中华皮肤科杂志 ›› 2020, Vol. 53 ›› Issue (10): 801-806.doi: 10.35541/cjd.20200180

• 论著 • 上一篇    下一篇

miRNA-188-5p表达下调通过调控PTEN/Akt途径抑制皮肤鳞状细胞癌细胞的增殖和侵袭能力

程赛1    夏永华1    张孟杰1    张彩凤2    刘冬1    胡华1    杨留中3   

  1. 1新乡医学院第一附属医院皮肤性病科，河南卫辉  453100；2新乡医学院第一附属医院消化内科，河南卫辉  453100；3新乡医学院第一附属医院肿瘤科，河南卫辉  453100
  • 收稿日期:2020-02-26 修回日期:2020-07-22 发布日期:2020-09-30
  • 通讯作者: 夏永华 E-mail:751195651@qq.com
  • 基金资助:
    河南省教育厅科技攻关项目（2011A320017）；新乡医学院博士科研项目启动基金（xyyfy2019BS-001）

Downregulation of microRNA-188-5p suppresses proliferative and invasive abilities of cutaneous squamous cell carcinoma cells by regulating the PTEN/Akt pathway

Cheng Sai1, Xia Yonghua1, Zhang Mengjie1, Zhang Caifeng2, Liu Dong1, Hu Hua1, Yang Liuzhong3   

  1. 1Department of Dermatology and Venereology, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, Henan, China; 2Department of Gastroenterology, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, Henan, China; 3Department of Oncology, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, Henan, China
  • Received:2020-02-26 Revised:2020-07-22 Published:2020-09-30
  • Contact: Xia Yonghua E-mail:751195651@qq.com
  • Supported by:
    Science and Technology Project of Henan Education Department （2011A320017）; Start?up Fund for MD. Research in Xinxiang Medical College （xyxyfy2019BS-001）

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摘要： 【摘要】 目的 探讨miRNA-188-5p（miR-188-5p）在皮肤鳞状细胞癌（鳞癌）组织和细胞中的表达，分析其表达下调对皮肤鳞癌细胞增殖和侵袭能力的影响。方法 2012年11月至2018年10月在河南省新乡医学院第一附属医院收集50例手术切除的皮肤鳞癌组织及50例癌旁正常皮肤组织。实时荧光定量PCR（qPCR）检测皮肤鳞癌组织、癌旁正常皮肤组织、皮肤鳞癌细胞系SCL-1、A431、HSC-5和人永生化角质形成细胞株HaCaT细胞中miR-188-5p的表达。培养的A431和HSC-5细胞分别分为2组：miR-188-5p抑制剂组和阴性对照组，对转染miR-188-5p抑制剂的细胞和阴性对照组细胞，通过qPCR检测miR-188-5p的相对表达（2-△△Ct），并以CCK8法和Transwell小室分别检测各组细胞的增殖活性和侵袭能力。双荧光素酶报告实验检测miR-188-5p和PTEN的相互作用，Western印迹法检测PTEN、总Akt（t-Akt）和磷酸化Akt（p-Akt）的表达。两独立样本比较采用t检验。结果 皮肤鳞癌组织中miR-188-5p的相对表达（5.213 ± 3.138）显著高于癌旁正常皮肤组织（1.010 ± 0.364，t = 9.187，P < 0.001）。SCL-1、A431和HSC-5细胞中miR-188-5p的表达（3.858 ± 0.163、7.068 ± 0.262和4.572 ± 0.413）均显著高于HaCaT细胞（1.079 ± 0.300，t = 17.890、21.110和8.737，均P < 0.05）。与阴性对照组相比，miR-188-5p抑制剂组A431和HSC-5细胞miR-188-5p表达显著下调（均P < 0.01），细胞增殖和侵袭能力下降（均P < 0.05）。双荧光素酶报告实验显示，miR-188-5p表达下调显著上调A431和HSC-5细胞中PTEN的表达，但抑制p-Akt的表达。结论 miR-188-5p在皮肤鳞癌组织和细胞中高表达，且miR-188-5p表达下调可能通过调控PTEN/Akt途径，抑制皮肤鳞癌细胞增殖活性和侵袭能力。

关键词: 癌, 鳞状细胞, 微RNAs, 细胞增殖, 细胞侵袭, 微小RNA-188-5p, PTEN

Abstract: 【Abstract】 Objective To determine the expression of microRNA-188-5p（miR-188-5p） in cutaneous squamous cell carcinoma （CSCC） tissues and cells, and to assess the effect of its downregulation on the proliferation and invasion of CSCC cells. Methods From November 2012 to October 2018, 50 surgically resected CSCC tissue specimens and 50 paracancerous normal skin tissue specimens were collected from the First Affiliated Hospital of Xinxiang Medical College in Henan Province. Real-time fluorescence-based quantitative PCR （qPCR） was employed to determine the expression of miR-188-5p in CSCC tissues, paracancerous normal skin tissues, CSCC cell lines SCL-1, A431 and HSC-5, and a human immortalized keratinocyte line HaCaT. Cultured A431 and HSC-5 cells were both divided into 2 groups: miR-188-5p inhibitor group and negative control group, which were transfected with a miR-188-5p inhibitor and its negative control respectively. Then, qPCR was performed to determine the relative expression level of miR-188-5p （expressed as 2-△△Ct）, and cell counting kit-8 （CCK8） and Transwell assays were conducted to assess cellular proliferative activity and invasive ability respectively in the above groups. Dual-luciferase reporter assay was performed to investigate interactions between miR-188-5p and phosphatase and tensin homologue deleted on chromosome 10 （PTEN）, and Western blot analysis to determine the protein expression of PTEN, total Akt （t-Akt） and phosphorylated Akt （p-Akt）. Two independent samples were compared by using t test. Results The relative expression level of miR-188-5p was significantly higher in the CSCC tissues （5.213 ± 3.138） than in the paracancerous normal skin tissues （1.010 ± 0.364, t = 9.187, P < 0.001）, and significantly higher in the SCL-1, A431 and HSC-5 cells （3.858 ± 0.163, 7.068 ± 0.262 and 4.572 ± 0.413, respectively） than in the HaCaT cells （1.079 ± 0.300, t = 17.890, 21.110 and 8.737, respectively, all P < 0.05）. Compared with the negative control group, the miR-188-5p inhibitor group showed significantly decreased miR-188-5p expression in both A431 and HSC-5 cells （both P < 0.01）, and decreased proliferative activity and invasive ability of both A431 and HSC-5 cells (all P < 0.05）. Dual-luciferase reporter assay showed that the downregulation of miR-188-5p significantly increased the expression of PTEN, but inhibited the expression of p-Akt in A431 and HSC-5 cells. Conclusion MiR-188-5p is highly expressed in CSCC tissues and cells, and the downregulation of miR-188-5p may inhibit the proliferative activity and invasive ability of CSCC cells by regulating the PTEN/Akt pathway.

Key words: Carcinoma, squamous cell, MicroRNAs, Cell proliferation, Cell invasion, microRNA-188-5p, PTEN