Identifying Protein Targets of a Tumor Suppressor MicroRNA in Human Metastatic Breast Cancer Cells

Abstract

MicroRNAs (miRs) are small non-coding RNAs that are predicted to regulate more than 50% of all human protein-coding genes by targeting mRNAs for degradation or translational repression. Most targets of miRs are identified by assessing changes in the transcriptome. While transcriptome analysis is successful in capturing mRNAs that are targeted for degradation, it fails to identify targets regulated via translational repression in which mRNA abundance is unaltered. Therefore, a method to systematically identify targets at the proteomic level is needed. Bio-orthogonal non-canonical amino acid tagging (BONCAT) is a method that labels the cellular proteome using the cell's endogenous machinery, co-translationally incorporating non-canonical amino acids into newly synthesized proteins. Azidohomoalanine, a methionine analogue, has been used to tag newly synthesized proteins, presenting azide groups at methionine positions. An alkyne-functionalized purification tag can be attached via copper-catalyzed azide-alkyne cycloaddition to selectively enrich newly synthesized proteins from the pre-existing protein pool, reducing sample complexity and making the proteome amenable to temporal characterization by mass spectrometry. Here we combine BONCAT with stable isotope labeling by amino acids in cell culture (SILAC), to identify protein targets of miR-126 in the human metastatic breast cancer cell line mda-mb-231. MiR-126 has been proposed to suppress metastasis, and is down-regulated in many types of cancers; however, only a few of its targets have been identified. The results of this study will help elucidate the mechanism of suppression and provide insight into the design of epigenetic therapies that restore expression of tumor suppressor genes.

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