showed that the 10

expression level of miR-432-3p was inversely correlated with KEAP1 mRNA level in primary ESCC 11

tumors. This suggests that the high expression of miR-432-3p and down-regulation of miRNAs that 12

negatively regulate NRF2, as well as somatic mutations in NRF2 or KEAP1, may be a possible 13

mechanism of NRF2 stabilization, indicating the presence of multiple pathways in NRF2 stabilization 14

in cancer. Hence, determination of the genetic status of NRF2 and KEAP1 and the expression status 15

of NRF2-regulating miRNAs including miR-432-3p in a large cohort of tumor samples, together with 16

elucidating the mechanism of up-regulation of miR-432-3p is needed to understand the various 17

mechanisms of NRF2 stabilization and to further develop a method for the classification of patients 18

with NRF2-stabilized tumors for overcoming therapeutic resistance. 19

So far, it has been reported that three miRNAs, miR-200a, mir-141, and mir-125b-5p, target KEAP1 20

in MDA-MD-231 (breast cancer cell line), A2780 (ovarian cancer cell line), and primary mouse 21

hepatocytes, respectively (35-37). However, among these miRNAs, miR-200a and miR-141, were 22




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not highly ranked as candidates that positively regulate the NRF2 pathway in our miRNA library 1

screening using HeLa cells (31). The miRNA-mediated regulation of KEAP1 expression may be 2

dependent on the cell-type or tissue-type. Furthermore, although we found that the transfection of 3

miR-125b-5p and other candidate miRNAs including miR-125a-5p and miR-297 could also induce an 4

increase of NRF2 expression in HeLa cells and the two ESCC cell lines (KYSE170 and KYSE770), 5

we did not observe a down-regulation of KEAP1 expression in these three cell lines transfected with 6

the miRNAs. This suggests that these miRNAs may be involved in NRF2 stabilization through an 7

unknown mechanism other than that involving KEAP1. 8

Administration of TS-miRNAs is expected to be useful strategy for cancer therapy (25,40). 9

Indeed, clinical trials on the administration of miR-34a, which can directly target several oncogenes 10

including BCL2, are currently on-going in hepatocellular carcinoma and hematopoietic tumors (38-11

39). We recently demonstrated that in vivo therapeutic administration of miR-507 and miR-634, 12

which can negatively regulate NRF2, enhanced chemotherapy-induced cytotoxicity against NRF2-13

stabilized cancers (28,31). On the other hand, the therapeutic effect of anti-miRs in inhibiting the 14

expression of oncogenic miRNAs such as miR-10b and miR-155 has also been demonstrated in 15

xenograft tumors in vivo (29-30). Our in vitro results in this study suggest that the inhibition of 16

endogenous miR-432-3p may overcome chemoresistance through the up-regulation of KEAP1 and 17

down-regulation of NRF2 in ESCC cells. Furthermore, we showed that gene editing by the 18

CRISPR/Cas9 system on the stem-loop structure of primary miR-432-3p can effectively inhibit 19

endogenous miR-432-3p expression probably due to the failure of miR-432-3p biogenesis. However, 20

transfection with commercially obtained anti-miR-432-3p was not effective for the inhibition of 21

endogenous miR-432-3p expression (data not shown). Thus, the improvement of chemical 22




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modification and sequence-based design of anti-miR-432-3p to more effectively inhibit the 1

endogenous miR-432-3p expression is required to achieve therapeutic inhibition of the NRF2-2

mediated oncogenic pathway via the up-regulation of KEAP1 in patients with NRF2-stabilized 3

tumors. 4

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ACKNOWLEDGEMENTS 1

The authors thank Ayako Takahashi and Rumi Mori (Tokyo Medical and Dental University, Japan) 2

for their technical assistance. 3

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FIGURE LEGENDS 1

Figure 1. Identification of KEAP1 as a direct target of miR-432-3p. 2

A. Western blotting analysis of transfected HeLa cells. The cells were transfected with 20 nM of 3

negative control-miRNA (miR-NC) or miR-432-3p. After 2 days, the cell lysates were 4

subjected to SDS-PAGE and immunoreacted with the indicated antibodies. 5

B. Expression analysis by qRT-PCR in transfected HeLa cells. The cells were transfected as 6

described in A and after 2 days total RNA was extracted. Bar, SD. 7

C. Stability assay of KEAP1 mRNA in the miR-432-3p- or miR-NC-transfected HeLa cells. The 8

cells were transfected as described in A, and after 2 days, the cells were treated with 9

actinomycin D (ActD). Total RNA was extracted at the indicated times. The t1/2 indicates the 10

half-life of KEAP1 mRNA. The error bars are not visualized due to too small. Bar, SD. 11

D. Western blotting analysis of exogenously transfected Flag-KEAP1 and endogenous KEAP1. 12

HeLa cells were transfected with miR-432-3p or miR-NC, and after 24 hours, the cells were 13

transfected with empty vector (vec.) or Flag-KEAP1 plasmids. After 24 hours, the cell lysates 14

were subjected to SDS-PAGE and immunoreacted with the indicated antibodies. The anti-15

KEAP1 antibody detects both Flag-KEAP1 (arrow) and endogenous KEAP1 (range). 16

E. Three regions (R1, R2, and R3) containing candidate binding sites of miR-432-3p within the 17

coding region of the KEAP1 gene. Candidate binding sites for the seed sequence of miR-18

432-3p within the coding region and corresponding mutant sequences are indicated in each 19

column. 20

F. Western blotting analysis of Flag-KEAP1 WT or its mutants. The cells were transfected as 21

described in D and the cell lysates were subjected to SDS-PAGE and immunoreacted with 22




27

the indicated antibodies. Images of western blots are indicated in the upper panels. The anti-1

KEAP1 antibody detects both Flag-KEAP1 (arrow) and endogenous KEAP1 (range). The 2

relative expression level of Flag-KEAP1 protein in miR-432-3p-transfected cells to that in the 3

miR-NC-transfected cells was indicated as a graph in the lower panel. Bar, SD. *p<0.05. 4

G. Luciferase assay using reporter plasmids. HeLa cells were transfected with miR-NC or miR-5

432-3p, and after 24 hours, the cells were transfected with a reporter plasmid (empty vector, 6

R1, R2, or R3). After 24 hours of transfection with reporter plasmids, firefly or Renilla 7

luciferase activities were measured. The luciferase activity in the miR-432-3p-transfected 8

cells relative to that in the miR-NC-transfected cells is indicated on the vertical axis of the 9

graph. Bar, SD. *p<0.05. 10

H. Luciferase assay using reporter plasmids. HeLa cells were transfected with miR-NC or miR-11

432-3p, and after 24 hours, the cells were transfected with a reporter plasmid (empty vector, 12

R1 WT, or R1 Mut). After 24 hours of transfection with reporter plasmids, firefly or Renilla 13

luciferase activities were measured. The luciferase activity is indicated as described in G. 14

Bar, SD. *p<0.05. **p<0.01. 15

16

Figure 2. Decrease in sensitivity to CDDP by the overexpression of miR-432-3p. 17

A. Effect of miR-432-3p overexpression on cell survival in response to CDDP treatment. The 18

cells were transfected with 20 nM or 2 nM of miR-NC or miR-432-3p. After 24 hours, the cells 19

were treated with CDDP (15 µM in HeLa cells, 10 µM in KYSE170 cells, and 60 µM in 20

KYSE770 cells) for 24 hours. Cell survival rates were measured by CV staining. An increase 21

in NRF2 protein level was confirmed by western blotting as shown in the upper panels. The 22




28

relative rate of cell survival of CDDP-treated cells to that of the untreated cells is indicated as 1

a graph in the lower panel. Bar, SD. **p<0.01. ***p<0.001. 2

B. Effect of siRNA-mediated inhibition of KEAP1 on cell survival in response to CDDP 3

treatment. KYSE770 cells were transfected with 20 nM of negative control-siRNA (control-4

siRNA) or KEAP1-siRNA. After 24 hours, the cells were treated with 60 µM of CDDP for 24 5

hours. Cell survival rates were measured by CV staining. Decrease in KEAP1 protein level 6

and increase in NRF2 protein level were confirmed by western blotting as shown in the upper 7

panels. The relative rate of cell survival of CDDP-treated cells to that of the untreated cells is 8

indicated as a graph in the lower panel. Bar, SD. *p<0.05. 9

C. Effect of siRNA-mediated inhibition of NRF2 on cell survival in response to CDDP treatment. 10

KYSE770 cells were transfected with 20 nM of negative control-miRNA (miR-NC) or miR-11

432-3p, and after 5 hours, the cells were transfected with control-siRNA or KEAP1-siRNA. 12

After 24 hours of transfection with siRNA, the cells were treated with 60 µM of CDDP for 24 13

hours. Cell survival rates were measured by CV staining. Increase or decrease in NRF2 14

protein level was confirmed by western blotting as shown in the upper panels. The relative 15

rate of cell survival of CDDP-treated cells to that of the untreated cells is indicated as a graph 16

in the lower panel. Bar, SD. ***p<0.001. 17

18

Figure 3. Increase in sensitivity to CDDP by the inhibition of miR-432-3p. 19

A. The three edited sequences within the mir-432 locus in a subline of KYSE2270 cells 20

generated by the CRISPR/Cas9 system. 21

B. Expression analysis of miR-432-3p in the miR-432-KO or WT cells by qRT-PCR. Bar, SD. 22




29

C. Expression analysis of KEAP1 mRNA in the miR-432-KO or WT cells by qRT-PCR. Bar, SD. 1

D. Stability assay of KEAP1 mRNA in the miR-432-KO or WT cells. The cells were treated with 2

actinomycin D (ActD) and total RNA was extracted at the indicated times. The t1/2 indicates 3

the half-life of KEAP1 mRNA. Bar, SD. 4

E. Western blotting analysis of the miR-432-KO or WT cells. The cell lysates were subjected to 5

SDS-PAGE and immunoreacted with the indicated antibodies. 6

F. Luciferase assay using reporter plasmids. The cells were transfected with reporter plasmids 7

(empty vector or ARE-containing reporter vector) and internal control vector. After 48 hours 8

of transfection, firefly or Renilla luciferase activities were measured. The relative luciferase 9

activity is indicated on the vertical axis in graph. Bar, SD. ***p<0.001. 10

G. Expression analysis of NQO1 mRNA in the miR-432-KO or WT cells by qRT-PCR. Bar, SD. 11

H. ChIP-qPCR analysis for binding of NRF2 to the NQO1 promoter region in miR-432-KO or 12

WT cells. qRT-PCR was performed on DNAs immunoprecipitated with rabbit polyclonal anti-13

NRF2 antibody or rabbit IgG antibody (negative control) using primers that flank the ARE 14

region in the NQO1 promoter. Bar, SD. ***p<0.001. 15

I. Cell survival assay of miR-432-KO or WT cells in response to CDDP treatment. The cells 16

were treated with 30 µM of CDDP for 24 hours. Cell survival rates were measured by CV 17

staining. The relative rate of cell survival of CDDP-treated cells to that of the untreated cells 18

was indicated as a graph. Bar, SD. **p<0.01. 19

20

Figure 4. Expression analysis of miR-432-3p in primary ESCC tumor samples. 21




30

A. Relative expression level of miR-432-3p in 84 primary ESCC samples relative to that in 1

corresponding non-cancerous tissues. Bar, SD. 2

B. Negative correlation between miR-432-3p and KEAP1 mRNA expression levels in 58 primary 3

tumor tissues. 4




Published OnlineFirst July 31, 2017.Mol Cancer Res Burak Akdemir, Yasuaki Nakajima, Johji Inazawa, et al. KEAP1

Induces NRF2 Stabilization by Directly TargetingmiR-432

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