2. 化合物評価
  3. トランスクリプトーム解析による分子プロファイリング

Molecular profiling based on the transcriptome analysis

(担当:清宮啓之、松浦正明、牛嶋 大、冨田章弘、馬島哲夫)
(Members in charge: Hiroyuki Seimiya, Masaaki Matsuura, Masaru Ushijima, Akihiro Tomida, Tetsuo Mashima)




Functional molecules in cells regulate specific signaling networks and globally controls the downstream gene expression. When a specific inhibitor blocks the function of the target molecule, expression for the relevant set of the target genes or molecular pathways will be altered.

This means that even as for the chemical compounds, whose target molecules are completely unknown, we can obtain the gene expression signatures of the compound-treated cells, evaluate their similarities with those of various known signaling inhibitors, and predict the intracellular target molecular pathways of the compounds.

We will perform the transcriptome analysis of the cells treated with the test compounds and obtain the gene expression signatures. Then, we will compare the signatures of the test compounds with those available in various gene expression database. Through these analyses, we will provide information that is useful for prediction of the intracellular target molecular pathways of the test compounds. We will also support analysis of RNAs extracted by the applicants themselves from their original human cultured cells that are treated with the test compounds.

Materials and Methods

まず、解析に用いる化合物の処理条件等を決定する。細胞増殖を抑制する化合物の場合、ヒト結腸がん細胞株HT-29や当該化合物に対して選択的な応答性を示す(と推定される)細胞を6-well plateに播種し、20時間培養後、化合物を添加し、さらに6時間培養する。化合物の処理濃度は、GI50(50%増殖阻害濃度)値の3~10倍で、48時間接触で80%以上増殖阻害する濃度を基本とする。細胞増殖抑制以外の作用を示す化合物の場合、当該作用を発揮するのに必要な濃度も考慮して、化合物の処理細胞や処理濃度等の条件を決定する。

化合物処理した細胞より、RNAを抽出し、Agilent 2100 Bioanalyzerを用い、クオリティチェックする。その後、GeneChip Human Genome U133 Plus 2.0 Arrayを用い、マイクロアレイによる網羅的遺伝子発現解析を行う。


First, the conditions for the treatment with the test compounds will be determined. In case of the compounds that inhibit cell growth, human colorectal cancer HT-29 cells or other cell lines that selectively respond (or are predicted to respond) to the compounds will be seeded onto a 6-well plate, cultured for 20 h, and then treated with the test compound for 6 h. In general, the concentration of the compound for the experiment is set 3 to 10 times higher than the GI50 value (concentration at which the cell growth is inhibited 50%), which inhibits the cell growth more than 80% when treated for 48 h. In case of the test compounds that induce other phenotypes than cell growth inhibition, the concentration required for such effect is considered to determine the conditions of the analysis, such as cell line to examine and the concentration of the test compound.

Cells are treated with the test compound as described above and RNA is extracted. After the qualitative validation of the extracted RNA with an Agilent 2100 Bioanalyzer, the sample is subjected to transcriptome analysis with the GeneChip Human Genome U133 Plus 2.0 Array.

The obtained gene expression data are analyzed with the gene signature database related with various chemical compounds to predict the mode-of-action of the samples or to evaluate the novelty of the mode-of-action.


  1. Mashima T, Ushijima M, Matsuura M, Tsukahara S, Kunimasa K, Furuno A, Saito S, Kitamura M, Soma-Nagae T, Seimiya H, Dan S, Yamori T, Tomida A.
    “Comprehensive transcriptomic analysis of molecularly targeted drugs in cancer for target pathway evaluation.” Cancer Sci. 106: 909-20 (2015)
  2. Hirashima K, Seimiya H. “Telomeric repeat-containing RNA/G-quadruplex-forming sequences cause genome-wide alteration of gene expression in human cancer cells in vivo.” Nucleic Acids Res. 43: 2022-32 (2015)
  3. Ushijima M, Mashima T, Tomida A, Dan S, Saito S, Furuno A, Tsukahara S, Seimiya H, Yamori T, Matsuura M. “Development of a gene expression database and related analysis programs for evaluation of anticancer compounds.” Cancer Sci. 104: 360-8 (2013)