Label-free Quantitative Analysis of Protein Expression Alterations in miR-26a-Knockout HeLa Cells using SWATH-MS Technology
| Autor: | Li Li, Lixin Ma, Yun Zhu, Xiangbin Kong, Tianqing Meng, Zhaowei Teng, Yan Hu, Hexiao Shen |
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| Rok vydání: | 2019 |
| Předmět: |
Proteomics
0301 basic medicine lcsh:Medicine Article Mass Spectrometry HeLa Gene Knockout Techniques 03 medical and health sciences 0302 clinical medicine Western blot microRNA medicine Humans Luciferases lcsh:Science Cell Proliferation Gene Editing Regulation of gene expression Multidisciplinary Base Sequence Staining and Labeling biology medicine.diagnostic_test Cell Cycle lcsh:R Proteins Reproducibility of Results Cell cycle biology.organism_classification Cell biology Blot MicroRNAs 030104 developmental biology Gene Expression Regulation lcsh:Q CRISPR-Cas Systems 030217 neurology & neurosurgery HeLa Cells Signal Transduction |
| Zdroj: | Scientific Reports, Vol 9, Iss 1, Pp 1-13 (2019) Scientific Reports |
| ISSN: | 2045-2322 |
| Popis: | MicroRNAs (miRNAs) bind to the 3ʹ-untranslated region of target mRNAs in a sequence-specific manner and subsequently repress gene translation. Human miR-26a has been studied extensively, but the target transcripts are far from complete. We first employed the CRISPR-Cas9 system to generate an miR-26a-knockout line in human cervical cancer HeLa cells. The miR26a-knockout line showed increased cell growth and altered proliferation. Proteomics technology of sequential window acquisition of all theoretical mass spectra (SWATH-MS) was utilized to compare the protein abundance between the wild-type and the knockout lines, with an attempt to identify transcripts whose translation was influenced by miR-26a. Functional classification of the proteins with significant changes revealed their function in stress response, proliferation, localization, development, signaling, etc. Several proteins in the cell cycle/proliferation signaling pathway were chosen to be validated by western blot and parallel reaction monitoring (PRM). The satisfactory consistency among the three approaches indicated the reliability of the SWATH-MS quantification. Among the computationally predicted targets, a subset of the targets was directly regulated by miR-26a, as demonstrated by luciferase assays and Western blotting. This study creates an inventory of miR-26a-targeted transcripts in HeLa cells and provides fundamental knowledge to further explore the functions of miR-26a in human cancer. |
| Databáze: | OpenAIRE |
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