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  

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

作　　者：郑云鹏[1] 李旭阳[1] 蔡丙杰[1] 李冬芹[1] 尹光文[1] Zheng Yunpeng;Li Xuyang;Cai Bingjie;Li Dongqin;Yin Guangwen(Department of Dermatology,The First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院皮肤科,450052

出　　处：《中华皮肤科杂志》2020年第6期415-423,共9页Chinese Journal of Dermatology

基　　金：河南省医学科技攻关计划项目(SB201901040)。

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�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 g

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 

分 类 号：R739.5[医药卫生—肿瘤]