MicroRNA-211 Regulates Oxidative Phosphorylation and Energy Metabolism in Human Vitiligo

Abstract

Vitiligo is a common chronic skin disorder characterized by loss of epidermal melanocytes and progressive depigmentation. Vitiligo has complex immune, genetic, environmental, and biochemical causes, but the exact molecular mechanisms of vitiligo development and progression, particularly those related to metabolic control, are poorly understood. In this study we characterized the human vitiligo cell line PIG3V and the normal human melanocyte line HEM-l by RNA sequencing, targeted metabolomics, and shotgun lipidomics. Melanocyte-enriched microRNA-211, a known metabolic switch in nonpigmented melanoma cells, was severely down-regulated in vitiligo cell line PIG3V and skin biopsy samples from vitiligo patients, whereas its predicted targets PPARGC1A, RRM2, and TAOK1 were reciprocally up-regulated. microRNA-211 binds to PGC1-α 3' untranslated region locus and represses it. Although mitochondrial numbers were constant, mitochondrial complexes I, II, and IV and respiratory responses were defective in vitiligo cells. Nanoparticle-coated microRNA-211 partially augmented the oxygen consumption rate in PIG3V cells. The lower oxygen consumption rate, changes in lipid and metabolite profiles, and increased reactive oxygen species production observed in vitiligo cells appear to be partly due to abnormal regulation of microRNA-211 and its target genes. These genes represent potential biomarkers and therapeutic targets in human vitiligo.

Figure 1.. Differential miR-211 expression in vitiligo and normal melanocytes and human tissue samples.

(a) miR-211 expression in healthy skin (normal skin pool from 5 individuals) and vitiligo lesions (n=11) using qRT-PCR analysis. Graph shows fold change in miR-211 expression in each patient compared to normal skin pool.(b) miR-211 expression in non-lesional (NL), peri-lesional (PL), and lesional (L) regions from three patients with vitiligo Graph shows fold change in miR-211 expression in each patient compared to the corresponding NL region.(c) miR-211 expression in primary melanocytes (HEM-l) and vitiligo (PIG3V) cells. Graph shows fold change in miR-211 expression compared to HEM-l cells. Student’s t-test was performed to detect differences between the samples as indicated. P values :*< 0.05 ;**< 0.01; ***<0.001; ****<0.0001

Figure 2.. Increased PGC1-α expression in vitiligo cells compared to normal melanocytes.

(a-b) HEM-l and PIG3V cells were analyzed for PGC1-α expression by qRT-PCR (a) and western blot analysis (b). (c) Immunofluorescent detection of PGC-1α (middle panel), nuclei (DAPI, left panel) and merged images (right panel) in PIG3V and HEM-l cells. 20× magnification, scale bar represents 100uM. Graph plot depicts relative fluorescence intensity of PGC1-α expression per cell. (d) PIG3V cells were treated with either miR-211-CNP or control CNP and analyzed for miR-211(left panel) and PGC1-α (right panel) expression 24 h post treatment and compared to untreated PIG3V cells. (e) PIG3V cells were transfected with luciferase expression vectors (pcDNA6-Luc) containing either PGC1-α-3’UTR or PGC1-α-3’UTR-miR211 del (miR-211 binding site deleted) sequences and treated with either miR211-CNP or control CNP at 2uM concentration 24 h post transfection. Graph shows relative fold change in luciferase activity 48hrs post transfection and compared to PIG3V cells transfected with PGC1-α-3’UTR alone. (f) Stable PIG3V lines were generated by infecting PIG3V cells with both control or miR-211 containing lentivirus and PGC1-α expression was analyzed by western blotting. Results shown are mean ± SDM and representative of at least three independent experiments. Student’s t-test was performed to detect significant differences. P values: **<0.01; ***<0.001; ****<0.0001

Figure 3.. Expression of putative miR-211 target genes is increased in vitiligo lesions.

qPCR analysis of (a) PGC1-α, (b) RRM2, and (c) TAOK1 was analyzed in healthy skin (normal skin pool from 5 individuals) and vitiligo lesions (n=11) by qRT-PCR. Results shown are mean ± SDM. Student’s t-test was performed to detect differences in normal skin pool and individual patient samples. P values: *<0.05; **<0.01; ***<0.001; ****P<0.0001

Figure 4.. Reduced oxidative capacity in vitiligo melanocytes compared to normal melanocytes.

(a and b) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analyzed using the Seahorse XF analyzer in HEM-l and PIG3V cells. (c) Ratio of mitochondrial to nuclear DNA content in HEM-l and PIG3V cells. (d) Mitochondrial content in HEM-l and PIG3V cells was analyzed by staining the cells with MitoTracker® Green. 20× magnification, scale bar represents 25uM (e) Mitochodrial complex expression in HEM-l and PIG3V cells by western blotting. (f) OCR in PIG3V cells treated either with miR211-CNP or control CNP at 2uM concentration. (g) Intracellular ROS levels were measured in 40,000 HEM-l or PIG3V cells using the OxiSelect^™ Intracellular ROS Assay Kit with hydrogen peroxide as positive control. (h) PIG3V cells were transfected with either scrambled siRNA (siNEG) or siRNA against PGC1-α and analyzed for PGC1-α expression and intracellular ROS levels. Results shown are mean ± SDM and representative of at least three independent experiments. P values: **<0.01; ***<0.001

Figure 5.. Lipidomics and metabolomics analysis of HEM-I and PIG3V cells.

(a-c) Graphs depict cumulative changes in fatty acid chains for different lipid groups. (a) Cardiolipin and phosphatidylglycerol. (b) Phosphatidic acid, diacylglycerol, and triacylglycerol. (c) Phosphatidylcholine, sphingomyelin, and phosphatidylserine. (d-e). Graphs depict significant changes in: (d) organic acids; (e) amino acids in HEM-1 and PIG3V cell lysates. P values: *<0.05; **<0.01; ***<0.001; ****<0.0001