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.(Doctoral Degree) 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。
ᶓ⣠➽⫗⭘࠾ࡅࡿࡀࢇ≉␗ⓗ࢚ࢿࣝࢠ࣮௦ㅰࢆ
ᶆⓗࡍࡿ
microRNA ࡢྠᐃࡑࡢไᚚ
MicroRNAs that regulate cancer-specific energy metabolism in rhabdomyosarcoma cells
2019
i
┠ḟ
➨1 ❶ ⥴ゝ 1 ➨2 ❶ ◊✲ࡢ⫼ᬒ┠ⓗ ➨1 ⠇ ᑠඣࡀࢇᶓ⣠➽⫗⭘ 4 ➨2 ⠇ microRNA miRNA ⸆ 6 ➨3 ⠇ Warburg ຠᯝ microRNA 8 ➨3 ❶ ➽≉␗ⓗ miR-1 miR-133b ࡢᶓ⣠➽⫗⭘⣽⬊࠾ࡅࡿ ⣽⬊ቑṪᢚไຠᯝ ➨1 ⠇ ᗎ 9 ➨2 ⠇ miR-1 miR-133b ࡢṇᖖ⤌⧊ཬࡧᶓ⣠➽⫗⭘⣽⬊ᰴ࠾ࡅࡿ Ⓨ⌧ࣞ࣋ࣝࡢ᳨ド 10 ➨3 ⠇ ᶓ⣠➽⫗⭘⣽⬊ᰴᑐࡍࡿ miR-1 miR-133b ࡢ ⣽⬊ቑṪᢚไຠᯝ 12 ➨4 ⠇ miR-1 miR-133b ࡼࡿࡀࢇ⣽⬊ࡢ࢚ࢿࣝࢠ࣮௦ㅰไᚚ 13 ➨5 ⠇ miR-1, miR-133b ཬࡧ siR-PTBP1 ࡼࡿ࣮࢜ࢺࣇࢪ࣮ࡢ᳨ド 16 ➨6 ⠇ miR-1 miR-133b ࡢᶓ⣠➽⫗⭘≉␗ⓗ࡞࣓࢟ࣛ㑇ఏᏊ࡛࠶ࡿPAX3-FOXO1 ᑐࡍࡿస⏝ 20
➨7 ⠇ PAX3-FOXO1 ࡢࢧࣞࣥࢩࣥࢢࡼࡿᶵ⬟ࡢ᳨ド 22
➨8 ⠇ miR-1 miR-133b ࡢ in vivo ࠾ࡅࡿᢠ⭘⒆ຠᯝࡢ᳨ド 23
➨4 ❶ ⥲ᣓ 24
ii
ㅰ ㎡ 27
ᐇ㦂ࡢ㒊 28
␎ㄒ㞟 35
1 ➨1 ❶ ⥴ゝ
ᶓ⣠➽⫗⭘ (rhabdomyosarcoma; RMS) ࡣࠊᑠඣᮇ࡛᭱ࡶⓎ㢖ᗘࡢ㧗࠸㌾㒊 ᝏᛶ⭘⒆࡛࠶ࡿࠋRMS ࡣࠊ⌮⤌⧊Ꮫⓗ⫾ඣᆺ㸸embryonal RMS (ERMS) ⬊ᕢᆺ㸸alveolar RMS (ARMS) ࡢ 2 ✀㢮ูࡉࢀࡿࠋ≉ࠊARMS ࡣࠊᰁⰍయ ㌿ᗙ t(2;13)(q35;q14) ⏤᮶ࡢ≉ᚩⓗ࡞࣓࢟ࣛ㑇ఏᏊ PAX3-FOXO1 ࢆⓎ⌧ࡋ࡚࠾ ࡾࠊணᚋⰋ࡛࠶ࡿࠋ་⒪ࡢ㐍ṌࡶRMS ࡢ⒵⋡ࡣྥୖࡋ࡚ࡁ࡚࠸ࡿࡀࠊ ARMS ᑐࡍࡿຠᯝⓗ࡞⸆ࡣ࡞ࡃࠊ᪂ࡋ࠸⒪ἲࡢ㛤Ⓨࡀᮃࡲࢀ࡚࠸ࡿࠋ୍ ᪉ࠊ௦ㅰ⏘≀ࡢ ᐃࢆࡍࡿ࣓ࢱ࣮࣒࣎ࣟゎᯒࡢ㐍Ṍࡼࡾࠊࡀࢇ≉␗ⓗ࡞࢚ࢿ ࣝࢠ࣮௦ㅰᶵᵓࡋ࡚Warburg ຠᯝࡀὀ┠ࡉࢀ࡚࠸ࡿࠋWarburg ຠᯝࡣࠊዲẼⓗ ᮲௳ୗ࠾࠸࡚ࡶゎ⢾⣔ࢆ✚ᴟⓗ⏝ࡍࡿ⌧㇟࡛࠶ࡿࠋWarburg ຠᯝࡢ⥔ᣢ 㔜せ࡞㑇ఏᏊࡋ࡚polypyrimidine tract-binding protein 1 (PTBP1) ࡀ࠶ࡿࠋPTBP1 ࡣࠊゎ⢾⣔ࡢᚊ㏿㓝⣲࡛࠶ࡿPyruvate kinase muscle (PKM) isoform ࡢ㑅ᢥⓗࢫࣉ ࣛࢩࣥࢢࡢᙺࢆᢸ࠺ splicer ࡋ࡚ᶵ⬟ࡋࠊPKM2 ඃⓎ⌧ࡉࡏࡿࡇ࡛ ゎ⢾⣔ࡼࡿ࢚ࢿࣝࢠ࣮௦ㅰࢆಁ㐍ࡋ࡚ࠊWarburg ຠᯝࡢᡂ❧ᐤࡋ࡚࠸ࡿࠋ ࡇࡢࡇࡽࠊPTBP1 ࡣࡀࢇ㑇ఏᏊ⪃࠼ࡽࢀࡿࠋࡇࢀࡲ࡛ࠊᡃࠎࡣࠊPTBP1 ࢆᶆⓗࡍࡿmicroRNA (miRNA, miR) ⩌ࢆ PTBP1 㛵㐃 miRs ࡋ࡚ Warburg ຠ ᯝ࠾ࡅࡿᶵ⬟╔┠ࡋ◊✲ࢆ㐍ࡵ࡚ࡁࡓࠋ⭠ࡀࢇࠊ⫶ࡀࢇࠊ⭤⬔ࡀࢇ⣽⬊ ࠾࠸࡚ࠊPTBP1 ࢆᶆⓗࡋ࡚Ⓨ⌧ࢆᢚไࡍࡿࡇ࡛ࠊ㢧ⴭ࡞⣽⬊ቑṪᢚไຠᯝ ࢆ♧ࡍࡇࢆሗ࿌ࡋ࡚ࡁࡓࠋࡑࡇ࡛ࠊᮏ◊✲࡛ࡣ᪂つ࡞ࢻࣛࣂ࣮ࡀࢇ㑇ఏᏊ࡛ ࠶ࡿPTBP1 ࢆᶆⓗࡍࡿ miR-1 miR-133b ↔Ⅼࢆ࠶࡚ࡓࠋmiR-1 miR-133b ࡣ➽≉␗ⓗⓎ⌧ࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾࠊreal-time RT-PCR ἲ᳨࡚ド ࡋࡓࡇࢁࠊṇᖖ㦵᱁➽⤌⧊➽⤌⧊ࢆ᭷ࡍࡿ⮚ჾ㧗Ⓨ⌧ࡋ࡚࠸ࡿࡇࡀ☜ ㄆ࡛ࡁࡓࠋࡲࡓࠊERMS ⣽⬊ᰴࡢ RD KYM-1ࠊARMS ⣽⬊ᰴࡢ Rh30 Rh41
2 ࠾࠸࡚ࠊṇᖖ㦵᱁➽⤌⧊ẚ㍑ࡋ࡚ࠊࡇࢀࡽࡢmiRNA ࡢⓎ⌧ࡣ㢧ⴭపୗࡋ ࡚࠸ࡿࡇࡀศࡗࡓࠋmiRNA ࡛ࡢ⒪ࢆ⪃࠼ࡿ࠺࠼࡛ࠊ⏕యෆᮏ᮶༑ศ Ꮡᅾࡍࡿ miRNA ࡀࡀࢇ࡞ࡢᝈ࡛పୗࡋ࡚࠸ࡿሙྜࠊࡑࡢ㊊ࡋ࡚࠸ࡿ miRNA ࢆ⿵ࡋඖࡢ≧ែᡠࡍࡇ࡛ࠊస⏝ࡢᑡ࡞࠸ࡼࡾຠᯝⓗ࡞⒪ࡀྍ ⬟࡞ࡿࠋࡀࢇ࡛ࡣ≉ࠊࡇࢀࡽࡢmiRNA ࡣࡀࢇᢚไⓗാࡃࡇࡀከ࠸ࠋ miR-1 miR-133b ࡣࠊṇᖖ➽⤌⧊࡛ከࡃࠊᶓ⣠➽⫗⭘⣽⬊࡛ᑡ࡞࠸ࡇࡽࠊ ࡀࢇᢚไⓗാࡃ⪃࠼ࡽࢀࡿࠋࡇࡢ⪃࠼ࢆࡶࠊmiR-1 miR-133b ࡢ RMS ᑐࡍࡿ᪂ࡓ࡞RNA ⸆ࡢࢩ࣮ࢬࡢྍ⬟ᛶࡀ࠶ࡿ⪃࠼ࠊࡇࢀࡽࡢ miRNA ࡢ Warburg ຠᯝ࣓࢟ࣛ㑇ఏᏊ PAX3-FOXO1 ࡢస⏝ࡘ࠸᳨࡚ウࡋࡓࠋ
3
ᮏᏛㄽᩥࡣࠊୗグࡢཎⴭㄽᩥࢆࡶసᡂࡋᒱ㜧ᏛᏛ㝔㐃ྜ⸆་⒪ ሗ◊✲⛉ᥦฟࡋࡓࡶࡢ࡛࠶ࡿࠋ
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.,
4 ➨2 ❶ ◊✲ࡢ⫼ᬒ┠ⓗ ➨1 ⠇ ᑠඣࡀࢇᶓ⣠➽⫗⭘ ᑠඣࡀࢇࡣᡂேẚ࡚㢖ᗘࡣᑡ࡞ࡃࠊࡍ࡚ࡢࡀࢇࡢ࠺ࡕ࡛ᑠඣࡀࢇࡀ༨ ࡵࡿྜࡣ 1㸣⛬ᗘ࡛࠶ࡿࡀࠊࡇࡶࡢẼࡼࡿṚᅉࡢ➨㸯࡞ࡗ࡚࠸ࡿࠋ ࡲ ࡓ ࠊ ᑠ ඣ ࡀ ࢇ ࡢ 㒊 ศ ࡣ ⓑ ⾑ ࠊ ⬻ ⭘ ⒆ ࡛ ࠶ ࡾ ࠊ ᶓ ⣠ ➽ ⫗ ⭘ (rhabdomyosarcoma; RMS) ࡣࠊẚ㍑ⓗࡲࢀ࡞ࡀࢇ࡛࠶ࡿࡀᑠඣࡀࢇࡢ࠾ࡼࡑ 3.5㸣ࢆ༨ࡵ࡚࠸ࡿ1ࠋ⫗⭘ࡣ㌟ࡢ㦵ࡸ㌾㒊⤌⧊ (⬡⫫ࠊ➽⫗࡞) ࡽⓎ⏕ࡍ ࡿᝏᛶ⭘⒆ࡢ⥲⛠࡛࠶ࡾࠊRMS ࡣᑠඣᮇ࡛᭱ࡶ㢖ᗘࡢ㧗࠸㌾㒊ᝏᛶ⭘⒆࡛࠶ࡿࠋ ⫗⭘⯡࡛࠸࠼ࡿࡇ࡛ࡣ࠶ࡿࡀࠊࡢᅛᙧ⭘⒆ẚ㍑ࡋࠊRMS ࠾࠸࡚ࡶ㑇 ఏᏊኚ␗ࡢᩘࡀᑡ࡞࠸2ࠋࡉࡽࠊRMS ࡣ⌮⤌⧊Ꮫⓗ⫾ඣᆺ㸸embryonal RMS
(ERMS) ⬊ᕢᆺ㸸alveolar RMS (ARMS) ࡢ 2 ✀㢮ูࡉࢀࡿ3ࠋ≉ࠊARMS
࡛ࡣᰁⰍయ㌿ᗙ t(2;13)(q35;q14) ⏤᮶ࡢ≉ᚩⓗ࡞࣓࢟ࣛ㑇ఏᏊ࡛࠶ࡿ PAX3-FOXO1 ࡢⓎ⌧ࡀ☜ㄆࡉࢀ࡚࠾ࡾ (Figure 1) 4ࠊணᚋⰋ࡛࠶ࡿ5ࠋPAX3-FOXO1
ࡣࠊ⣽⬊࿘ᮇࡢஹ㐍ࠊศᢚไࢆㄏᑟࡋ4ࠊணᚋⰋࢆᘬࡁ㉳ࡇࡍࠋ་⒪ᢏ⾡ࡢ
㐍ṌࡶRMS ࡢ⏕Ꮡ⋡ࡣྥୖࡋ࡚ࡁ࡚࠾ࡾࠊᖺ㱋ࡸཎⓎ㒊ࡼࡗ࡚␗࡞
ࡿࡀࠊ⌧ᅾࠊ5 ᖺ⏕Ꮡ⋡ࡣ 70%࡛࠶ࡿࠋࡋࡋ࡞ࡀࡽࠊ࣓࢟ࣛ㑇ఏᏊࢆⓎ⌧ࡍࡿ ARMS ࠾࠸࡚ࠊ5 ᖺ⏕Ꮡ⋡ࡣ 60%ࡲ࡛ୗࡀࡿ (National Cancer Institute ࡢ Physician Data Query ࡼࡾᢤ⢋)ࠋࡋࡋ࡞ࡀࡽࠊ⒪ἲࡣ⌧ᅾࡢࡇࢁࡕࡽࡢ ᆺ࡛ࡶඹ㏻࡛ࠊ᭱ึᡭ⾡࡛ษ㝖ྍ⬟࡛࠶ࢀࡤษ㝖ᚋᢠࡀࢇ⒪ࢆ⾜࠺ࠋ ࡁࡉࡸሙᡤࡼࡾ᭱ึษ㝖࡛ࡁ࡞࠸ሙྜࡣᢠࡀࢇ⒪ࢆ⾜࠸ࠊ⭘⒆⦰ᑠࢆ ࡣࡾࠊᡭ⾡ࠊᨺᑕ⥺࡞ࡢᒁᡤ⒪ἲࡢᚋࠊ⾡ᚋᏛ⒪ἲࢆ⾜࠺ࠋᶓ⣠➽⫗⭘ ᭷ຠ࡞ᢠࡀࢇࡣࣅࣥࢡࣜࢫࢳࣥࠊࢡࢳࣀ࣐ࢩࣥDࠊࢩࢡࣟ࣍ࢫࣇ࣑ࢻࠊ ࣍ࢫࣇ࣑ࢻࠊ࢚ࢺ࣏ࢩࢻ࡛࠶ࡿࠋ⌧ᅾࠊᶆ‽ⓗࢃࢀ࡚࠸ࡿࡢࡀࣅࣥࢡࣜ ࢫࢳࣥࠊࢡࢳࣀ࣐ࢩࣥDࠊࢩࢡࣟ࣍ࢫࣇ࣑ࢻࡢ 3 ࢆే⏝ࡋࡓ VAC ⒪ἲ
5 ࡛࠶ࡿࠋVAC ⒪ἲࡣࠊ1970 ᖺ௦㛤Ⓨࡉࢀࡓ᪉ἲ࡛ࠊࡑࢀ௨๓ẚ⒪ᡂ⦼ ࡣྥୖࡋࡓࠋ㏆ᖺ࡛ࡣࠊᶓ⣠➽⫗⭘⒪࠾࠸࡚ࠊ7 ࡲ࡛ 5 ᖺ⏕Ꮡ⋡ࢆୖࡆࡿ ࡇ㈉⊩ࡋࡓࠋࡋࡋ࡞ࡀࡽࠊ⣙40 ᖺࠊࡼࡾ᭷ຠ࡞ᢠࡀࢇ⒪ἲࡣぢࡘ ࡗ࡚࠸࡞࠸ࠋ Figure 1. ᰁᰁⰍయ㌿ᗙࡼࡿ⬊ᕢᆺᶓ⣠➽⫗⭘ࡢ࣓࢟ࣛ㑇ఏᏊPAX3-FOXO1
6 ➨2 ⠇ microRNA miRNA ⸆
microRNA (miRNA, miR) ࡣࠊ⏕యෆᏑᅾࡍࡿࢤࣀ࣒ࢥ࣮ࢻࡉࢀࡓ 21-25 ሷ ᇶᑐࡢnon-cording RNA ࡛࠶ࡿࠋࡇࡢ miRNA ࡣ mRNA ࡽࢱࣥࣃࢡ㉁ࢆⓎ⌧ࡉ ࡏ ࡿ ⩻ ヂ ࢆ ไ ᚚ ࡍ ࡿ ാ ࡁ ࢆ ᣢ ࡗ ࡚ ࠾ ࡾ ࠊ ࡇ ࡢ ⤌ ࡳ ࡣ RNA ᖸ ΅ (RNA interference; RNAi) ࡤࢀ࡚࠸ࡿ 6,7ࠋࡋࡋ࡞ࡀࡽࠊmiRNA ࡀⓎ⌧␗ᖖࢆ㉳
ࡇࡍࡇࡼࡾ⩻ヂࡢไᚚࡀ㐓⬺ࡉࢀࠊࡀࢇ࡞ࡢẼࡢⓎᐦ᥋㛵ࢃࡗ ࡚࠸ࡿࡇࡀศࡗ࡚ࡁࡓ8ࠋRNAi ࡢ⤌ࡳࡣࠊmiRNA ࡀ⣽⬊ෆධࡿ࢞
ࢻ㙐ࡢࡳࡀ RNA ㄏᑟࢧࣞࣥࢩࣥࢢ」ྜయ (RNA induced silencing complex; RISC) ࡤࢀࡿࢱࣥࣃࢡ」ྜయ⤖ྜࡍࡿࠋࡑࡋ࡚ࠊࡑࡢ RISC-miRNA 」ྜ యࡀᶆⓗmRNA ሷᇶ㓄ิ౫Ꮡⓗ 3’㠀⩻ヂ㡿ᇦ (untranslated region; UTR) ⤖ྜࡍࡿࡇ࡛ࠊ⩻ヂᢚไࢆ⾜ࡗ࡚࠸ࡿ (Figure 2)9ࠋmiRNA - mRNA ࡢ 3’-UTR
ࡢ⤖ྜࡣ┦⿵࡛࠶ࡿᚲせࡣ࡞ࡃ㸪miRNA ࡢ 2-7 ሷᇶࡢ seed sequence ࡀ㔜せ ࡛࠶ࡾࠊseed sequence ┦⿵ⓗ࡞㓄ิ⤖ྜࡍࡿࠋࡑࡢࡓࡵ㸪୍ࡘࡢ miRNA ࡣ
ᩘ༑ࡽᩘⓒࡢ mRNA ࢆᶆⓗࡍࡿ⪃࠼ࡽࢀ࡚࠾ࡾ㸪ࣄࢺ࠾࠸࡚ࡣ
mRNA ࡢ⣙ 60%ࡀ miRNA ไᚚࡉࢀࡿ᥎ᐃࡉࢀ࡚࠸ࡿ10ࠋ
2018 ᖺ 10 ᭶Ⅼ࡛ࠊ⣙ 2600 ࢆ㉸࠼ࡿ miRNA ࡀࣄࢺ࠾࠸࡚ྠᐃࡉࢀ࡚࠸ ࡿ (miRbase Release 22.1 (http://www.mirbase.org/) ࡼࡾᘬ⏝)ࠋከࡃࡢ miRNA ࡘ ࠸࡚ࡢ≉␗ⓗ࡞⏕≀Ꮫⓗᶵ⬟ࡣࠊ࠸ࡲࡔࡣ⌮ゎࡉࢀ࡚࠸࡞࠸ࡀࠊ⏕≀ࡢ Ⓨ⏕11,12ࠊ⣽⬊ࡢቑṪ13,14ࠊศ15-17ࠊ⣽⬊Ṛ18,19࠸ࡗࡓ⏕⌧㇟ࢆไᚚࡋ࡚ ࠸ࡿࡇࡀศࡗ࡚ࡁࡓࠋࡑࡋ࡚ࠊࡇࢀࡽࡢmiRNA ࡢㄪ⠇ᶵᵓࢆ⏝ࡋ miRNA ࢆ⿵ࡍࡿ⿵⒪ἲࡢ◊✲ࡀ㐍ࡵࡽࢀ࡚࠸ࡿ20ࠋmiRNA ࡢ⿵⒪ἲࡣ⏕యෆ ᮏ᮶ᏑᅾࡍࡿmiRNA ࡀࠊࡀࢇ࡞ࡢᝈ࡛పୗࡋ࡚࠸ࡿሙྜࠊࡑࡢ㊊ࡋ࡚࠸ ࡿmiRNA ࢆ⿵ࡋ࡚ඖࡢ≧ែᡠࡑ࠺ࡍࡿ᪉ἲ࡛ࠊస⏝ࡀᑡ࡞ࡃࡼࡾຠᯝ ⓗ࡞⒪⪃࠼ࡽࢀࡿࠋ
7 ࡲࡓࠊ᰾㓟་⸆ࡢ⌧≧ࡣࠊ2018 ᖺ 8 ᭶ୡ⏺ึࡢ siRNA ་⸆ရࡋ࡚ࢺࣛࣥ ࢫࢧࣞࢳࣥᆺᐙ᪘ᛶ࣑ࣟࢻ࣮ࢩࢫ (hATTR ࣑ࣟࢻ࣮ࢩࢫ) ࡢ⒪⸆ ࡛࠶ࡿ patisiran ࡀ⡿ᅜ࣭Ḣᕞ࡛ᢎㄆࢆྲྀᚓࡋ࡚࠸ࡿࠋsiRNA ࢆ⏝࠸ࡓ⒪ࡣࠊ miRNA ࢆ⏝࠸ࡓ⒪ࡣ␗࡞ࡾࠊཎᅉ㑇ఏᏊࢆ≺ࡗ࡚㜼ᐖࡍࡿࡓࡵᚑ᮶ࡢ⒪ ἲ࡛࠶ࡿศᏊᶆⓗ⒪࡛ࠊ୍ࡘࡢᶆⓗࢆ㜼ᐖࡍࡿࡇࡼࡿ⸆⪏ᛶࡀၥ㢟 ࡞ࡿᠱᛕࡀ࠶ࡿࠋ Figure 2. RNA ᖸᖸ΅ࡢ⤌ࡳ
8 ➨3 ⠇ Warburg ຠᯝ microRNA
㏆ᖺࠊࡀࢇ≉␗ⓗ࡞࢚ࢿࣝࢠ࣮௦ㅰᶵᵓࡋ࡚ Warburg ຠᯝࡀὀ┠ࡉࢀ࡚ ࠸ࡿࠋWarburg ຠᯝࡣࠊࡀࢇ⣽⬊ࡀዲẼⓗ᮲௳ୗ࡛ࡶゎ⢾⣔ࢆ⏝ࡋ࡚࢚ࢿ ࣝࢠ࣮⏕⏘ࢆ⾜ࡗ࡚࠸ࡿ⌧㇟࡛࠶ࡿ21,22ࠋࡲࡓࠊWarburg ຠᯝࢆಁ㐍ࡍࡿ㑇ఏᏊ
ࡋ࡚polypyrimidine tract-binding protein 1 (PTBP1) ࡀ࠶ࡾࠊPTBP1 ࡢᏑᅾୗ࡛ ゎ⢾⣔ࡢᚊ㏿㓝⣲࡛࠶ࡿPyruvate kinase muscle (PKM) isoform ࡢⓎ⌧ࢆ PKM2 ඃ ࡉࡏࡿࡇ࡛ࠊWarburg ຠᯝࡀᡂ❧ࡍࡿ (Figure 3) 23,24ࠋ
Figure 3. ࡀࡀࢇ≉␗ⓗ࡞࢚ࢿࣝࢠ࣮௦ㅰࠕWarburg ຠᯝࠖ25
ࡑࡋ࡚ࠊᡃࠎࡣࠊࡇࢀࡲ࡛PTBP1 ࢆᶆⓗࡍࡿ miRNA ⩌ࢆ PTBP1 㛵㐃 miRs ࡋ࡚Warburg ຠᯝ࠾ࡅࡿᶵ⬟ࢆ୰ᚰ◊✲ࢆ㐍ࡵ࡚ࡁࡓ26,27ࠋ
ࡑࡇ࡛ᮏㄽᩥ࡛ࡣࠊPTBP1 㛵㐃 miRs ࡢෆࠊ➽⫗≉␗ⓗศᕸࡍࡿ 1 miR-133b 16,28,29㛵ࡋ࡚ࠊRMS ࠾ࡅࡿ Warburg ຠᯝࡢᙳ㡪ཬࡧ࣓࢟ࣛ㑇ఏᏊ
9 ➨3 ❶ ➽≉␗ⓗ miR-1 miR-133b ࡢᶓ⣠➽⫗⭘⣽⬊࠾ࡅࡿ⣽⬊ቑṪᢚไຠ ᯝ ➨1 ⠇ ᗎ RMS ࡣࠊᵝࠎ࡞᪂つᢠࡀࢇࡀ㛤Ⓨࡉࢀࡿᗘຠᯝࡢ᳨ドࡀ࡞ࡉࢀ࡚ࡁࡓࡀࠊ ᡂᯝࢆ࠶ࡆࡽࢀࡓ⸆ࡣ࡞ࡃࠊࡇࢀࡲ࡛ࡢ⒪ᡓ␎ࡣ␗࡞ࡗࡓ⒪ἲࡀᚲせ ࡛࠶ࡿ⪃࠼ࡽࢀࡿࠋࡑࡇ࡛ᮏ❶࡛ࡣࠊࡀࢇ≉␗ⓗ࡞࢚ࢿࣝࢠ࣮௦ㅰ࡛࠶ࡿ Warburg ຠᯝࢆไᚚࡍࡿ PTBP1 ࢆᶆⓗࡍࡿ miRNA ࡘ࠸࡚ࠊࣄࢺ RMS ⣽⬊ ᰴᑐࡍࡿቑṪᢚไຠᯝࢆ᳨ドࡋࠊቑṪᢚไ࣓࢝ࢽࢬ࣒ࡢゎ᫂ࢆヨࡳࠊRNA ⸆ࡢࢩ࣮ࢬࡋ࡚ࡢྍ⬟ᛶࢆ᳨ウࡋࡓࠋ
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➨2 ⠇ miR-1 miR-133b ࡢṇᖖ⤌⧊ཬࡧᶓ⣠➽⫗⭘⣽⬊ᰴ࠾ࡅࡿⓎ⌧ࣞ࣋ ࣝࡢ᳨ド
TargetScan Release 7.1 (http://www.targetscan.org/) ࢹ࣮ࢱ࣮࣋ࢫࡼࡾࠊPTBP1 ࢆ ᶆⓗࡍࡿmiRNA ࡢ୰࡛ࠊ➽≉␗ⓗ࡞ miR-1 miR-133b ࡢṇᖖ⤌⧊࠾ࡅࡿ Ⓨ⌧ࢆ☜ㄆࡍࡿࡓࡵࠊreal-time RT-PCR ࢆ⾜ࡗࡓࠋண㏻ࡾࡑࢀࡽࡢⓎ⌧ࡣࠊ➽ ⤌⧊ཬࡧ➽⤌⧊ࢆ᭷ࡍࡿ⮚ჾ≉␗ⓗ࡛࠶ࡿࡇࡀ☜ㄆ࡛ࡁࡓ (Figure 4A)ࠋࡉ
ࡽࠊ➽⤌⧊≉␗ⓗⓎ⌧ࡀ㞟୰ࡋ࡚࠸ࡿࡇࡀ☜ㄆ࡛ࡁࡓࡓࡵࠊRMS ⣽⬊ᰴ
(ERMS ⣽⬊ᰴ: RD KYM-1ࠊARMS ⣽⬊ᰴ: Rh30 Rh41) ࠾ࡅࡿⓎ⌧ࡶྠ ᵝ☜ㄆࡋࡓࡇࢁࠊṇᖖ㦵᱁➽ (Skeletal muscle; SKM) ⤌⧊ẚࠊ㢧ⴭⓎ ⌧ࡀపୗࡋ࡚࠸ࡿࡇࡀศࡗࡓ (Figure 4B)ࠋࡇࢀࡽࡢ⤖ᯝࡼࡾࠊ1 miR-133b ࡣṇᖖ⤌⧊࠾࠸࡚➽⤌⧊≉␗ⓗⓎ⌧ࡀ㞟୰ࡋ࡚࠾ࡾࠊࡉࡽࠊ⫗⭘
ࡍࡿࡇ࡛ࡇࢀࡽࡢ miRNA ࡢⓎ⌧ࡀపୗࡍࡿࡇࡀ♧ࡉࢀࡓࠋࡇࡢࡇࡽࠊ
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Figure 4. ࣄࢺṇṇᖖ⤌⧊ࣄࢺᶓ⣠➽⫗⭘⣽⬊࠾ࡅࡿmiR-1 miR-133b ࡢ
Ⓨ⌧㔞ẚ㍑
(A)ᵝࠎ࡞ࣄࢺṇᖖ⤌⧊࠾ࡅࡿ miR-1 miR-133b ࡢⓎ⌧ศᕸ
(B)ࣄࢺ RMS ⣽⬊ᰴ (RD KYM-1ࠊRh30 Rh41) ࣄࢺṇᖖ SKM ⤌⧊ࡢ miR-1 miR-133b ࡢⓎ⌧㔞ࡢẚ㍑
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➨3 ⠇ ᶓ⣠➽⫗⭘⣽⬊ᰴᑐࡍࡿ miR-1 miR-133b ࡢ⣽⬊ቑṪᢚไຠᯝ miR-1 miR-133b ࡢ⣽⬊ቑṪᢚไຠᯝࢆ᳨ドࡍࡿࡓࡵࠊRMS ⣽⬊ᰴࡇࢀࡽ ࡢ miRNA ࢆ Lipofectamine RNAiMAX ࢆ⏝࠸࡚ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ⣽
⬊⏕Ꮡ⋡ࡣࠊࡑࢀࡒࢀࡢ⣽⬊ᰴ࠾ࡅࡿຠᯝ᭱㛫ࡋ࡚ࠊERMS ⣽⬊ᰴ࠾
࠸࡚ࡣ72 㛫ᚋࠊARMS ⣽⬊ᰴ࠾࠸࡚ࡣ 48 㛫ᚋࢺࣜࣃࣥࣈ࣮ࣝⰍ⣲
㝖ヨ㦂ἲࡼࡾ ᐃࡋࡓࠋࡑࡢ⤖ᯝࠊࡕࡽࡢmiRNA ࡶࠊࡍ࡚ࡢ⣽⬊ᰴ࠾
࠸࡚ࠊscramble RNA ࢆ⏝࠸ࡓ control ẚ⃰ᗘ౫Ꮡⓗ㢧ⴭ࡞⣽⬊ቑṪᢚไຠ ᯝࢆ♧ࡋࡓ (Figure 5)ࠋ
Figure 5. ࣄࣄࢺᶓ⣠➽⫗⭘⣽⬊ᑐࡍࡿmiR-1 miR-133b ࡢ⣽⬊ቑṪᢚไຠᯝ
ࣄࢺRMS ⣽⬊ᰴ miR-1 miR-133b (10, 20 nM) ࢆᑟධࡋࡓ㝿ࡢ⏕⣽⬊⋡ࡢホ ౯
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➨4 ⠇ miR-1 miR-133b ࡼࡿࡀࢇ⣽⬊ࡢ࢚ࢿࣝࢠ࣮௦ㅰไᚚ
miR-1 miR-133b ࡣࠊWarburg ຠᯝ㛵㐃ࡍࡿ PTBP1 ࢆᶆⓗࡍࡿ miRNA ࡋ࡚ሗ࿌ࡀ࠶ࡿ30,31ࠋࡑࡇ࡛ࠊWarburg ຠᯝ㛵㐃ࡍࡿ㑇ఏᏊࡢࢱࣥࣃࢡ㉁ࡢ Ⓨ⌧ኚࢆ࢚࢘ࢫࢱࣥࣈࣟࢵࢺ᳨࡚ドࡋࡓࠋ☜ࠊࡕࡽࡢmiRNA ࡶࠊࡍ ࡚ࡢ⣽⬊ᰴ࠾࠸࡚ࠊPTBP1 ࡢⓎ⌧㔞ࡢపୗࡀ☜ㄆ࡛ࡁࡓ (Figure 6A)ࠋࡉࡽ ࠊࡇࢀࡽࡢ miRNA ࡀᮏ PTBP1 ࢆᶆⓗࡋ࡚࠸ࡿࡢࢆ☜ㄆࡍࡿࡓࡵࠊࣝ ࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮ࢵࢭࠊࣥࢱࢦࢽࢫࢺࡋ࡚1 anti-miR-133b ࢆ⏝࠸᳨࡚ドࡋࡓࠋࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮ࢵࢭ࠾࠸࡚ࠊPTBP1 ࡢ3’UTR ࡢࢩ࣮ࢻ㓄ิ࣑࣮ࣗࢸ࣮ࢩࣙࣥ (Mut) ࢆຍ࠼ࡿࡇࡼࡾࠊ㓄ิࡀ ṇᖖ (Wild) ࡢሙྜ࠾ࡅࡿࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢᢚไࡀ࢟ࣕࣥࢭࣝࡉࢀࡿࡇ ࡀ☜ㄆ࡛ࡁࡓ (Figure 6B)ࠋࡲࡓࠊanti-miR-1 anti-miR-133b ࡼࡗ࡚ࠊPTBP1 ࡢⓎ⌧㔞ࡢపୗࡢ࢟ࣕࣥࢭࣝࡀ☜ㄆ࡛ࡁࡓ (Figure 6C)ࠋࡇࢀࡽࡢ⤖ᯝࡼࡾࠊࡇ ࢀࡽࡢmiRNA ࡀ RMS ⣽⬊ᰴ࠾࠸࡚ࡶ PTBP1 ࢆ┤᥋ᶆⓗࡋ࡚Ⓨ⌧ᢚไࢆ⾜ ࡗ࡚࠸ࡿࡇࡀ♧ࡉࢀࡓࠋ PTBP1 ࢆࡇࢀࡽࡢ miRNA ࡀᶆⓗࡋ࡚࠸ࡿࡇࡀ☜ㄆ࡛ࡁࡓࡓࡵࠊPTBP1 ࡀ ࢫࣉࣛࢩࣥࢢࡋ࡚࠸ࡿゎ⢾⣔ࡢᚊ㏿㓝⣲࡛࠶ࡿPKM ࡢ 2 ࡘࡢࢯࣇ࢛࣮࣒ ࡢⓎ⌧ࢆ࢚࢘ࢫࢱࣥࣈࣟࢵࢺ࡚ゎᯒࡋࡓࠋࡑࡢ⤖ᯝࠊࡀࢇඃⓎ⌧ࡋ࡚࠸ ࡿPKM2 ࡽ PKM1 Ⓨ⌧㔞ࡢẚࡀࢩࣇࢺࡋ࡚࠸ࡿࡇࡀศࡗࡓ (Figure 6A)ࠋ PKM1 ࢩࣇࢺࡍࡿࡇࡀศࡗࡓࡓࡵࠊTCA cycle ࡘ࠸࡚࣓ࢱ࣮࣒࣎ࣟゎᯒ ࢆ⏝࠸᳨࡚ドࡋࡓࠋࡕࡽࡢmiRNA ࡶ TCA cycle ࢆಁ㐍ࡉࡏࡿࡇࡀศࡗࡓ (Figure 6D)ࠋࡉࡽࠊࡑࡢ㝿ࡢ ATP ࡢ⣽⬊ෆ㔞ࢆ ᐃࡋࡓ⤖ᯝࠊPKM1 ࡢⓎ⌧ቑ ຍక࠸ࠊATP ⏘⏕ࡀ㢧ⴭቑຍࡍࡿࡇࡶศࡗࡓ (Figure 6D)ࠋ
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Figure 6. ࣄࣄࢺᶓ⣠➽⫗⭘⣽⬊࠾ࡅࡿmiR-1 miR-133b ࡢ PTBP1 ᶆⓗ᳨ド
(A)ࣄࢺ RMS ⣽⬊ᰴ miR-1 miR-133b (10, 20 nM) ࢆᑟධࡋࡓ㝿ࡢ Warburg ຠᯝ㛵㐃㑇ఏᏊ࡛࠶ࡿPTBP1, PKM1 ࠾ࡼࡧ PKM2 ࡢࢱࣥࣃࢡ㉁Ⓨ⌧
(B)ࣄࢺ RMS ⣽⬊ᰴ࡛ miR-1 miR-133b ࡢ PTBP1 ࡢ 3’UTR ⤖ྜ㓄ิ࠾ࡅࡿ ࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮ࢵࢭ
(C)ࣄࢺ RMS ⣽⬊ᰴ࡛ miR-1 miR-133b ᑐࡍࡿ antagomiR ࡼࡿ࢟ࣕࣥࢭࣝ ᐇ㦂
(D)ࣄࢺ RMS ⣽⬊ᰴ miR-1 miR-133b ࢆᑟධࡋࡓ㝿ࡢ࣓ࢱ࣮࣒࣎ࣟゎᯒ (E)ࣄࢺ RMS ⣽⬊ᰴ miR-1 miR-133b ࢆᑟධࡋࡓ㝿ࡢ⣽⬊ෆ ATP 㔞
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ᶆⓗࡋ࡚࠸ࡿPTBP1 ࡢᶵ⬟ࢆ᳨ドࡍࡿࡓࡵࠊsiRNA ࡼࡿࢧࣞࣥࢩࣥࢢ ࢆ⾜ࡗࡓࠋPTBP1 ࡢࣀࢵࢡࢲ࢘ࣥࡣࠊmiRNA ࢆᑟධࡋࡓྠᵝࠊ㢧ⴭ࡞⣽ ⬊ቑṪᢚไຠᯝࢆ♧ࡋࡓ (Figure 7A)ࠋࡉࡽࠊࡀࢇඃⓎ⌧ࡋ࡚࠸ࡿ PKM2 ࡽPKM1 Ⓨ⌧㔞ࡢẚࡀࢩࣇࢺࡋ࡚࠸ࡓ (Figure 7B)ࠋຍ࠼࡚ࠊ࣓ࢱ࣮࣒࣎ࣟ ゎᯒࡼࡿTCA cycle ࡢಁ㐍 (Figure 7C)ࠊ⣽⬊ෆࡢ ATP 㔞ࡶቑຍࡍࡿࡇࡶ☜ ㄆ࡛ࡁࡓ (Figure 7D)ࠋ௨ୖࡢ⤖ᯝࡼࡾࠊmiR-1 miR-133b ࡣࠊPTBP1 ࢆᶆⓗ㑇 ఏᏊࡋ࡚Ⓨ⌧ᢚไࢆࡋ࡚ࠊ⣽⬊ቑṪᢚไຠᯝࢆ♧ࡋ࡚࠸ࡿࡇࡀศࡗࡓࠋ ࡉࡽࠊࡇࢀࡽࡢ miRNA ࡣࠊPKM2 ࡽ PKM1 Ⓨ⌧㔞ࡢẚࢆࢩࣇࢺࡉࡏࠊ TCA cycle ࢆಁ㐍ࡉࡏࠊࡑࡢ⤖ᯝࠊATP ⏘⏕ࢆቑຍࡉࡏࡿࡇࡶ♧ࡉࢀࡓࠋ
Figure 7. ࣄࣄࢺᶓ⣠➽⫗⭘⣽⬊࠾ࡅࡿPTBP1 ࣀࢵࢡࢲ࢘ࣥࡢຠᯝ (A)ࣄࢺ RMS ⣽⬊ᰴ siR-PTBP1 (0.5, 5 nM) ࢆᑟධࡋࡓ㝿ࡢ⏕⣽⬊⋡ࡢホ౯ (B)ࣄࢺ RMS ⣽⬊ᰴ siR-PTBP1 ࢆᑟධࡋࡓ㝿ࡢ Warburg ຠᯝ㛵㐃㑇ఏᏊ ࡛࠶ࡿPTBP1, PKM1 ཬࡧ PKM2 ࡢࢱࣥࣃࢡ㉁Ⓨ⌧ (C)ࣄࢺ RMS ⣽⬊ᰴ siR-PTBP1 ࢆᑟධࡋࡓ㝿ࡢ࣓ࢱ࣮࣒࣎ࣟゎᯒ (D)ࣄࢺ RMS ⣽⬊ᰴ siR-PTBP1 ࢆᑟධࡋࡓ㝿ࡢ⣽⬊ෆ ATP 㔞
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➨5 ⠇ miR-1, miR-133b ཬࡧ siR-PTBP1 ࡼࡿ࣮࢜ࢺࣇࢪ࣮ࡢ᳨ド
miR-1 miR-133b ࡢ⣽⬊ቑṪᢚไຠᯝࢆゎ᫂ࡍࡿࡓࡵࠊ⣽⬊ࡢ⾲⌧ᆺࡍ࡞ࢃ ࡕ⣽⬊Ṛࡘ࠸᳨࡚ドࡋࡓࠋ⣽⬊Ṛࡣࠊࡁࡃศ㢮ࡋ࡚ࣉࣟࢢ࣒ࣛ⣽⬊Ṛ㠀 ࣉࣟࢢ࣒ࣛ⣽⬊Ṛศࡅࡽࢀࡿࠋࡉࡽࠊࣉࣟࢢ࣒ࣛ⣽⬊ṚࡣᙧែᏛୖࡢ㐪࠸ ࡽࠊ࣏ࢺ࣮ࢩࢫࠊ࣮࢜ࢺࣇࢪ࣮ࠊࢿࢡࣟࣉࢺ࣮ࢩࢫࡢ3 ࡘศ㢮ࡍࡿࡇ ࡀ࡛ࡁࡿ32ࠋmiR-1 miR-133b ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡍࡿࡇ࡛ࠊPTBP1 ࢆᶆⓗ㑇ఏᏊࡍࡿⓎ⌧ࡢᢚไࢆࡋࡓ⣽⬊ቑṪᢚไຠᯝࢆ♧ࡋࡓࡇࡽࠊ Warburg ຠᯝࢆไᚚࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡑࡇ࡛ࠊࡀࢇ⣽⬊ࡣࠊTCA cycle ಁ㐍ࡼࡿάᛶ㓟⣲✀ (Reactive Oxygen Species; ROS) ࡢⓎ⏕ᑐᛂࡍࡿ ࡓࡵ࣮࢜ࢺࣇࢪ࣮ࢆᘬࡁ㉳ࡇࡋ࡚࠸ࡿࡢ࡛ࡣ࡞࠸⪃࠼ࡓࠋ࣮࢜ࢺࣇ ࢪ࣮ࡣTCA cycle ࢆ⏝ࡍࡿࡇ࡛Ⓨ⏕ࡍࡿ ROS ࡼࡗ࡚ㄏᑟࡉࢀࡿࡇ33,34
ࡀศࡗ࡚࠾ࡾࠊ࣮࢜ࢺࣇࢪ࣮ࡘ࠸᳨࡚ドࡋࡓࠋ ࣮࢜ࢺࣇࢪ࣮ࡣࠊ୍⯡ᰤ㣴㣚㣹࡞ࡢࢫࢺࣞࢫ᮲௳ୗ࡛άᛶࡉࢀࠊ⣽⬊ ࡢ⏕Ꮡಁ㐍ᶵᵓࡋ࡚ാࡃࠋࡲࡓࠊ࣮࢜ࢺࣇࢪ࣮ࡣ⣽⬊ෆࡢ␗ᖖ࡞ࢱࣥࣃࢡ㉁ ࡸᦆയࢆཷࡅࡓ࢜ࣝ࢞ࢿࣛࢆศゎࡋ࡚ࠊ⣽⬊ࡢᜏᖖᛶࢆ⥔ᣢࡍࡿࡓࡵࡢ⏕⌮ⓗ ࡞⣽⬊ᶵ⬟࡛ࡶ࠶ࡿࠋࡑࡢ୍᪉࡛ࠊ⣽⬊ᙉ࠸ࢫࢺࣞࢫࡀ㛗ᮇ㛫ຍࢃࡗࡓ㝿ࡣ ࣮࢜ࢺࣇࢪ࣮ࡀஹ㐍ࡋࠊ⣽⬊Ṛㄏᑟᶵᵓࡋ࡚స⏝ࡍࡿࠋ࣮࢜ࢺࣇࢪ࣮ࡢ㛤 ጞึᮇᙧᡂࡉࢀࡿ࣮࢜ࢺࣇࢦࢯ࣮࣒ࡢᙧᡂ LC3B ࡢϩࡀᚲ㡲࡛࠶ࡿࡇ ࡽࠊLC3B ࡢϨࡽϩࡢ⛣⾜ࡀ࣮࢜ࢺࣇࢦࢯ࣮࣒࣐࣮࣮࢝ࡋ࡚⏝ࡉࢀ ࡚࠸ࡿ35,36ࠋ࣮࢜ࢺࣇࢦࢯ࣮࣒ࡣࣜࢯࢯ࣮࣒⼥ྜࡋࠊ࣮࢜ࢺࣜࢯࢯ࣮࣒࡞ ࡿࠋ࣮࢜ࢺࣇࢦࢯ࣮࣒ෆࡢࢱࣥࣃࢡ㉁ࡸ࢜ࣝ࢞ࢿࣛࡣࣜࢯࢯ࣮࣒ෆࡢຍỈศ ゎ㓝⣲࡛ศゎࡉࢀࠊ࣑ࣀ㓟࡞ࡗ࡚ࢫࢺࣞࢫᛂ⟅ᚲせ࡞ࢱࣥࣃࢡ㉁ࡢྜᡂ ⏝ࡉࢀࡿ 35ࠋࡑࡇ࡛ࠊLC3B ࡢⓎ⌧ࢆ࢚࢘ࢫࢱࣥࣈࣟࢵࢺ࡚☜ㄆࡋࡓࠋ ࣮࢜ࢺࣇࢪ࣮ࢆᘬࡁ㉳ࡇࡋ࡚࠸ࡿ⪃࠼ࡽࢀࡿLC3B ࡢϨࡽϩࡢ⛣⾜ࡀࠊ
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miR-1,miR-133b (Figure 8A) ཬࡧ siR-PTBP1 (Figure 8B) ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥ
ࡋࡓ࡚ࡢRMS ⣽⬊ᰴ࡛☜ㄆ࡛ࡁࡓࠋࡉࡽࠊᙧែᏛⓗ࣮࢜ࢺࣇࢪ࣮ࢆ᳨
ドࡍࡿࡓࡵࠊ㟁Ꮚ㢧ᚤ㙾࡚Rh30 ⣽⬊ᰴ࡚ᙧែࢆᙳࡋࡓࠋࡑࡢ⤖ᯝࠊ࢜
࣮ࢺࣇࢪ࣮ࡢᙧែᏛⓗ≉ᚩ࡛࠶ࡿ✵⬊ᙧᡂࡀࠊmiR-1, miR-133b (Figure 8C) ཬ ࡧsiR-PTBP1 (Figure 8D) ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࢧࣥࣉࣝ࠾࠸࡚☜ㄆࡉ ࢀࡓࠋࡇࢀࡽࡢ⤖ᯝࡽࠊmiR-1 miR-133b ࡣࠊPTBP1 ࡢⓎ⌧ᢚไࢆࡋ࡚࢜ ࣮ࢺࣇࢪ࣮ࢆㄏᑟࡋ࡚࠸ࡿࡇࡀ♧ࡉࢀࡓࠋ
ḟࠊ࣮࢜ࢺࣇࢪ࣮ࡣࣉࣟࢢ࣒ࣛ⣽⬊Ṛࡢ୍ࡘࡋ࡚⪃࠼ࡽࢀ࡚࠸ࡿࡀࠊ⣽ ⬊Ṛࢆ㉳ࡇࡍࡶࡢ (autophagic cell death) ⏕Ꮡᐤࡍࡿࡶࡢ (autophagic cell survival) ࡀᏑᅾࡍࡿ 37ࠋࡕࡽࡢ࣮࢜ࢺࣇࢪ࣮ࡀ㉳ࡁ࡚࠸ࡿࡢ᳨ドࡍࡿࡓ
ࡵࠊ࣮࢜ࢺࣇࢪ࣮ࣥࣄࣅࢱ࣮࡛࠶ࡿ3-Methyladenine (3-MA) ࢆ⏝࠸ࡓࠋRD Rh30 ࡢࡑࢀࡒࢀࡢ⣽⬊ᰴ࠾࠸࡚ࠊ3-MA ࢆ⏝࠸࡚࣮࢜ࢺࣇࢪ࣮ࢆ㜼ᐖࡍ ࡿࡇ࡛⏕⣽⬊ᩘࡀᅇࡋࡓࡓࡵࠊࡕࡽࡢ miRNA (Figure 8E) ࡶ siR-PTBP1 (Figure 8F) ࡶ⣽⬊Ṛࢆᘬࡁ㉳ࡇࡍ autophagic cell death ࡛࠶ࡿࡇࡀ☜ㄆ࡛ࡁࡓࠋ ࡉࡽࠊ࣮࢜ࢺࣇࢪ࣮ࡣTCA cycle ಁ㐍ࡼࡗ࡚Ⓨ⏕ࡍࡿ ROS ࡼࡗ࡚ㄏ ᑟࡉࢀࡿࡓࡵࠊmiR-1, miR-133b ཬࡧ siR-PTBP1 ࠾࠸࡚ࡶ ROS ࡼࡿ࣮࢜ࢺ
ࣇࢪ࣮ㄏᑟ࡛࠶ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࡢ࡛ࠊᢠ㓟࡛࠶ࡿN-ࢭࢳࣝ-L-ࢩ
ࢫࢸࣥ (N-acetyl-L-cysteine; NAC) ࢆ⏝࠸࡚ ROS ࢆ㜼ᐖࡋࡓ㝿࣮࢜ࢺࣇ ࢪ࣮ࡀኚࡍࡿࡢ᳨ドࡋࡓࠋRh30 ⣽⬊ᰴ࠾࠸࡚ࠊmiR-1, miR-133b (Figure 8G) ཬࡧ siR-PTBP1 (Figure 8H) ࡑࢀࡒࢀ NAC ࢆే⏝ࡍࡿࡇ࡛ࠊ࣮࢜ࢺࣇࢪ ࣮ࡀᢚไࡉࢀࠊ⏕⣽⬊ᩘࡀᅇࡍࡿࡇࡀศࡗࡓࠋ
௨ୖࡢ⤖ᯝࡼࡾࠊmiR-1 miR-133b ࡼࡿ⣽⬊ቑṪᢚไຠᯝࡣࠊPTBP1 ࡢⓎ ⌧ᢚไࢆࡋࡓTCA cycle ಁ㐍ࡼࡿ ROS ࡢⓎ⏕㉳ᅉࡋࡓ autophagic cell death ࡼࡿ⣽⬊Ṛ࡛࠶ࡿࡇࡀ♧ࡉࢀࡓࠋ
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Figure 8. ࣄࣄࢺᶓ⣠➽⫗⭘⣽⬊࠾ࡅࡿmiR-1, miR-133b ཬࡧ siR-PTBP1 ࡢ
࣮࢜ࢺࣇࢪ᳨࣮ド
(A, B)ࣄࢺ RMS ⣽⬊ᰴ miR-1, miR-133b (10, 20 nM; A) ཬࡧ siR-PTBP1 (0.5, 5 nM; B) ࢆᑟධࡋࡓ㝿ࡢ࣮࢜ࢺࣇࢪ࣮࣐࣮࣮࡛࢝࠶ࡿ LC3B ࡢ ࢱࣥࣃࢡ㉁Ⓨ⌧
(C, D)Rh30 ⣽⬊ᰴ miR-1, miR-133b (20 nM; C) ཬࡧ siR-PTBP1 (5 nM; D) ࢆ ᑟධࡋ࡚48 㛫ᚋࡢ㟁Ꮚ㢧ᚤ㙾ࡼࡿᙧែᏛⓗ᳨ド
N: fragmented nuclei, AV: autophagic vesicles, Aly: autolysosome.
(E, F)RD Rh30 ⣽⬊ᰴ࣮࢜ࢺࣇࢪ࣮㜼ᐖ 3-MA (0.5 mM) ࢆ 5 㛫 ฎ⌮ᚋࠊmiR-1, miR-133b (20 nM; E) ཬࡧ siR-PTBP1 (5 nM; F) ࢆᑟධࡋ࡚ 72 48 㛫ᚋࡢ⏕⣽⬊ᩘホ౯ LC3B ࡢࢱࣥࣃࢡ㉁Ⓨ⌧
(G, H)Rh30 ⣽⬊ᰴ miR-1, miR-133b (20 nM; G) ཬࡧ siR-PTBP1 (5 nM; H) ࢆ ᑟධࡋ࡚24 㛫ᚋࠊROS 㜼ᐖ NAC (3 mM) ࢆῧຍࡋ࡚ࡉࡽ
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➨ 6 ⠇ miR-1 miR-133b ࡢᶓ⣠➽⫗⭘≉␗ⓗ࡞࣓࢟ࣛ㑇ఏᏊ࡛࠶ࡿ PAX3-FOXO1 ᑐࡍࡿస⏝
miR-1 miR-133b ࡢ RMS ⣽⬊ᰴ࠾ࡅࡿ⣽⬊ቑṪᢚไຠᯝࡀࠊPTBP1 ࡢⓎ ⌧ࡢᢚไࢆࡋࡓautophagic cell death ࡛࠶ࡿࡇࡀศࡗࡓࡓࡵࠊARMS ࠾ ࠸࡚≉ᚩⓗ࡞࣓࢟ࣛ㑇ఏᏊ࡛࠶ࡿ PAX3-FOXO1 ࡢࡼ࠺࡞ᙳ㡪ࢆ࠼ࡿ᳨ ドࢆ⾜ࡗࡓࠋ
ࡲࡎࠊRMS ⣽⬊ᰴ࠾࠸࡚ PAX3-FOXO1 ࡢⓎ⌧ࢆ☜ㄆࡍࡿࡓࡵࠊreal-time PCR ࢆ⾜ࡗ࡚ࠊmRNA 㔞ࢆ ᐃࡋࡓࠋARMS ⣽⬊ᰴ࡛ PAX3-FOXO1 ࡢⓎ⌧ࡀ㢧ⴭ ஹ㐍ࡋ࡚࠸ࡿࡇࡀศࡗࡓ (Figure 9A)ࠋmRNA ࡛ࣞ࣋ࣝ PAX3-FOXO1 ࡢ㑇ఏ ᏊࡢⓎ⌧ࡀ☜ㄆ࡛ࡁࡓࡓࡵࠊࢱࣥࣃࢡ㉁࡛ࣞ࣋ࣝⓎ⌧ࡀ࠺ኚࡍࡿࡢࢆ࢘ ࢚ࢫࢱࣥࣈࣟࢵࢺ᳨࡚ドࡋࡓࠋmiR-133b ࢆᑟධࡋࡓ㝿ࠊRh30 Rh41 ࡢ୧ ⣽⬊࡛PAX3-FOXO1 ࡢⓎ⌧ࡢపୗࡀ☜ㄆ࡛ࡁࡓ (Figure 9B, 9C)ࠋࡇࡢ⤖ᯝࡽࠊ miR-133b ࡣࠊARMS ≉ᚩ࡛࠶ࡿ࣓࢟ࣛ㑇ఏᏊ PAX3-FOXO1 ࢆⓎ⌧పୗࡉࡏࡿ ࡇࡀ♧ࡉࢀࡓࠋ
ḟࠊmiR-133b ࡀࡢࡼ࠺ PAX3-FOXO1 ࡢⓎ⌧పࡉࡏ࡚࠸ࡿࡢࡘ࠸࡚ ᳨ドࢆ⾜ࡗࡓࠋFigure 9C ࡢ RD KYM-1 ࠾࠸࡚ࡶ miR-133b ࡛ FOXO1 ࡢⓎ ⌧ࡀඹ㏻ࡋ࡚పୗࡋ࡚࠸ࡿࡇࡀศࡗࡓࠋࡇࡢࡇࡽࠊmiR-133b ࡀ FOXO1 ࢆ┤᥋ᶆⓗࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋseed sequence ࡀ 7mer ௨ୖ࣐ࢵࢳࡍ
ࡿࡇࡀᇶ‽࡛࠶ࡿࢹ࣮ࢱ࣮࣋ࢫ࠾࠸࡚ࠊFOXO1 ࢆᶆⓗࡋ࡚࠸ࡿࡇࡣ☜ ㄆ࡛ࡁ࡞ࡗࡓࠋࡋࡋࠊ6mer ࠾࠸࡚ࡶ miRNA ࡀຠᯝࢆⓎࡍࡿ࠸࠺ሗ࿌ 38,39ࡀ࠶ࡾࠊFOXO1 ࡢ 3’UTR ࠾࠸࡚㓄ิࢆ☜ㄆࡋ࡚ࡳࡓࡇࢁࠊ2 ᡤ࡛⤖ ྜྍ⬟࡞㓄ิࢆⓎぢࡋࡓࠋࡑࡇ࡛ࠊࡑࢀࡽࡢ㓄ิ࡛ࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮ ࢵࢭࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊ1 ᡤ࡛ࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢపୗࡢ࢟ࣕࣥࢭࣝࡀ ☜ㄆ࡛ࡁࡓࡓࡵࠊmiR-133b ࡣ FOXO1 ࢆᶆⓗࡋ࡚࠸ࡿྍ⬟ᛶࡀ㧗࠸⪃࠼ࡽ ࢀࡿ (Figure 9D)ࠋࡉࡽࠊanti-miR-133b ࢆ⏝࠸ࡿࡇ࡛ࠊmiR-133b ࡛Ⓨ⌧ࡀᢚ
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ไࡉࢀ࡚࠸ࡓFOXO1 PAX3-FOXO1 ࡢⓎ⌧ࡀᅇࡍࡿࡇࡀศࡗࡓ (Figure 9E)ࠋࡇࢀࡽࡢ⤖ᯝࡼࡾࠊmiR-133b ࡣࠊFOXO1 ARMS ࡢ≉ᚩⓗ࡞࣓࢟ࣛ㑇ఏ Ꮚ࡛࠶ࡿPAX3-FOXO1 ࢆᶆⓗࡋ࡚Ⓨ⌧ᢚไࡍࡿࡇࡀ♧ࡉࢀࡓࠋ
Figure 9. ࣓࢟࢟ࣛ㑇ఏᏊ PAX3-FOXO1 ᑐࡍࡿ miR-133b ࡢస⏝
(A)ࣄࢺ SKM ⤌⧊ࣄࢺ RMS ⣽⬊ᰴ࠾ࡅࡿ PAX3-FOXO1 ࡢ mRNA Ⓨ⌧㔞 (B)ࣄࢺ ARMS ⣽⬊ᰴ miR-1, miR-133b (10, 20 nM) ཬࡧ siR-PTBP1 (0.5, 5 nM)
ࢆᑟධࡋ 48 㛫ᚋࡢ PAX3 ࡢᢠయࢆ⏝࠸ࡓ PAX3-FOXO1 ࡢࢱࣥࣃࢡ㉁Ⓨ⌧ (C)ࣄࢺ RMS ⣽⬊ᰴ miR-1, miR-133b (10, 20 nM) ཬࡧ siR-PTBP1 (0.5, 5 nM)
ࢆᑟධࡋࡓ㝿ࡢ FOXO1 ࡢᢠయࢆ⏝࠸ࡓ FOXO1 PAX3-FOXO1 ࡢ ࢱࣥࣃࢡ㉁Ⓨ⌧
(D)miR-133b ࡢ FOXO1 ࡢ 3’UTR ⤖ྜ㓄ิ࠾ࡅࡿࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮ ࢵࢭ
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➨7 ⠇ PAX3-FOXO1 ࡢࢧࣞࣥࢩࣥࢢࡼࡿᶵ⬟ࡢ᳨ド
miR-133b ࡀ PAX3-FOXO1 ࢆ┤᥋ᶆⓗࡋ࡚࠸ࡿࡇࡀศࡗࡓࡓࡵࠊPAX3-FOXO1 ࡢᶵ⬟ࢆ᳨ドࡍࡿࡓࡵࠊPAX3-ࢆ┤᥋ᶆⓗࡋ࡚࠸ࡿࡇࡀศࡗࡓࡓࡵࠊPAX3-FOXO1 ࡢࢧࣞࣥࢩࣥࢢࢆ⾜ࡗࡓࠋsiR-PF240ࢆARMS ⣽⬊ᰴࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡍࡿࠊ㢧ⴭ࡞⣽⬊ቑṪᢚไຠᯝ ࢆ♧ࡋࡓ (Figure 10A)ࠋࡇࡢ⣽⬊ቑṪᢚไຠᯝࡣࠊ⣽⬊࿘ᮇࡢṆࡼࡿࡶࡢ࡛ ࠶ࡿ࠸࠺ሗ࿌ࡀ࠶ࡿ40ࠋWarburg ຠᯝࡢ㛵ಀࢆ᳨ドࡍࡿࡓࡵࠊ㛵㐃ࡍࡿࢱ ࣥࣃࢡ㉁ࡢⓎ⌧ࢆ࢚࢘ࢫࢱࣥࣈࣟࢵࢺ࡚☜ㄆࡋࡓࠋ⯆῝࠸ࡇࠊPTBP1 ࡢ Ⓨ⌧ࡀపୗࡋ࡚࠸ࡿࡇຍ࠼ࠊPKM2 ඃࡽ PKM1 ඃࢩࣇࢺࡋ࡚࠸ࡿ ࡇࡀ☜ㄆ࡛ࡁࡓ (Figure 10B)ࠋࡇࢀࡽࡢ⤖ᯝࡼࡾࠊARMS ࡢ≉ᚩⓗ࡞࣓࢟ࣛ㑇 ఏᏊ࡛࠶ࡿPAX3-FOXO1 ࡢୗὶ PTBP1 ࡀ࠶ࡾࠊWarburg ຠᯝࡢ㛵㐃ࡀ♧၀ ࡉࢀࡓࠋ Figure 10. ࣄࣄࢺ⬊ᕢᆺᶓ⣠➽⫗⭘⣽⬊࠾ࡅࡿPAX3-FOXO1 ࣀࢵࢡࢲ࢘ࣥࡢ ຠᯝ
(A)ࣄࢺ ARMS ⣽⬊ᰴ siR-PF2 (0.5, 5 nM) ࢆᑟධࡋࡓ㝿ࡢ⏕⣽⬊ᩘࡢホ౯ (B)ࣄࢺ ARMS ⣽⬊ᰴ siR-PF2 (0.5, 5 nM) ࢆᑟධࡋࡓ㝿ࡢ Warburg ຠᯝ㛵㐃
23
➨8 ⠇ miR-1 miR-133b ࡢ in vivo ࠾ࡅࡿᢠ⭘⒆ຠᯝࡢ᳨ド
miR-1 miR-133b ࡀࠊin vitro ࠾࠸࡚ࠊ⣽⬊ቑṪᢚไຠᯝࢆ♧ࡍࡇࡀ☜ㄆ
࡛ࡁࡓࡢ࡛ࠊࢾ࣮ࢻ࣐࢘ࢫࡢ⓶ୗRh30 ⣽⬊ࢆ⛣᳜ࡋࡓᢸ⒴࣐࢘ࢫࣔࢹࣝࢆ⏝
࠸ࡓ in vivo ࠾࠸࡚ࡶᢠ⭘⒆ຠᯝࢆ♧ࡍࡢ᳨ドࢆ⾜ࡗࡓࠋLipofectamine RNAiMAX ࢆ⏝࠸࡚ࠊ⭘⒆࿘㎶ᒁᡤᢞࡋࡓࠋࡑࡢ⤖ᯝࠊscramble RNA ࢆ⏝ ࠸ࡓ control ẚࠊ⭘⒆ࡢࢧࢬࡀᑠࡉࡃ࡞ࡗ࡚࠸ࡿࡇࡀ᫂ࡽ࡞ࡗࡓ (Figure 11A)ࠋ⭘⒆ࢧࢬࡢ⤒ⓗ࡞ኚࡶ miR-1 miR-133b ࢆᢞࡋ࡚࠸ࡿ᪉ ࡀ᭷ព⭘⒆ࡢቑࡀᢚไࡉࢀ࡚࠸ࡿࡇࡀศࡗࡓ (Figure 11B)ࠋ⭘⒆ࡽᢳ ฟࡋࡓࢱࣥࣃࢡ㉁ࡢⓎ⌧ࢆ☜ㄆࡋ࡚ࡶ in vitro ྠᵝࡢ⤖ᯝࡀᚓࡽࢀࡓ (Figure 11C)ࠋࡇࢀࡽࡢ⤖ᯝࡼࡾࠊ⏕యෆ࠾࠸࡚ࡶ miR-1 miR-133b ࡀᢠ⭘⒆ຠᯝࢆ ♧ࡍࡇࡀศࡗࡓࠋ
Figure 11. Rh30 ᢸᢸ⒴࣐࢘ࢫࣔࢹࣝ࠾ࡅࡿmiR-1 miR-133b ࡢᢠ⭘⒆ຠᯝ
(A)Control, miR-1 ཬࡧ miR-133b ࡢᢞᚋࡢฟ⭘⒆┿ (B)⭘⒆ࢧࢬࡢ⤒ⓗኚ
(C)Control, miR-1 ཬࡧ miR-133b ᢞ⭘⒆࠾ࡅࡿ Warburg ຠᯝ㛵㐃㑇ఏᏊ ࡛࠶ࡿPTBP1, PKM1, PKM2 ཬࡧ PAX3-FOXO1 ࡢࢱࣥࣃࢡ㉁Ⓨ⌧
24 ➨4 ❶ ⥲ᣓ
RMS ⣽⬊࠾࠸࡚ PTBP1 㛵㐃 miRs ࡛࠶ࡿ➽≉␗ⓗ miR-1 miR-133b ࡀ Warburg ຠᯝస⏝ࡋᢠࡀࢇ miRNA ࡋ࡚ാ࠸࡚࠸ࡿࡇࡀ♧ࡉࢀࡓࠋࡲࡓࠊ RMS ࠾ࡅࡿࡇࢀࡽࡢ miRNA ࡣࠊṇᖖ㦵᱁➽⤌⧊ẚ㍑ࡋࠊⓎ⌧ࡀ㢧ⴭప ୗࡋ࡚࠾ࡾࠊ⿵⒪ἲࡀᮇᚅ࡛ࡁࡿࠋࡉࡽࠊmiR-133b ࡣࠊARMS ≉ᚩⓗ࡞ ࣓࢟ࣛ㑇ఏᏊ࡛࠶ࡿ PAX3-FOXO1 ࡢ┤᥋ⓗ࡞స⏝ࢆㄆࡵࡓࠋࡇࢀࡲ࡛ࠊ➽≉ ␗ⓗ࡞miRs ᶓ⣠➽⫗⭘࠾ࡅࡿ࠸ࡃࡘࡢሗ࿌ࡀ࠶ࡿࡀࠊWarburg ຠᯝࡢほ Ⅼࡽ᳨ドࡋࡓࡶࡢࡣ࡞ࡃࠊ࣓࢟ࣛ㑇ఏᏊ┤᥋ⓗస⏝ࡋ࡚࠸ࡿሗ࿌ࡶ࡞࠸ࠋ
miR-1 miR-133b ࡣ Warburg ຠᯝࢆᡂ❧ࡉࡏࡿୖ࡛㔜せ࡞ࡀࢇ㑇ఏᏊ⪃࠼ ࡽࢀࡿPTBP1 ࢆᶆⓗ㑇ఏᏊࡋ࡚Ⓨ⌧ࢆᢚไࡋࠊ㑅ᢥⓗࢫࣉࣛࢩࣥࢢࡼࡗ ࡚ࡀࢇඃⓎ⌧ࡋ࡚࠸ࡓ PKM2 ࡽ PKM1 Ⓨ⌧㔞ࡢẚࢆࢩࣇࢺࡉࡏ Warburg ຠᯝࢆ◚ࡓࢇࡋࠊautophagic cell death ࡼࡿ⣽⬊Ṛㄏᑟࡋࡓࠋࡉࡽ ࠊ⯆῝࠸ࡇࠊmiR-133b ࠾࠸࡚ࡣࠊARMS ≉ᚩⓗ࡞࣓࢟ࣛ㑇ఏᏊ࡛ ࠶ࡿ PAX3-FOXO1 ࡶᶆⓗ㑇ఏᏊࡋ࡚Ⓨ⌧ࢆ┤᥋ⓗᢚไࡋ࡚࠸ࡿࡇࡀศ ࡗࡓ (Figure 12)ࠋ ࡀࢇ࠾࠸࡚Ⓨ⌧ࡀஹ㐍ࡋ࡚࠸ࡿPTBP1 ࢆᶆⓗࡋ࡚⣽⬊Ṛㄏᑟࡍࡿࡇ ࡽࠊࡇࢀࡽࡢmiRs ࡣ᰾㓟་⸆ࡢࢩ࣮ࢬࡢྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋࡉࡽ miR-133b ࡣ ARMS ࡢ≉ᚩⓗ࡞࣓࢟ࣛ㑇ఏᏊ࡛࠶ࡿ PAX3-FOXO1 ࡶᶆⓗ㑇ఏᏊࡋ ࡚࠸ࡿࡇࡽRMS ≉␗ⓗ࡞⸆ࡢᒎ㛤ࡀ⪃࠼ࡽࢀࡿࠋ ࡇࢀࡲ࡛ PAX3-FOXO1 ࡀ⣽⬊࿘ᮇṆࡢ㜼ᐖศㄏᑟࡢᢚไᐤࡋ࡚ ࠸ࡿ࠸࠺ሗ࿌ࡀ࠶ࡿ40ࠋࡇࡢ࣓࢟ࣛ㑇ఏᏊࢆࢧࣞࣥࢩࣥࢢࡍࡿࠊ⣽⬊࿘ᮇ ࡢG1 ᮇ࡛ࡢṆຍ࠼ࠊ➽ศࡢ࣐࣮࣮࡛࢝࠶ࡿ Myogenin ࡢஹ㐍ࡀᘬࡁ㉳ࡇ ࡉࢀࡿࡇࡀศࡗ࡚࠸ࡿ40ࠋᅇࠊmiR-133b ࡀ PAX3-FOXO1 ࢆ┤᥋ⓗᶆⓗ
25 ࡋ࡚࠸ࡿࡇࢆ♧ࡏࡓࡇ࡛ࠊ⣽⬊࿘ᮇࡢṆࠊศㄏᑟࡶᘬࡁ㉳ࡇࡋ࡚࠸ࡿ ྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡶࡋࠊ⣽⬊࿘ᮇṆࠊศㄏᑟࢆᘬࡁ㉳ࡇࡋ࡚࠸ࡓሙྜࠊ miR-133b ࡣ PAX3-FOXO1 ࣓࢟ࣛ㑇ఏᏊࡢⓎ⌧ࢆ┤᥋ⓗᢚไࡍࡿࡇ࡛⣽⬊࿘ ᮇࢆṆࡉࡏ࡚➽ศㄏᑟࡋࠊࡉࡽⓎ⌧ஹ㐍ࡋ࡚࠸ࡿPTBP1 ࡢⓎ⌧ᢚไࢆ ࡋ࡚⣽⬊Ṛㄏᑟࡍࡿࡇࡶ࡛ࡁࡿࡓࡵࠊ⿵⒪ἲࡋ࡚ࡁ࡞ྍ⬟ᛶࢆ ⛎ࡵ࡚࠸ࡿ⪃࠼ࡽࢀࡿࠋ ࡉࡽࠊࣄࢺ៏ᛶ㦵㧊ᛶⓑ⾑࠾࠸࡚≉ᚩⓗ࡞࣓࢟ࣛ㑇ఏᏊ࡛࠶ࡿ BCR-ABL ࡀ Warburg ຠᯝ࠾࠸࡚㔜せ࡞㑇ఏᏊ࡛࠶ࡿ PTBP1 ࡢୖὶ⨨ࡋࠊ࢝ࢫ ࢣ࣮ࢻࢆᙧᡂࡋ࡚࠸ࡿ࠸࠺ሗ࿌ࡀ࠶ࡿ 41ࠋࢻࣛࣂ࣮㑇ఏᏊ⪃࠼ࡽࢀࡿ࢟ ࣓ࣛ㑇ఏᏊࡀ Warburg ຠᯝࡢୖὶᏑᅾࡋ࡚࠸ࡿ࡞ࡿࠊ࣓࢟ࣛ㑇ఏᏊࡢⓎ ⌧ᢚไࡣ Warburg ຠᯝࡲ࡛ᙳ㡪ࢆཬࡰࡍࠋᅇࡢ◊✲࡛ࡣࠊ࣓࢟ࣛ㑇ఏᏊࡀ PTBP1 ┦స⏝ࡋ࡚࠸ࡿࡇࡀศࡗࡓࠋሗ࿌࠶ࡗࡓ࠾ࡾࠊRMS ࠾࠸ ࡚ࡶ࣓࢟ࣛ㑇ఏᏊࡀୖὶ࠶ࡗࡓሙྜࠊmiR-133b ࡣࡑࡢ୧᪉ࢆᶆⓗࡋ࡚࠾ࡾ RMS ≉␗ⓗ㧗࠸ຠᯝࢆ♧ࡍඹ siR-PF2 ࡢే⏝⒪ἲࡣ㧗࠸┦ຠᯝࢆᮇ ᚅ࡛ࡁࡿ⪃࠼ࡽࢀࡿࠋ
Figure 12. ࣄࣄࢺ⬊ᕢᆺᶓ⣠➽⫗⭘⣽⬊࠾ࡅࡿmiR-1 miR-133b ࡢ Warburg
26 ➨5 ❶ ⤖ㄒ
➽≉␗ⓗmiR-1 miR-133b ࡣࠊRMS ⣽⬊ᰴ࠾࠸࡚ Warburg ຠᯝࡸ࣓࢟ࣛ 㑇ఏᏊࡢⓎ⌧ࢆࡶㄪ⠇ࡋࠊᢠࡀࢇస⏝ࢆ♧ࡍࡇࡀศࡗࡓࠋࡉࡽࠊin vivo ࠾࠸࡚ࡶྠᵝࡢຠᯝࢆ♧ࡋࡓࡇࡽࠊࡇࢀࡽࡢmicroRNA ࡢ⿵⒪ἲࡀ RMS ࠾࠸࡚᭷ຠ࡛࠶ࡿྍ⬟ᛶࡀ♧ࡉࢀࡓࠋ
27 ㅰ ㎡ ᮏ◊✲ࡢ㐙⾜࠾ࡼࡧㄽᩥࡢసᡂ࠶ࡓࡾࠊ⤊ጞࡈ᠓⠜࡞ࡿࡈᣦᑟࠊࡈ㠴᧡ࢆ ㈷ࡾࡲࡋࡓᒱ㜧ᏛᏛ㝔㐃ྜ⸆་⒪ሗ◊✲⛉ ㉥ᑿ ᖾ༤ ᩍᤵᚰࡼ ࡾឤㅰࡢពࢆ⾲ࡋࡲࡍࠋ ᮏ◊✲ࢆ㐍ࡵࡿ࠶ࡓࡾࠊከ࡞ࡈ༠ຊࡈຓゝࢆ࠸ࡓࡔࡁࡲࡋࡓி㒔ᗓ❧ ་⛉Ꮫ ⣽ ᩍᤵࠊᐑᆅ ᩍᤵࠊ⳥ᆅ 㢧 ≉௵ຓᩍࠊ᠕⩏ ሿᏛ ᭮ᡃ ᭸⩏ ᩍᤵࠊ▼ 㯞ᕼ ᢏ⾡ဨࠊ㜰་⛉Ꮫ ఀ⸨ ⿱Ꮚ ຌປᩍᤵࠊ㇂ཱྀ 㧗ᖹ ຓᩍཌࡃ࠾♩⏦ࡋୖࡆࡲࡍࠋ ࡲࡓࠊᮏㄽᩥࡢసᡂ㝿ࡋࠊࡈຓゝ࠾ࡼࡧࡈᰯ㜀ࢆ㈷ࡾࡲࡋࡓᒱ㜧ᏛᏛ 㝔㐃ྜ⸆་⒪ሗ◊✲⛉ ➉᳃ ὒ ᩍᤵࠊụ⏣ ᑗ ᩍᤵࠊ⏣୰ ᏹᖾ ᩍᤵ῝ࡃឤㅰ࠸ࡓࡋࡲࡍࠋ ᮏ◊✲ࡢ㐙⾜࠶ࡓࡾࠊ◊✲άືࢆඹࡋ࡚ࡁࡓᒱ㜧ᏛᏛ㝔㐃ྜ⸆་ ⒪ሗ◊✲⛉㉥ᑿ◊✲ᐊࡢⓙᵝᚰࡼࡾ࠾♩⏦ࡋୖࡆࡲࡍࠋ ᭱ᚋࠊ⤊ጞ⚾ࡢ◊✲⏕άࡢບࡳ࡞ࡗࡓᐙ᪘ឤㅰࡋࡲࡍࠋ ᮏ◊✲ࡀᶓ⣠➽⫗⭘ࡢᝈ⪅ࡉࢇࡑࡢࡈᐙ᪘ࡢᨭࡢ୍ຓ࡞ࡿࡇࢆ㢪 ࠸ㅰ㎡࠸ࡓࡋࡲࡍࠋ
28 ᐇ㦂ࡢ㒊
⣽⬊ᇵ㣴
ࣄࢺ⫾ඣᆺᶓ⣠➽⫗⭘ (ERMS) ⣽⬊ᰴࡢRDKYM-1ࡣࠊJCRB (Japanese Collection of Research Bioresources) ࡽ㉎ධࡋࠊࣄࢺ⬊ᕢᆺᶓ⣠➽⫗⭘
(ARMS) ⣽⬊ᰴࡢRh30Rh41ࡣࠊி㒔ᗓ❧་⛉Ꮫᑠඣ⛉Ꮫᩍᐊࡢ⣽ᩍᤵ ⳥ᆅ㢧≉௵ຓᩍᥦ౪ࡋ࡚࠸ࡓࡔ࠸ࡓࠋᇵᆅࡣࠊRDࡀEagle’s minimal
essential mediumࠊKYM-1ࡀDulbecco’s modified Eagle’s mediumHam’s F12 mediumࢆ1:1࡛ΰࡐࡓࡶࡢࠊRh30Rh41ࡀRPMI-1640ࠊࢆࡑࢀࡒࢀ⏝࠸ࡓࠋ⣽ ⬊ࡢᇵ㣴ࡣࠊࡑࢀࡒࢀࡢᇵᆅ8%FBSࢆຍ࠼ࠊ37Υࠊ5%CO2ࡢ᮲௳ୗ࡛⾜ࡗ ࡓࠋ 㑇ఏᏊᑟධᐇ㦂㸦ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥ㸧 ⣽⬊ࢆ0.5105ಶ/mLࡢ⣽⬊ᐦᗘ࡛6࢙࢘ࣝࣉ࣮ࣞࢺ✀ࡋࠊ୍ᬌᇵ㣴ࡋ ࡓࠋ24㛫ᚋࠊ1࢙࢘ࣝᙜࡓࡾ50ȣLࡢOpti-MEM (Invitrogen)ࠊ0.8 ȣLࡢ Lipofectamine RNAiMAX (Invitrogen)ࠊmicroRNAࡢ⃰ᗘࡀ10, 20 nM࡞ࡿࡼ࠺ ΰྜࡋࠊ15ศᐊ ࡛㟼⨨ࡋࡓࠋsiRNA࠾࠸࡚ࡶࠊྠᵝࠊ⃰ᗘࡀ0.5, 5 nM ࡞ࡿࡼ࠺ΰྜࡋࡓࠋRNAࡣࠊhomo sapiens (has)-miR-1, has-miR-133b (Ambion), siR-PTBP1, siR-PF2 (Invitrogen) ཬࡧcontrol RNA (Hokkaido System Sciences) ࢆ⏝࠸ࡓࠋࡲࡓࠊRh30, Rh41⣽⬊㛵ࡋ࡚ࡣࠊ6࢙࢘ࣝࣉ࣮ࣞࢺ୰ࡢ ᇵ㣴ᾮࢆࢫࣆ࣮ࣞࢱ࣮ࡼࡗ࡚㝖ཤࡋࠊFBSࢆྵࡲ࡞࠸RPMI-1640ࢆຍ࠼ ࡓࠋ15ศ㟼⨨ࡉࡏࡓΰྜᾮࢆྛ࢙࢘ࣝ50 ȣLࡎࡘୗࡋࡓࠋ5㛫ᇵ㣴ࡋࡓ ᚋࠊᇵ㣴ᾮ୰ࡢ⃰ᗘࡀ8%࡞ࡿࡼ࠺FBSࢆῧຍࡋࡓࠋ
29 miR-1ࡢ㓄ิࡣ5’-UGGAAUGUAAAGAAGUAUGUAU-3’ࠊ miR-133bࡢ㓄ิࡣ5’-UUUGGUCCCCUUCAACCAGCUA-3’ࠊ siR-PTBP1ࡢ㓄ิࡣ5’-AUCUCUGGUCUGCUAAGGUCACUUC-3’ࠊ siR-PF2ࡢ㓄ิࡣ5’-CCUCUCACCUCAGAAUUCA-3’ࠊ control RNAࡢ㓄ิࡣ5’-GUAGGAGUAGUGAAAGGCC-3’࡛࠶ࡿࠋ ࣮࢜ࢺࣇࢪ࣮㜼ᐖ ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡢ
5㛫๓࣮࢜ࢺࣇࢪ࣮㜼ᐖ࡛࠶ࡿ3-Methyladenine (3-MA; Calbiochem)ࢆᇵᆅຍ࠼ࡓࠋ
ROS㜼ᐖ
ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡢ24㛫ᚋROS㜼ᐖ࡛࠶ࡿN-acetyl-L-cysteine (NAC; Sigma Aldrich)ࢆᇵᆅຍ࠼ࡓࠋ
Anti-miR-1anti-miR-133b
miR-1ࡶࡋࡃࡣmiR-133bࡢࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࠊanti-miR-1ࡶࡋࡃࡣ anti-miR-133b (Ambion) ࢆྠࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ
⣽⬊⏕Ꮡホ౯
30
ࣜࣃࣥࣈ࣮ࣝ (Life technologies) ➼㔞ΰྜࡋࠊ⾑⌫ィ⟬┙࡚⏕⣽⬊ᩘࢆィ ᩘࡋࡓࠋࢥࣥࢺ࣮ࣟࣝ⣽⬊ᑐࡍࡿྜ (%)ࢆ⣽⬊⏕Ꮡ⋡ࡋࡓࠋ᥋╔⣽⬊ࡢ ሙྜࡣࠊᇵ㣴ࡋࡓ⣽⬊ࢆࢺࣜࣉࢩ࡚ࣥ㐟㞳ࡉࡏࠊィᩘࡋࡓࠋ
RNAᢳฟ
RNAᢳฟࡣࠊNucleoSpin microRNA isolation kit (TaKaRa) ࢆ⏝࠸࡚〇ရࣉࣟ ࢺࢥ࣮ࣝ࠾ࡾ⾜ࡗࡓࠋRNAࡢရ㉁ࡣࠊ࣒࣍ࣝࣝࢹࣄࢻࢤࣝ㟁ẼὋືἲࡼ ࡾ☜ㄆࡋࡓࠋ
ᐃ㔞RT-PCR microRNA ࡢᐃ㔞
miR-1 miR-133b ࡢⓎ⌧ࣞ࣋ࣝࢆ᳨ドࡍࡿࡓࡵࠊTaqMan MicroRNA Assays (Applied Biosystems)ࠊTHUNDERBIRD Probe qPCR Mix (TOYOBO) ࢆ⏝࠸࡚〇ရ ࣉࣟࢺࢥ࣮ࣝ࠾ࡾ⾜ࡗࡓࠋᶆ‽ࡍࡿࡓࡵࠊRNU6B ࡶྠ⾜ࡗࡓࠋ
mRNA ࡢᐃ㔞
PAX3,PAX3-FOXO1 ࡢ mRNA Ⓨ⌧ࣞ࣋ࣝࢆ᳨ドࡍࡿࡓࡵࠊPrimeScript® RT reagent Kit (TaKaRa)ࠊTHUNDERBIRD SYBR qPCR Mix (TOYOBO)ࢆ⏝࠸࡚〇ရ ࣉࣟࢺࢥ࣮ࣝ࠾ࡾ⾜ࡗࡓࠋᶆ‽ࡍࡿࡓࡵࠊglyceraldehyde-3-phosphate dehydrogenase (GAPDH )ࡶྠ⾜ࡗࡓࠋ
PAX3 ࡢࣉ࣐࣮ࣛࡣࠊsense: 5’-GAGACTGGCTCCATACGTCC-3’ PAX3-antisense: 5’-ACGGTGTTTCGATCACAGAC-3’ࠊPAX3-FOXO1 ࡢࣉ࣐࣮ࣛࡣࠊ
31 PAX3-sense FOXO1-antisense:5’-TGAACTTGCTGTGTAGGGACAG-3’ࠊGAPDH ࡢ ࣉ ࣛ ࣐ ࣮ ࡣ ࠊGAPDH-sense:5’-CCACCCATGGCAAATTCCATGGCA-3’ GAPDH-antisense:5’-TCTAGACGGCAGGTCAGGTCCACC-3’ࢆ⏝࠸ࡓࠋ ࢱࣥࣃࢡ㉁ᢳฟࠊ⃰ᗘ ᐃ࣭ㄪ〇 ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࢆ⾜ࡗࡓ⣽⬊ࢆࠊࢫࢡ࣮ࣞࣃ࣮࡛ࡣࡀࡋᅇࡋࡓᚋࠊ PBS ࡛Ὑίࡋࡓࠋࢱࣥࣃࢡ㉁ᢳฟᾮ ࠙Protein lysis buffer <10 mM Tris-HCl (pH7.4), 0.1% SDS, 1% NP-40, 0.1% ࢹ࢜࢟ࢩࢥ࣮ࣝ㓟ࢼࢺ࣒ࣜ࢘,150 mM NaCl,1 mM EDTA>: Protease Inhibitor Cocktail (nacalai tesque): Phosphatase Inhibitor Cocktail solution ϩ: solution Ϫ (Sigma-Aldrich Co.)= 50 : 1 : 1 : 1ࠚ ࢆㄪᩚࡋࠊᅇࡋࡓ⣽
⬊࣌ࣞࢵࢺຍ࠼ࠊ࣎ࣝࢸࢵࢡࢫ࡛⁐ゎࡉࡏࠊịୖ࡛20 ศ㟼⨨ࡋࡓࠋࡑ
ࡢᚋࠊ13000 rpm, 20 min, 4Υ࡛㐲ᚰࡋࠊୖΎࢆᅇࡋࠊࢱࣥࣃࢡ㉁ࢧࣥࣉࣝ ࡋࡓࠋࢱࣥࣃࢡ㉁ࡢ⃰ᗘ ᐃࡣࠊDC Protein Assay Kit (BIO-RAD)ࢆ⏝࠸࡚⾜ࡗ ࡓࠋ⃰ᗘ ᐃࡋࡓࢱࣥࣃࢡ㉁ࢆ5SDS sample buffer <62.5 mM Tris-HCl (pH6.8), 2% SDS, 10% ࢢࣜࢭ࣮ࣟࣝ, 50 mM DTT, 0.01% ࣈࣟࣔࣇ࢙ࣀ࣮ࣝࣈ࣮ࣝ>ΰ ࡋ࡚10 ȣg / 10 ȣL ㄪ〇ࡋࠊ98Υ࡛ 5 ศຍ⇕ฎ⌮ࡋࡓᚋࠊịୖ࡛ 5 ศ㟼⨨ ࡋࡓࠋ
Western blotting
ࢱࣥࣃࢡ㉁ࡣࠊSDS࣏ࣜࢡ࣑ࣜࣝࢻࢤࣝ㟁ẼὋືࡼࡾ10㹼12.5%ࡢ࣏ࣜ ࢡ࣑ࣜࣝࢻࢤ࡛ࣝ࠶ࡿSuper Sep Ace (Wako) ࢆ⏝࠸࡚ศ㞳ࡋࠊTransfer membrane (MILLIPURE) ࢚ࣞࢡࢺࣟࣈࣟࢵࢺࡋࡓࠋࡑࡢᚋࠊ5%ࢫ࣒࣑࢟ࣝࢡ
32
⁐ᾮ࡛ࣈࣟࢵ࢟ࣥࢢࢆ⾜࠸ࠊྛ≉␗ⓗ࡞୍ḟᢠయᾐࡋ୍ᬌᛂࡉࡏࡓࠋࡇࡢ ◊✲࡛⏝ࡋࡓᢠయࡣࠊᢠࣄࢺLC3B ᢠయࠊ ᢠࣄࢺFOXO1 ᢠయࠊᢠࣄࢺ PTBP1 ᢠయ (CST)ࠊᢠࣄࢺPKM1 ᢠయࠊᢠࣄࢺPKM2 ᢠయ (Novus)ࠊᢠࣄࢺ PAX3 ᢠయ (abcam)࡛࠶ࡿࠋ⩣᪥ࠊḟᢠయ (Anti-mouse IgG HRP-linked antibody ࡲࡓࡣ Anti-rabbit IgG HRP-linked antibody (CST)) ࡛ᛂࡉࡏࡓࠋࢱࣥ ࣃࢡ㉁Ⓨ⌧㔞ࡣࠊLuminata Forte Western HRP Substrate (MILLIPORE) ࢆ⏝ࡋ ⺯ගⓎගࡉࡏࠊLuminescent image analyzer LAS-4000 UV mini (Fujifilm) ᳨࡚ ฟࡋࡓࠋࢱࣥࣃࢡ㉁Ⓨ⌧㔞ࡢcontrolࡣࠊᢠࣄࢺGAPDH ᢠయ (CST) ࢆྠࡌ⭷ ࡛ᛂࡉࡏ᳨ฟࡋࡓࠋ ༙ᐃ㔞ゎᯒἲ ගᏛ⃰ᗘࡣࠊImageQuant TLゎᯒࢯࣇࢺ࢙࢘ࢆ⏝࠸࡚ࠊ᳨ฟࡉࢀࡓࣂࣥࢻࡢ ᩘ್ࡼࡾ⾜ࡗࡓࠋ ࣝࢩࣇ࢙࣮ࣛࢮࢵࢭ ࣄࢺPTBP1ࡢ3’UTRࡢmiR-1miR-133bࡢࡑࢀࡒࢀࡢ⤖ྜ㡿ᇦࢆluciferase reporter pMIR-control vector (Applied Biosystems) ⤌ࡳ㎸ࡴࡇ࡛ࢭࣥࢧ࣮࣋ࢡ
ࢱ࣮ࢆసᡂࡋࠊࡇࢀࡽࡢmiRsࡼࡾᶆⓗࡉࢀ࡚࠸ࡿ㓄ิࡢホ౯ࢆ⾜ࡗࡓࠋ
PTBP1ࡢmRNAࢆቑᖜࡉࡏࡿࡓࡵࠊRNAࡢ㏫㌿ࢆPrimeScript RT Reagent Kit (TaKaRa) ࢆ⏝࠸࡚⾜ࡗࡓࠋࡑࡇࡽࠊ┠ⓗࡢ㓄ิࢆቑᖜࡍࡿࡓࡵࡢࣉ࣐ࣛ ࣮ࢆసᡂࡋࠊPCRࢆ⾜࠸ࠊࡑࡢ㓄ิࢆ࣋ࢡࢱ࣮⤌ࡳ㎸ࢇࡔࠋࡉࡽࠊࡇࢀࡽ ࡢmiRsࡢ⤖ྜ㡿ᇦ (miR-1: CAUUCCA, miR-133b: GGACCAAA)3ሷᇶࡢኚ␗ (miR-1: CAGGAC, miR-133b: GGCUUAAA) ࢆຍ࠼ࡓ㓄ิࢆᣢࡘࢭࣥࢧ࣮࣋ࢡࢱ
33
࣮ࢆࠊPrimeSTAR Mutagenesis Basal Kit (TaKaRa) ࢆ⏝࠸࡚సᡂࡋࡓࠋኚ␗ࢆධ ࢀࡓࢭࣥࢧ࣮࣋ࢡࢱ࣮ࡢ㓄ิࡣࠊᒱ㜧Ꮫࡢ⏕⛉Ꮫ◊✲ࢭࣥࢱ࣮࡛ࡢDNA sequencingࡼࡾ☜ㄆࡋࡓࠋ⣽⬊ࢆ96࢙࢘ࣝࣉ࣮ࣞࢺ0.5104ಶ/100ȣLࡎࡘ
✀ࡋࠊ24㛫ᚋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࢆ⾜ࡗࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥ ࡣࠊࢭࣥࢧ࣮࣋ࢡࢱ࣮ (0.5 ȣg / well)20 nMࡢmiR-1miR-133bཬࡧnon-specific control miRNA (Dharmacon) ࢆࠊ㝧࢜ࣥᛶࡢ࣏ࣜࢯ࣮࣒࡛࠶ࡿࠊ Lipofectamine RNAiMAXࢆ⏝࠸࡚⾜ࡗࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡢ48㛫ᚋ ࠊDual-Glo Luciferase Assay System (Promega) ࡼࡾࠊࣝࢩࣇ࢙࣮ࣛࢮάᛶࢆ ᐃࡋࡓࠋFirefly luciferase activityࡣRenilla luciferase activityࡼࡾつ᱁ࡋࡓࠋ
࣓ࢱ࣮࣒࣎ࣟゎᯒ
Rh30⣽⬊ࢆᐇ㦂๓᪥6-wellࣉ࣮ࣞࢺୖ0.5105 cells/ 1mLࡢ⣽⬊ᐦᗘ࡛
ࡁࠊ5% CO2ࠊ37Υ࡛ᇵ㣴ࡋࡓࠋmiR-1, miR-133b, siR-PTBP1ࡲࡓࡣControl RNA
ࢆࡑࢀࡒࢀࡢwellῧຍࡋࡓࠋ48㛫ᚋཧ⪃ᩥ⊩42ࡢᡭ㡰ᚑࡗ࡚ࢧࣥࣉࣝࢆ స〇ࡋࡓࠋị෭ࡋࡓ5%࣐ࣥࢽࢺ࣮࡛ࣝ⣽⬊ࢆ2ᅇὙίࡋࠊࡑࡢᚋ⣽⬊25 mM ࡢ࣓ࢳ࢜ࢽࣥࢫࣝ࣍ࣥࠊ2-࣓ࢱ࣍ࣜࣀ࢚ࢱࣥࢫࣝ࣍ࣥ㓟ࠊD-ࢩࣙ࢘ࣀ࢘-10-ࢫ ࣝ࣍ࣥ㓟 (Wako)࣓ࢱࣀ࣮ࣝࡢΰྜᾮࢆ1 wellᙜࡓࡾ250 μLຍ࠼ࠊ10ศ㟼⨨ࡋ ࡓࠋࡑࡢᚋࠊ᪂ࡋ࠸࢚ࢵ࣌ࣥࢳ࣮ࣗࣈᾮࢆ࡚ᅇࡋࡓࠋࡑࡢෆࡢ400 μLࢆ ᪂ࡋ࠸࢚ࢵ࣌ࣥࢳ࣮ࣗࣈධࢀࠊࡑࡇ400 μLࡢࢡ࣒ࣟࣟ࣍ࣝ (Wako)ࠊ200 μL ࡢmilliQỈࢆධࢀࡼࡃΰࡐࠊ10,000 gࠊ3ศࠊ4Υ࡛㐲ᚰࡋࡓࠋ400 μLࡢỈᒙ ࢆUltrafreeMC-PLHCC 250/pk for Metabolome Analysis (Merck) ࡚ࠊ9,100 gࠊ2㛫ࠊ20Υ࡛㐲ᚰࡋ࡚ࢧࣥࣉࣝࡽࢱࣥࣃࢡࢆ㝖ཤࡋࡓࠋࡑࡢᚋࠊࢁ㐣ࡋ ࡓࢧࣥࣉࣝࢆ࢚ࣂ࣏࣮ࣞࢱ࣮࡚2㛫ࠊ42Υ࡛⃰⦰ࡋࡓࠋ100 mMࡢ3-࣑ࣀ
34
ࣆࣟࣜࢪࣥࠊ10 mMࡢ1,3,5-࣋ࣥࢮࣥࢺࣜ࢝ࣝ࣎ࣥ㓟 (Wako) ࢆmilliQỈΰࡐ ࡓᾮࢆ1ࢧࣥࣉࣝᙜࡓࡾ25 μLຍ࠼ࠊᨩᢾࡋࡓࠋAglient CE Capllary
Electrophoresis System (Aglient Technologis, CA, USA) ࢆ⏝࠸࡚CE-TOFMS࡚௦ ㅰ⏘≀ࡢ᳨ฟࢆ⾜ࡗࡓࠋࢹ࣮ࢱࡢゎᯒࡣMetabolome Analysis and Screening Tool for Easy and Rapid HANDling of Sample data; Master Hands (ver. 2.17.2.15, Keio University, ᒣᙧ, ᪥ᮏ)ࢆ⏝࠸ࡓࠋ
Rh30ᢸ⒴࣐࢘ࢫࣔࢹࣝࡢసᡂmiR-1miR-133bࡢᢞᐇ㦂
BALB/cSlc-nu/nuࢾ࣮ࢻ࣐࢘ࢫࢆJapan SLCࡽ㉎ධࡋࡓࠋRh30⣽⬊ࢆ2106
ಶ / 100 ȣLࡢྜ࡛࣐࢘ࢫࡢ⫼୰⛣᳜ࡋࡓࠋ12᪥ᚋࠊ⭘⒆ࡀ࠶ࡿ⛬ᗘࡁ ࡃ࡞ࡗࡓࡇࢁ࡛ࠊscramble RNA, miR-1ཬࡧmiR-133bࢆ0.2 nmolࡎࡘ50 ȣlࡢ Opti-MEM୰1 ȣlࡢLipofectamine RNAiMAXΰྜࡉࡏ4᪥࠾ࡁᒁᡤᢞࡋ
ࡓࠋࡑࢀࡒࢀࡢࢢ࣮ࣝࣉ3༉ࡎࡘ⏝ࡋࡓࠋ ⭘⒆ࡢࡁࡉࡣࠊ0.5236L1(L2)2ࡢබᘧ࡛ồࡵࡓࠋ(L1ࡣࠊ⭘⒆ࡢ㛗㍈ࠊL2ࡣ⭘ ⒆ࡢ▷㍈ࢆ⾲ࡍ) ࡇࡢ⟬ฟ᪉ἲࡣࠊ㐣ཤࡢᩥ⊩࡛ሗ࿌ࡉࢀ࡚࠸ࡓࡶࡢࢆཧ⪃ ࡋࡓࠋ⭘⒆ࡢホ౯ࡣ⭘⒆⛣᳜ࡽ24᪥ᚋ࣐࢘ࢫࢆᒕẅࡋ⾜ࡗࡓࠋฟ⭘⒆ࡣ ࢱࣥࣃࢡ㉁ࡢᢳฟ⏝ࡋࡓࠋື≀ᐇ㦂ࣉࣟࢺࢥ࣮ࣝࡣࠊື≀◊✲ጤဨᒱ 㜧Ꮫࡢື≀⚟♴ࡼࡾᢎㄆࡉࢀࡓࠋ ⤫ィᏛⓗゎᯒ ᐇ㦂ᡂ⦼ࡣࠊᖹᆒ್sᶆ‽೫ᕪ࡛♧ࡋࡓࠋ⣽⬊⏕Ꮡホ౯࠾ࡼࡧ microRNA mRNA ࡢⓎ⌧ࣞ࣋ࣝࡢ⤫ィᏛⓗ࡞ẚ㍑ࡣࠊStudent's t-test ࡼࡾ⾜ࡗࡓ
35 ␎ㄒ㞟
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
36 ཧ⪃ᩥ⊩
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