title: Proteomic screening and identification of microRNA-128 targets in glioma cells.
abstract: Brain-enriched miR-128 is repressed in glioma cells, and could inhibit the proliferation of gliomas by targeting genes such as E2F3a and BMI1. To identify more targets of miR-128 in glioblastoma multiforme, the pulse stable isotope labeling with amino acids in cell culture (pSILAC) technique was used to test its impact on whole protein synthesis in T98G glioma cells. We successfully identified 1897 proteins, of which 1459 proteins were quantified. Among them, 133 proteins were downregulated after the overexpression of miR-128. Through predictions using various bioinformatics tools, 13 candidate target genes were chosen. A luciferase assay validated that 11 of 13 selected genes were potential targets of miR-128, and a mutagenesis experiment confirmed CBFB, CORO1C, GLTP, HnRNPF, and TROVE2 as the target genes. Moreover, we observed that the expression of CORO1C, TROVE2, and HnRNPF were higher in glioma cell lines compared to normal brain tissues and presented a tendency toward downregulation after overexpression of miR-128 in T98G cells. Furthermore, we have validated that CORO1C, TROVE2, and HnRNPF could inhibit glioma cell proliferation. In sum, our data showed that the integration of pSILAC and bioinformatics analysis was an efficient method for seeking the targets of miRNAs, and plentiful targets of miR-128 were screened and laid the foundation for research into the miR-128 regulation network.
authors.affiliation: The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
authors.affiliation: The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China.
authors.forename: Shan
authors.initials: S
authors.lastname: Wang
authors.affiliation: The State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, P. R. China.
authors.forename: Jiawei
authors.initials: J
authors.lastname: Zeng
authors.affiliation: The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China.
authors.forename: Xiangbin
authors.initials: X
authors.lastname: Ruan
authors.affiliation: The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Han
authors.affiliation: The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yin
authors.affiliation: The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China.
authors.forename: Jiangang
authors.initials: J
authors.lastname: Yuan
authors.affiliation: The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China.
authors.forename: Boqin
authors.initials: B
authors.lastname: Qiang
authors.affiliation: The State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, P. R. China.
authors.forename: Wantao
authors.initials: W
authors.lastname: Ying
authors.affiliation: The State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, P. R. China.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Qian
authors.affiliation: The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China.
authors.forename: Xiaozhong
authors.initials: X
authors.lastname: Peng
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D001932:Brain Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D053263:Gene Regulatory Networks; D005910:Glioma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D007553:Isotope Labeling / Q000379:methods; D035683:MicroRNAs / Q000235:genetics*; D008856:Microscopy, Fluorescence; D020543:Proteome / Q000235:genetics* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D034622:RNA Interference; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D020413:3' Untranslated Regions; C545189:MIRN128 microRNA, human; D035683:MicroRNAs; D020543:Proteome
keywords: Cell biology; Glioma; Quantitative proteomics; miR-128; pSILAC