Effects of Wnt5A gene overexpression on cytoskeletal proteins of melanocytes

Abstract

Objective To evaluate the effect of Wnt5A gene overexpression on cytoskeletal proteins of melanocytes after the plasmid containing the Wnt5A gene is transfected into primary melanocytes. Methods In vitro cultured primary human melanocytes were divided into three groups: blank control group receiving no treatment, negative control group transfected with endotoxin-free pcDNA3.1 (+) empty vector by Lipo3000 in Opti-MEM medium, Wnt5A plasmid group transfected with endotoxin-free pcDNA3.1 (+) vector containing the Wnt5A gene by Lipo3000 in Opti-MEM medium. After the trans-fection, quantitative PCR (qPCR) was performed to measure the mRNA expression of Wnt5A, ras-related C3 botulinum toxin substrate 1 (Rac1) , filamentous actin (F-actin) and β-tubulin, Western blot analysis to determine the protein expression of Wnt5A, receptor tyrosine kinase like orphan receptor 2 (ROR2) , Rac1, F-actin and β-tubulin, and an immunofluorescence assay (IFA) to observe the expression of cytoskeletal proteins. Results qPCR showed significant differences in the mRNA expression of the Wnt5A gene and its downstream genes Rac1 and F-actin among the Wnt5A plasmid group, negative control group and blank control group (F= 1 374.179, 112.576, 66.458, respectively, all P 0.05) . Additionally, the Wnt5A plasmid group showed significantly higher mRNA expression of Wnt5A, Rac1 and F-actin compared with the blank control group and negative control group (all P 0.05) . IFA showed no obvious difference in the fluorescence intensity of β-tubulin or F-actin between the Wnt5A group and the two control groups, but melanocytes showed larger size and increased number of dendrites, and the cytoskeleton changed dramatically with varying fluorescence intensity of F-actin, fuzzy texture, fractured or locally clustered tonofilaments in the Wnt5A group. Conclusion The overexpression of the Wnt5A gene in melanocytes can regulate the mRNA and protein expression of cytoskeletal proteins, make melanocytes larger and more dendritic, and cause changes in the cytoskeleton, which may facilitate the transportation of melanosomes, and participate in the occurrence of hyperpigmented diseases. Key words: Melanocytes; Wnt signaling pathway; Cytoskeleton; Actins; Tubulin; rac1 GTP-binding protein; Wnt5A protein