Analysis of lncRNAs expression in UVB-induced stress responses of melanocytes

• 120 mj/cm2 UVB induced TYR mRNA expression and increased tyrosinase activity.
• LncRNAs in melanocytes undergo significant changes following irradiation with UVB.
• Some lncRNAs expression changes induced by UVB are dependent on ROS generation.
• UVB/ROS/lnc-CD1D-2:1 axis may be involved in the the synthesis of melanin.

BackgroundLong non-coding RNAs (lncRNAs) have close relationships with oxidative stress, nutritional deficiency, DNA damage and other types of cellular stress responses. Previous studies have demonstrated that some non-coding RNAs in melanocytes such as microRNAs can change and contribute to the synthesis of melanin or the development of melanoma after stimulation with UV. However, as an important component of non-coding RNAs, it is unclear what changes occur in lncRNAs during UV-induced stress responses in melanocytes.ObjectiveTo explore changes in the expression of long non-coding RNAs (lncRNAs) in melanocytes following UVB-induced stress, and to explore if lncRNAs are involved in the synthesis of melanin.MethodsPrimary melanocytes were irradiated by 20 mJ/cm2 UVB. The MTT method was used to detect cell proliferation. Quantitative real-time PCR was carried out to analyze expression of tyrosinase (TYR) and lncRNAs. Dopa colorimetry was performed to analyze TYR activity. The expression profile of lncRNAs and mRNAs were confirmed using an Agilent Human lncRNA 4 × 180 K chip. Intracellular ROS levels were detected by flow cytometry. ROS scavenger (NAC) was employed to inhibit the ROS level. TYR mRNA expression and activity were re-analysed after transfecting of lnc-CD1D-2:1 siRNA and lnc-SGCG-5:4 siRNA in UVB-irradiated melanocytes to confirm the roles of the two lncRNAs in the synthesis of melanin. phospho-ERK, phospho-p38, and phospho-JNK expressions were detected by Western Blot.ResultsCell proliferation of the 20 mJ/cm2 UVB-irradiated melanocytes decreased to 91% of that of the control cells. Twenty-four hours after irradiation with 20 mJ/cm2 UVB, TYR mRNA expression and activity of the irradiated cells were significantly increased relative to the control group. Chip detection data showed that after irradiation with 20 mJ/cm2 UVB, the expression of 807 lncRNAs and 69 stress response-related genes had changed by more than two-fold. Expression levels of Lnc-GKN2-1:1, lnc-CD1D-2:1, and lnc-SGCG-5:4 and ROS content were significantly increased after UVB irradiation. NAC reduced UVB-induced ROS generation and inhibited UVB-induced upregulation of lnc-GKN2-1:1 and lnc-CD1D-2:1. Lnc-CD1D-2:1 siRNA significantly suppressed the UVB-induced TYR mRNA expression and tyrosinase activation. Lnc-CD1D-2:1 siRNA inhibited UVB-induced p38 phosphorylation.ConclusionsLncRNAs in melanocytes undergo significant changes following irradiation with 20 mJ/cm2 UVB, suggestting that lncRNAs participate in the UVB-induced stress response. Some lncRNAs expression changes induced by UVB are dependent on ROS generation. ROS-mediated production of lnc-CD1D-2:1 may be involved in the melanogenesis induced by UVB.