Identification of host factors targeted by miR-130a. (A) Northern blot analysis of miR-130a precursor and its mature form in HepG2 cell lines stably transfected with a miR-130a expression vector. (B) As implicated by bioinformatic analysis in Table 2, RT-qPCR analysis detected the reduction of PGC1α and PPARγ mRNAs (in asterisk) in stable miR-130a expressing HepG2 cell lines. (C) Northern blot analysis detected concurrent reduction of PGC1α (2.3 kb) and PPARγ (1.4 kb) mRNAs in stable miR-130a expressing cell lines. HNF4α and tubulin were included as a control. (D) Western blot analysis detected concurrent reduction of PGC1α (91 kDa), and PPARγ (54 kDa) proteins in stable miR-130a expressing cells. (E) HepG2 cells were transfected with LNA-miR-130a or a LNA scramble control. The increased PGC1α mRNA and protein levels were measured by real-time RT-qPCR (upper panel) and Western blot analysis (lower panel), respectively. (F) MiR-130a was shown to directly target at the 3′ UTR (nucleotides 2025–2031) of PGC1α by compensatory mutagenesis. Mutation sites of the miR-130a seed sequence mutant and the PGC1α target site mutant were underlined in sequence alignment. The reduction of luciferase activity was observed when wild-type miR-130a was in combination with wild-type PGC1α 3′ UTR, or when seed mutant miR-130a was in combination with target site mutant of PGC1α. (G) Cotransfection of the PGC1α 3′ UTR luciferase reporter with increasing amounts of a miR-130a expression vector resulted in gradually decreasing reporter activity in a dose–response manner (left panel). Conversely, treatment with increasing amounts of a LNA-miR-130a plasmid resulted in gradually increasing reporter activity in a dose–response manner (right panel). (**) P < 0.05.
