miR-4521—A novel biomarker for human lentigos

Lentigos are hyperpigmented skin lesions that can vary in size and shape, typically appearing on the face, hands and shoulders, usually on sun exposed skin. Currently, the molecular pathogenesis of lentigos remains unclear. As a first step in identifying potential biomarkers of lentigos that may drive lentigo formation, we performed laser-capture microdissection (LCM) on the lesional epidermis of lentigos from five archived patient samples and, as a control, from the epidermis of three unremarkable skin samples matching for site, sex and age (Table S1). Total RNA was extracted from the micro-dissected lentigo and control tissue samples to perform transcriptomic microarray analysis (ThermoFisher Human transcriptome Array 2.0.) to identify potential biomarkers of lentigos. Bioinformatics analysis of data obtained from the whole transcriptome array (ThermoFisher TAC4.0 software package, Figure S1) identified coding and non-coding transcripts that were significantly differentially expressed in lentigos; some of these differentially expressed transcripts are associated with melanogenesis which is consistent with previous studies (Table S2).^{1, 2} Multiple non-coding RNAs were found to be differentially regulated in the top 10 differentially expressed genes, including small nucleolar RNA species (snoRNA) and micro-RNA species (miR) (Tables S2 and S3). Studies have shown evidence that microRNAs play important biological roles ranging from development, differentiation, tumour progression or suppression.³ In our data, one of the most highly expressed microRNAs was miR-4521, which is recently reported to act as a “tumor suppressor miR” to decrease the expression of pro-oncogenic targets such as forkhead box protein M1 and insulin-like growth factor 2.⁴ Therefore, we further characterized the expression pattern of has-miR-4521 to validate it as a biomarker of lentigo.

The expression of miR-4521 in lentigos was evaluated by qRT-PCR and miR RNA in situ hybridization. miR-4521 expression level was 3.61 higher than that of control epidermis by qRT-PCR (Figure 1A, Mann–Whitney test, lentigo n = 6, control n = 4, p < 0.05). miR-4521 RNA in situ hybridization assays demonstrated stronger positive signals for the miR-4521 anti-sense probe in the epidermis between the stratum basalis and the stratum granulosum (Figure 1 E,G and H) compared to matched control skin (Figure 1D,F). In addition, the signal intensity of the anti-sense probe in the lentigo epidermis (Figure 1H) was higher than that of adjacent un-remarkable epidermis (Figure 1G) and matched control (Figure 1F). Together, these data indicate that miR-4521 expression is increased in lentigos primarily in the lesional keratinocytes. Melanocyte numbers were determined in all samples and there was no statistically significant difference noted (lentigo; 2.74 ± 0.37 SE /0.1 mm, control; 3.71 ± 0.21 SE / 0.1 mm, Mann–Whitney test, p > 0.05, specimens described in Table S1). In silico analysis of potential miR-4521 targets indicate that miR-4521 may potentially regulate proteosomal degradation and autophagy as GABA Type A Receptor Associated Protein Like 2 appears to represent a potential target (Table S4). Further studies on the expression of miR-4521 and its impact on GABA Type A Receptor Associated Protein Like 2 function may provide helpful information on the molecular pathogenesis of lentigos.

Conceptualization: HM, YSH, AFK, OC, YS, TI, JS. Data Curation: HM, YSH, YS, TI, JS. Formal Analysis: HM, YSH, AFK, YS, JS. Funding Acquisition: HM, TI, JS. Investigation: HM, YSH, AFK, YS, JS. Methodology: HM, YSH, AFK, YS, JS. Project Administration: HM, YSH, YS, JS. Resources: HM, YSH, YS, TI, JS. Software: HM, YSH, JS. Supervision: HM, TI, JS. Validation: HM, YSH, AFK, OC, YS, TI, JS. Visualization: HM, YSH, YS, TI, JS. Writing Original Draft Preparation: HM, YSH, AFK, OC, YS, TI, JS. Writing Review and Editing: HM, YS, JS.

The authors declare no potential conflicts of interest regarding this publication.