In vitro effect of low-concentration hydrogen peroxide on autophagy in human melanocytes and screening for autophagy-related lncRNAs

Abstract

Objective To evaluate the effect of the treatment with low-concentration hydrogen peroxide (H2O2) on the adhesive function of and autophagy in human melanocytes, and to screen long noncoding RNAs (lncRNAs) related to autophagy. Methods Melanocytes were isolated from foreskins of healthy males after circumcision, and subjected to cultivation. Melanocytes at exponential growth phase were divided into 3 groups: control group receiving no treatment, H2O2 group treated with 400 μmol/L H2O2, and H2O2 + NAC group pretreated with 4 mmol/L NAC for 2 hours followed by the treatment with 400 μmol/L H2O2. After 5-day treatment, immunofluorescence study was performed to determine the expression of E-cadherin, microtubule-associated protein 1 light chain 3 (LC3) and p62, and Western blot analysis to determine the expression of autophagy-related protein LC3 and p62. Cell structures and autophagosomes were observed by transmission electron microscopy, and autophagy-related lncRNAs were screened using gene chip technology. Statistical analysis was done with Graphpad Prism 6 software using one-way analysis of variance for comparison among groups, and Tukey′s test for multiple comparisons. Results Under the confocal microscopy, the H2O2 group showed significantly decreased fluorescence intensity of E-cadherin and LC3 in the melanocytes and decreased number of autophagosomes in melanocytes, but significantly increased fluorescence intensity of p62 compared with the control group and H2O2 + NAC group. Western blot analysis showed that the LC3-Ⅱ/LC3-Ⅰ ratio in the melanocytes was significantly lower in the H2O2 group (0.604 ± 0.012) than in the control group (1.200 ± 0.081, q=7.718, P 0.05) . Gene chip technology showed that 18 autophagy-related lncRNAs were associated with premature senescence of melanocytes and differentially expressed in the H2O2 group compared with the control group, and the autophagy-related lncRNA NONHSAT190308.1 (> 10-fold increase) was screened out. Conclusion Low-concentration H2O2 can decrease the expression of E-cadherin and the level of autophagy in melanocytes, and can up-regulate the expression of autophagy-associated lncRNA NONHSAT190308.1. Key words: Vitiligo; Melanocytes; Cell adhesion; Autophagy; Oxidative stress; Hydrogen peroxide; Microtubule-associated proteins; E-cadherin; LncRNA