PMC

miR-663 retrograde regulation of OXPHOS and its role in tumorigenesis. A summary figure explains the regulatory mechanism underlying retrograde regulation of miR-663 and its role in controlling mitochondrial function and tumorigenesis.

Inhibition of miR-663 reprograms OXPHOS gene expression. A, protein expression of subunits for each OXPHOS complex in mitochondria from miR-663–modulated MCF7 cells (top blot) and MDA-MB-231 cells (bottom blot). B, semi-quantitative PCRs of respiratory complex subunits using mRNA isolated from miR-663–modulated MCF7 cells. All PCRs were run at the same time as their corresponding actin controls and represent two biological replicates.

Inhibition of miR-663 alters OXPHOS enzyme activity. Isolated mitochondria were used to conduct enzymatic activity assays of OXHPOS Complexes I–V in MCF7 cells expressing a microRNA control vector, an miR-663 expression vector, or an anti–miR-663 vector. Activities were normalized to total protein. *, p < 0.05; **, p < 0.01. Assays represent two biological replicates run in triplicate. Error bars depict S.D.

Mitochondrial dysfunction induces hypermethylation of miR-663 promoter. A, methylation-specific PCR assessing methylation of the miR-663 promoter in Rho⁰ cells compared with WT or Cybrid cells and after treatment of WT cells with rotenone or antimycin A. M denotes methylated DNA amplified by primers specific for methylated DNA. U denotes unmethylated DNA amplified by primers specific for unmethylated DNA. B, bisulfite sequencing depicts 21 CpG dinucleotides present in the miR-663 promoter. Each row represents the sequencing results of a single clone. Each black circle represents a methylated CpG dinucleotide, and each white circle represents an unmethylated CpG dinucleotide. C, the bar graphs on top show real-time PCR of primary miR-663 in 143B cells after treatment with rotenone or antimycin A in the presence or absence of 5-aza-2′-deoxycytidine. The lower panels show MSP with rotenone or antimycin A in the presence or absence of 5-aza-2′-deoxycytidine. D, cellular methyltransferase activity after inhibition of Complex I (rotenone) or III (antimycin). *, p < 0.05; **, p < 0.01. Error bars represent S.D. Real-time PCR data represent the average of three biological replicates. MSP and bisulfite sequencing results are representative of two biological replicates. Methyltransferase activity data represent two biological replicates run in triplicate.

Inhibition of miR-663 destabilizes supercomplexes. A, semi-quantitative PCRs of respiratory complex assembly factors using mRNA isolated from miR-663–modulated MCF7 cells. All PCRs were done at the same time as their corresponding actin control. B, luciferase assays assessing direct interactions between miR-663 and the 3′-UTRs of predicted miR-663 targets. C, BN-PAGE of OXPHOS supercomplexes in MCF7 cells (left blot) and MDA-MB-231 cells (right blot) stably expressing either a microRNA control vector, an miR-663 expression vector, or an anti–miR-663 vector. Mitochondria were solubilized in 1% digitonin. Blots were probed with an OXPHOS antibody mixture (MitoSciences) including antibodies against NDUFB8 (Complex I), SDHB (Complex II), UQCRC2 (Complex III), COXII (Complex IV), and ATP5A (Complex V). D, Blue Native blots showing supercomplex stability after CRISPR disruption of subunits or assembly factors for Complexes I–IV in MCF7 cells. Mitochondria were solubilized in 1% digitonin or 1% Triton as noted. Antibodies are indicated below each blot and dilutions of 1:1000 were used for all. E, primary miR-663 transcript expression by real-time PCR using cDNA prepared from CRISPR transfected MCF7 cells. *, p < 0.05; **, p < 0.01. BN-PAGE and semi-quantitative PCR data are representative of two biological replicates. Real-time PCR data represent three biological replicates. Luciferase assays represent two biological replicates run in quadruplicate. Error bars depict S.D.

miR-663 is regulated by mitochondrial dysfunction and DNA methylation. A, top panel shows quantification of mtDNA content after depletion of mtDNA in MCF7 cells. Depletion was achieved by activating a doxycycline-inducible dominant negative POLG mutant, D1135A. Bottom panel shows real-time PCR of the primary miR-663 transcript in MCF7 cells showing down-regulation of miR-663 after depletion of mtDNA. B and C, MCF7 cells (B) and MDA-MB-231 (C) treated with rotenone also had reduced expression of miR-663. Middle panels show COBRA analyses in which bisulfite converted DNA was digested with BstUI. Lower molecular weight bands indicate CGCG sequences that were methylated. Bottom panels show bisulfite sequencing results in breast cancer cells treated with DMSO or rotenone. D, median beta values from 533 TCGA breast tumors analyzed on the Infinium HumanMethylation450 BeadChip platform show the methylation status at nine CpG loci across the miR-663 promoter region. Each locus on the bead chip array is denoted by its distance from the beginning of the miR-663 precursor transcript. *, p < 0.05; ***, p < 0.001. Real-time PCR data represent the average of three biological replicates. Error bars represent S.D. Error bars for tumor methylation beta values are S.E.

miR-663 controls mitochondria-to-nucleus retrograde signaling. A, results of a microRNA microarray using RNA from 143B parental (WT) cells, parental cells devoid of mtDNA (Rho⁰), and Rho⁰ cells with restored mtDNA (Cybrid). The graph represents -fold change of the six most modulated microRNAs in the parental and cybrid lines relative to the Rho⁰ line. B, confirmation of the microarray results showing down-regulation of miR-663. Real-time PCR confirmation of down-regulated miR-663 at the mature transcript level in Rho⁰ cells compared with WT (parental) and cybrid cells. C, real-time PCR of the primary transcript and the mature miR-663 molecule shows down-regulation of both forms after inducing mitochondrial dysfunction at each complex in the electron transport chain. 143B cells were treated for 12 h with DMSO or inhibitors of mitochondrial oxidative phosphorylation (OXPHOS) complexes. Rot = rotenone (Complex I, 100 nm), mal = malonate (Complex II, 10 mm), AA = antimycin A (Complex III, 20 μm), KCN = potassium cyanide (Complex IV, 1 mm), and OM = oligomycin (Complex IV, 12.5 μm). # denotes p < 0.01 comparing primary transcript between DMSO and treatments, and * denotes p < 0.05 comparing mature transcript between DMSO and treatments. D, assessment of reactive oxygen species by detection of oxidized CM-H₂DCFDA in 143B cells after treatment with rotenone or antimycin A. E, expression of primary miR-663 expression in 143B cells treated with H₂O₂. F, expression of primary miR-663 in 143B cells treated with rotenone alone or rotenone in combination with N-acetylcysteine. *, p < 0.05; **, p < 0.01 relative to DMSO. All real-time PCR data represent the average of three biological replicates. Error bars represent S.D.

miR-663 regulates cellular growth, invasion, and tumor progression. A and B, after stable transfection of MCF7 and MDA-MB-231 breast cancer cell lines with the microRNA control, miR-663 expression vector, or an anti–miR-663 vector, tumorigenic assays were carried out to assess the role of miR-663 in cellular invasion (A) and cellular proliferation (B). C and D, tumor growth (C) and tumor weight (D) were assessed in mouse subcutaneous xenografts stably expressing a microRNA control, miR-663 expression vector, or an anti–miR-663 vector. E, analysis of TCGA tumors (bar graph) showing mature miR-663 expression by tumor stage I–IV (tumor sample n = 82, 121, 390, 150, and 13 for normal, stage I, stage II, stage III, and stage IV, respectively). Kaplan-Meier curves (right plot) derived from TCGA data showing 10-year survival of patients with group stage II breast tumors, stratified by high (n = 15) or low (n = 18) mature miR-663 expression. F, analysis of TCGA tumors (bar graph) showing mature miR-663 expression by tumor metastatic stage (tumor sample n = 82, 690, and 15 for normal, M0, and M1, respectively). Survival of patients (right plot) with metastatic stage 0 breast tumors stratified by high (n = 11) and low (n = 22) mature miR-663 expression. Patients were stratified by high (expression values >0) and low (expression values of 0) mature miR-663 expression. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Cellular invasion and proliferation assays present at least two biological replicates. Proliferation assays were run in quadruplicate. In vitro assay error bars represent S.D. Xenograft and primary tumor error bars denote S.E.