中华皮肤科杂志 ›› 2019, Vol. 52 ›› Issue (7): 494-497.doi: 10.3760/cma.j.issn.0412-4030.2019.07.010

• 研究报道 • 上一篇    下一篇

微小RNA-373表达下调对皮肤鳞状细胞癌A431细胞周期和凋亡的影响

夏永华1    张彩凤2    李敏1    刘冬1    胡华1    张孟杰1    程赛1   

  1. 1新乡医学院第一附属医院皮肤科，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
  • 收稿日期:2018-07-05 修回日期:2019-01-06 出版日期:2019-07-15 发布日期:2019-07-01
  • 通讯作者: 夏永华 E-mail:751195651@qq.com
  • 基金资助:
    河南省教育厅科学技术研究重点项目（2011A320017）; 新乡医学院科研培育基金（2013QN118）

Effect of downregulation of microRNA-373 expression on cell cycle and apoptosis of a cutaneous squamous cell carcinoma cell line A431

Xia Yonghua1, Zhang Caifeng2, Li Min1, Liu Dong1, Hu Hua1, Zhang Mengjie1, Cheng Sai1   

  1. 1Department of Dermatology, 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
  • Received:2018-07-05 Revised:2019-01-06 Online:2019-07-15 Published:2019-07-01
  • Contact: Xia Yonghua E-mail:751195651@qq.com
  • Supported by:
    Key Program for Science and Technology Research of Henan Educational Committee （2011A320017）; Scientific Research Cultivation Fund of Xinxiang Medical University （2013QN118）

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摘要： 【摘要】 目的 探讨微小RNA-373（miR-373）表达下调对皮肤鳞状细胞癌（鳞癌）A431细胞周期和凋亡的影响。方法 将对数生长期A431细胞分为3组，miR-373抑制剂组和阴性对照组分别转染miR-373抑制剂和阴性对照miRNA，未处理组不做任何处理。转染后48 h，采用实时PCR检测各组miR-373的表达水平；采用CCK-8试剂分析miR-373表达下调对A431细胞增殖的影响，流式细胞仪检测不同处理组A431细胞周期分布和凋亡的变化，最后采用比色法检测各组胱天蛋白酶3（caspase-3）活性的变化。两组之间数据比较采用t检验，3组及以上采用单因素方差分析，两两组间比较采用LSD-t检验。结果 miR-373抑制剂组miR-373表达水平为0.120 ± 0.036，显著低于未处理组（1.002 ± 0.022）和阴性对照组（1.037 ± 0.028），t值分别为36.21、34.83，均P < 0.001。miR-373抑制剂组在48、72和96 h细胞增殖活性均显著低于未处理组和阴性对照组，F值分别为10.805、13.720和30.907，P值分别为0.038、0.010和0.001）。miR-373抑制剂组G0/G1期比例为64.69% ± 1.18%，显著高于未处理组（52.74% ± 0.66%）和阴性对照组（53.80% ± 0.80%），t值分别为15.51和13.24，均P < 0.001；总凋亡细胞比例为22.69% ± 1.24%，显著高于未处理组（9.62% ± 1.14%）和阴性对照组（9.66% ± 0.97%），均P < 0.001；caspase-3活性为1.238 ± 0.057，显著高于未处理组（0.413 ± 0.028）和阴性对照组（0.437 ± 0.036）（均P < 0.001）。结论 miR-373可能在皮肤鳞癌细胞周期调控和凋亡诱导中发挥重要作用。

关键词: 肿瘤, 鳞状细胞； 微RNAs； 细胞周期； 细胞凋亡； 半胱氨酸天冬氨酸蛋白酶3； 微小RNA-373

Abstract: 【Abstract】 Objective To evaluate the effect of downregulation of microRNA （miR）-373 expression on cell cycle and apoptosis of a cutaneous squamous cell carcinoma （CSCC） cell line A431. Methods A431 cells at exponential growth phase were classified into 3 groups: miR-373 inhibitor group and negative control group transfected with miR-373 inhibitor and negative control miRNA respectively, and untreated group receiving no treatment. At 48 hours after the transfection, real-time PCR was performed to determine the expression of miR-373 in the above 3 groups, cell counting kit-8 （CCK-8） assay to evaluate the effect of downregulated expression of miR-373 on the proliferation of A431 cells, flow cytometry to investigate the distribution of cell cycle and changes in apoptosis of A431 cells in different treatment groups, and colorimetric analysis to detect the changes in caspase-3 activity in different treatment groups. Statistical analysis was carried out with SPSS 17.0 software by using two-sample t test for the comparison between two groups, one-way analysis of variance （ANOVA） for the comparison among 3 groups, and least significant difference （LSD）-t test for multiple comparisons. Results The expression of miR-373 was significantly lower in the miR-373 inhibitor group （0.120 ± 0.036） than in the untreated group （1.002 ± 0.022） and negative control group （1.037 ± 0.028, LSD-t = 36.21，34.83, respectively, both P < 0.001）. At 48, 72 and 96 hours, the miR-373 inhibitor group showed significantly decreased proliferative activity of A375 cells compared with the untreated group and negative control group （F = 10.805, 13.720 and 30.907 respectively, P = 0.038, 0.010 and 0.001 respectively）. The proportion of A375 cells in G0/G1 phase was significantly higher in the miR-373 inhibitor group （64.69% ± 1.18%） than in the untreated group （52.74% ± 0.66%, t = 15.51, P < 0.001） and negative control group （53.80% ± 0.80%, t = 13.24, P < 0.001）. The proportion of total apoptotic cells and activity of caspase-3 in the miR-373 inhibitor group were 22.69% ± 1.24% and 1.238 ± 0.057 respectively, which were significantly higher than those in the untreated group （9.62% ± 1.14%, 0.413 ± 0.028 respectively, both P < 0.001）and negative control group （9.66% ± 0.97%, 0.437 ± 0.036 respectively, both P < 0.001）. Conclusion MiR-373 may play an important role in the regulation of cell cycle and induction of apoptosis of the CSCC cell line A431.

Key words: Neoplasms, squamous cell, MicroRNAs, Cell cycle, Apoptosis, Caspase 3, microRNA-373