Title 横紋筋肉腫におけるがん特異的エネルギー代謝を標的とするmicroRNAの同定とその制御( 本文(Fulltext) )

Author(s) 杉戸, 信彦

Report No.(DoctoralDegree) 博士(薬科学) 連創博甲第46号

Issue Date 2019-03-25

Type 博士論文

Version ETD

URL http://hdl.handle.net/20.500.12099/77972

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

microRNA

MicroRNAs that regulate cancer-specific energy metabolism

in rhabdomyosarcoma cells

2019

i

1 1

2

1 4

2 microRNA miRNA 6

3 Warburg microRNA 8

3 miR-1 miR-133b

1 9

2 miR-1 miR-133b

10

3 miR-1 miR-133b

12

4 miR-1 miR-133b 13

5 miR-1, miR-133b siR-PTBP1 16

6 miR-1 miR-133b

PAX3-FOXO1 20

7 PAX3-FOXO1 22

8 miR-1 miR-133b in vivo 23

4 24

5 26

ii

27

28

35

36

1

1

(rhabdomyosarcoma; RMS)

RMS embryonal RMS (ERMS)

alveolar RMS (ARMS) 2 ARMS

t(2;13)(q35;q14) PAX3-FOXO1

RMS

ARMS

Warburg Warburg

Warburg

polypyrimidine tract-binding protein 1 (PTBP1) PTBP1

Pyruvate kinase muscle (PKM) isoform

splicer PKM2

Warburg

PTBP1 PTBP1

microRNA (miRNA, miR) PTBP1 miRs Warburg

PTBP1

PTBP1 miR-1 miR-133b miR-1 miR-133b

real-time RT-PCR

ERMS RD KYM-1 ARMS Rh30 Rh41

2

miRNA

miRNA

miRNA

miRNA

miRNA

miR-1 miR-133b

miR-1 miR-133b RMS

RNA miRNA

Warburg PAX3-FOXO1

3

Cancer-specific energy metabolism in rhabdomyosarcoma cells isregulated by

microRNA.,

Nobuhiko Sugito, Kohei Taniguchi, Yuki Kuranaga, Maki Ohishi, Tomoyoshi Soga, Yuko

Ito, Mitsuru Miyachi, Ken Kikuchi, Hajime Hosoi, and Yukihiro Akao.,

Nucleic Acid Therapeutics; 27(6): 365-377 (2017).

4

2

1

1

(rhabdomyosarcoma; RMS)

3.5 1 ( )

RMS

RMS

2 RMS embryonal RMS

(ERMS) alveolar RMS (ARMS) 2 3 ARMS

t(2;13)(q35;q14) PAX3-

FOXO1 (Figure 1) 4 5 PAX3-FOXO1

4

RMS

5 70%

ARMS 5 60% (National Cancer Institute

Physician Data Query )

D

D 3 VAC

5

VAC 1970

7 5

40

Figure 1. PAX3-FOXO1

6

2 microRNA miRNA

microRNA (miRNA, miR) 21-25

non-cording RNA miRNA mRNA

RNA (RNA

interference; RNAi) 6,7 miRNA

8 RNAi miRNA

RNA (RNA induced silencing complex;

RISC) RISC-miRNA

mRNA 3’ (untranslated region; UTR)

(Figure 2)9 miRNA - mRNA 3’-UTR

miRNA 2-7 seed sequence

seed sequence miRNA

mRNA

mRNA 60% miRNA 10

2018 10 2600 miRNA

(miRbase Release 22.1 (http://www.mirbase.org/) ) miRNA

11,12 13,14 15-17 18,19

miRNA miRNA

20 miRNA

miRNA

miRNA

7

2018 8 siRNA

(hATTR )

patisiran siRNA

miRNA

Figure 2. RNA

8

3 Warburg microRNA

Warburg

Warburg

21,22 Warburg

polypyrimidine tract-binding protein 1 (PTBP1) PTBP1

Pyruvate kinase muscle (PKM) isoform PKM2

Warburg (Figure 3) 23,24

Figure 3. Warburg 25

PTBP1 miRNA PTBP1 miRs

Warburg 26,27

PTBP1 miRs miR-1 miR-

133b 16,28,29 RMS Warburg

9

3 miR-1 miR-133b

1

RMS

Warburg PTBP1 miRNA RMS

RNA

10

2 miR-1 miR-133b

TargetScan Release 7.1 (http://www.targetscan.org/) PTBP1

miRNA miR-1 miR-133b

real-time RT-PCR

(Figure 4A)

RMS

(ERMS : RD KYM-1 ARMS : Rh30 Rh41)

(Skeletal muscle; SKM)

(Figure 4B) miR-1 miR-

133b

miRNA

miRNA

11

Figure 4. miR-1 miR-133b

(A) miR-1 miR-133b

(B) RMS (RD KYM-1 Rh30 Rh41) SKM

miR-1 miR-133b

12

3 miR-1 miR-133b

miR-1 miR-133b RMS

miRNA Lipofectamine RNAiMAX

ERMS

72 ARMS 48

miRNA

scramble RNA control

(Figure 5)

Figure 5. miR-1 miR-133b

RMS miR-1 miR-133b (10, 20 nM)

13

4 miR-1 miR-133b

miR-1 miR-133b Warburg PTBP1 miRNA

30,31 Warburg

miRNA

PTBP1 (Figure 6A)

miRNA PTBP1

anti-miR-1 anti-miR-

133b PTBP1

3’UTR (Mut)

(Wild)

(Figure 6B) anti-miR-1 anti-miR-133b PTBP1

(Figure 6C)

miRNA RMS PTBP1

PTBP1 miRNA PTBP1

PKM 2

PKM2 PKM1 (Figure 6A)

PKM1 TCA cycle

miRNA TCA cycle

(Figure 6D) ATP PKM1

ATP (Figure 6D)

14

Figure 6. miR-1 miR-133b PTBP1

(A) RMS miR-1 miR-133b (10, 20 nM) Warburg

PTBP1, PKM1 PKM2

(B) RMS miR-1 miR-133b PTBP1 3’UTR

(C) RMS miR-1 miR-133b antagomiR

(D) RMS miR-1 miR-133b

(E) RMS miR-1 miR-133b ATP

15

PTBP1 siRNA

PTBP1 miRNA

(Figure 7A) PKM2

PKM1 (Figure 7B)

TCA cycle (Figure 7C) ATP

(Figure 7D) miR-1 miR-133b PTBP1

miRNA PKM2 PKM1

TCA cycle ATP

Figure 7. PTBP1

(A) RMS siR-PTBP1 (0.5, 5 nM)

(B) RMS siR-PTBP1 Warburg

PTBP1, PKM1 PKM2

(C) RMS siR-PTBP1

(D) RMS siR-PTBP1 ATP

16

5 miR-1, miR-133b siR-PTBP1

miR-1 miR-133b

3

32 miR-1 miR-133b PTBP1

Warburg TCA

cycle (Reactive Oxygen Species; ROS)

TCA cycle ROS 33,34

LC3B

LC3B

35,36

35 LC3B

LC3B

17

miR-1,miR-133b (Figure 8A) siR-PTBP1 (Figure 8B)

RMS

Rh30

miR-1, miR-133b (Figure 8C)

siR-PTBP1 (Figure 8D)

miR-1 miR-133b PTBP1

(autophagic cell death) (autophagic cell

survival) 37

3-Methyladenine (3-MA) RD

Rh30 3-MA

miRNA (Figure 8E) siR-PTBP1

(Figure 8F) autophagic cell death

TCA cycle ROS

miR-1, miR-133b siR-PTBP1 ROS

N- -L-

(N-acetyl-L-cysteine; NAC) ROS

Rh30 miR-1, miR-133b (Figure 8G)

siR-PTBP1 (Figure 8H) NAC

miR-1 miR-133b PTBP1

TCA cycle ROS autophagic cell death

18

19

Figure 8. miR-1, miR-133b siR-PTBP1

(A, B) RMS miR-1, miR-133b (10, 20 nM; A) siR-PTBP1

(0.5, 5 nM; B) LC3B

(C, D)Rh30 miR-1, miR-133b (20 nM; C) siR-PTBP1 (5 nM; D)

48

N: fragmented nuclei, AV: autophagic vesicles, Aly: autolysosome.

(E, F)RD Rh30 3-MA (0.5 mM) 5

miR-1, miR-133b (20 nM; E) siR-PTBP1 (5 nM; F)

72 48 LC3B

(G, H)Rh30 miR-1, miR-133b (20 nM; G) siR-PTBP1 (5 nM; H)

24 ROS NAC (3 mM)

24 LC3B

20

6 miR-1 miR-133b PAX3-FOXO1

miR-1 miR-133b RMS PTBP1

autophagic cell death ARMS

PAX3-FOXO1

RMS PAX3-FOXO1 real-time PCR

mRNA ARMS PAX3-FOXO1

(Figure 9A) mRNA PAX3-FOXO1

miR-133b Rh30 Rh41

PAX3-FOXO1 (Figure 9B, 9C)

miR-133b ARMS PAX3-FOXO1

miR-133b PAX3-FOXO1

Figure 9C RD KYM-1 miR-133b FOXO1

miR-133b FOXO1

seed sequence 7mer

FOXO1

6mer miRNA

38,39 FOXO1 3’UTR 2

1

miR-133b FOXO1

(Figure 9D) anti-miR-133b miR-133b

21

FOXO1 PAX3-FOXO1 (Figure

9E) miR-133b FOXO1 ARMS

PAX3-FOXO1

Figure 9. PAX3-FOXO1 miR-133b

(A) SKM RMS PAX3-FOXO1 mRNA

(B) ARMS miR-1, miR-133b (10, 20 nM) siR-PTBP1 (0.5, 5 nM)

48 PAX3 PAX3-FOXO1

(C) RMS miR-1, miR-133b (10, 20 nM) siR-PTBP1 (0.5, 5 nM)

FOXO1 FOXO1 PAX3-FOXO1

(D)miR-133b FOXO1 3’UTR

(E) RMS miR-133b antagomiR

22

7 PAX3-FOXO1

miR-133b PAX3-FOXO1 PAX3-

FOXO1 PAX3-FOXO1 siR-

PF240 ARMS

(Figure 10A)

40 Warburg

PTBP1

PKM2 PKM1

(Figure 10B) ARMS

PAX3-FOXO1 PTBP1 Warburg

Figure 10. PAX3-FOXO1

(A) ARMS siR-PF2 (0.5, 5 nM)

(B) ARMS siR-PF2 (0.5, 5 nM) Warburg

PTBP1, PKM1 PKM2

23

8 miR-1 miR-133b in vivo

miR-1 miR-133b in vitro

Rh30

in vivo Lipofectamine

RNAiMAX scramble RNA

control

(Figure 11A) miR-1 miR-133b

(Figure 11B)

in vitro (Figure

11C) miR-1 miR-133b

Figure 11. Rh30 miR-1 miR-133b

(A)Control, miR-1 miR-133b

(B)

(C)Control, miR-1 miR-133b Warburg

PTBP1, PKM1, PKM2 PAX3-FOXO1

24

4

RMS PTBP1 miRs miR-1 miR-133b

Warburg miRNA

RMS miRNA

miR-133b ARMS

PAX3-FOXO1

miRs Warburg

miR-1 miR-133b Warburg

PTBP1

PKM2 PKM1

Warburg autophagic cell death

miR-133b ARMS

PAX3-FOXO1

(Figure 12)

PTBP1

miRs miR-

133b ARMS PAX3-FOXO1

RMS

PAX3-FOXO1

40

G1 Myogenin

40 miR-133b PAX3-FOXO1

25

miR-133b PAX3-FOXO1

PTBP1

BCR-

ABL Warburg PTBP1

41

Warburg

Warburg

PTBP1 RMS

miR-133b

RMS siR-PF2

Figure 12. miR-1 miR-133b Warburg

26

5

miR-1 miR-133b RMS Warburg

in vivo

microRNA RMS

27

28

(ERMS) RD KYM-1 JCRB (Japanese

Collection of Research Bioresources)

(ARMS) Rh30 Rh41

RD Eagle’s minimal

essential medium KYM-1 Dulbecco’s modified Eagle’s medium Ham’s F12

medium 1:1 Rh30 Rh41 RPMI-1640

8%FBS 37 5%CO2

0.5 105 /mL 6

24 1 50 L Opti-MEM (Invitrogen) 0.8 L

Lipofectamine RNAiMAX (Invitrogen) microRNA 10, 20 nM

15 siRNA 0.5, 5 nM

RNA homo sapiens (has)-miR-1, has-miR-133b

(Ambion), siR-PTBP1, siR-PF2 (Invitrogen) control RNA (Hokkaido System

Sciences) Rh30, Rh41 6

FBS RPMI-1640

15 50 L 5

8% FBS

29

miR-1 5’-UGGAAUGUAAAGAAGUAUGUAU-3’

miR-133b 5’-UUUGGUCCCCUUCAACCAGCUA-3’

siR-PTBP1 5’-AUCUCUGGUCUGCUAAGGUCACUUC-3’

siR-PF2 5’-CCUCUCACCUCAGAAUUCA-3’

control RNA 5’-GUAGGAGUAGUGAAAGGCC-3’

5 3-

Methyladenine (3-MA; Calbiochem)

ROS

24 ROS N-acetyl-L-cysteine

(NAC; Sigma Aldrich)

Anti-miR-1 anti-miR-133b

miR-1 miR-133b anti-miR-1

anti-miR-133b (Ambion)

30

(Life technologies)

(%)

RNA

RNA NucleoSpin microRNA isolation kit (TaKaRa)

RNA

RT-PCR

microRNA

miR-1 miR-133b TaqMan MicroRNA Assays

(Applied Biosystems) THUNDERBIRD Probe qPCR Mix (TOYOBO)

RNU6B

mRNA

PAX3,PAX3-FOXO1 mRNA PrimeScript® RT

reagent Kit (TaKaRa) THUNDERBIRD SYBR qPCR Mix (TOYOBO)

glyceraldehyde-3-phosphate

dehydrogenase (GAPDH )

PAX3 PAX3-sense: 5’-GAGACTGGCTCCATACGTCC-3’ PAX3-

antisense: 5’-ACGGTGTTTCGATCACAGAC-3’ PAX3-FOXO1

31

PAX3-sense FOXO1-antisense:5’-TGAACTTGCTGTGTAGGGACAG-3’ GAPDH

GAPDH-sense:5’-CCACCCATGGCAAATTCCATGGCA-3’

GAPDH-antisense:5’-TCTAGACGGCAGGTCAGGTCCACC-3’

PBS Protein lysis buffer <10 mM Tris-HCl (pH7.4),

0.1% SDS, 1% NP-40, 0.1% ,150 mM NaCl,1 mM

EDTA>: Protease Inhibitor Cocktail (nacalai tesque): Phosphatase Inhibitor Cocktail

solution : solution (Sigma-Aldrich Co.)= 50 : 1 : 1 : 1

20

13000 rpm, 20 min, 4

DC Protein Assay Kit (BIO-RAD)

5 SDS sample buffer <62.5 mM Tris-HCl (pH6.8),

2% SDS, 10% , 50 mM DTT, 0.01% >

10 g / 10 L 98 5 5

Western blotting

SDS 10 12.5%

Super Sep Ace (Wako) Transfer

membrane (MILLIPURE) 5%

32

LC3B FOXO1

PTBP1 (CST) PKM1 PKM2 (Novus)

PAX3 (abcam) (Anti-mouse IgG HRP-linked

antibody Anti-rabbit IgG HRP-linked antibody (CST))

Luminata Forte Western HRP Substrate (MILLIPORE)

Luminescent image analyzer LAS-4000 UV mini (Fujifilm)

control GAPDH (CST)

ImageQuant TL

PTBP1 3’UTR miR-1 miR-133b luciferase

reporter pMIR-control vector (Applied Biosystems)

miRs

PTBP1 mRNA RNA PrimeScript RT Reagent Kit

(TaKaRa)

PCR

miRs (miR-1: CAUUCCA, miR-133b: GGACCAAA) 3

(miR-1: CAGGAC, miR-133b: GGCUUAAA)

33

PrimeSTAR Mutagenesis Basal Kit (TaKaRa)

DNA

sequencing 96 0.5 104 /100 L

24

(0.5 g / well) 20 nM miR-1 miR-133b non-

specific control miRNA (Dharmacon)

Lipofectamine RNAiMAX 48

Dual-Glo Luciferase Assay System (Promega)

Firefly luciferase activity Renilla luciferase activity

Rh30 6-well 0.5 105 cells/ 1mL

5% CO2 37 miR-1, miR-133b, siR-PTBP1 Control RNA

well 48 42

5% 2 25 mM

2- D- -10-

(Wako) 1 well 250 μL 10

400 μL

400 μL (Wako) 200 μL

milliQ 10,000 g 3 4 400 μL

UltrafreeMC-PLHCC 250/pk for Metabolome Analysis (Merck) 9,100

g 2 20

2 42 100 mM 3-

34

10 mM 1,3,5- (Wako) milliQ

1 25 μL Aglient CE Capllary

Electrophoresis System (Aglient Technologis, CA, USA) CE-TOFMS

Metabolome Analysis and Screening

Tool for Easy and Rapid HANDling of Sample data; Master Hands (ver. 2.17.2.15,

Keio University, , )

Rh30 miR-1 miR-133b

BALB/cSlc-nu/nu Japan SLC Rh30 2 106

/ 100 L 12

scramble RNA, miR-1 miR-133b 0.2 nmol 50 l

Opti-MEM 1 l Lipofectamine RNAiMAX 4

3

0.5236L1(L2)2 (L1 L2

)

24

microRNA

mRNA Student's t-test

35

RMS rhabdomyosarcoma

ERMS embryonal RMS

ARMS alveolar RMS

PTBP1 polypyrimidine tract-binding protein 1

PKM pyruvate kinase muscle

miRNA, miR microRNA

PCR polymerase chain reaction

RNAi RNA interference

RISC RNA induced silencing complex

UTR untranslated region

PAX3 paired box gene 3

FOXO1 forkhead box protein O1

ROS reactive oxygen species

3-MA 3-Methyladenine

NAC N-acetyl-L-cysteine

GAPDH glyceraldehyde-3-phosphate dehydrogenase

36

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