title: Analysis of lncRNAs expression in UVB-induced stress responses of melanocytes.
abstract: BACKGROUND
  Long 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.
  
  
  OBJECTIVE
  To 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.
  
  
  METHODS
  Primary melanocytes were irradiated by 20mJ/cm(2) 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 4×180K 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.
  
  
  RESULTS
  Cell proliferation of the 20mJ/cm(2) UVB-irradiated melanocytes decreased to 91% of that of the control cells. Twenty-four hours after irradiation with 20mJ/cm(2) 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 20mJ/cm(2) 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.
  
  
  CONCLUSIONS
  LncRNAs in melanocytes undergo significant changes following irradiation with 20mJ/cm(2) 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.
authors.affiliation: Department of Plastic and Reconstructive Surgery, Third Xiangya Hospital of Central South University, Changsha 410013, China.
authors.forename: Qinghai
authors.initials: Q
authors.lastname: Zeng
authors.affiliation: Department of Oncology, Third Xiangya Hospital of Central South University, Changsha 410013, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Dermatology, Xiangya Hospital of Central South University, Changsha 410013, China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Medical Genetics, Central South University, Changsha 410078, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Xia
authors.affiliation: Institute of Medical Physics and Engineering, Department of Engineering Physics, Tsinghua University, Beijing 100084, China.
authors.forename: Jintian
authors.initials: J
authors.lastname: Tang
authors.affiliation: Department of Oncoplastic and Reconstructive Surgery, The Affiliated Tumor Hospital of Xiangya Medical School, Central South University, Changsha 410013, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhou
authors.affiliation: School of Pharmaceutical sciences of Central South University, Changsha 410013, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Genetics and Birth Health of Hunan Province, Family Planning Institute of Hunan Province, Changsha 410126, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Central Laboratory, Third Xiangya Hospital of Central South University, Changsha 410013, China.
authors.forename: Lihua
authors.initials: L
authors.lastname: Huang
authors.affiliation: Central Laboratory, Third Xiangya Hospital of Central South University, Changsha 410013, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Xiang
authors.affiliation: Department of Oncology, Third Xiangya Hospital of Central South University, Changsha 410013, China. Electronic address: caokehui168@aliyun.com.
authors.forename: Ke
authors.initials: K
authors.lastname: Cao
authors.affiliation: Department of Plastic and Reconstructive Surgery, Third Xiangya Hospital of Central South University, Changsha 410013, China. Electronic address: doctorzhoujianda@163.com.
authors.forename: Jianda
authors.initials: J
authors.lastname: Zhou
mesh_terms: D055610:Antigens, CD1d / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D049109:Cell Proliferation / Q000528:radiation effects; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000528:radiation effects; D006801:Humans; D008543:Melanins / Q000096:biosynthesis; D008544:Melanocytes / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects*; D014442:Monophenol Monooxygenase / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress / Q000528:radiation effects; D062085:RNA, Long Noncoding / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D049031:Sarcoglycans / Q000235:genetics; D014466:Ultraviolet Rays / Q000009:adverse effects*
chemical_list: D055610:Antigens, CD1d; C526877:CD1D protein, human; D002352:Carrier Proteins; C503073:GKN2 protein, human; D008543:Melanins; D062085:RNA, Long Noncoding; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C526573:SGCG protein, human; D049031:Sarcoglycans; D014442:Monophenol Monooxygenase
keywords: Melanin; Melanocytes; Melanoma; UVB; lncRNA