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1 Korean Journal of Oral and Maxillofacial Pathology 2017;41(2): ISSN: (Print); (Online) Available online at mir-4708 의 multidrug resistance protein-1 발현조절을통한 paclitaxel 민감성증가 최장열 1), 유지원 2), 안상건 1)* 조선대학교 1) 치과대학구강병리학교실, 2) 치의학전문대학원구강내과학교실 <Abstract> mir-4708 Enhances Paclitaxel Sensitivity through Modulating Expression of Multidrug Resistance Protein-1 Jang-Yeol Choi 1), Ji-won Ryu 2), Sang-Gun Ahn 1)* 1) Department of Pathology, College of Dentistry, 2) Department of Oral Medicine, School of Dentistry, Chosun University, Gwangju 61452, Rep of Korea. Multidrug resistance (MDR) remains one of the most significant obstacles in various cancer treatment, and this process often involves dysregulation of the number of micro-rnas. The aim of this study was to explore the role of mir-4708 on the regulation of MDR-1 expression and the regulation of multidrug resistance (MDR) to chemotherapeutic drugs. Luciferase reporter assays demonstrated that mir-4708 directly binds MDR-1 3 -UTR and down-regulated reporter luciferase activity. The mrna and protein expression levels of MDR1 were significantly decreased following mir-4708 overexpression. Additionally, the accumulation of rhodamine-123 in paclitacel resistant FaDu cells following mir-4708 transfection was significantly increased compared with control, indicating that the efflux capacity was reduced. These results demonstrated that mir-4708 could be involved in the regulation of MDR via targeting MDR-1 and may provide a potential strategy for reversing drug resistance in oral cancer. Key words: mir-4708, FaDu, MDR1, Paclitaxel Ⅰ. INTRODUCTION 항암화학요법을이용한암치료에있어서큰장애가되는요소중의하나는항암제내성이다. 암환자에대한항암화학요법이반복적, 장기적으로사용하게되면서항암부작용과항 * Correspondence: Sang-Gun Ahn, PhD, Department of Pathology, School of Dentistry, Chosun University, 309 Pilmun-Daero, Dong-gu, Gwangju 61452, Rep of Korea. Tel: ; Fax: ahnsg@chosun.ac.kr ORCID : Received: Mar ; Revised: Apr ; Accepted: Apr 암내성이발생한다. 또한서로다른여러항암제에대한광범위한교차내성을보이는다제내성 (multidrug resistance) 으로인해사용가능한항암제의범위가제한됨으로서암치료의중요한문제점으로대두되고있다 1,2). 항암제내성은환자및종양들사이의유전적차이등을포함한다양한인자들에의해유발될수있으나, 현재보고된세포내항암제내성기작의중요한분자생물학적기전을보면, MDR 유전자에의해세포내약물축적을능동적으로유출함으로서세포내약물축적억제유도가주기전으로알려져있다. 다제내성은또한항암제의

2 제한적인흡수, ceramide 와같은막지질들의변화등을통해세포내약제의축적을제한, apoptosis 억제, DNA 손상의복구와약제의비독성화를유도및세포주기를변화시킴으로항암제에대한내성을부가적으로유도할수있다 3). 다제내성에의한항암화학요법의여러문제점을극복하기위해다제내성기전, 내성세포의특성및내성극복과관련된연구가광범위하게진행되고있으며, 그중에서다제내성을유발하는유전자 (ATP-binding cassette(abc) transporter; MDR-1, MRP, 그리고 BCRP) 들에대한연구가내성극복과관련되어빠르게진행되고있다. 지금까지 48개의 human ABC 유전자가확인되었고, 그중에서 multidrug resistance 1 (MDR-1 또는 ABCB1) 에의한내성은다제내성기전중에서많은연구가진행되고있다 4-7). 최근, MDR1 의과발현은종양세포내 paclitaxel 축적을감소시켜다제내성에관여한다는연구결과가있으며 8), A549 lung adenocarcinoma 세포에서 MDR1의 hypermethylation 이 cisplatin 에대한내성억제 9) 및간암세포주인 HepG2 세포에서 MDR1의 antisense RNA를이용한 MDR1의발현억제가항암제내성감소를유도함이보고되었다 10). 또한다양한세포내신호전달기전에관여하는 microrna (mirna) 가약물내성조절에중요하게관여하고있는것이보고되고있으며, 한예로, 위암세포에서 mir-129가 MDR-1을조절하여 cisplatin 내성억제에관여하며 11), mir-186 은난소암세포에서 MDR1과직접적으로결합하여이들발현을억제, paclitaxel 과 cisplatin 에대한내성을저해시켰다 12). 또한 mir-873, mir-27a, mir-145 그리고 mir-302 은각각난소암세포, 간암세포, 장상피세포, 유방암세포에서직, 간접적으로 MDR-1 발현조절을통해다제성약물내성에관여하고있음이보고되고있다 13-16). 그러나최근 MDR-1 을표적으로한다제내성억제연구에서 in vitro에서뚜렷한효과를보였던 verapamil 과 cyclosporin-a 가임상연구에서실패사례를보고됨으로서논란의여지가남아있다 17). 그러나, MDR-1 의발현이여러종양에서항암치료에대한반응과예후에관련이있는다수의연구들을통해 MDR-1을표적으로한내성극복치료를시도하고있거나 MDR-1 의기질에대한저해제를탐색하는연구가지속적으로 수행되고있다. 이연구에서구강암 FaDu 세포에서 mirna 인 hsa-mir p이 MDR-1 유전자의발현에미치는영향을확인하였다. Luciferase 분석을통해 hsa-mir p가 MDR-1 의 3 -UTR 부위에결합함을확인하였고, hsa-mir p의발현유도가 MDR-1 mrna 와단백질의발현을감소시키는것을확인하였다. 또한 hsa-mir p 의과발현에의한 MDR-1 의감소는 paclitaxel 내성 FaDu 세포 (FaDu-PTX) 내 Rhodamine 123의축적을증가시킴을확인하였다. II. MATERIALS and METHODS 1. Cell Culture 사람인후두암세포인 FaDu와 paclitaxel 내성 FaDu (FaDu-PTX) 세포는 10% fetal bovine serum (FBS), 100units/mL penicillin, and 100ug/mL streptomcin 을함유한 Dulbecco's modified Eagle's medium (DMEM, welgen, korea) 을사용하였고, 5% CO2, 37 에서배양하였다. 2. Plasmid construction mirna expression vector 인 psuper plasmid에 hsa-mir p를 cloning 하기위해 hsa-mir-4708 primer을합성하였다 ; Forward: CATAGATCTTTTAGGAGAGAGATGCCGCCTT GCTCCTTGAACAGGAGG 와 Hsa-mir-4708 primer Reverse: CATAAGCTTTTTAGTATCAGAGAGATGCCGCCTTGCTCCTC CTGTTCAAGGA (Macrogen, Korea). PCR 조건은 95 에서 3분간 denaturation, 95 20초, 35~55 20초, 72 20초, 35 cycle, 72 에서 5분간 extension 을통해얻은 PCR 생산물을 BglII와 HindIII로 digestion 하여 psuper basic vector에 ligation 하였다. MDR1 3'-UTR는 pmir luciferase report vector에 cloning 하기위해 MDR1 3'-UTR wild type 과 mutant를특이 primer 를합성하여사용하였다. MDR1 3'-UTR, Forward: CATACTA GTTGAGAGACATCATCAAGTGGAGA, Reverse: CAT AAGC 56

3 TTTCACAGGCAGTTTGGACAAG를이용하여 95, 5min, 9 5, 30sec - 56, 30sec - 72, 1min 30cycle, 72, 5min 조건으로 PCR 하였다. MDR1 3'-UTR mutant type 은 Forward: GGACTGTAACTGACTTAAAAGATTATAGA, Reverse: TCTA TAACTTTTAAGTCAGTTACAGTCC를통해합성하였고, MDR1 3'-UTR wild type과 mutant type을 pmir luciferase vector (Ambion) 에 cloning 하여 plasmid를제작하였다. 3. Luciferase Assay 6 well plate 에 2.5 x 10 5 cells/well로 seeding한 FaDu 세포에 psuper basic vector 또는 psuper/hsa-mir-4708 와 pmir/mdr1 3'-UTR wild type 또는 mutant를각각 co-transfection 하고 48시간후 cell lysis buffer로 lysis하였다. 단백질은 Bradford assay (Bio-rad) 를통하여정량하였고, luciferase assay는 promega (USA) 의 luciferase assay system (E1500) 을사용하였다. Luminometer 에서측정하고값을단백질정량값으로나누어 normalization 하였다. 4. RT-PCR analysis 각 transfection한세포는 RNAiso solution(takara, Japan) 을이용하여제조사의방법에따라 total RNA를얻었다. total RNA는 cdna 합성 kit(promaga) 를이용하여 cdna 를합성하였고, PCR과 qrt-pcr 에사용하였다. 사용한 primer 는 Table 1과같다. mirna 의 qrt-pcr을위한 cdna합성은 Mir-XTM mirna First-Strand Synthesis and SYBRR qrt-pcr kit(clontech) 을사용하였고제조사의방법에따라합성하여사용하였다. 5. Western blot 단백질은 transfection 후 48시간동안배양한세포를 RIPA buffer 를이용하여단백질을분리하여 Bradford assay를통하여단백질정량후 SDS-PAGE 를시행하였고, PVDF membrane 에 transfer 하였다. Western blot은 5% skim milk/tbst에 1시간반응, 1st anti-body (Santa cruz, MDR1: sc-55510, β-actin: sc-47778) 4 overnight, 10분간 3번 TBST로 washing하고 2nd anti-body (goat anti-mouse IgG) 1시간상온반응하였다. 6. Rhodamine accumulation FaDu-PTX 세포에 psuper-4708과 psuper basic을 Transfection 하고 4시간후 paclitaxel (PTX) 을처리하여 48시간동안배양하였다. 2시간동안 12 μm 의 rhodamine 123( Rho123) 을처리하고 PBS로 washing 후 fluorometer에서 fluorescence 를측정하였다. 측정값은세포수로 normalize 하였다. 7. Statistical analysis 데이터는 EXCEL 을이용하여평균과표준편차를구한후 t-test 를이용하여상호작용을검증하였다. 이연구의통계학적유의수준은 *p<0.05, **p<0.01로하였다. Table 1. Specific primers for MDR1 and hsa-mir p. mirna 3' universal antisense, U6 primers: supplied by mirna cdna synthesis kit. 57

4 III. RESULTS 이전연구에서 HOX family 중에하나인 HOXC6 가 MDR-1 과 BCL-2 의조절을통해다제내성기전에중요한역할을하고 있음을보고하였다 18). 따라서 HOXC6 과발현에따른 mirna 의변화를확인하기위해 microrna array를시행한결과 HOXC6 가과발현된구강암 FaDu 세포에서 hsa-mir-4708가 3.16배감소하였다 (Table 2). microrna array 결과를확인하 Table 2. mir p significantly differentially expressed between FaDu-pcDNA4.A and FaDu-HOXC6 cell lines. Pc4: pcdna4.a plasmid fc: fold change Fig. 1. Down-regulation of mir-4708 in HOXC6 overexpressed FaDu cells. The mir-4708 levels were quantified by qrt-pcr analysis in vector or pcdna4-hoxc6 transfected FaDu cells. **P<0.01. b a Fig. 2. mir p binds to MDR1 3'-UTR. (a) Sequence alignment of mir-4708 target site in MDR1 3'-UTR. (b) MDR1 3'-UTR wild-type or mutant was co-transfected with psuper-basic or psuper/mir-4708 into FaDu cells. The luciferase activity was determined by luminometer, and then normailzed. **p<

기위해 mir-4708 specific primer 를사용한 qrt-pcr 의결과 HOXC6 가과발현되는 FaDu 세포에서 mir-4708 이 microrna array 결과와유사하게의미있는감소를관찰하였다 (Fig. 1, p<0.01)).

mir-4708-3p 와 MDR1 의 3 UTR 간에결합을확인하기위 a 해 mir-4708을 psuper basic vector에유전자재조합하였고, MDR-1 의 3 -UTR 부위에 mir-4708 예상결합염기서열을포함한 wild type과예상결합위치의염기서열을결실시킨 mutant type의 DNA를삽입시킨 luciferase

psuper-mir-4708와 MDR-1 의 3 -UTR wild type luciferase vector (pmir-mdr1 wt) 또는 MDR-1의 3 -UTR mutant luciferase vector (pmir-mdr1 mut) 를 FaDu 세포주에 co-transfection 하여 Luciferase assay

MDR1 mrna expression levels were quantified by RT-PCR and qrt-pcr analysis.

5 기위해 mir-4708 specific primer 를사용한 qrt-pcr 의결과 HOXC6 가과발현되는 FaDu 세포에서 mir-4708 이 microrna array 결과와유사하게의미있는감소를관찰하였다 (Fig. 1, p<0.01)). Hsa-miR-4708 에의해조절되는타겟유전자를 TargetScan, mirbase 등의 database를통해조사한결과 mir-4708이 MDR1 의 3 UTR 의 위치와결합할수있음을확인하였다. mir p 와 MDR1 의 3 UTR 간에결합을확인하기위 a 해 mir-4708을 psuper basic vector에유전자재조합하였고, MDR-1 의 3 -UTR 부위에 mir-4708 예상결합염기서열을포함한 wild type과예상결합위치의염기서열을결실시킨 mutant type의 DNA를삽입시킨 luciferase report vector를제작하였다 (Fig. 2a). psuper-mir-4708와 MDR-1 의 3 -UTR wild type luciferase vector (pmir-mdr1 wt) 또는 MDR-1의 3 -UTR mutant luciferase vector (pmir-mdr1 mut) 를 FaDu 세포주에 co-transfection 하여 Luciferase assay 를실행한결과 pmirb Fig. 3. Expression of MDR1 down-regulated by mir-4708 expression. (a) FaDu cells were transfected with psuper basic vector or psuper/mir-4708 for 48 h. MDR1 mrna expression levels were quantified by RT-PCR and qrt-pcr analysis. (b) Total proteins were subjected to western blot analysis using antibodies against The graphs show the quantitative evaluation of MDR1 protein expression. *p<0.05, **p<0.01. a b Fig. 4. Effect of mir-4708 on cell viability and Rhodamine123 accumulation. The FaDu-PTX cells were transfected psuper-basic or psuper/hsa-mir-4708 with 400 nm PTX. (a) Representative drug uptake is shown depicting the levels of Rho123 accumulation in FaDu-PTX cells. (b) Cell viability was assessed by the MTT assay, as described in the Materials and Methods. The results of three experiments are summarized. **p<

6 MDR1 wild type과 psuper-mir-4708가 co-transfection한세포에서 luciferase activity가 DNA 농도의존적으로감소하였으며, 3 μg psuper-mir-4708를처리한실험군에서 MDR-1의 3 -UTR luciferase activity 가 40 % 가량감소한것을확인하였다. 반면 MDR-1의 3 -UTR mutant type에서는같은농도의 DNA transfection 조건에서변화하지않았다 (Fig. 2b). MiR-4708 이 MDR-1 mrna 와단백질발현에미치는영향을확인하기위해 FaDu 세포에 psuper-mir-4708과 control vector를 transfection하고 24시간후 MDR1 의 mrna level을 RT-PCR, qrt-pcr analysis 그리고 wester blot analysis 를시행하였다. FaDu 세포에서 mir-4708를 transfection FaDu 세포에서 MDR-1의 mrna level이 control 또는 vector transfection 세포에비해 25% 감소함을 RT-PCR 과 qrt-pcr 을통해확인하였다 (Fig. 3a). 또한 mir-4708이 MDR-1 단백질생성에미치는영향을확인하기위해 FaDu 세포에 psuper-mir-4708를 transfection하고 48시간후 anti-mdr1 antibody와함께 Western blot을수행한결과 qrt-pcr 결과와마찬가지로 control 에비해 33% 정도의 MDR1 단백질의감소를확인하였다 (Fig. 3b). mir-4708 에의한 MDR-1 의발현억제가세포내약물축적을유도하는지알아보기위해 paclitaxel (PTX) 에대한저항성을갖고있는 FaDu-PTX 세포에서 Rhodamine123 의세포내축적을조사하였다. mir-4708을과발현시킨 FaDu-PTX 세포에서 Rhodamine123의세포내축적이 control 세포에비해 54% 증가하였으며 (Fig. 4a), 또한 mir-4708에의한 MDR-1 의발현억제가항암제인 paclitaxel 에대한민감도가증가하는지를세포생존율을통해확인하였다. 400 nm PTX만처리한 control 과 PTX 와 mir vector 를함께처리된세포에서의세포생존률간의의미있는유의성이관찰되지않았으나 mir-4708과 PTX가함께처리된 FaDu-PTX 세포에서세포생존율이유의성있게 42% 감소하는것을확인하였다 (Fig. 4b). IV. DISCUSSION 암치료에있어항암제내성은항암치료에주요문제점이 며, 항암제를이용한항암치료실패의원인되고있다. 현재, 세포막에존재하는 ATP-binding cassette (ABC) membrane transporter 들이항암제내성을조절하는주요기전중하나로여겨지고있으며, 4) ATP-binding cassette (ABC)-membrane transporter 중 ABCG2 (BCRP) 는유방암과두경부암에서항암제내성에관여하고 19,20), 뿐만아니라 ABCC1 (MRP-1), ABCB1 (MDR-1) 등여러 ATP-binding cassette (ABC) family들이구강암에서항암제내성에직간접적으로관여하고있음이알려져있다 21). MDR-1 은다재내성관련가장많이연구된 ABC transporter 중하나로종양억제인자인 p53과 Fos, Jun과복합체를형성다양한세포조절기능을가지는 AP-1 이 MDR-1의발현을조절하는전사인자로보고되고있으며 22), CCAAT/enhancerbinding protein family 인 C/EBPβ, NFIL6 (nuclear factor for interleukin-6) 그리고염증과연관된 NF-κB 또한 MDR-1 promoter 에결합 MDR1/ABCB1 발현을조절하고있음이보고되었다 23,24). 본연구자의이전연구에서 homeodomain 을포함하는전사인자인 HOXC6 가구강암에서 MDR-1 의전사를조절하고있음을입증하였다 18). 최근 ATP-binding cassette (ABC)-membrane transporter 들의발현에 HOXC6 를포함한다른 HOX family 유전자들이관여하고있음이보고되고있다. 특히, human myelogenous leukemia K562/ADM 세포에서 HOXB4 의 knockdown 은 P-gp, MRP1, BCRP의발현을억제하고, HOXA10 의 knockdown 은 P-gp와 MRP1의발현을억제시켜약물내성을감소시킨다는보고가있었으며 25, 26), 또한 pancreatic cancer 세포에서 homeobox gene MSX2는 ABCG2 발현조절과관련성이확인되었다 27). 따라서 HOX family 유전자또한항암제내성조절기전간의매우밀접한연관성이있음을시사하고있다. 본연구에서구강암 FaDu 세포에서항암제내성에직, 간접적으로관련된 HOXC6 과발현에따른 microrna array 를시행한결과 hsa-mir-4708 의발현감소를확인하였으며, qrt-pcr 을통해이들발현이감소함을다시한번확인하였다 (Table 2, Fig. 1). Persson et. al., 은유방암조직에서 mir-4708 을포함하는여러 mirna 가 ERBB2/Her2 유전자의발현과함께유도됨으로써임상표지인자및잠재적종양유전자마커로나타날 60

7 수있음을보여주고있다 28). 따라서 mir-4708 이구강암항암제내성에관여하고있는지를조사하였다. mirnas database (TargetScan, mirbase) 를통해 hsa-mir p의예상 target 유전자를조사한결과 ATP-binding cassette, sub-family B, member 1 (MDR-1/ABCB1, NM_ ) 와 ATP-binding cassette, sub-family C, member 12 (MRP/ABCC12, NM_033226) 의 3'-untranslated region (3'-UTR) 에결합할수있음을확인하였다. mir-4708 와 MDR-1 의직접적인결합을보기위해 MDR-1의 3'-UTR을 luciferase vector 에 subcloning 하여 luciferase assay 한결과 luciferase activity 가 control 과비교하여약 40 % 감소하는것을확인하였다 (Fig. 2). 이는 mir-4708이 MDR-1 3'-UTR에결합하여이들전사를억제함을확인할수있었고, 이와상응해서 mir-4708 과발현된 FaDu 세포에서 mrna 와단백질의발현이각각 ±25%, ±33% 의감소를확인함으로서 mir-4708이 MDR-1 3'-UTR 에결합하여 MDR-1 조절에직접적으로관여함을입증하였다 (Fig. 3). 또한 paclitaxel 저항성을가지는 FaDu-PTX 세포에서 mir-4708의과발현은 Rhodamine123의세포내축적을증가시킬뿐아니라 paclitaxel 에대한세포민감성을증가시켜줌으로서 mir-4708 이 MDR-1 조절은통해항암제내성기전을조절할수있음을입증하였다 (Fig. 4). 결론적으로본연구결과는구강암세포에서화학요법에의한항암제내성의획득에 mirna-4708 조절장애의중요성을나타내는증거를제공하고있으며, 또한구강암세포항암제내성을저해할수있는 mirna 기반치료전략및개발대한기반을제공할수있을것이다. Ⅴ. ACKNOWLEDGMENTS This work was supported by basic science research program through the National Research Foundation of Korea (NRF) funded by ministry of science, ICT and future planning (no ) and funded by the Korean government MSIP (no ). REFERENCES 1. Nooter K, Stoter G: Molecular mechanisms of multidrug resistance in cancer chemotherapy. Pathol Res Pract 1996;192: Partridge AH, Burstein HJ, Winer EP: Side effects of chemotherapy and combined chemohormonal therapy in women with early-stage breast cancer. J Natl Cancer Inst Monogr 2001;30: Gottesman MM, Fojo T, Bates SE: Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2002;2: El-Awady R, Saleh E, Hashim A: The Role of Eukaryotic and Prokaryotic ABC Transporter Family in Failure of Chemotherapy. Front Pharmacol 2017;7: Chang FW, Fan HC, Liu JM: Estrogen Enhances the Expression of the Multidrug Transporter Gene ABCG2- Increasing Drug Resistance of Breast Cancer Cells through Estrogen Receptors. Int J Mol Sci 2017;18: Guan GF, Zhang DJ, Zheng Y: Significance of ATP-binding cassette transporter proteins in multidrug resistance of head and neck squamous cell carcinoma. Oncol Lett 2015;10: Lu JF, Pokharel D, Bebawy M: MRP1 and its role in anticancer drug resistance. Drug Metab Rev 2015;47: Szakács G, Paterson JK, Gottesman MM: Targeting multidrug resistance in cancer. Nat Rev Drug Discov 2006;5: Li A, Song J, Lai Q: Hypermethylation of ATP-binding cassette B1 (ABCB1) multidrug resistance 1 (MDR1) is associated with cisplatin resistance in the A549 lung adenocarcinoma cell line. Int J Exp Pathol 2016 [Epub ahead of print]. 10. Chan JY, Chu AC, Fung KP: Inhibition of P-glycoprotein expression and reversal of drug resistance of human hepatoma HepG2 cells by multidrug resistance gene (mdr1) antisense RNA. Life Sci 2000;67: Lu C, Shan Z, Li C, Yang L: MiR-129 regulates cisplatin-resistance in human gastric cancer cells by targeting P-gp. Biomed Pharmacother 2017;86:

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Jkbcs016(92-97).hwp

Jkbcs016(92-97).hwp Expression of bcl-2 and Apoptosis and Its Relationship to Clinicopathological Prognostic Factors in Breast Cancer - A Study with Long Term Follow-up correlated with the survival rate.(journal of Korean