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. 2022 Jan 24;30(4):1645–1660. doi: 10.1016/j.ymthe.2022.01.032

Figure 6.

Figure 6

c-Myc upregulates miR-373-3p to downregulate MFN1 and transcriptionally upregulates Drp1

(A) Transcriptional activity of the DRP1 promoter was assessed by luciferase reporter assay in Huh6 cells following c-Myc knockdown. shctrl, control shRNA; shc-Myc, shRNA against c-Myc. (B) Transcriptional activities of serially truncated DRP1 promoter constructs were assessed by luciferase reporter assay in Huh6 cells. (C) The putative binding site for c-Myc was predicted by in silico analysis. (D) The transcriptional activity of the mutant DRP1 promoter was assessed using a luciferase reporter assay in Huh6 cells. (E) Plasmid-based ChIP-PCR analyses of c-Myc binding to the DRP1 promoter in Huh6 cells. WT, wild type; MU, mutant. (F) Venn diagram of the predicted miRNAs. Two classic databases were used to screen for miRNAs that could regulate MFN1. The yellow region contains 56 miRNAs screened from the TargetScan database, and the blue region contains 92 miRNAs screened from the miRDB database. The green region represents the intersection of the two databases, including five miRNAs with identical predictions. (G) Quantitative real-time reverse-transcription PCR (quantitative real-time RT-PCR) analyses of expression levels of the five predicted miRNAs in Huh6 cells treated as indicated. (H–K) Representative confocal microscopy images and analyses of mitochondrial morphology in Huh6 cells treated as indicated. Scale bars: 10μm. TSB-NC, negative control of miRNA target site blocker; TSB-MFN1, a target site blocker that selectively prevents miR-373-3p binding to the 3′ UTR of MFN1. (L and M) Cell proliferation was analyzed by CCK-8 in Huh6 cells with treatment as indicated. (N) Tumor volume curves of subcutaneous xenograft tumor models developed from Huh6 cells with treatment as indicated. Tumor volume was measured using vernier calipers every 5 days from day 10 after subcutaneous implantation. (O and P) Images of tumors from sacrificed mice were obtained on day 30 after injection. Tumor weights of different groups were compared. (Q) The percentage of PCNA-positive cells in subcutaneous xenograft tumors. Data are expressed as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01.