The Mechanism Research Of Mir-423-5p Regulated Human Melanocyte Oxidative Stress Injury Induced By Hydrogen Peroxide

Vitiligo is a common chronic depigmentation skin disorder, mainly caused by the melanocyte absence or loss of function. The pathogenesis of vitiligo has not been fully elucidated. In recent years a large number of studies have shown that oxidative stress damage in vitiligo melanocytes play an important role.Usually, the oxidation and antioxidant system is under the dynamic balance in normal body, and any resaon to break this balance will lead to oxidative stress injury. There is considerable evidence indicated that vitiligo patients have the oxidation and antioxidant system imbalance. A large number of directly and indirectly evidence confirmed that oxidative stress play an important role in vitiligo's pathogenesis, such as: excessive H₂O₂ was accumulated in vitiligo patients'epidermis, SOD activity and lipid peroxidation levels were significantly increased in vitiligo patients's peripheral blood erythrocyte, MDA was concentrated in vitiligo patients's plasma, DNA strand breaks and oxidized DNA bases levels were increased in vitiligo patients's peripheral mononuclear cells, MSR expression and activity was significantly decreased in vitiligo patients'lesion, Nrf2 nuclear distribution of plasma was imbalanced in vitiligo patients, and the relathionship between lots of oxidative stress-related gene polymorphisms and susceptibility to vitiligo was also widly reported.microRNA (miRNA), a class of about 21-22 nt non-coding, single-stranded small RNA molecules, could transcriptional downreuglate the expression of its target gene, through specific mRNA base pairing between its own sequence and its target gene. Previous studies have shown that miRNA molecules were significantly involved in cell oxidative stress. Various miRNA molecules have been found differentially expressed in H₂O₂, ionizing radiation, etoposide or the other factors induced oxidative stress. In cardiac disease, neurodegenerative disease and other oxidative stress related diseases, the abnormal expression of miRNA molecules play an anti-oxidation or promoting oxidative stress role in two-way adjustment function, which suggested that miRNA plays an important role in cell stress response against and oxidative stress. So, search melanocyte oxidative stress-related miRNA molecules and investigate the possible function and mechanism of miRNA in human melanocytes oxidative stress could provide new ideas and new theories for vitiligo mechanism research and clinical drug invention.Methods:We first used the appropriate H₂O₂ concentration to treat immortalized human epidermal melanocytes PIG1 and immortalized vitiligo melanocyte PIG3V, and used the miRNA chip to analysis the differential expressed miRNAs between the H₂O₂ treated and untreated human melanocytes. Next, we used the Quantitative RT-PCR to validate their expression. In order to observe the effect about choosed miRNAs on the processing of H₂O₂ induced melanocyte oxidative stress injury, we used miRNAs'specific precursors and inhibitors to transiently transfect PIG1 and PIG3V melanocytes, then examined cell viability, apoptosis, intracellular ROS, MDA, SOD and GSTs expression level in transfected groups and control groups. To further search the miRNAs direct target genes, bioinformatics software was used to predict miRNA target genes. Reporter gene assay was used to validate the relationship between miRNAs and their targets. RT-PCR and Western Blot methods were used to further validate the possible signal pathway. Gene transfection, Western Blot and cell viability analysis were used to further confirm the possible signal pathway of miR-423-5p/GSTM1 in H₂O₂ induced human melanocytes oxidative injury.Results:1. Through miRNA expression profile analysis, we found that compared with the H₂O₂ untreated PIG1 cells, 7 miRNAs were overexpressed and 3 miRNAs were lowexpressed in H₂O₂ treated PIG1 cells. Compared with untreated PIG3V cells, 3 miRNAs were overexpressed and 5 miRNAs were lowexpressed in treated PIG3V cells. After carefully examining the intersection of two experimental results, we found three miRNAs (miR-93, miR320b and miR-423-5p) changed in the same direction in H₂O₂ treated PIG1 cells and PIG3V cells. Quantitative RT-PCR results showed that miR-93 and miR-320b expression decreased in treated PIG1 cells, which is inconsistent with the CHIP result. While miR-423-5p was overexpressed in both PIG1 and PIG3V cells after H₂O₂ treated, especially in PIG3V cells.2. Quantitative PCR results showed that miR-423-5p specific precursor and inhibitor can successfully increase or decrease endogenous miR-423-5p expression in PIG1 and PIG3V cells. In H₂O₂ treated PIG1 and PIG3V cells, upreulation miR-423-5p with its specific precursor increased the intracellular ROS and MDA level, decreased SOD, GSTs activity, inhibited cell proliferation and promoted H₂O₂ induced cell apoptosis. Oppositely, in H₂O₂ treated PIG1 and PIG3V cells, downregulation miR-423-5p with its specific inhibitor could decrease the intracellular ROS and MDA level, increase SOD, GSTs activity, promote cell proliferation and inhibit H₂O₂ induced cell apoptosis.3. Biological software analysis predicted that GSTM1 was a possible target gene of miR-423-5p. Dual-luciferase reporter gene assay showed that miR-423-5p could down-regulate GSTM1 expression by directly targeting its 3'-UTR. In H₂O₂ treated PIG1 cells, cells transfected with miR-423-5p specific inhibitor downreulation miR-423-5p could upregulate GSTM1 protein expression compared with cells transfected with the inhibitor control. After treated by H₂O₂, the GSTM1 mRNA expression showed no significant change in PIG1 cells, when transfected with miR-423-5p specific precursor or inhibitor. Further, compared with untransfected or transfected with pcDNA3.1(+) control vector, upregulation GSTM1 expression in miR-423-5p specific inhibitor transfected H₂O₂ treated PIG1 cells could partly reverse miR-423-5p inhibited cell proliferation.Conclusion:1. miR-423-5p was differentially expressed in human melanocytes before and after H₂O₂ treatment, suggesting miR-423-5p may be involved in hydrogen peroxide induced human melanocyte oxidative stress.2. miR-423-5p could exacerbate the melanocyte oxidation-antioxidant imbalance under oxidative stress state, increase melanocytes oxidative stress level, decreased melanocytes antioxidant capacity, inhibit cell proliferation, and increased hydrogen peroxide induced apoptosis ratio. These results all indicated that miR-423-5p is one of the important risk factors in melanocyte oxidative stress injury. 3. GSTM1 was a direct target gene of miR-423-5p in the process of melanocytes oxidative stress. GSTM1 gene belongs to GST gene family; GST is one of the most important enzymes against oxidative stress in human body. Our results showed that miR-423-5p could decrease the melanocytes oxidative stress ability and increase oxidative stress induced melanocytes injury, this function was partly through negatively regulating GSTM1 to achieve.