lncRNA DLX6-AS1通过靶向miR-16-5p/NUCKS1调控皮肤鳞状细胞癌细胞A431增殖、迁移和侵袭  

lncRNA DLX6-AS1 regulates the proliferation,migration and invasion of a cutaneous squamous cell carcinoma cell line A431 by targeting miR-16-5p/NUCKS1

作　　者：郑云鹏[1] 蔡丙杰[1] 李旭阳[1] 李冬芹[1] 尹光文[1] Zheng Yunpeng;Cai Bingjie;Li Xuyang;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8期607-615,共9页Chinese Journal of Dermatology

基　　金：郑州大学第一附属医院院内青年创新基金。

摘　　要：目的研究长链非编码生长停滞特异性蛋白6反义RNA1(lncRNA DLX6-AS1)对皮肤鳞状细胞癌细胞A431增殖、迁移和侵袭的影响及潜在机制。方法以双荧光素酶报告系统实验验证lncRNA DLX6-AS1与miR-16-5p、miR-16-5p与核酪蛋白激酶和细胞周期蛋白依赖性激酶底物1(NUCKS1)mRNA的靶向结合。通过单独或联合转染lncRNA DLX6-AS1抑制物(si-DLX6-AS1)、miR-16-5p抑制物(anti-miR-16-5p)、NUCKS1抑制物(si-NUCKS1)和相应对照(DLX6-AS1-NC、anti-miR-NC、NUCKS1-NC)调控A431细胞目的基因的表达,采用qRT-PCR检测A431细胞lncRNA DLX6-AS1、miR-16-5p、NUCKS1 mRNA的表达;Western印迹检测NUCKS1、细胞周期蛋白D1(Cyclin D1)抗体、基质金属蛋白酶(MMP)2和MMP9蛋白水平;CCK8实验检测细胞存活率;Transwell实验检测细胞迁移和侵袭能力。两组间比较采用独立样本t检验。结果双荧光素酶报告系统实验显示,lncRNA DLX6-AS1靶向结合miR-16-5p,miR-16-5p靶向结合NUCKS1。si-DLX6-AS1组A431细胞miR-16-5p表达水平(3.01±0.31)高于DLX6-AS1-NC组(1.02±0.10,t=18.33,P<0.001);NUCKS1、Cyclin D1、MMP2和MMP9蛋白相对水平均低于DLX6-AS1-NC组(均P<0.05),细胞存活率、迁移和侵袭细胞数亦低于DLX6-AS1-NC组(均P<0.05)。si-NUCKS1组A431细胞NUCKS1、Cyclin D1、MMP2和MMP9蛋白相对水平均低于NUCKS1-NC组(均P<0.05),细胞存活率、迁移和侵袭细胞数亦低于NUCKS1-NC组(均P<0.05)。低表达lncRNA DLX6-AS1后,anti-miR-16-5p组A431细胞miR-16-5p表达(0.34±0.04)低于anti-miR-NC组(1.00±0.12,t=15.65,P<0.05),Cyclin D1、MMP2和MMP9相对表达水平均高于anti-miR-NC组(均P<0.05),细胞存活率、迁移和侵袭细胞数均高于anti-miR-NC组(均P<0.05)。低表达lncRNA DLX6-AS1、敲减miR-16-5p后,si-NUCKS1组A431细胞NUCKS1、Cyclin D1、MMP2和MMP9蛋白相对水平均低于NUCKS1-NC组(P<0.05),细胞存活率、迁移和侵袭细胞数均低于NUCKS1-NC组(P<0.05)。结论lncRNA DLX6-AS1可通过靶向miR-16-5p/NUCKS1调控A431细胞增殖、迁移�Objective To investigate effects of long non-coding growth stasis specific protein 6 antisense RNA1(lncRNA DLX6-AS1)on the proliferation,migration and invasion of a cutaneous squamous cell carcinoma cell line A431,and to explore the underlying mechanisms.Methods A dual-luciferase reporter system was used to verify the targeting relationship between lncRNA DLX6-AS1 and miR-16-5p,as well as between miR-16-5p and nuclear ubiquitous casein and cyclin-dependent kinase substrate 1(NUCKS1)mRNA.Cultured A431 cells were divided into several groups:si-DLX6-AS1 group and DLX6-AS1-NC group transfected with lncRNA DLX6-AS1 inhibitor and its negative control respectively;anti-miR-16-5p group and anti-miR-NC group transfected with miR-16-5p inhibitor and its negative control respectively;si-NUCKS1 group and NUCKS1-NC group transfected with NUCKS1 inhibitor and its negative control respectively;si-DLX6-AS1+anti-miR-16-5p group transfected with lncRNA DLX6-AS1 inhibitor followed by miR-16-5p inhibitor,and si-DLX6-AS1+anti-miR-NC group transfected with lncRNA DLX6-AS1 inhibitor followed by anti-miR-NC;si-DLX6-AS1+anti-miR-16-5p+si-NUCKS1 group transfected with lncRNA DLX6-AS1 inhibitor,miR-16-5p inhibitor and NUCKS1 inhibitor,and si-DLX6-AS1+anti-miR-16-5p+NUCKS1-NC group transfected with lncRNA DLX6-AS1 inhibitor,miR-16-5p inhibitor and NUCKS1-NC.After the above treatment,real-time fluorescence-based quantitative PCR(qRT-PCR)was performed to measure the mRNA expression of lncRNA DLX6-AS1,miR-16-5p and NUCKS1 in A431 cells,Western blot analysis to determine the protein expression of NUCKS1,Cyclin D1 antibody,matrix metalloproteinase(MMP)2 and MMP9,cell counting kit-8(CCK8)assay to detect cell survival rate,and Transwell assay to evaluate cell migratory and invasive abilities.Two-independent-sample t test was used for comparisons between two groups.Results Dual-luciferase reporter assay showed targeted binding of lncRNA DLX6-AS1 to miR-16-5p,as well as of miR-16-5p to NUCKS1.Compared with the DLX6-AS1-NC group,the si-DLX6-AS1 group

关 键 词：肿瘤 鳞状细胞 RNA 非翻译小片段 细胞增殖 细胞凋亡 肿瘤侵润 lncRNA DLX6-AS1 miR-16-5p NUCKS1 

分 类 号：R739.5[医药卫生—肿瘤]