中华皮肤科杂志 ›› 2020, Vol. 53 ›› Issue (6): 415-423.doi: 10.35541/cjd.20191053

• 论著 • 上一篇    下一篇

lncRNA LEF1-AS1通过靶向miR-612调控皮肤鳞状细胞癌细胞株增殖、凋亡、迁移侵袭的体外实验研究

郑云鹏    李旭阳    蔡丙杰    李冬芹    尹光文   

  1. 郑州大学第一附属医院皮肤科  450052
  • 收稿日期:2019-11-06 修回日期:2020-03-27 发布日期:2020-06-01
  • 通讯作者: 尹光文 E-mail:gwyin67@126.com
  • 基金资助:
    河南省医学科技攻关计划项目（SB201901040）

LncRNA LEF1-AS1 regulates proliferation, apoptosis, migration and invasion of cutaneous squamous cell carcinoma cells by targeting miR-612: an in vitro experimental study

Zheng Yunpeng, Li Xuyang, Cai Bingjie, Li Dongqin, Yin Guangwen   

  1. Department of Dermatology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
  • Received:2019-11-06 Revised:2020-03-27 Published:2020-06-01
  • Contact: Yin Guangwen E-mail:gwyin67@126.com
  • Supported by:
    Medical Science and Technique Program of Henan Province （SB201901040）

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摘要： 【摘要】 目的 探讨长链非编码RNA（lncRNA）LEF1-AS1对皮肤鳞状细胞癌细胞增殖、凋亡、迁移、侵袭的影响及其机制。方法 通过干扰皮肤鳞状细胞癌SCC13细胞LEF1-AS1的表达和过表达、抑制miR-612的表达，将SCC13细胞分为转染LEF1-AS1干扰序列、无义序列的干扰LEF1-AS1组、干扰对照组，转染miR-612过表达序列、无义序列的miR-612过表达组、过表达对照组，以及转染LEF1-AS1干扰序列与miR-612抑制序列的干扰抑制组，和转染LEF1-AS1干扰序列与miR-612抑制无义序列的干扰抑制对照组。采用qRT-PCR法检测各组细胞miR-612的相对表达，CCK8法检测细胞增殖，流式细胞仪分析细胞的凋亡情况，Transwell试验检测细胞的迁移、侵袭能力，Western印迹检测细胞周期蛋白依赖激酶1（cyclinD1）、细胞周期蛋白依靠性激酶抑制剂（p21）、Bcl-2家族蛋白（Bcl-2）、Bcl-2相关X蛋白（Bax）、基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）的表达。用LncBase Predicted v.2 在线预测网站预测lncRNA LEF1-AS1与miR-612之间的互补序列，将互补/非互补序列用于构建荧光素酶报告基因质粒，分别与miR-612过表达及过表达对照基因共转染SCC13细胞，验证lncRNA LEF1-AS1与miR-612的结合能力。两组间比较采用t检验，多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。结果 miR-612过表达组细胞增殖能力、迁移及侵袭细胞数均低于过表达对照组（均P < 0.05），凋亡率高于过表达对照组（P < 0.05）。干扰LEF1-AS1组、干扰对照组、干扰抑制组、干扰抑制对照组miR-612的相对表达差异有统计学意义（F = 150.78，P < 0.001），24、48、72 h时的增殖能力差异有统计学意义（均P < 0.05），凋亡率、迁移及侵袭细胞数差异均有统计学意义（均P < 0.05）。干扰LEF1-AS1组细胞中miR-612表达、凋亡率高于干扰对照组，48、72 h细胞增殖能力、迁移细胞数和侵袭细胞数低于干扰对照组（均P < 0.05），而干扰抑制组细胞中miR-612表达、细胞凋亡率低于干扰抑制对照组，48、72 h细胞增殖能力、迁移细胞数和侵袭细胞数高于干扰抑制对照组（均P < 0.05）。Western印迹显示，4组细胞cyclinD1、p21、Bcl-2、Bax、MMP-2、MMP-9蛋白相对水平差异均有统计学意义（均P < 0.001），干扰LEF1-AS1组cyclinD1、Bcl-2、MMP-2、MMP-9蛋白相对水平均高于干扰对照组，p21、 Bax蛋白相对水平低于干扰对照组（均P < 0.05）；而干扰抑制组cyclinD1、Bcl-2、MMP-2、MMP-9蛋白相对水平均高于干扰抑制对照组，p21、 Bax蛋白相对水平低于干扰抑制对照组（均P < 0.05）。共转染互补序列后，miR-612过表达组细胞荧光活性低于过表达对照组（t = 21.19，P < 0.001）；而共转染非互补序列后，miR-612组细胞荧光活性与过表达对照组差异无统计学意义（t = 0.28，P = 0.78）。结论 lncRNA LEF1-AS1调控皮肤鳞状细胞癌细胞的增殖、凋亡、迁移和侵袭，机制与靶向miR-612相关。

关键词: 肿瘤, 鳞状细胞； RNA, 非翻译小片段； 细胞增殖； 细胞凋亡； lncRNA LEF1-AS1； miR-612

Abstract: 【Abstract】 Objective To evaluate the effects of long non-coding RNA （lncRNA） LEF1-AS1 on proliferation, apoptosis, migration and invasion of cutaneous squamous cell carcinoma cells, and to explore their mechanisms. Methods Cutaneous squamous cell carcinoma SCC13 cells were divided into si-LEF1-AS1 group transfected with lncRNA LEF1-AS1 interference oligonucleotides （si-LEF1-AS1）, si-NC group transfected with lncRNA LEF1-AS1 nonsense oligonucleotides （si-NC）, miR-612 group transfected with miR-612-overexpressing oligonucleotides, miR-NC group transfected with miR-612 nonsense oligonucleotides （miR-NC）, si-LEF1-AS1 + anti-miR-612 group transfected with si-LEF1-AS1 and oligonucleotides against miR-612, and si-LEF1-AS1 + anti-miR-NC group transfected with si-LEF1-AS1 and miR-612 nonsense oligonucleotides. Quantitative reverse transcription （qRT）-PCR was performed to determine the relative expression of miR-612 in SCC13 cells, cell counting kit-8 （CCK8） assay to evaluate cellular proliferative activity, flow cytometry to detect cell apoptosis, Transwell assay to assess migratory and invasive abilities of SCC13 cells, and Western blot analysis to determine protein expression of cyclin-dependent kinase 1 （cyclinD1）, cyclinD1 inhibitor p21, Bcl-2 family protein （Bcl-2）, Bcl-2 related X protein （Bax）, matrix metalloproteinase 2 （MMP-2） and MMP-9. The online bioinformatics database LncBase predicted v.2 was employed to predict the complementary sequence between lncRNA LEF1-AS1 and miR-612, and luciferase reporter gene plasmids were constructed by using the complementary/non-complementary sequence, which were co-transfected with miR-612-overexpressing oligonucleotides （miR-612 overexpression group） or miR-NC （overexpression control group） into SCC13 cells in order to verify the binding ability of lncRNA LEF1-AS1 to miR-612. Statistical analysis was carried out by using t test for comparison between two groups, one-way analysis of variance for comparison among multiple groups, and least significant difference （LSD）-t test for multiple comparisons. Results Compared with the miR-NC group, miR-612 group showed significantly decreased cellular proliferative ability, number of migratory cells and invasive cells （all P < 0.05）, but a significantly increased apoptosis rate （P < 0.05）. The relative expression of miR-612 （F = 150.78, P < 0.001）, cellular proliferative activity at 24, 48, 72 hours （all P < 0.05）, apoptosis rate and number of migratory and invasive cells （all P < 0.05） significantly differed among the si-LEF1-AS1 group, si-NC group, si-LEF1-AS1 + anti-miR-612 group and si-LEF1-AS1 + anti-miR-NC group. Compared with the si-NC group, the si-LEF1-AS1 group showed significantly increased expression of miR-612 and apoptosis rates, but significantly decreased cellular proliferative activity at 48, 72 hours, and number of migratory and invasive cells （all P < 0.05）; compared with the si-LEF1-AS1 + anti-miR-NC group, the si-LEF1-AS1 + anti-miR-612 group showed significantly decreased expression of miR-612 and apoptosis rates, but significantly increased cellular proliferative activity at 48, 72 hours, and number of migratory and invasive cells （all P < 0.05）. Western blot analysis showed that the relative protein expression of cyclinD1, p21, Bcl-2, Bax, MMP-2 and MMP-9 significantly differed among the si-LEF1-AS1 group, si-NC group, si-LEF1-AS1 + anti-miR-612 group and si-LEF1-AS1 + anti-miR-NC group （all P < 0.001）; compared with the si-NC group, the si-LEF1-AS1 group showed significantly increased protein expression of cyclinD1, Bcl-2, MMP-2 and MMP-9, but significantly decreased protein expression of p21 and Bax （all P < 0.05）; compared with the si-LEF1-AS1 + anti-miR-NC group, the si-LEF1-AS1 + anti-miR-612 group showed significantly increased protein expression of cyclinD1, Bcl-2, MMP-2 and MMP-9, but significantly decreased protein expression of p21 and Bax （all P < 0.05）. After co-transfection with complementary sequences, the fluorescence activity was significantly lower in the miR-612 overexpression group than in the overexpression control group （t = 21.19, P < 0.001）; after co-transfection with non-complementary sequences, no significant difference was observed in the fluorescence activity between the miR-612 overexpression group and overexpression control group （t = 0.28, P = 0.78）. Conclusion lncRNA LEF1-AS1 regulates the proliferation, apoptosis, migration and invasion of cutaneous squamous cell carcinoma cells, likely by targeting miR-612.

Key words: Neoplasms, squamous cell, RNA, small untranslated, Cell proliferation, Apoptosis, LncRNA LEF1-AS1, miR-612

引用本文

郑云鹏 李旭阳 蔡丙杰 李冬芹 尹光文. lncRNA LEF1-AS1通过靶向miR-612调控皮肤鳞状细胞癌细胞株增殖、凋亡、迁移侵袭的体外实验研究[J]. 中华皮肤科杂志, 2020,53(6):415-423. doi:10.35541/cjd.20191053

Zheng Yunpeng, Li Xuyang, Cai Bingjie, Li Dongqin, Yin Guangwen. LncRNA LEF1-AS1 regulates proliferation, apoptosis, migration and invasion of cutaneous squamous cell carcinoma cells by targeting miR-612: an in vitro experimental study[J]. Chinese Journal of Dermatology, 2020, 53(6): 415-423.doi:10.35541/cjd.20191053