MiR-30a-5p Alters Epidermal Terminal Differentiation during Aging by Regulating BNIP3L/NIX-Dependent Mitophagy

Abstract

Chronological aging is characterized by an alteration in the genes' regulatory network. In human skin, epidermal keratinocytes fail to differentiate properly with aging, leading to the weakening of the epidermal function. MiR-30a is particularly overexpressed with epidermal aging, but the downstream molecular mechanisms are still uncovered. The aim of this study was to decipher the effects of miR-30a overexpression in the human epidermis, with a focus on keratinocyte differentiation. We formally identified the mitophagy receptor BNIP3L as a direct target of miR-30a. Using a 3D organotypic model of reconstructed human epidermis overexpressing miR-30a, we observed a strong reduction in BNIP3L expression in the granular layer. In human epidermal sections of skin biopsies from donors of different ages, we observed a similar pattern of BNIP3L decreasing with aging. Moreover, human primary keratinocytes undergoing differentiation in vitro also showed a decreased expression of BNIP3L with age, together with a retention of mitochondria. Moreover, aging is associated with altered mitochondrial metabolism in primary keratinocytes, including decreased ATP-linked respiration. Thus, miR-30a is a negative regulator of programmed mitophagy during keratinocytes terminal differentiation, impairing epidermal homeostasis with aging.

Keywords: BNIP3L; aging; keratinocyte; miR-30a; mitochondria; mitophagy.

Figure 1

miR-30a interactions with BNIP3L. (A) Schematic of the BNIP3L 3’UTR region showing the putative miR-30a binding sites. (B) Relative mRNA expression of BNIP3L in keratinocytes 24 h after transfection of miR-30a-3p or miR-30a-5p mimics or a scrambled sequence (NC: Negative Control). qPCR analysis was normalized to TBP and RPL13A housekeeping genes using the 2^−ΔΔCt quantification method (mean ± SD, n = 6; * p < 0.05 and **** p < 0.0001). Exact p-values were determined using the one-way ANOVA and Tukey post-hoc tests. (C) Normalized luciferase activity (red firefly/green renilla) after co-transfections of HEK293T cells with the Wild-Type (WT) or Mutant (Mut) reporter plasmids and miRNA mimics (median ± SD, n = 4 or n = 8; **** p < 0.0001). Exact p-values were determined using the one-way ANOVA and Tukey post-hoc tests. (D) Schematic of a portion of BNIP3L 3’UTR region with the three putative complementary pairing sites with miR-30a-5p. The mutated sites, obtained by inverse PCR and characterized by Sanger sequencing, are also indicated below the original sequence. (E) Western blot analysis of keratinocytes of BNIP3L (25 kDa) expression 36 h after transfection of miR-30a-5p mimics or a scrambled sequence (NC: Negative Control). Western blot analysis of Vinculin (116 kDa) was used as a loading normalizer. (F) Quantification of BNIP3L expression normalized by the expression of Vinculin (mean ± SD, n = 3; * p < 0.05). Exact p-value was determined using the Student’s t-test.

Figure 2

Effect of miR-30a on BNIP3L expression in the granular layer of reconstructed epidermis. (A) Immunofluorescent staining of BNIP3L in reconstructed human epidermis overexpressing miR-30a or a scrambled sequence by stable lentiviral transduction. Representative photographs of 3 independent replicates are shown. (B) Quantification of the surface of the epidermis with a positive signal for BNIP3L in each condition (median ± SD, n = 6; **** p < 0.0001). Exact p-value was determined using the Student’s t-test.

Figure 3

BNIP3L expression in human skin biopsies from individuals at different ages. (A) Immunofluorescent staining of BNIP3L and KRT14 in human skin sections. Representative photographs of 6 individuals from two different age groups: adult, or aged people (top, from left to right: 38, 35 and 42 years old; bottom, from left to right: 61, 71 and 78 years old). Inserts are a magnification of the selected area showing BNIP3L-positive cells (white arrowheads) (B) Mathematical correlation between the percentage of BNIP3L-positive cells from the whole epidermis and the chronological age of the individual from which the skin biopsy has been sampled (n = 21). Exact p-value was determined using the Pearson correlation test. (C) Mathematical correlation between the total BNIP3L fluorescence intensity from the whole epidermis and the chronological age of the individual from which the skin biopsy has been sampled (n = 21). Exact p-value was determined using the Pearson correlation test.

Figure 4

Human keratinocyte differentiation, BNIP3L expression and mitochondria retention in vitro with aging. (A) Relative mRNA expression of differentiation markers and BNIP3L in keratinocytes during a kinetic of differentiation in 2D culture. Three different age groups were tested (young group: n = 6, from 3 to 10 years old, mean age at 5; adult group: n = 6, from 26 to 46 years old, mean age at 36.5; aged group: n = 5, from 68 to 92 years old, mean age at 79) and followed over the time (0, 2, 4, 6 and 9 days after reaching confluency). qPCR analysis was normalized to TBP and RPL13A housekeeping genes (mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001). Exact p-values were determined using the two-way ANOVA and Tukey post-hoc tests. (B) Mitochondria content per cell estimated by qPCR at day 0. qPCR analysis was performed by normalizing the relative content of mitochondrial genes (ND1 and TL1) with the content of nuclear genes (HBB and SERPINA1) using the 2^−ΔΔCt quantification method in the three groups (mean ± SD; young group: n = 6; adult group: n = 6; aged group: n = 5). (C) Evolution of the content of mitochondria expressed as a percentage of the quantity at day 0 (median ± SD; * p < 0.05, ** p < 0.01). Exact p-values were determined using the two-way ANOVA and Tukey post-hoc tests.

Figure 5

Mitochondrial metabolic activity of keratinocytes from different age groups and after overexpression of miR-30a. Major aspects of mitochondrial coupling and respiratory control were measured using the Seahorse Bioanalyzer and determined by the sequential additions of oligomycin, an ATP synthase inhibitor, FCCP, a protonophoric uncoupler, and rotenone and antimycin A, two inhibitors of the electron transport chain. sbl: miR-scrambled; 30a: miR-30a. Basal respiration (A), ATP-linked respiration (B), maximal respiration (C), reserve capacity (D), non-mitochondrial respiration (E) and proton leak (F) were determined by measuring the Oxygen Consumption Rate (OCR) in the culture media (median ± SD; young group: n = 6; adult group: n = 6; aged group: n = 5; sbl group: n = 3; 30a group: n = 3; ** p < 0.01, *** p < 0.001 and **** p < 0.0001). Exact p-values were determined using the one-way ANOVA and Tukey post-hoc tests for comparison between ages and paired Student’s t-test for comparison between cells transfected with either scramble miRNA sequence or miR-30a.