中华皮肤科杂志 ›› 2023, Vol. 56 ›› Issue (1): 29-34.doi: 10.35541/cjd.20220175

• 论著 • 上一篇    下一篇

miRNA-193b-5p抑制转录调节因子CITED2表达对黑素合成的影响

杨荷丹1    丁徽1    方富民1    郑慧颖1    张晓丽1    刘杏1    葛一平1    杨寅1    林彤2   

  1. 1中国医学科学院、北京协和医学院皮肤病医院激光科，南京  210042；2中国医学科学院、北京协和医学院皮肤病医院  江苏省皮肤病与性病分子生物学重点实验室，南京  210042
  • 收稿日期:2022-03-15 修回日期:2022-07-30 发布日期:2023-01-03
  • 通讯作者: 林彤；杨寅；葛一平 E-mail:ddlin@hotmail.com; yushiyy@163.com; gypfeng@hotmail.com
  • 基金资助:
    国家自然科学基金（82103705）；中国医学科学院医学与健康科技创新工程项目（CIFMS-2021-I2M-1-001）

Effect of miRNA-193b-5p-mediated decreased expression of transcriptional regulator CITED2 on melanogenesis

Yang Hedan1, Ding Hui1, Fang Fumin1, Zheng Huiying1, Zhang Xiaoli1, Liu Xing1, Ge Yiping1, Yang Yin1, Lin Tong2   

  1. 1Department of Cosmetic Laser Surgery, Hospital of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China; 2Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Hospital of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China
  • Received:2022-03-15 Revised:2022-07-30 Published:2023-01-03
  • Contact: Lin Tong; Yang Yin; Ge Yiping E-mail:ddlin@hotmail.com; yushiyy@163.com; gypfeng@hotmail.com
  • Supported by:
    National Natural Science Foundation of China （82103705）; CAMS Innovation Fund for Medical Sciences （CIFMS-2021-I2M-1-001）

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摘要： 【摘要】 目的 初步探讨miRNA（miR）-193b-5p对黑素合成的影响及可能机制。方法 从健康男性包皮环切术后废弃的正常包皮组织中分离培养人原代黑素细胞。分别转染miR-NC模拟物（miR-NC mimics组）和miR-193b-5p模拟物（miR-193b-5p mimics组）至人原代黑素细胞及人黑素瘤MNT1细胞，转染48 h时实时定量PCR（RT-qPCR）检测miR-193b-5p的过表达效率，转染72 h时Western印迹检测黑素合成相关蛋白酪氨酸酶和小眼畸形相关转录因子的表达，转染1周时采用氢氧化钠法测定细胞中黑素含量。通过TargetScan网站预测miR-193b-5p的靶基因并采用双荧光素酶报告实验验证，RT-qPCR及Western印迹检测miR-193b-5p过表达后该靶基因mRNA和蛋白表达水平的变化。两组间比较采用两独立样本t检验。结果 人原代黑素细胞及人黑素瘤MNT1细胞中，miR-193b-5p mimics组miR-193b-5p的表达水平均显著高于miR-NC mimics组，t值分别为65.57、22.49，均P < 0.001，且miR-193b-5p mimics组黑素含量（0.091 ± 0.007、0.130 ± 0.004）显著低于miR-NC mimics组（0.117 ± 0.002、0.188 ± 0.032），t值分别为5.98、3.24，P值分别 < 0.01、 < 0.05。Western印迹检测显示，人原代黑素细胞及人黑素瘤MNT1细胞中，miR-193b-5p mimics组黑素合成相关蛋白酪氨酸酶及小眼畸形相关转录因子的表达均显著低于miR-NC mimics组（均P < 0.05）。通过TargetScan预测及双荧光素酶报告实验验证，转录调节因子CITED2的3′非翻译区存在miR-193b-5p的结合位点。人原代黑素细胞及人黑素瘤MNT1中上调miR-193b-5p表达后，CITED2 mRNA及蛋白的表达水平均显著低于miR-NC mimics组（均P＜0.05）。结论 miR-193b-5p过表达可下调黑素合成相关蛋白酪氨酸酶及小眼畸形相关转录因子的表达，抑制黑素合成，该过程与靶向抑制CITED2的表达有关。

关键词: 黑素细胞, 黑素瘤细胞, 黑素合成, CITED2, miRNA-193b-5p

Abstract: 【Abstract】 Objective To investigate the effect of miRNA （miR）-193b-5p on melanogenesis and its possible mechanisms. Methods Human primary melanocytes were isolated from discarded normal foreskin tissues of healthy males after circumcision, and cultured in vitro. miR-NC mimics （miR-NC mimic group） and miR-193b-5p mimics （miR-193b-5p mimic group） were transfected into human primary melanocytes and human MNT1 melanoma cells, separately. After transfection, real-time quantitative PCR （RT-qPCR） was performed to determine the overexpression efficiency of miR-193b-5p at 48 hours, Western blot analysis to determine the expression of melanogenesis-related proteins tyrosinase （TYR） and microphthalmia-associated transcription factor （MITF） in human primary melanocytes and human MNT1 melanoma cells at 72 hours, and the melanin content in the above cells was determined by a sodium hydroxide solubilization method at 1 week. The target gene of miR-193b-5p was predicted by using Targetscan algorithms and verified by dual-luciferase reporter assay, and RT-qPCR and Western blot analysis were performed to analyze changes in mRNA and protein expression of the target gene respectively after the overexpression of miR-193b-5p. Two-independent-samples t test was used for comparisons between two groups. Results In human primary melanocytes and human MNT1 melanoma cells, the miR-193b-5p expression levels were significantly higher in the miR-193b-5p mimic groups than in the miR-NC mimic groups （t = 65.57, 22.49, respectively, both P < 0.001）, and the melanin content was significantly lower in the miR-193b-5p mimic groups （0.091 ± 0.007, 0.130 ± 0.004, respectively） than in the miR-NC mimic groups （0.117 ± 0.002, 0.188 ± 0.032, t = 5.98, 3.24, P < 0.01, < 0.05, respectively）. Western blot analysis showed that the expression of melanogenesis-related proteins TYR and MITF in both human primary melanocytes and human MNT1 melanoma cells was significantly lower in the miR-193b-5p mimic groups than in the miR-NC mimic groups （all P < 0.01）. TargetScan analysis and dual-luciferase reporter assay revealed a binding site for miR-193b-5p in the 3′ untranslated region of the transcriptional regulator CITED2. After up-regulation of miR-193b-5p expression in human primary melanocytes and human MNT1 melanoma cells, the CITED2 mRNA and protein expression levels significantly decreased compared with the miR-NC mimic groups （all P < 0.05）. Conclusion miR-193b-5p overexpression can down-regulate the expression of melanogenesis-related proteins TYR and MITF, and then inhibit melanogenesis, which may be related to the targeted inhibition of CITED2 expression.

Key words: Melanocytes, Melanoma cells, Melanogenesis, CITED2, miRNA-193b-5p