Title
新規がん抑制遺伝子型microRNAの探索と創薬への展開( 内
容と審査の要旨(Summary) )
Author(s)
中本, 航介
Report No.(Doctoral
Degree)
博士(農学) 甲第687号
Issue Date
2018-03-13
Type
博士論文
Version
ETD
URL
http://hdl.handle.net/20.500.12099/75245
※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。㹙㸯㸯㹛 Ặ ྡ㸦ᮏ㸦ᅜ㸧⡠㸧 ୰ᮏ ⯟㸦ឡ▱┴㸧 Ꮫ ࡢ ✀ 㢮 ༤ኈ㸦㎰Ꮫ㸧 Ꮫ グ ␒ ྕ ㎰༤⏥➨㸴㸶㸵ྕ Ꮫ ᤵ ᖺ ᭶ ᪥ ᖹᡂ㸱㸮ᖺ㸱᭶㸯㸱᪥ ◊ ✲ ⛉ ཬ ࡧ ᑓ ᨷ 㐃ྜ㎰Ꮫ◊✲⛉ ⏕≀㈨※⛉Ꮫᑓᨷ ◊✲ᣦᑟࢆཷࡅࡓᏛ ᒱ㜧Ꮫ Ꮫ ㄽ ᩥ 㢟 ┠ ᪂つࡀࢇᢚไ㑇ఏᏊᆺmicroRNA ࡢ᥈⣴⸆ࡢ ᒎ㛤 ᑂ ᰝ ጤ ဨ ᰝ ᒱ㜧Ꮫ ᩍᤵ ᰗ℩ ➗Ꮚ ᰝ ᒱ㜧Ꮫ ᩍ ᤵ ୖ㔝 ⩏ோ ᰝ 㟼ᒸᏛ ᩍ ᤵ Ἑྜ ┿࿃
ㄽ ᩥ ࡢ ෆ ᐜ ࡢ せ ᪨
microRNA (miRNA) ࡣࠊ20-24 ሷᇶ⛬ᗘࡢෆᅉᛶᮏ㙐 RNA ࡛࠶ࡾࠊ⩻ヂ࡛ࣞ࣋ࣝࢱࣥࣃ ࢡ㉁ࡢⓎ⌧ㄪ⠇ࢆᢸ࠺⣽⬊ෆㄪ⠇ᅉᏊ࡛࠶ࡿࠋ⣽⬊ෆ࡛⏕ᡂࡋࡓmiRNA ᮏ㙐ࡣࠊArgonaute ࡤࢀࡿࢱࣥࣃࢡ㉁ඹRNA-induced silencing complex (RISC) ࢆᙧᡂࡍࡿࠋ⥆࠸࡚ࠊ୍᪉ ࡢ㙐ࡀゎ㞳ࡋࡓᚋࠊ࢞ࢻ㙐ࡀᶆⓗ mRNA ⤖ྜࡍࡿࡇ࡛ࢱࣥࣃࢡ㉁ࡢ⩻ヂࢆᢚไࡋ ࡚࠸ࡿࠋ㏆ᖺࠊmiRNA ࡢⓎ⌧␗ᖖࡀ⣽⬊ࡢࡀࢇ㛵ࡋ࡚࠸ࡿࡇࡀ᫂ࡽ࡞ࡾࠊࡀࢇ ⣽⬊࠾ࡅࡿmiRNA ࡢᶵ⬟ゎᯒࡀ┒ࢇ⾜ࢃࢀ࡚࠸ࡿࠋᮏ◊✲࡛ࡣࠊගࢡࣟࢫࣜࣥࢡἲࡼ ࡾᶆⓗmRNA ࢆᤕ⋓ྍ⬟࡞᪂つ RNA ࣉ࣮ࣟࣈࡢ㛤ⓎࠊmiRNA ࡢ⸆ᛂ⏝ࢆᣦྥࡋࡓ cRGD ࢥࣥࢪࣗࢤ࣮ࢺࡢ㛤Ⓨࢆ┠ⓗࡋࡓࠋ
1) ᪂つගᛂᛶ miRNA ࣉ࣮ࣟࣈࡢ㛤Ⓨ
ሷᇶ㒊ࢆගᛂᛶศᏊ࡛࠶ࡿࢺࣜࣇ࣓ࣝ࢜ࣟࢳࣝࣜࣝࢪࢪ࡛ࣜࣥ⨨ࡋࡓ✀㢮ࡢࢾ ࢡࣞ࢜ࢩࢻࢼࣟࢢࢆタィ࣭ྜᡂࡋࡓࠋࢾࢡࣞ࢜ࢩࢻࢼࣟࢢࢆᑟධࡋࡓRNA ࣉ࣮ࣟࣈ┦ ⿵㙐RNA ࡢᮏ㙐 RNA ࢆྵࡴ⁐ᾮࢆ UV ↷ᑕࡋࡓᚋࠊ⏕ᡂ≀ࢆ PAGE ཬࡧ MALDI-TOF/MS ࡼࡾゎᯒࡋࡓࡇࢁࠊRNA ࣉ࣮ࣟࣈ┦⿵㙐 RNA ࡢ㛫ࢡࣟࢫࣜࣥࢡࡀᙧᡂࡉࢀ࡚࠸ࡿ ࡇࡀ☜ㄆࡉࢀࡓࠋ⥆࠸࡚ࠊࢾࢡࣞ࢜ࢩࢻࢼࣟࢢࣅ࢜ࢳࣥศᏊࢆᑟධࡋࡓmiRNA-145 ࣉ ࣮ࣟࣈࢆྜᡂࡋࠊࡑࡢᶆⓗmRNA ࡢࣅ࢜ࢳࣥࣛ࣋ࣝࢆヨࡳࡓࠋmiRNA-145 ࣉ࣮ࣟࣈࢆ⣽⬊ ᑟධࡋࠊUV ↷ᑕࡼࡾᶆⓗ mRNA ࢆࣅ࢜ࢳࣥࣛ࣋ࣝࡋࡓࠋࣛ࣋ࣝࡉࢀࡓᶆⓗ mRNA ࢆࣇࢽࢸ࣮⢭〇ࡋࠊmiR-145 ࡢ᪤▱ᶆⓗ㑇ఏᏊ࡛࠶ࡿ FSCN1 mRNA ࡢⓎ⌧㔞ࢆᐃ㔞ࡋ ࡓࠋࡑࡢ⤖ᯝࠊ⢭〇RNA ୰ࡢ FSCN1 mRNA 㔞ࡀᖜቑຍࡋ࡚࠾ࡾࠊྜᡂࡋࡓ miRNA-145 FSCN1 mRNA ࡢ㛫ࢡࣟࢫࣜࣥࢡࡀᙧᡂࡉࢀ࡚࠸ࡿࡇࡀ☜ㄆࡉࢀࡓࠋ୍᪉࡛ࠊᅇྜ ᡂࡋࡓ miRNA-145 ࡀᶆⓗࡋ࡞࠸ c-MYC mRNA 㔞ࡣኚࡀぢࡽࢀ࡞ࡗࡓࡇࡽࠊ miRNA-145 ࣉ࣮ࣟࣈࡣᶆⓗ mRNA 㑅ᢥⓗࢡࣟࢫࣜࣥࢡᙧᡂࢆࡍࡿࡇࡀ☜ㄆࡉࢀࡓࠋ
2) cRGD-ࢥࣥࢪࣗࢤ࣮ࢺࡢྜᡂࡑࡢ㑇ఏᏊⓎ⌧ᢚไ⬟
miRNA ་⸆㐺⏝ྍ⬟࡞ࣜ࢞ࣥࢻࢥࣥࢪࣗࢤ࣮ࢺᆺࢹࣜࣂ࣮ࣜࢩࢫࢸ࣒ࡢ㛤Ⓨࢆ⾜ࡗࡓࠋ ࣜ࢞ࣥࢻศᏊࡣࠊࡀࢇ⣽⬊࡛ཷᐜయࡢⓎ⌧㔞ࡀቑຍࡋ࡚࠸ࡿcyclic RGD (cRGD) ࣌ࣉࢳࢻࢆ 㑅ᢥࡋࠊpost-synthetic modification ἲࡼࡾࠊඛࡎࠊcRGD-siRNA ࢥࣥࢪࣗࢤ࣮ࢺࢆྜᡂࡋࡓࠋ ᮏ◊✲࡛ࡣࠊࡼࡾ⡆౽cRGD ࢥࣥࢪࣗࢤ࣮ࢺࡢᵓ㐀ࢆ᭱㐺ࡍࡿⅭࠊcRGD ࢥࣥࢪࣗࢤ࣮ࢩ ࣙࣥࣘࢽࢵࢺཬࡧࢫ࣮࣌ࢧ࣮ࣘࢽࢵࢺࡢ 2 ✀㢮ࡢ࣑ࢲࢺయࢆ⏝࠸࡚ siRNA ࢆྜᡂࡋࠊ cRGD ࡢᑟධᩘཬࡧ cRGD 㛫ࡢ㊥㞳ࡀ⣽⬊ෆྲྀࡾ㎸ࡳ࠼ࡿᙳ㡪ࢆホ౯ࡋࡓࠋࡑࡢ⤖ᯝࠊ cRGD ࢆ㐃⥆ࡋ࡚ᑟධࡋࡓ siRNA ࡣࠊ⣽⬊ᑟධヨ⸆↓ࡋ࡛⣽⬊ෆྲྀࡾ㎸ࡲࢀࡿࡇࡀ☜ㄆ ࡉࢀࡓࠋࡲࡓࠊ㐃⥆ࡍࡿcRGD 㛫ࢫ࣮࣌ࢧ࣮ࣘࢽࢵࢺࢆᑟධࡍࡿࡇ࡛ࠊ⣽⬊ෆࡢྲྀࡾ㎸ ࡳࡀᖜୖ᪼ࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋࡇࢀࡣࠊࢫ࣮࣌ࢧ࣮ࣘࢽࢵࢺࡢᑟධࡼࡾcRGD ࡀ」ᩘࡢࣥࢸࢢࣜࣥ┦స⏝ࡍࡿከ౯ຠᯝࡼࡿࡶࡢ⪃࠼ࡽࢀࡿࠋ ࡋࡋ࡞ࡀࡽࠊタィࡋࡓ cRGD-siRNA ࢥࣥࢪࣗࢤ࣮ࢺࡣࠊ༢⊂࡛ࡣ㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ♧ ࡉ࡞ࡗࡓࠋࡑࡇ࡛ࠊcRGD-RNA ࢥࣥࢪࣗࢤ࣮ࢺࡢ⣽⬊ᑟධຠ⋡ࢆྥୖࡉࡏࡿ┠ⓗ࡛ࠊ᪂ࡓ ࣏࣑ࣜࣥಟ㣭ᆺ cRGD-siRNA ࢥࣥࢪࣗࢤ࣮ࢺࢆྜᡂࡋࡓࠋࡑࡢ⤖ᯝࠊࣜࣥ㓟⏤᮶ࡢ㈇㟁 Ⲵࢆ୰ࡋࡓ࣏࣑ࣜࣥಟ㣭ᆺcRGD ࢥࣥࢪࣗࢤ࣮ࢺࡣ 250 nM ࡛⣙ 40%ࡢ㑇ఏᏊⓎ⌧ᢚไຠ ᯝࢆ♧ࡋࠊ࣏࣑ࣜࣥಟ㣭ࡀ siRNA ࢥࣥࢪࣗࢤ࣮ࢺࡢᑟධຠ⋡ࢆ㧗ࡵࡿࡇࡀ᫂ࡽ࡞ࡗ ࡓࠋ miRNA ་⸆ရࡢ㛤Ⓨ࠾࠸࡚ࠊ་⸆ရೃ⿵࡞ࡿࡀࢇᢚไ㑇ఏᏊᆺ miRNA ࡢ᥈⣴ࢻࣛ ࢵࢢࢹࣜࣂ࣮ࣜࢩࢫࢸ࣒ࡢ㛤Ⓨࡣᴟࡵ࡚㔜せ࡞◊✲ㄢ㢟࡛࠶ࡿࠋᮏ◊✲࡛㛤Ⓨࡋࡓ᪂つගᛂ ᛶmiRNA ࣉ࣮ࣟࣈࡣ miRNA ࡢᶆⓗ mRNA ࢆ⥙⨶ⓗࣛ࣋ࣝࡍࡿࡇࡀྍ⬟࡛࠶ࡾࠊ་⸆ ရೃ⿵࡞ࡿmiRNA ࡢ᥈⣴࠾࠸࡚ᙉຊ࡞ࢶ࣮ࣝ࡞ࡾᚓࡿࠋࡲࡓࠊ᪂ࡓ⪃ࡋࡓ࣏ࣜ ࣑ࣥಟ㣭ᆺ cRGD ࢥࣥࢪࣗࢤ࣮ࢺࡣࠊᚑ᮶ࡢࣜ࢞ࣥࢻࢥࣥࢪࣗࢤ࣮ࢺࢆୖᅇࡿ㑇ఏᏊⓎ⌧ᢚ ไຠᯝࢆ♧ࡋࠊmiRNA ་⸆ရࡶ㐺⏝ྍ⬟࡞᪂つࢻࣛࢵࢢࢹࣜࣂ࣮ࣜࢩࢫࢸ࣒ࡋ࡚ᮇᚅ࡛ ࡁࡿࠋ
ᑂ ᰝ ⤖ ᯝ ࡢ せ ᪨
ᮏ◊✲ࡣࠊ㏆ᖺὀ┠ࡀ㧗ࡲࡗ࡚࠸ࡿmiRNA ་⸆ရࡢᐇ⏝ྥࡅ࡚⾜ࡗࡓ୍㐃ࡢ◊✲ࢆ ࡲࡵࡓࡶࡢ࡛࠶ࡿࠋmicroRNA (miRNA) ࡣ 20-24 ሷᇶ⛬ᗘࡢෆᅉᛶᮏ㙐 RNA ࡛࠶ࡾࠊ⩻ヂ࡛ࣞ࣋ࣝࢱࣥࣃ ࢡ㉁ࡢⓎ⌧ㄪ⠇ࢆᢸ࠺⣽⬊ෆㄪ⠇ᅉᏊ࡛࠶ࡿࠋ㏆ᖺࠊmiRNA ࡢⓎ⌧␗ᖖࡀ⣽⬊ࡢࡀࢇ 㛵ࡋ࡚࠸ࡿࡇࡀ᫂ࡽ࡞ࡾࠊࡾࢃࡅࠊࡀࢇᢚไ㑇ఏᏊᆺmi-RNA ࡢ᥈⣴ཬࡧ་⸆ရ ࡢᛂ⏝◊✲ࡀ┒ࢇ⾜ࢃࢀ࡚࠸ࡿࠋࡋࡋ࡞ࡀࡽࠊmiRNA ࡢᶆⓗ mRNA ࡢ⤖ྜࣃࢱ࣮ ࣥࡣ࣑ࢫ࣐ࢵࢳࢆྵࡳከᵝ࡛࠶ࡾࠊࡑࡢᶵ⬟ゎᯒࡣከࡃࡢ㛫㈝⏝ࢆᚲせࡍࡿࠋࡲ ࡓࠊmiRNA ࡢ་⸆ရࡢᛂ⏝࠾࠸࡚ࡣࠊ⤌⧊≉␗ⓗ࡞ࢹࣜࣂ࣮ࣜࢩࢫࢸ࣒ࡢ㛤Ⓨࡀྍ Ḟ࡛࠶ࡿࠋ ᮏ◊✲࡛ࡣࠊගࢡࣟࢫࣜࣥࢡᛂࡼࡾᶆⓗmRNA ࢆᤕ⋓ྍ⬟࡞᪂つ RNA ࣉࣟ̾ࣈࡢ㛤 ⓎࠊmiRNA ࡢ་⸆ရᛂ⏝ࢆ┠ᣦࡋࡓ cRGD ࢥࣥࢪࣗࢤ࣮ࢺࢹࣜࣂ࣮ࣜࢩࢫࢸ࣒ࡢ㛤Ⓨࢆ ⾜ࡗࡓࠋ
ඛࡎࠊ᪂つගᛂᛶmiRNA ࣉ࣮ࣟࣈࡢ㛤Ⓨࢆ⾜ࡗࡓࠋሷᇶ㒊ࢆගᛂᛶศᏊ࡛࠶ࡿࢺࣜ ࣇ࣓ࣝ࢜ࣟࢳࣝࣜࣝࢪࢪ࡛ࣜࣥ⨨ࡋࡓ✀㢮ࡢࢾࢡࣞ࢜ࢩࢻࢼࣟࢢࢆタィ࣭ྜᡂࡋ ࡓࠋࢼࣟࢢࢆᑟධࡋࡓRNA ࣉ࣮ࣟࣈ┦⿵㙐 RNA ࡢᮏ㙐 RNA ࢆྵࡴ⁐ᾮࢆ UV ↷ ᑕࡋࡓᚋࠊ⏕ᡂ≀ࢆPAGE ཬࡧ MALDI-TOF/MS ࡼࡾゎᯒࡋࡓࡇࢁࠊRNA ࣉ࣮ࣟࣈ┦ ⿵㙐RNA ࡢ㛫ࢡࣟࢫࣜࣥࢡࡀᙧᡂࡉࢀ࡚࠸ࡿࡇࡀ☜ㄆࡉࢀࡓࠋ⥆࠸࡚ࠊࢼࣟࢢ ࣅ࢜ࢳࣥศᏊࢆᑟධࡋࡓmiRNA-145 ࣉ࣮ࣟࣈࢆྜᡂࡋࠊࡑࡢᶆⓗ mRNA ࡢࣅ࢜ࢳࣥࣛ࣋ࣝ ࢆヨࡳࡓࠋmiRNA-145 ࣉ࣮ࣟࣈࢆ⣽⬊ᑟධࡋࠊUV ↷ᑕࡼࡾᶆⓗ mRNA ࢆࣅ࢜ࢳࣥࣛ ࣋ࣝࡋࡓࠋࣛ࣋ࣝࡉࢀࡓᶆⓗmRNA ࢆࣇࢽࢸ࣮⢭〇ࡋࠊmiRNA-145 ࡢ᪤▱ᶆⓗ 㑇ఏᏊ࡛࠶ࡿFSCN1 mRNA ཬࡧ miRNA-145 ࡀᶆⓗࡋ࡞࠸ c-MYC mRNA 㔞ࡢⓎ⌧㔞ࢆᐃ㔞 ࡋࡓࠋࡑࡢ⤖ᯝࠊ⢭〇RNA ୰ࡢ FSCN1 mRNA 㔞ࡢࡳࡀᖜቑຍࡋ࡚࠾ࡾࠊྜᡂࡋࡓ miRNA-145 FSCN1 mRNA ࡢ㛫㑅ᢥⓗࢡࣟࢫࣜࣥࢡࡀᙧᡂࡉࢀ࡚࠸ࡿࡇࡀ☜ㄆࡉ ࢀࡓࠋ
⥆࠸࡚miRNA ་⸆㐺⏝ྍ⬟࡞ࣜ࢞ࣥࢻࢥࣥࢪࣗࢤ࣮ࢺᆺࢹࣜࣂ࣮ࣜࢩࢫࢸ࣒ࡢ㛤Ⓨࢆ ⾜ࡗࡓࠋࣜ࢞ࣥࢻศᏊࡣࠊࡀࢇ⣽⬊࡛ཷᐜయࡢⓎ⌧㔞ࡀቑຍࡋ࡚࠸ࡿcyclic RGD (cRGD) ࣌ࣉࢳࢻࢆ㑅ᢥࡋࠊpost-synthetic modification ἲࡼࡾ cRGD-siRNA ࢥࣥࢪࣗࢤ࣮ࢺࢆྜᡂࡋ ࡓࠋcRGD ࡢᑟධᩘཬࡧ cRGD 㛫ࡢ㊥㞳ࡀ⣽⬊ෆྲྀࡾ㎸ࡳ࠼ࡿᙳ㡪ࢆホ౯ࡋࡓࠋࡑࡢ⤖ ᯝࠊcRGD ࢆ㐃⥆ࡋ࡚ᑟධࡋࡓ siRNA ࡣࠊ⣽⬊ᑟධヨ⸆↓ࡋ࡛⣽⬊ෆྲྀࡾ㎸ࡲࢀࡿࡇࡀ ☜ㄆࡉࢀࡓࠋࡲࡓࠊ㐃⥆ࡍࡿcRGD 㛫ࢫ࣮࣌ࢧ࣮ࣘࢽࢵࢺࢆᑟධࡍࡿࡇ࡛ࠊ⣽⬊ෆࡢ ྲྀࡾ㎸ࡳࡀᖜୖ᪼ࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋࡇࢀࡣࠊࢫ࣮࣌ࢧ࣮ࣘࢽࢵࢺࡢᑟධࡼ ࡾcRGD ࡀ」ᩘࡢࣥࢸࢢࣜࣥ┦స⏝ࡍࡿከ౯ຠᯝࡼࡿࡶࡢ⪃࠼ࡽࢀࡿࠋ୍᪉ࠊタ ィࡋࡓcRGD-siRNA ࢥࣥࢪࣗࢤ࣮ࢺ༢⊂࡛ࡣ㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ♧ࡉ࡞ࡗࡓⅭࠊRNA ࢥࣥࢪࣗࢤ࣮ࢺࡢ⣽⬊ᑟධຠ⋡ࢆྥୖࡉࡏࡿ┠ⓗ࡛ࠊ᪂ࡓ࣏࣑ࣜࣥಟ㣭ᆺ cRGD-siRNA ࢥࣥࢪࣗࢤ࣮ࢺࢆྜᡂࡋࡓࠋࡑࡢ⤖ᯝࠊࣜࣥ㓟⏤᮶ࡢ㈇㟁Ⲵࢆ୰ࡋࡓ࣏࣑ࣜࣥಟ 㣭ᆺcRGD ࢥࣥࢪࣗࢤ࣮ࢺࡣ 250 nM ࡛⣙ 40%ࡢ㑇ఏᏊⓎ⌧ᢚไຠᯝࢆ♧ࡋࠊ࣏࣑ࣜࣥಟ 㣭ࡀsiRNA ࢥࣥࢪࣗࢤ࣮ࢺࡢᑟධຠ⋡ࢆ㧗ࡵࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ ௨ୖࡘ࠸࡚ࠊᑂᰝጤဨဨ୍⮴࡛ᮏㄽᩥࡀᒱ㜧ᏛᏛ㝔㐃ྜ㎰Ꮫ◊✲⛉ࡢᏛㄽᩥ ࡋ࡚༑ศ౯್ࡢ࠶ࡿࡶࡢㄆࡵࡓࠋ ࠙Ꮫㄽᩥࡢᇶ♏࡞ࡿᏛ⾡ㄽᩥࠚ
1) Title: Diazirine-containing RNA photo-cross-linking probes for capturing microRNA targets
㞧ㄅྡ: The Journal of Organic Chemistry, 2014, 79, 2463-2472 ⴭ⪅: Kosuke Nakamoto, Yoshihito Ueno.
2) Title: Labeling of target mRNAs using a photo-reactive microRNA probe 㞧ㄅྡ: Chemical Communications, 2016, 52, 6720-6722.
