LncRNA LEF1-AS1 regulates proliferation, apoptosis, migration and invasion of cutaneous squamous cell carcinoma cells by targeting miR-612: an in vitro experimental study
10.35541/cjd.20191053
- VernacularTitle: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
- Author:
Yunpeng ZHENG
1
;
Xuyang LI
;
Bingjie CAI
;
Dongqin LI
;
Guangwen YIN
Author Information
1. 郑州大学第一附属医院皮肤科 450052
- From:
Chinese Journal of Dermatology
2020;53(6):415-423
- CountryChina
- Language:Chinese
-
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.
