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- 1. つくば遺伝子研究所/紀和実験動物研究所 遺伝子発現解析 私たちは、遺伝子発現解析のパイプランを可能な限り実装し、その有効性について検 証しています。解析方法としては、6種類、さらに、遺伝子レベル、エキソンレベル での解析が行えるように、組み立てています。 また、細胞のがん化や、継代によるゲノムの再構成を検出するために、遺伝子発現 レベルの解析では、融合遺伝子(fusion gene)からの発現の検出、そして、ゲノムレベ ルでは、私たちが開発したPED (BMC Bioinformatics. 2019 Jun 28;20(1):362.)プログラムを用いて解析 を行っています。 今回ご紹介するのは、STAR-Cufflinks-Cuffdiff-Rを用いて、「先発薬投与と後 発薬投与における遺伝子発現にみられる差異」を検討してみまし た。
- 4. 実験動物を用いて、遺伝子発現レベルでの差異についての調査 先発薬 後発薬 肝臓からRNAを抽出 先発薬区、後発薬区の肝臓RNA mRNA 4000万リードの解析 (100bp pair-end) コンピュータによる解析で、25,801個の遺伝子及 び725,857個のエキソンの発現量の変化を解析。 体重換算で、人で10mg相当量(5μg/マウス(30g)in 0.5%CMC)をマウスの胃にゾンデで朝に1回4日間連 続投与。薬品添付文書では、薬品の血中濃度は、約６ 時間維持されていることから、採材は、最終投与後７ 時間で行った。 各実験区C57BL/6J Kwl 8週齢 ♂ ６匹を使用 0.5%CMC(コントロール)
- 5. • 臓器の摘出 １．頚椎脱臼による安楽死後、直ちに肝臓、腎臓を切除し回収する。 ２．回収した肝臓、腎臓は、クライオチューブに入れ直ちに液体窒素タンクにて凍結する。 • 肝臓からのRNAの抽出 プロトコール） １．サンプル ①先発薬（シンバスタチン） in 0.5%CMC 肝臓 ３組織 ②後発薬（シンバスタチン） in 0.5&CMC 肝臓 ３組織 ２．常法に従ってRNAを抽出 ３．抽出されたRNAの品質 純度およびRIN値(分解度) • RNAシークエンシング(配列解析) TruSeq Stranded mRNA Library Prep Kitを用い、取得した総RNAから特異的にmRNAを濃縮して、これら mRNAを鋳型としてcDNAを合成後、シークエンス用ライブラリーを作製した。 次に、cDNAライブラリーを次世代シークエンサー（NovaSeq6000）にて100bp, Paired-end, １サンプル4000万リー ドでシークエンスした。 つくば遺伝子研究所
- 6. we TopHat2 Cufflinks Cuffdiff R FeatureCount DESeq２ BaySeq R R STAR Cufflinks Cuffdiff R FeatureCount DESeq２ BaySeq R R RNAseqを用いる遺伝子発現解析手法 リアルタイム RT-PCR In silico analysis Wet analysis つくば遺伝子研究所
- 7. STAR Cufflinks Cuffdiff R シンバスタチンの先発薬と後発薬(ジネリック)投与による、肝臓での遺伝子発現の解析 を用いて、遺伝子レベルで解析 先発薬 後発薬 主成分分析 クラスター解析 (無根系統樹) 肝臓の遺伝子発現において、後発薬は、先発薬とは異なったパターンを示す結果が得られた。
- 8. シンバスタチンの先発薬と後発薬(ジネリック)投与による、肝臓での遺伝子発現の差異 どんな遺伝子の発現が変化しているのか? 薬物間相互作用に差異が生じる可能性は否定出来ない gene_id 遺伝子名 p値 q値 (FDR) log2.fold _change 集計発現量比 (後発薬／先発 薬) 先発薬 発現量 試料１ 先発薬 発現量 試料２ 先発薬 発現量 試料３ 後発薬 発現量 試料１ 後発薬 発現量 試料２ 後発薬 発現量 試料３ 先発薬 集計発現量 後発薬 集計発現量 ENSMUSG00000037025 forkhead box A2 5.00E-05 0.005534 3.57667 11.93122277 12.1426 11.1492 14.8343 20.3344 418.687 13.8888 12.7005 151.533 ENSMUSG00000031574 steroidogenic acute regulatory protein 5.00E-05 0.005534 2.83702 7.145425875 1.57887 1.04636 1.57207 9.33274 13.1248 7.47391 1.39732 9.98446 ENSMUSG00000025488 cytochrome c oxidase subunit 8B 0.00125 0.083656 2.34383 5.076485321 4.86581 0.439844 0 13.8318 5.5445 7.45377 1.75945 8.93185 ENSMUSG00000061780 complement factor D (adipsin) 5.00E-05 0.005534 2.32013 4.99377216 2.25576 1.37456 0.540244 9.43703 4.58814 6.78997 1.38832 6.93297 ENSMUSG00000028295 Small integral membrane protein 8 5.00E-05 0.005534 2.23666 4.713046776 15.8666 14.8301 15.3122 12.7382 13.9983 189.564 15.3285 72.2438 ENSMUSG00000070565 RAS protein activator like 2 5.00E-05 0.005534 2.19167 4.568339912 1.53079 1.68477 1.5872 1.71465 2.21518 17.9657 1.60068 7.31241 ENSMUSG00000056328 myosin, heavy polypeptide 1, skeletal muscle, adult 5.00E-05 0.005534 2.04018 4.112968435 4.96778 0.308885 0.872736 13.0475 6.16387 5.98754 2.03971 8.38927 ENSMUSG00000057003 myosin, heavy polypeptide 4, skeletal muscle 5.00E-05 0.005534 1.88861 3.702782991 3.46787 0.103921 0.770706 5.38907 4.54322 6.06097 1.44011 5.3324 ENSMUSG00000021453 growth arrest and DNA- damage-inducible 45 gamma 5.00E-05 0.005534 1.86414 3.640508556 3.75514 6.29 24.4003 72.7076 22.5911 30.2523 11.4747 41.7736 ENSMUSG00000061816 myosin, light polypeptide 1 5.00E-05 0.005534 1.84101 3.582607513 9.50541 0.290513 1.51814 18.5468 11.6955 10.1057 3.75144 13.44 ENSMUSG00000032648 muscle glycogen phosphorylase 5.00E-05 0.005534 1.77501 3.422403825 2.47458 0.226681 0.869379 5.82743 2.52487 3.8276 1.18511 4.05591 ENSMUSG00000031097 troponin I, skeletal, fast 2 5.00E-05 0.005534 1.77389 3.419747959 17.4731 1.39085 4.53214 36.4977 21.9975 21.2043 7.76305 26.5476 ENSMUSG00000007877 titin-cap 5.00E-05 0.005534 1.75746 3.381023397 9.21897 0.311583 2.17704 19.3174 9.46407 10.6436 3.8829 13.1282 ENSMUSG00000043013 one cut domain, family member 1 5.00E-05 0.005534 1.74336 3.348140338 5.18739 1.18835 4.44715 9.85034 8.42876 17.8282 3.59709 12.0436 ENSMUSG00000009733 transcription factor CP2 2.00E-04 0.018871 1.73442 3.327456948 2.66305 2.41505 2.86546 14.4546 8.87258 3.12205 2.64619 8.80506 ENSMUSG00000066687 zinc finger and BTB domain containing 16 5.00E-05 0.005534 1.65041 3.139228403 2.43259 3.29003 0.856894 5.91387 3.59762 11.1523 2.1951 6.8909 ENSMUSG00000024430 calcium-binding tyrosine- (Y)-phosphorylation regulated (fibrousheathin 2) 5.00E-05 0.005534 1.61595 3.065133689 7.6063 4.75056 4.16971 16.0211 15.9252 18.619 5.50151 16.8629 ENSMUSG00000030399 creatine kinase, muscle 5.00E-05 0.005534 1.61205 3.056858982 17.2619 0.714133 2.85211 29.5108 15.6005 18.2813 6.90693 21.1136 ENSMUSG00000018893 myoglobin 5.00E-05 0.005534 1.60469 3.041303934 21.6212 0.742349 3.51349 37.8062 20.8153 19.7385 8.58053 26.096 ENSMUSG00000052131 aldo-keto reductase family 1, member B7 5.00E-05 0.005534 1.59561 3.022222757 1.43379 0.848481 1.14148 3.65634 3.63988 3.03327 1.13956 3.444 ENSMUSG00000017300 troponin C2, fast 5.00E-05 0.005534 1.5926 3.015923846 18.5328 0.598467 3.92818 33.8858 16.8657 18.5087 7.64718 23.0633 ENSMUSG00000033196 myosin, heavy polypeptide 2, skeletal muscle, adult 5.00E-05 0.005534 1.58986 3.010201369 3.46979 0.084039 0.588824 4.53389 3.20064 4.66976 1.37355 4.13465 ENSMUSG00000026103 glutaminase 5.00E-05 0.005534 1.54727 2.922635676 1.04796 0.79113 1.24251 1.99622 1.43727 5.55473 1.02607 2.99883 ENSMUSG00000030785 cytochrome c oxidase subunit 6A2 5.00E-05 0.005534 1.54679 2.921663446 4.65808 0.250213 0.80167 7.71963 4.65151 4.24026 1.89377 5.53296 ENSMUSG00000022346 myelocytomatosis oncogene 5.00E-05 0.005534 1.47686 2.783422666 2.90197 2.04328 2.87182 18.0244 1.64363 2.1502 2.60257 7.24404 ENSMUSG00000030672 myosin light chain, phosphorylatable, fast skeletal muscle 5.00E-05 0.005534 1.40044 2.639820804 19.5009 2.38709 5.20035 33.2297 19.8679 18.1653 8.9915 23.736 ENSMUSG00000032578 cytokine inducible SH2- containing protein 0.00065 0.050216 1.39073 2.622113255 5.10508 0.598091 3.24722 10.232 4.45655 8.71016 2.97242 7.79401 ENSMUSG00000087687 PET100 homolog 0.00225 0.123922 1.35898 2.565037646 1.71858 1.3547 2.37974 7.8661 1.9384 4.19362 1.81585 4.65774 ENSMUSG00000032077 BUD13 homolog 5.00E-05 0.005534 1.29014 2.445517858 4.82751 4.65028 9.11281 15.769 14.7422 14.908 6.19231 15.1434 ENSMUSG00000020776 Fas (TNFRSF6) binding factor 1 0.0016 0.097448 1.25065 2.379486056 0.700672 1.34494 1.7355 2.91727 2.99795 3.08249 1.261 3.00055 ENSMUSG00000026208 desmin 5.00E-05 0.005534 1.18842 2.279030133 4.05113 1.7844 1.9242 7.24203 4.1961 6.21659 2.58114 5.88248 ENSMUSG00000020475 phosphoglycerate mutase 2 0.00135 0.088295 1.18336 2.271050836 2.95194 0.551197 0.513966 4.00091 1.97943 3.11374 1.33387 3.0293 ENSMUSG00000022003 solute carrier family 25, member 30 0.00045 0.037536 1.17704 2.261123825 2.71336 7.94449 11.2433 19.2064 22.558 7.79113 7.30676 16.5214 ENSMUSG00000005716 parvalbumin 3.00E-04 0.026564 1.17447 2.257099471 3.859 0.224519 0.477996 3.44028 3.15343 3.64649 1.51274 3.41441 ENSMUSG00000021622 creatine kinase, mitochondrial 2 5.00E-05 0.005534 1.11018 2.158725793 6.25253 0.221602 1.13003 7.07645 4.52083 4.7416 2.52162 5.44349 ENSMUSG00000002831 perilipin 4 0.00175 0.105467 1.06442 2.091328943 0.998585 1.21322 1.125 1.80909 2.6969 2.46541 1.11232 2.32623 ENSMUSG00000074623 predicted gene 826 0.0021 0.117918 1.05099 2.071951165 3.7744 1.6838 2.53733 3.22108 5.90922 7.37779 2.65975 5.51088 ENSMUSG00000023034 nuclear receptor subfamily 4, group A, member 1 0.0016 0.097448 1.03842 2.053976962 0.332632 1.11037 1.8378 2.91956 1.49848 2.32977 1.0944 2.24789 ENSMUSG00000028680 polo like kinase 3 5.00E-05 0.005534 1.00927 2.012892324 8.19012 8.6121 14.543 23.8452 18.7703 20.4492 10.4428 21.0203 ENSMUSG00000060600 enolase 3, beta muscle 4.00E-04 0.034361 1.00003 2.000041589 9.79748 4.12747 5.06462 17.1345 10.7007 10.0866 6.31606 12.6324 ENSMUSG00000058672 tubulin, beta 2A class IIA 5.00E-05 0.005534 -1.04292 0.485344147 31.9767 11.9737 10.9829 8.97264 11.0334 6.57647 18.2658 8.86515 ENSMUSG00000052837 jun B proto-oncogene 5.00E-05 0.005534 -1.0739 0.475033117 17.8723 50.1378 26.5791 16.0128 13.748 15.254 31.5914 15.007 ENSMUSG00000019874 fatty acid binding protein 7 0.003 0.152801 -1.09849 0.467005032 3.55318 1.46561 2.8124 1.08829 1.62653 0.931417 2.60478 1.21645 ENSMUSG00000074794 arrestin domain containing 3 5.00E-05 0.005534 -1.16649 0.44550391 45.1293 57.5143 27.4016 21.475 23.3465 13.1305 43.3698 19.3214 ENSMUSG00000025004 cytochrome P450, family 2, subfamily c, polypeptide 40 5.00E-05 0.005534 -1.17429 0.443101772 24.97 4.73317 45.4264 11.2394 17.5969 4.34393 24.9778 11.0677 ENSMUSG00000022508 B cell leukemia/lymphoma 6 5.00E-05 0.005534 -1.20299 0.434374103 8.1387 29.4724 17.3675 12.8392 6.12254 5.0043 18.3664 7.97787 ENSMUSG00000019726 lysosomal trafficking regulator 5.00E-05 0.005534 -1.23037 0.426208125 12.7974 3.19225 3.05472 2.58977 2.49968 2.99716 6.32711 2.69667 ENSMUSG00000037379 spondin 2, extracellular matrix protein 1.00E-04 0.01037 -1.25086 0.42019765 2.41299 2.96774 2.7938 0.972782 1.88651 0.571816 2.72516 1.1451 ENSMUSG00000072596 eosinophil-associated, ribonuclease A family, member 2 6.00E-04 0.047632 -1.44522 0.367236153 3.69605 2.98915 2.76079 1.11275 1.01269 1.33947 3.14623 1.15541 ENSMUSG00000095562 LncRNA 0.00275 0.143236 -1.48363 0.357587943 2.79273 1.52425 8.40777 0.860326 0.693242 2.98394 4.23456 1.51422 ENSMUSG00000092008 cytochrome P450, family 2, subfamily c, polypeptide 69 5.00E-05 0.005534 -1.49509 0.354758707 10.2159 2.81622 37.148 4.97855 10.2012 2.56242 16.6912 5.92136 p値::群間差が偶然生じる可能性を示す尺度。例えば、p値が0.01（p=0.01）というのは、この結果を偶然生じることが100回に1回あることを意味する。p値が小さくなるほど 、それだけ群間差の可能性が高くなる。
- 9. Gene Ontology解析 Molecular Function Molecular-level activities performed by gene products. Molecular function terms describe activities that occur at the molecular level, such as “catalysis” or “transport”. GO molecular function terms represent activities rather than the entities (molecules or complexes) that perform the actions, and do not specify where, when, or in what context the action takes place. Molecular functions generally correspond to activities that can be performed by individual gene products (i.e. a protein or RNA), but some activities are performed by molecular complexes composed of multiple gene products. Examples of broad functional terms are catalytic activity and transporter activity; examples of narrower functional terms are adenylate cyclase activity or Toll-like receptor binding. To avoid confusion between gene product names and their molecular functions, GO molecular functions are often appended with the word “activity” (a protein kinase would have the GO molecular function protein kinase activity). Qvalue上位100件のGOデータのみを対象として実行している（それ以上になると図が複雑になりすぎて目視できなくなるため）。図中で黄色く着色されているのが、上位100件に該当するGOterm No. GOID Term No. GOID Term No. GOID Term No. GOID Term 1 GO:0070888E-box binding 40 GO:0005085 guanyl-nucleotide exchange factor activity 79 GO:0031432titin binding 118 GO:0044877 protein-containing complex binding 2 GO:0030676 Rac guanyl-nucleotide exchange factor activity 41 GO:0042289 MHC class II protein binding 80 GO:0001221 transcription cofactor binding 119 GO:0048037cofactor binding 3 GO:0000978 RNA polymerase II proximal promoter sequence-specific DNA binding 42 GO:0031267small GTPase binding 81 GO:0001085 RNA polymerase II transcription factor binding 120 GO:0097159 organic cyclic compound binding 4 GO:0005089 Rho guanyl-nucleotide exchange factor activity 43 GO:0051393alpha-actinin binding 82 GO:0020037heme binding 121 GO:1901363 heterocyclic compound binding 5 GO:0048365Rac GTPase binding 44 GO:0051015actin filament binding 83 GO:0003677DNA binding 122 GO:0097367 carbohydrate derivative binding 6 GO:0000977 RNA polymerase II regulatory region sequence-specific DNA binding 45 GO:0001067 regulatory region nucleic acid binding 84 GO:0035639 purine ribonucleoside triphosphate binding 123 GO:0043167ion binding 7 GO:0000987 proximal promoter sequence- specific DNA binding 46 GO:0043565 sequence-specific DNA binding 85 GO:0000166nucleotide binding 124 GO:0036094 small molecule binding 8 GO:0005524ATP binding 47 GO:0003690 double-stranded DNA binding 86 GO:0046872metal ion binding 125 GO:0008289lipid binding 9 GO:0003945 N-acetyllactosamine synthase activity 48 GO:0032553ribonucleotide binding 87 GO:0031406 carboxylic acid binding 126 GO:0003824catalytic activity 10 GO:0005088 Ras guanyl-nucleotide exchange factor activity 49 GO:0017076 purine nucleotide binding 88 GO:0016757 transferase activity, transferring glycosyl groups 127 GO:0005198 structural molecule activity 11 GO:0017048Rho GTPase binding 50 GO:0046914 transition metal ion binding 89 GO:0016772 transferase activity, transferring phosphorus- containing groups 128 GO:0140110 transcription regulator activity 12 GO:0001012 RNA polymerase II regulatory region DNA binding 51 GO:0033293 monocarboxylic acid binding 90 GO:0016746 transferase activity, transferring acyl groups 129 GO:0098772 molecular function regulator 13 GO:0000976 transcription regulatory region sequence-specific DNA binding 52 GO:0016758 transferase activity, transferring hexosyl groups 91 GO:0016788 hydrolase activity, acting on ester bonds 130 GO:0005488binding 14 GO:0008144drug binding 53 GO:0016773 phosphotransferase activity, alcohol group as acceptor 92 GO:0016705 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen 131 GO:0003674 molecular_functio n 15 GO:0032559adenyl ribonucleotide binding 54 GO:0016301kinase activity 93 GO:0004497 monooxygenase activity 16 GO:0035250UDP-galactosyltransferase activity 55 GO:0016775 phosphotransferase activity, nitrogenous group as acceptor 94 GO:0016614 oxidoreductase activity, acting on CH-OH group of donors 17 GO:0004674 protein serine/threonine kinase activity 56 GO:0046912 transferase activity, transferring acyl groups, acyl groups converted into alkyl on transfer 95 GO:0000981 DNA-binding transcription factor activity, RNA polymerase II- specific 18 GO:0009922fatty acid elongase activity 57 GO:0016747 transferase activity, transferring acyl groups other than amino-acyl groups 96 GO:0042802 identical protein binding 19 GO:0004465lipoprotein lipase activity 58 GO:0016298lipase activity 97 GO:0046983 protein dimerization activity 20 GO:0017016Ras GTPase binding 59 GO:0052689 carboxylic ester hydrolase activity 98 GO:0005102 signaling receptor binding 21 GO:0051371muscle alpha-actinin binding 60 GO:0008391 arachidonic acid monooxygenase activity 99 GO:0019899enzyme binding 22 GO:0044212 transcription regulatory region DNA binding 61 GO:0008401 retinoic acid 4- hydroxylase activity 100 GO:0005516calmodulin binding 23 GO:1990837 sequence-specific double- stranded DNA binding 62 GO:0016712 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen 101 GO:0019904 protein domain specific binding 24 GO:0032555purine ribonucleotide binding 63 GO:0016713 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced iron- sulfur protein as one donor, and incorporation of one atom of oxygen 102 GO:0008092 cytoskeletal protein binding 25 GO:0030554adenyl nucleotide binding 64 GO:0016709 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NAD(P)H as one donor, and incorporation of one atom of oxygen 103 GO:0008134 transcription factor binding 26 GO:0005506iron ion binding 65 GO:0008395 steroid hydroxylase activity 104 GO:0046906tetrapyrrole binding 27 GO:0005504fatty acid binding 66 GO:0016616 oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor 105 GO:0003676nucleic acid binding 28 GO:0008378galactosyltransferase activity 67 GO:0001228 DNA-binding transcription activator activity, RNA polymerase II-specific 106 GO:1901265 nucleoside phosphate binding 29 GO:0008194UDP-glycosyltransferase activity 68 GO:0001227 DNA-binding transcription repressor activity, RNA polymerase II-specific 107 GO:0043168anion binding 30 GO:0004672protein kinase activity 69 GO:0042803 protein homodimerization activity 108 GO:0043169cation binding 31 GO:0004111creatine kinase activity 70 GO:0042287MHC protein binding 109 GO:0043177organic acid binding 32 GO:0102338 3-oxo-lignoceronyl-CoA synthase activity 71 GO:0051427 hormone receptor binding 110 GO:0140096 catalytic activity, acting on a protein 33 GO:0102336 3-oxo-arachidoyl-CoA synthase activity 72 GO:0051020GTPase binding 111 GO:0016740transferase activity 34 GO:0102337 3-oxo-cerotoyl-CoA synthase activity 73 GO:0070080titin Z domain binding 112 GO:0016787hydrolase activity 35 GO:0004312fatty acid synthase activity 74 GO:0050693LBD domain binding 113 GO:0016491 oxidoreductase activity 36 GO:0004806triglyceride lipase activity 75 GO:0031433telethonin binding 114 GO:0005200 structural constituent of cytoskeleton 37 GO:0008392 arachidonic acid epoxygenase activity 76 GO:0003779actin binding 115 GO:0008307 structural constituent of muscle 38 GO:0070330aromatase activity 77 GO:0051373FATZ binding 116 GO:0003700 DNA-binding transcription factor activity 39 GO:0018685alkane 1-monooxygenase activity 78 GO:0042805actinin binding 117 GO:0005515protein binding
- 11. 先発薬 www.info.pmda.go.jp › interview 後発薬 image.packageinsert.jp › pdf ここに示した夾雑物の差異が、肝臓の遺伝子発現に影響を及ぼしている可能性があると思われます