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Control of microRNA expression by mitochondrial function

ABSTRACT: Normal cellular function requires communication between mitochondria and the nucleus, termed mitochondria-to-nucleus retrograde signaling. Interruption of this mechanism has been implicated in the dysregulation of many cancer-related pathways, including cell death programs and tumor suppressor networks. Many proteins are known modulators of retrograde signaling, but whether microRNAs (miRNAs) are also involved is unknown. We conducted a miRNA microarray analysis using RNA from a parental cell line, a Rho0 line lacking mitochondrial DNA (mtDNA) and a Rho0 line with restored mtDNA. We found that miR-663 was down-regulated in the mtDNA-depleted Rho0 line. mtDNA restoration reversed this miRNA to parental levels, suggesting that it is an epigenetically-regulated mediator of retrograde signaling. We further demonstrated by methylation specific PCR and bisulfite sequencing that miR-663 is epigenetically regulated by pharmacological disruption of oxidative phosphorylation (OXPHOS). Restoration of rotenone-suppressed miR-663 expression by N-acetylcysteine suggested that mitochondrial dysfunction–induced reactive oxygen species play a role in epigenetic miR-663 regulation. We noted that miR-663 regulates the expression of nuclear-encoded respiratory chain subunits, e.g. NDUFB8, SDHB, UQCRFS1, and COX4L1. miR-663 also regulated the OXPHOS assembly factors NDUFAF1, SDHAF2, UQCC2, and SCO1 and was required for respiratory supercomplex stability. Furthermore, using luciferase assays, we found that miR-663 directly regulates UQCC2. The miR-663 sponge reduced OXPHOS complex activity and increased in vitro cellular proliferation and promoted tumor development in mice. We also found that increased miR-663 expression in breast tumors consistently correlates with increased patient survival. We provide first evidence for miRNA mediating retrograde signaling, demonstrating its epigenetic regulation and its role in breast tumorigenesis.

ORGANISM(S): Betapolyomavirus hominis Mus musculus Human gammaherpesvirus 8 Human alphaherpesvirus 1 JC polyomavirus Rattus norvegicus Betapolyomavirus macacae Human immunodeficiency virus 1 Homo sapiens human gammaherpesvirus 4 Human betaherpesvirus 5

PROVIDER: GSE104277 | GEO | 2017/10/19

SECONDARY ACCESSION(S): PRJNA412221

REPOSITORIES: GEO

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Project description:The mitochondrial and nuclear genomes contribute to mitochondrial function, and when mitochondrial function is compromised, mitochondrial retrograde signaling alters nuclear gene expression. We performed gene expression profiling of engineered cells that had mitochondria containing a disease-associated mutation that causes mitochondrial dysfunction. By generating networks of transcription factors that targeted these genes, the authors revealed putative mitochondrial retrograde signaling pathways. One such pathway involved retinoic acid receptor alpha (RXRA), the mRNA for which was reduced in the mutant cells. Network analysis and experiments in cells suggested that mitochondrial dysfunction caused by the mutation initiated a positive feedback loop that aggravated mitochondrial dysfunction: Reduced RXRA abundance further compromised expression of genes encoding products involved in mitochondrial function and translation. This gene-transcription factor mapping-network approach may reveal targets for therapeutic intervention of diseases associated with mitochondrial dysfunction. To investigate retrograde signaling pathways induced by the mitochondrial dysfunction caused by the mt3243 mutation, we generated the three types of cybrid cells using a mitochondria-mediated transformation method. Cells lacking mtDNA (rho0) were fused with mtDNAs with the mt3243 mutation isolated from platelets of a diabetic patient with sensorineural hearing loss. We then isolated three types of cybrid cells: W cells had wild-type mtDNA (3243A homoplasmy), H cells had both the mutant and wild-type mtDNA (3243A/G heteroplasmy) with 70% of the mtDNA containing 3243G, and M cells with only mutant mtDNA (3243G homoplasmy). Gene expression profiles of cybrid cells were generated using Illumina HumanHT-12-v3-BeadChip (Illumina, San Diego, CA), which includes 49,896 probes corresponding to 25,202 annotated genes. According to the Illumina protocols, three biological triplicates of each type of cybrid cells were analyzed. Total RNA (500ng) was isolated from cybrid cells using RNeasy Mini Kit (Qiagen, GmbH, Germany). RNA integrity number (RIN) was in the range of RIN = 9.2 and 10 when measured with an Agilent 2100 Bioanalyzer. RNA was reversely transcribed and amplified using IlluminaTotalPrep RNA amplification kit (Ambion, Austin, TX). In vitro transcription was then carried out to prepare cRNA. The cRNAs were hybridized to the array and then labeled with Cy3-streptavidin (Amersham Bioscience, Little Chalfont, UK). The fluorescent signal on the array was measured with a BeadStation 500 System (Illumina, San Diego, CA).

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Control of microRNA expression by mitochondrial function

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