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

Abstract: 【Abstract】 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 showed significantly increased miR-16-5p expression in A431 cells （3.01 ± 0.31 vs. 1.02 ± 0.10, t = 18.33, P < 0.001）, but significantly decreased protein expression of NUCKS1, Cyclin D1, MMP2 and MMP9 （all P < 0.05）, and significantly decreased cell survival rate and numbers of migratory and invasive cells （all P < 0.05）. Compared with the NUCKS1-NC group, the si-NUCKS1 group showed significantly decreased protein expression of NUCKS1, Cyclin D1, MMP2 and MMP9 in A431 cells （all P < 0.05）, and significantly decreased cell survival rate and numbers of migratory and invasive cells （all P < 0.05）. After inhibition of lncRNA DLX6-AS1 expression, the si-DLX6-AS1 + anti-miR-16-5p group showed significantly decreased miR-16-5p expression in A431 cells （0.34 ± 0.04） compared with the si-DLX6-AS1 + anti-miR-NC group （1.00 ± 0.12, t = 15.65, P < 0.05）, but significantly increased protein expression of Cyclin D1, MMP2 and MMP9, cell survival rate and numbers of migratory and invasive cells compared with the si-DLX6-AS1 + anti-miR-NC group （all P < 0.05）. After inhibition of lncRNA DLX6-AS1 expression and knockdown of miR-16-5p, the si-DLX6-AS1 + anti-miR-16-5p + si-NUCKS1 group showed significantly decreased protein expression of NUCKS1, Cyclin D1, MMP2 and MMP9 in A431 cells, as well as cell survival rate and numbers of migratory and invasive cells, compared with the si-DLX6-AS1 + anti-miR-16-5p + NUCKS1-NC group （all P < 0.05）. Conclusion lncRNA DLX6-AS1 can regulate the proliferation, migration and invasion of A431 cells by targeting miR-16-5p/NUCKS1, suggesting that lncRNA DLX6-AS1 may be a potential molecular target for the treatment of cutaneous squamous cell carcinoma.

Key words: Neoplasms, squamous cell, RNA, small untranslated, Cell proliferation, Apoptosis, Neoplasm invasiveness, lncRNA DLX6-AS1, miR-16-5p, NUCKS1