Chinese Journal Of Cell And Stem Cell >Archive >2020 >Volume 10 Number 3 June 2020 >Original Researches >contents

Journal content

Effects of MiR-363-3p targeting TWIST1 on the migration and invasion of A549 cells by regulating epithelial-mesenchymal transition

Li Yuehua, Cao Shuang


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【Abstract】 Objective To explore the molecular mechanism of miR-363-3p targeting TWIST1 regulates migration and invasion of non-small cell lung cancer A549 cells. Methods A549 cells cultured in vitro were divided into the blank control group (without any treatment), mimic control group (transfected with miR-363-3p mimic negative control), mimic group (transfected with miR- 363- 3p mimic). Different groups were set up in the later experiment, including mimic+pcDNA group (transfected with miR-363-3p mimic and pcDNA3.1 empty vector plasmid), mimic+TWIST1 group (transfected with miR-363-3p mimic and pcDNA3.1 TWIST1 overexpression plasmid).The expression of miR-363-3p was detected by RT-PCR. The expression of TWIST1 protein and epithelial-mesenchymal transition (EMT)-related proteins Vimentin and E-cadherin were detected by Western blot, and the migration and invasion of cells were detected by Transwell chamber. The targeting relationship between miR-363-3p and TWIST1 was examed by dual luciferase reporter gene assay. Independent sample t-test was used for comparison between the two groups, single factor analysis of variance was used for comparison between multiple groups, and LSD-t test was used for comparison between the two groups. Results Compared with the blank control group and mimic control group, the expression of miR-363-3p (1.00±0.08, 0.97±0.05 vs 3.82±0.45)in A549 cells of mimic group were increased, and the TWIST1 mRNA and protein expression levels were decreased (1.00 ±0.06, 0.98±0.06 vs 0.39±0.02), (0.81±0.05, 0.78±0.06 vs 0.42±0.02), the difference was statistically significant (P < 0.05). Compared with the blank control group and the mimic control group, the expression level of Vimentin protein in the mimic group A549 cells as well as the number of migration cells (85.75±5.45, 83.52±6.85 vs 53.05±4.50)and invasive cells (128.26±6.15, 125.95±8.05 vs 71.64±5.75) were decreased (0.58±0.04, 0.55±0.05 vs 0.36±0.03), while the expression level of E-cadherin protein was increased (0.22±0.02, 0.25±0.03 vs 0.47±0.03), the difference was statistically significant (all P < 0.05). Compared with the mimic control group, the expression level of Vimentin protein in the simulant group cells (0.55±0.05 vs 0.36±0.03)and the number of migrating cells (83.52±6.85 vs 53.05±4.50)as well as invasive cells (125.95±8.05 vs 71.64±5.75)were decreased, while the expression level of E-cadherin protein (0.25±0.03 vs 0.47±0.03)was increased(all P < 0.05). TWIST1 is a potential target gene of miR-363-3p. Compared with the mimic+pcDNA3.1 group, the expression level of Vimentin protein in A549 cells in the mimic+TWIST1 group was increased (0.32±0.02 vs 0.42±0.03), while the expression level of E-cadherin protein (0.56±0.04 vs 0.38±0.03) was decreased. The migration number(49.45±4.22 vs 67.52±5.05)and invasion number (72.45±5.73 vs 108.35±6.56)of A549 cells were increased (all P < 0.05). Conclusion MiR-363-3p could inhibit migration and invasion of A549 cells by regulating EMT through targeting TWIST1.
 

【Key words】 MiR-363-3p; Non-small cell lung cancer; EMT; Migration; Invasion;  TWIST1