2012 Fiscal Year Final Research Report

Proteomic analysis of micro RNA target proteins involved in cancer metastasis

Research Project

Project/Area Number	22501040
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Tumor diagnosis
Research Institution	独立行政法人医薬基盤研究所
Principal Investigator	HARA Yasuhiro 独立行政法人医薬基盤研究所, 創薬基盤研究部, 研究員 (70568617)
Co-Investigator(Kenkyū-buntansha)	TOMONAGA Takeshi 独立行政法人医薬基盤研究所, 創薬基盤究部, プロジェクトリーダー (80227644) MATSUBARA Hisahiro 千葉大学, 医学(系)・研究科(研究院), 教授 (20282486) ISHIHAMA Yasushi 京都大学, 薬学研究科(研究院), 教授 (30439244)
Project Period (FY)	2010 – 2012
Keywords	プロテオミクス解析
Research Abstract	MicroRNAs (miRNAs) are small noncoding RNAs that down-regulate gene expression and play important roles in tumorigenesis. Although a number of miRNA target proteins could be predicted, few of them have been validated experimentally and the precise mechanism of the inhibition of metastasis by miRNA has not been elucidated. Thus, we conducted a proteomic search for miRNA-target proteins. First, we focused on miR-31, recently, reported to inhibit metastasis of human breast cancer xenografts, and identification of its targets should be important for understanding the mechanism of cancer metastasis. Thus, we conducted a proteomic search for miR-31-target proteins. We over-expressed miR-31 in metastatic MDA-MB-231 human breast cancer cells using lentiviral vector and searched for differentially expressed proteins between miR-31?infected and empty vector?infected cells. Protein extracts from the cells were digested with trypsin and labeled with an isobaric tagging reagent, iTRAQ. The tryptic peptides were fractionated using a SCX column and identified by LC-MS/MS. A number of proteins with altered expression were identified by miR-31 over-expression. Next, we selected miR-145,miR-205,miR-206,miR-335 as candidates for miRNAs involved in metastasis inhibition. We over-expressed these miRNAs in MDA-MB-231 cells using lentiviral vector and determined whether they affect cells invasion by matrigel invasion assay. Results from invasion assays showed that these miRNAs were not able to suppress cells invasion.