SRSF3によるPKMアイソフォームの選択とワーバーグ効果の制御

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(1)Title. SRSF3によるPKMアイソフォームの選択とワーバーグ効果の制御( 本文(Fulltext) ). Author(s). 倉永, 祐希. Report No.(Doctoral Degree). 博士(薬科学) 連創博甲第45号. Issue Date. 2019-03-25. Type. 博士論文. Version. ETD. URL. http://hdl.handle.net/20.500.12099/77971. ※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。.

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(216) ȌΪ̯ȌΪĖxjj˽͓ʆɊ¢ˬýĹȄǍȉxZĹȄȌΪɂ ͅ Table 1 Table 2 ǷxZ Table 1. Clinicopathological features in colorectal cancer patients Case Age Sex a Site b. a. Size c. Depth d Stage e. 1. 41. M. RS. 70×45. SS. B. 2. 49. F. R. 50×70. SS. B. 3. 42. M. R. 30×20. SS. B. 4. 68. M. R. 65×60. SE. D. 5. 73. M. R. 40×40. MP. A. 6. 56. F. S. 27×24. SS. C. 7. 64. M. R. 28×22. MP. A. 8. 82. F. A. 33×28. MP. A. 9. 62. M. S. 55×40. SS. D. 10. 68. F. R. 35×35. SS. B. M, male; F, female. b Location of tumor: RS, rectosigmoid; R, rectum; S, sigmoid colon;. A, ascending colon.. c. Diameter in mm.. d. SS, Subserosa; SE, Serosa exposure; MP,. Mucosa propria. e Duckes’ system classification.. Table 2. Clinicopathological features in colorectal adenoma patients Case Sex a Site b Size c Grade d. a. M, male; F, female.. b. 1. F. S. 20. High. 2. F. S. 20. Low. 3. F. A. 15. Low. 4. M. S. 15. Low. 5. M. R. 10. High. Location of tumor: S, sigmoid colon; A, ascending colon; R,. rectum. c Diameter in mm. d Low, low-grade adenoma (mild and moderate atypia); High,. . .

(217) high-grade adenoma (severe atypia and carcinoma in situ). 2-1. ΕȯƐʣn¾éÎ±ǽˀȝ Protein lysis buffer (0.1% SDS (Wako)Y1% NP-40 (Wako)Y0.5% Å¬¯·´^å ǟÈÆä¨Ü (Wako) in Phosphate buffered salts (PBS) (Takara Bio Inc., Otsu, Japan)) 2% Protease inhibitor cocktail (Nakalai tesque, Kyoto, Japan)YPhosphatase inhibitor cocktail II m III (Sigma-Aldrich)¢ǆǁx¢ͷjZ¾éÎ±ǽˀȝĈ ¢ΕȯƐʣ˔ΐ˺YÖÂ¹å¢ͷj̢Ƚ×Þ¸È§ºxZ~Ƣ 20 ̵Ł̢ʿɱʼw|Y13,000 rpmY4°CY20 ̵ŁĎɑ̵΂xZĎɑ̵΂xȽ ɮ¢ĤȐxY¾éÎ±ǽµéÔåxZ 2-2. ΕȯƐʣn¾éÎ±ǽːΐ DC Protein assay kit (Biorad, Hercules, CA, USA) ¢ͷjƹZÔçÆ´^å ČǡͷxZ 2-3. ΕȯƐʣ SDS-PAGE ͷµéÔåǖɫ ːΐx¾éÎ±ǽ¢ SDS sample buffer (50 mM Tris-HCl (pH 6.8) (Tris; , HCl; Wako)Y2% SDS (Wako)Y10%²ä»ç^å (Sogma-Aldrich)Y100 mM DTT (Wako)Y 0.1%ÓçÞÒ©Ì^åÓå^ (Wako)) ǆΤx 1 μg/μL ˆɬxY98°C 5 ̵ŁȂ̳Ȥ΀xƢY̢Ƚ 5 ̵ŁɱʼxZ ¨©¹¾éÓçÂÆ͉ ,, ) m˗Ȁ SDS-PAGE YÙä¥±äå¥ÛÇ³å Super Sep Ace (Wako) ¢ͷj PowerPac basic (Biorad)³å 1 ͒ˢ 20 mA ː˛ΆɃƋĕYÚ^^ÍéÇoȗ̵ ̵΂zƹZć˫ƢY³å¢ blotting buffer (25 mM TrisY0.2 M ²ä· . .

(218) éY20%Ý¾Ì^å) ɔxZPVDF ÝéÓæé (Millipore Corporation, MA, USA) YÝ¾Ì^åɔxƢˇȢɥɔzuəɥĖȤ΀¢ƹZ~ ƢY ǬŤ blotting buffer 5 ̵˒˞ɔxZ͹ŲʖnY ǬYPVDF Ý éÓæéY³åY ǬȣșYTrans-blot SD Semi-Dry Electrophoretic Transfer Cell 15 V ː˛ìɃƋĕ 40 ̵Ł˗ȀxZ 3-2. ÝéÓæéÓçÂ¯é²mƱƘƱʣ̑đ ˗ȀƢY5%¹¯ÜÛå± in 0.1% Tween 20 ńͬ 50 mM Tris-HCl buffer (TBS-T) Blocking buffer ¢ǖɫxY1 ǴŁÓçÂ¯é²xZÓçÂ¯é²ȓΊƢ TBS-T äé¹xƢ 5 ̵Ł 3 ĤɽɄxYƱʣŋȇĈ (2% BSA, 0.01%¥¸ĖÈ Æä¨Ü in TBS-T) ŋȇxýǵƱʣɔx 4°C ý̓̑đw|Zͼ˸ TBS-T äé¹xƢ 5 ̵Ł 3 ĤɽɄxYBlocking buffer ŋȇx˶ǵƱʣ ɔxǼĔY1 ǴŁYɝkɃƋĕ̑đw|ZǡͷxƱʣ Table 3 Ƿ zˍiZ Table 3. Antibodies using this study. . Antibody. Company. Catalog number. PTBP1. Cell Signaling Technology. 8776. hnRNPA1. Cell Signaling Technology. 4296. LC3B. Cell Signaling Technology. 3868. PKM1. Novus Biologicals. NBP2-14833SS. PKM2. Novus Biologicals. NBP1-48308SS. SRSF3 (SRp20). MBL. RN080PW. ß-actin. Sigma-Aldrich. A5316. GAPDH. Cell Signaling Technology. 2118. HRP-conjugated anti mouse. Cell Signaling Technology. 7076. HRP-conjugated anti rabbit. Cell Signaling Technology. 7074. .

(219) 3-3. ÍéÇƐȝ ˶ǵƱʣƱƘƱʣ̑đoȓΊxYTBS-T äé¹xƢ 5 ̵Ł 3 Ĥ ɽɄxZLuminata Forte Western HRP Substrate (Millipore) ̎Ʀw|ƢY Luminescent image analyzer LAS-4000 UV mini (GE Healthcare UK Ltd., Buckinghamshire, England) ¢ͷjƐȝxZÍéÇģɲ ImageQuant TL Toolbox version 7.0 (GE Healthcare)¢ǡͷxZ. Å^¾»ÂÆģɲ o£ù˙ǥ̎ƚÅ^¾¢ȐȕxÅ^¾Õ^¹¢Ϳͷx C+' ̎ƚģ ɲ (D9EC?D;X >JJFI MMM ED9EC?D; EH= H;IEKH9; BE=?D >JCB¢ǡͷxZ ù˙ǥ˭˺ǾƖ Ĭǎ͊ 0.5×105 cells/mL ǎ͙͊˞ 6 ¨©åÔæ^ǼȋxZDLD1YWiDrYRh30 m RD ǎ͊Æãé¹Ò©±·âé¢ƹk 24 ǴŁʂ́ ȋxYÔæ^Æɷʾw|Z͏ƒŞǡͷx siRNA (Invitrogen) ̆Λ Table 4 ǷxZControl ̙˱ô RNA Hokkaido System Sciences (Sapporo, Japan) ƺ˺xZĬ siRNA Lipofectamine RNAiMAX (Invitrogen) ¢ǡͷxÀ¬éɪäÙ½^Ü¢Žɫw|Yǎ͊˴˭˺xZ ù˙ǥ˭˺¢ƹǎ͊ 72 ǴŁƢ»å¹±æ^Î^xqÆäÔ·é Ȥ΀ĤȐxYĬƐȺǡͷxZ. . .

(220) Table 4. Sequences of siRNAs using this study Gene. Sequences. PTBP1. 5’-AUCUCUGGUCUGCUAAGGUCACUUC-3’. hnRNPA1. Santa Cruz Biotechnology ƺ˺ Cat no. sc-270345. SRSF3. 5’-AGAACACUAUGUGGCUGCCGUGUAA-3’. Control (non-specific). 5’-GUAGGAGUAGUGAAAGGCC-3’. ̈ͺǎ͊n¾éÎ±ǽˀȝ͉ ΕȯƐʣn¾éÎ±ǽˀȝͷj Protein lysis buffer 2% Protease inhibitorYPhosphatase inhibitor cocktail II m III ¢ǆǁx¢ͷjZ¾ éÎ±ǽˀȝĈ¢ĤȐxǎ͊ÖæÂÆ˔ΐ˺YqĭΧxZ~Ƣ 20 ̵Ł̢ʿɱʼw|Y13,000 rpmY4°CY20 ̵ŁĎɑ̵΂xZĎɑ̵΂xȽ ɮ¢ĤȐxY¾éÎ±ǽµéÔåxZːΐn SDS-PAGE µéÔåǖɫ 2-2, -3 ǷxΕȯƐʣµéÔåˬ͵͇͉ƹZ . ̈ͺǎ͊n +' ˀȝ͉ +' ˀȝ NucleoSpin miRNA kit (MACHEREY-NAGEL, GmbH & Co. KG, Düren, Germany) ¢ǡͷxˀȝxZÔçÆ´^åČǡͷxZRNA ΐ UV spectrophotometry ːΐxZ +- G)+ ͉ PrimeScript RT reagent kit (Takara) ¢ǡͷxY37°C 15 ̵Y85°C 5 ̦Y4°C RNA µéÔåŖ˗Ȁ̑đ¢ƹjYˁż cDNA ¢ǁɫxZQuantitative reverse. . .

(221) transcription-PCR (qRT-PCR) ̑đ Universal SYBR select Master Mix (Applied Biosystems, Forester City, CA) m Table 5 Ôã§Ú^»ÂÆ¢ǡͷxZ GAPDH mRNA ΐ¢˴̯´éÆç^åxZ95°C 30 ̦ȥō̻ɪ¢ƹ ƢY95°C 5 ̦̻ɪ̑đm 60°C 60 ̦¥É^äé²[Ɏˈ̑đ¢ 40 µ§ ±åƹjY95°C 15 ̦Y60°C 30 ̦Y95°C 15 ̦¹ÄÂÔͱģűɿ¢̵ɲxZ ĬµéÔå̑đ 3 Ĥ{ƹjYΔΔCt m mRNA | Table 5. Sequences of primers using this study ;D;. ,;GK;D9;. )-) <EHM7H:. Q -. )-) H;L;HI;. Q - --- --- -. --. >D+') <EHM7H:. Q -- - -. Q. >D+') H;L;HI;. Q -- --- . ,+, <EHM7H:. Q. ,+, H;L;HI;. Q ---- -- Q. )$& <EHM7H:. Q - -- - - Q. )$& H;L;HI;. Q - - - Q. )$& <EHM7H:. Q -- Q. )$& H;L;HI;. Q ---. --- - Q. ---. )! <EHM7H:. Q -. )! H;L;HI;. Q -- . . Q. ---- Q. -. Q. - Q --. -. Q. - Q. »åÍ§¥ÐäÄ¦ ɯǎ͊ɧYÆäÎéÓå^ɋʅ̃ȫǯƖ͉̤ėxZ̈ͺƢǎ͊Ͷ Ĉ¢ÆäÎéÓå^(Life Technologies, Carlsbad, CA, USA)ˣΐǆǁxYƉŝƄ Ǟ̕ɯǎ͊ɧ¢ƄɧxZ´éÆç^åźʤzɯǎ͊ɧ(%)¢ǎ͊ɯʞ ΄xZ. . .

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(223) ´çÉ^Ò«^Ý^·âé¥Â»§ DLD-1 ǎ͊¢ǾƖʂ˸ 6-well Ôæ^ÆȽ 0.5×105 cells/ 1mL ǎ͙͊˞ ́p 5% CO2Y37°C ̈ͺxZͼ˸ siRNA-PTBP1, hnRNPA1, SRSF3 Control RNA (5 nM)¢~ well ˖ĘxZ48 ǴŁƢǎ͊¢ĤȐxY 2×102 cells ǎ͊¢ 0.4%¥®ç^¹ (Takara)¢ń£ 8%FBS ńͬ RPMI1680 ̈ ʻ (Wako)ǆ}YȽʊxZĕʊ 0.5%¥®ç^¹ (Takara)¢ń£ 8%FBS ńͬ RPMI1680 ̈ʻ (Wako)¢ǡͷxYĕʊYȽʊȣ 6 cm-Å¦Â· àȾ| 5% CO2Y37°C ̈ͺxZ2 ȔŁƢYÝ¾Ì^å (Sigma-Aldrich) 10 ̵Łɔzu´çÉ^¢ƟːxY5%°Ü¶ɾɋĈ(Muto pure chemicals Co., Ltd., ˡŢY˸͏) 30 ̵ŁɔzuɾɋxZ~ƢɥˮɥɽɄxYɾ ´çÉ^ɧ¢ɧlZ Ý¾Øç^Üģɲ DLD-1 ǎ͊¢ǾƖʂ˸ 6-well Ôæ^ÆȽ 0.5×105 cells/ 1mL ǎ͙͊˞ ́p 5% CO2Y37°C ̈ͺxZͼ˸ siRNA-PTBP1, hnRNPA1, SRSF3 Control RNA (5 nM)¢~ well ˖Ęx (Ĭ siRNA ˢ 6 well ͷó x)Z72 ǴŁƢǙƷ̶ƑȊȣȘʌǖx[46]Z̢Ιx 5%ÚéÉÆ ^åǎ͊¢ 2 ĤɽɄxZ~Ƣǎ͊ 25 mM ÝÀ¬Éé¹å×é; MetSulY2-Ý¾×äÌª¾é¹å×éǟ; MESYD-·â¨Ì¨-10-¹å×éǟ; CSA ¢ Ý¾Ì^å (j{ǯͧ Wako)ǆ}Ĉ¢ 1-well ˢ 250 µL ĘlY10 ̵ɱʼxZ~ƢɓxjªÂÖéÀà^ÓĈ¢ʃĤȐxZ~˴ 400 . .