Proteomics and RNA-seq of TGP-200c and LNA-200c in MIN6 cells
Global Proteomics: The effect of LNA-200c (100 nM) on the proteome was studied. Amongst 3,016 proteins detected, 29 were significantly affected (10 down-regulated and 19 up-regulated. Interestingly, none of these 29 proteins overlapped with those significantly affected by TGP-200c. Unlike TGP-200c, where global upregulation of miR-200c target proteins was observed, no significant general up-regulation of these proteins was observed as they fall below the false discovery rate of 1%, although an increase in abundance was observed for the direct target Vitamin K Epoxide Reductase Complex Subunit 1 Like 1 (VKORC1L1). Other proteins regulated by the miR-200 family, namely miR-200a and miR-249, were also affected, such as Dolichyl Pyrophosphate phosphatase 1 (DOLPP1), Mitogen Activated Protein Kinase 1 (MAPK1), Translin Associated Factor X (TSNAX), and Solute Carrier Family 7 Member 30 (SLC30A7); MAPK1 appears to function in cell survival in T2D (Favaro et al., 2012; Granata et al., 2007). Notably, proteomics analysis for LNA-200c could be confounded by the downregulation of all mature miR-200 family members by the oligonucleotide. Pathway analysis, however, showed that similar networks were affected by LNA-200c and TGP-200c, for example the regulation of macromolecule metabolism, apoptosis, metabolism, and insulin signaling. RNA-seq analysis: Among the 15,737 genes that were detected, only 23 transcripts (0.15%) were significantly affected by TGP-200c (with >2-fold change and p < 0.05; 10 down-regulated and 13 up-regulated). Among these 23 significantly affected transcripts, two, Evi2b and Gpr174, are predicted mRNA targets for miR-200c-5p (by TargetScan) (Agarwal et al., 2015) . For LNA-200c, 45 transcripts (0.29%) were significantly affected upon treatment (25 down-regulated and 20 up-regulated; Figure S6A). Among these 45, five mRNAs, Msln, Gm14288, Evi2b, Cldn2, Sox6, are predicted by TargetScan to be direct targets of miR-200c-3p (n = 1) or -5p (n = 4) (Agarwal et al., 2015). Of the 23 transcripts modulated by TGP-200c and the 45 affected by LNA-200c, five overlap: Evi2b (direct target of miR-200c), Gm15387, Gm6169, Gm4353, and Gm19680. Evidently, structure-binding ligands can have specificities competitive with modalities that recognize sequence.