中华皮肤科杂志 ›› 2022, Vol. 55 ›› Issue (7): 588-595.doi: 10.35541/cjd.20210595

• 研究报道 • 上一篇    下一篇

微小RNA-181b-5p对皮肤黑素瘤细胞株增殖和侵袭的作用及机制研究

夏利1    杨林洪1    许丽2    孙文国3    于亮1    翟婉芳1    王东霞1    邝小弯1   

  1. 1桂林市人民医院皮肤科，桂林  541000；2广东省第二人民医院皮肤科，广州  510220；3桂林医学院附属医院泌尿外科，桂林  541000
  • 收稿日期:2021-08-18 修回日期:2022-05-07 发布日期:2022-07-05
  • 通讯作者: 许丽 E-mail:sunny32844@163.com
  • 基金资助:
    广西壮族自治区卫计委自筹经费科研项目;桂林医学院中青年教职工科研能力提升项目;广西壮族自治区卫健委自筹经费科研项目

Effect of microRNA-181b-5p on the proliferation and invasion of cutaneous melanoma cells and its mechanisms

Xia Li1, Yang Linhong1, Xu Li2, Sun Wenguo3, Yu Liang1, Zhai Wanfang1, Wang Dongxia1, Kuang Xiaowan1   

  1. 1Department of Dermatology, Guilin People′s Hospital, Guilin 541000, Guangxi, China; 2Department of Dermatology, Guangdong Second Provincial General Hospital, Guangzhou 510220, China; 3Department of Urology, Affiliated Hospital of Guilin Medical College, Guilin 541000, Guangxi, China
  • Received:2021-08-18 Revised:2022-05-07 Published:2022-07-05
  • Contact: Xu Li E-mail:sunny32844@163.com
  • Supported by:
    Guangxi Zhuang Autonomous Region Health and Family Planning Commission Self-funded Research Project;Young and Middle-aged Faculty Research Ability Enhancement Project of Guilin Medical College;Guangxi Zhuang Autonomous Region Health Commission Self-funded Research Project

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摘要： 【摘要】 目的 探讨微小RNA（miRNA）-181b-5p是否通过靶向普列克底物蛋白（PLEK）抑制皮肤黑素瘤（CM）细胞的增殖及侵袭能力。方法 生物信息学分析CM核心基因；双荧光素酶报告基因实验验证miRNA-181b-5p和PLEK的靶向结合。采用RNA oligo和siRNA分别调控A375细胞miRNA-181b-5p和PLEK的表达，具体分组为：模拟物阴性对照组、miRNA-181b-5p模拟物组、抑制剂阴性对照组、miRNA-181b-5p抑制剂组、PLEK siRNA组、siRNA阴性对照组、miRNA-181b-5p抑制剂和对照siRNA共转染组以及miRNA-181b-5p抑制剂和PLEK siRNA3共转染组。上述各组用相应试剂处理细胞48 h后，采用qPCR检测A375细胞中miRNA-181b-5p和PLEK mRNA的表达，Western印迹检测PLEK蛋白的表达，Transwell小室侵袭实验检测A375细胞的侵袭能力；继续培养24 ~ 96 h后，CCK8实验检测A375细胞增殖能力。结果 PLEK为CM的核心基因，CM原位癌组织较癌旁组织高表达PLEK（P = 0.031），转移组织较原位癌组织高表达PLEK（P = 0.001）。与人表皮黑素细胞HEMa-LP相比，A375细胞高表达PLEK mRNA（3.884 ± 0.156比0.997 ± 0.010，t = 18.48，P < 0.001）及PLEK蛋白（2.840 ± 0.301比1.029 ± 0.094，t = 5.47，P = 0.005），低表达miRNA-181b-5p（0.333 ± 0.042比0.967 ± 0.069，t = 7.83，P = 0.001）。双荧光素酶报告基因实验显示，miRNA-181b-5p和PLEK靶向结合。与模拟物阴性对照相比，miRNA-181b-5p模拟物转染后A375细胞的存活率显著降低（48 h，t = 7.96，P = 0.015；72 h，t = 7.50，P = 0.002；96 h，t = 7.96，P = 0.001），侵袭能力也显著降低（t = 5.07，P = 0.007）；相反miRNA-181b-5p 抑制剂组A375细胞存活率高于抑制剂阴性对照组（24 h，t =5.38，P = 0.013；48 h，t = 5.36，P = 0.013；72 h，t =7.63，P = 0.005；96 h，t =5.99，P = 0.004），侵袭能力也高于抑制剂阴性对照组（t = 7.24，P = 0.002）；与对照siRNA组相比，PLEK siRNA转染后A375细胞的增殖能力降低（48、72、96 h时，P值分别为0.015、0.011、0.001），侵袭能力也降低（t = 4.93，P = 0.008）；与miRNA-181b-5p 抑制剂和对照 siRNA共转染组相比，miRNA-181b-5p 抑制剂和PLEK siRNA共转染组A375细胞的增殖率明显降低（24、48、72、96 h时，P值分别为0.042、0.042、0.037、0.017），侵袭能力也明显降低（t = 8.52，P = 0.001）。结论 miRNA-181b-5p抑制A375细胞增殖和侵袭能力，其机制涉及靶向下调PLEK的表达。

关键词: 黑色素瘤, 实验性, 微RNAs, 细胞增殖, 肿瘤侵润, miR-181b-5p, 普列克底物蛋白

Abstract: 【Abstract】 Objective To explore whether microRNA （miRNA）-181b-5p inhibits the proliferation and invasion of cutaneous melanoma cells by targeting pleckstrin （PLEK）. Methods Bioinformatics methods were used to analyze cutaneous melanoma-associated core genes; dual-luciferase reporter assay was performed to verify the targeted interaction between miRNA-181b-5p and PLEK. Oligo RNA and small interfering RNA （siRNA） were used to regulate the expression of miRNA-181b-5p and PLEK in A375 cells respectively in this experiment, and A375 cells were divided into the following groups in detail: mimic negative control group, miRNA-181b-5p mimic group, inhibitor negative control group, miRNA-181b-5p inhibitor group, PLEK siRNA group, siRNA negative control group, miRNA-181b-5p inhibitor + control siRNA co-transfection group and miRNA-181b-5p inhibitor + PLEK siRNA3 co-transfection group. After 48-hour treatment, qPCR was performed to determine the mRNA expression of miRNA-181b-5p and PLEK in A375 cells, Western blot analysis to determine the PLEK protein expression, and Transwell assay to assess the invasive ability of A375 cells; after additional 24-96 hours of culture, cell counting kit-8 (CCK8) assay was conducted to assess the proliferative ability of A375 cells. Results PLEK was the core gene for cutaneous melanoma. PLEK expression in the cutaneous melanoma in situ tissues was significantly higher than that in the paracancerous tissues （P = 0.031）, but lower than that in the metastatic tissues （P = 0.001）. Compared with human epidermal melanocytes HEMa-LP, the mRNA and protein expression of PLEK significantly increased in A375 cells （mRNA: 3.884 ± 0.156 vs. 0.997 ± 0.010, t = 18.48, P < 0.001; protein: 2.840 ± 0.301 vs. 1.029 ± 0.094, t = 5.47, P = 0.005）, but the miRNA-181b-5p expression significantly decreased in A375 cells （0.333 ± 0.042 vs. 0.967 ± 0.069, t = 7.83, P = 0.001）. Dual-luciferase reporter assay showed targeted binding of miRNA-181b-5p to PLEK. Compared with the mimic negative control group, the miRNA-181b-5p mimic group showed significantly decreased survival rate of A375 cells （48 hours: t = 7.96, P = 0.015; 72 hours: t = 7.50, P = 0.002; 96 hours: t = 7.96, P = 0.001）, and significantly decreased invasive ability of A375 cells（t = 5.07, P = 0.007）; on the contrary, the survival rate and invasive ability of A375 cells were significantly higher in the miRNA-181b-5p inhibitor group than in the inhibitor negative control group （survival rate: 24 hours, t =5.38, P = 0.013; 48 hours, t = 5.36, P = 0.013; 72 hours, t =7.63, P = 0.005; 96 hours, t = 5.99, P = 0.004; invasive ability: t = 7.24, P = 0.002）; compared with the siRNA negative control group, the proliferative and invasive ability of A375 cells significantly decreased in the PLEK siRNA group （proliferative ability: 48, 72, 96 hours, P = 0.015, 0.011, 0.001, respectively; invasive ability: t = 4.93, P = 0.008）; compared with the miRNA-181b-5p inhibitor + control siRNA co-transfection group, the miRNA-181b-5p inhibitor + PLEK siRNA co-transfection group showed significantly decreased proliferation rate and invasive ability of A375 cells （proliferation rate: 24, 48, 72, 96 hours, P = 0.042, 0.042, 0.037, 0.017, respectively; invasive ability: t = 8.52, P = 0.001）. Conclusion miRNA-181b-5p can inhibit the proliferation and invasion of cutaneous melanoma A375 cells, likely by down-regulating the PLEK expression.

Key words: Melanoma, experimental, MicroRNAs, Cell proliferation, Neoplasm invasiveness, miR-181b-5p, Pleckstrin