MicroRNA-101-3p Modulates Mitochondrial Metabolism via the Regulation of Complex II Assembly

MicroRNA-101 (miR-101) is a tumour suppressor that regulates cancer proliferation and apoptotic signalling. Loss of miR-101 increases the expression of the Polycomb Repressive Complex 2 (PRC2) subunit enhancer of zeste homolog 2 (EZH2), resulting in alterations to the epigenome and enhanced tumorigenesis. MiR-101 has also been shown to modulate various aspects of cellular metabolism, however little is known about the mechanisms involved. To investigate the metabolic pathways that are regulated by miR-101, we performed transcriptome and functional analyses of osteosarcoma cells overexpressing miR-101. We found that miR-101 downregulates multiple mitochondrial processes, including oxidative phosphorylation, pyruvate metabolism, the citric acid cycle and phospholipid metabolism. We also found that miR-101 overexpression disrupts the transcription of mitochondrial DNA (mtDNA) via the downregulation of the mitochondrial transcription initiation complex proteins TFB2M and Mic60. These alterations in protein expression disrupt mitochondrial function, with significant decreases in both basal (54%) and maximal (67%) mitochondrial respiration rates. Native gel electrophoresis revealed that this diminished respiratory capacity was associated with reduced steady-state levels of mature succinate dehydrogenase (complex II), with a corresponding reduction of complex II enzymatic activity. Furthermore, miR-101 overexpression reduced the expression of the SDHB subunit, with a concomitant disruption of the assembly of the SDHC subunit into mature complex II. Overall, we describe a new role for miR-101 as a modulator of mitochondrial metabolism via its regulation of multiple mitochondrial processes, including mtDNA transcription and complex II biogenesis. We performed RNA-seq on 143BTK- osteosarcoma cells (ATCC CRL-8303) transfected with either a scrambled negative control miRNA or miR-101 for 120 h with n=4 replicates