Abstract: 【Abstract】 Objective To investigate the effect of KRT5 knockdown in keratinocytes on melanin content in co-cultured melanocytes, and to explain mechanisms underlying formation of hyperpigmented lesions in reticulate pigmented anomaly of the flexures （Dowling-Degos disease, DDD）. Methods HaCaT cells with heterozygous mutations in the KRT5 gene were obtained by using clustered regularly interspaced short palindromic repeats （CRISPR）-CRISPR-associated protein 9 （Cas9） technology （experimental group）, and HaCaT cells transfected with non-targeting single guide RNA:Cas9 protein complex served as control group, both of which were in vitro co-cultured with primary human melanocyte cells (HEMn） separately. Immunofluorescence study was conducted to determine the expression of cytokeratin and melanosomes in co-cultured cells; melanin content was detected in melanocytes in different co-culture groups, which were obtained by differential trypsinization. Immunohistochemical study was performed to determine the expression of melanocyte-specific premelanosome protein 17 （Pmel17） in skin lesions in a patient with DDD carrying a KRT5 mutation and normal skin tissues in a healthy control. Results Sanger sequencing showed a heterozygous mutation （c.1delA） at the initiation codon of exon 1 of the KRT5 gene in HaCaT cells in the experimental group, but no mutation in the KRT5 gene in the control group. Western blot analysis showed that the KRT5 protein expression was significantly lower in the experimental group （0.60 ± 0.05） than in the control group （1.00 ± 0.00, t = 32.38, P = 0.001）. Compared with the co-culture system in the control group, the number of Pmel17-labeled melanosomes markedly increased with the melanin content elevated by 52.5% （t = -3.48, P = 0.025） in the HEMn cells co-cultured with HaCaT cells in the experimental group. Immunohistochemical study showed that the Pmel17 expression increased in the skin lesions in the DDD patient with KRT5 mutation compared with the normal skin tissues in the healthy control. Conclusion The effect of HaCaT cells with CRISPR-Cas9-induced KRT5 mutation on the co-cultured HEMn melanocytes was verified by the successfully established in vitro co-culture system, which provides a primary cell model for further studies on interaction mechanisms between keratinocytes and melanocytes, and on pathogenesis of skin pigmentation abnormalities.

Key words: Melanocytes, Keratinocyte, Coculture techniques, Keratin-5, Organisms, genetically modified, Hyperpigmentation, Reticulate pigmented anomaly of the flexures, CRISPR-Cas9