[SOX2-OT/SOX2 axis regulates lung cancer H520 cell migration via Gli1-mediated epithelial-mesenchymal transition]

Abstract

Objective: To explore the regulatory role of SOX2-OT in migration of lung squamous cell carcinoma H520 cells and the underlying mechanisms.

Methods: Wound- healing and Transwell migration assays were performed to examine the changes in migration and invasion capacity of lung squamous cell line H520, which expressed higher levels of SOX2-OT than other lung cancer cell lines, following RNA interference-mediated SOX2-OT knockdown. The transcription levels of epithelial-mesenchymal transition (EMT)-related components was detected by qRT-PCR and immunoblotting. Gli1 gain-of-function analysis was performed in H520 cells with SOX2-OT knockdown and the changes in EMT phenotype of the cells were examined. miR-200c mimic and inhibitor were used to analyze the mechanism by which SOX2-OT positively regulates Gli1 and the mediating role of SOX2.

Results: SOX2-OT knockdown significantly lowered the invasiveness and migration capacity of H520 cells and caused changes in EMT phenotype of the cells. Overexpression of Gli1, which was positively regulated by SOX2-OT, reversed the inhibitory effect of SOX2-OT knockdown on migration of H520 cells. Transfection of the cells with miR-200c inhibitor effectively reversed SOX2-OT knockdown-induced down-regulation of SOX2.

Conclusion: The SOX2-OT/SOX2 axis positively regulates migration of lung squamous H520 cells via Gli1-mediated EMT.

Keywords: Gli1; SOX2; SOX2-OT; epithelial-mesenchymal transition; lung squamous cell carcinoma.

Figure 1

Expression level of SOX2-OT mRNA in lung squamous cell carcinoma (LUSC) specimens and in different lung cancer cell lines. A: Scatter plot generated by GEPIA for comparing transcription levels of SOX2-OT between 486 LUSC and 338 adjacent normal tissues. B: qRT-PCR analysis showing aberrantly higher SOX2-OT expression in H520 cells than in the other cell lines. C: SOX2-OT transcription level in H520 cells after transfection with two shRNAs (#4 and #5) for SOX2-OT knockdown and the empty vector (SHV). β-actin was used as the internal control. All data are presented as Mean±SD if not specified otherwise. *P < 0.05, **P < 0.01.

Figure 2

Effect of SOX2-OT knockdown on invasive capacity of H520 cells. SOX2-OT knockdown significantly inhibits metastatic capacity of H520 cells as shown by wound healing assay (A, B; original magnification: ×100) and attenuates invasiveness of the cells as shown by Transwell assay (C, D; ×200). **P < 0.01.

Figure 3

Changes of expressions of EMT-related genes inH520 cells with SOX2-OT knockdown detected by qRT-PCR (A) and Western blotting (B, C) with β-actin and α- tubulin as the inner control, respectively. *P < 0.05, **P < 0.01.

Figure 4

Effect of modification of SOX2-OT expression on Gli1 expression in H520 cells. SOX2-OT knockdown in H520 cells down-regulates expressions of Gli1 mRNA and protein detected with qRT-PCR (A) and Western blotting (B, C). Transfection with Gli1-overexpressing plasmid (pcDNA3.1-Gli1) counteracts the inhibitory effect of SOX2-OT knockdown shown by qRT-PCR (D) and Western blotting (E, F). Cells co-transfected with SHV and pcDNA3.1 backbone served as the control. **P < 0.01.

Figure 5

Gli1 overexpression attenuates the inhibitory effect of SOX2-OT knockdown on cell invasion and EMT-related phenotype in H520 cells. A, B: Wound healing assay of H520 cells transfected with both shRNA against SOX2-OT (#4) and pcDNA 3.1- Gli1 (× 100). C, D: Transwell assay of H520 cells of H520 cells transfected with both shRNA against SOX2-OT (#4) and pcDNA 3.1-Gli1 (×200). E: mRNA expression levels of vimentin, Snail and E-cadherin in H520 cells with different treatments detected with qRT-PCR. **P < 0.01.

Figure 6

SOX2-OT positively modulates SOX2 by sponging miR-200c in H520 cells. A: Bioinformatic analysis of the binding sites of miR-200c on SOX2 and SOX2-OT predicted by StarBase. B: Transcription of SOX2 and SOX2-OT is inhibited by miR-200c mimic and elevated by miR-200c inhibitor. C: miR- 200c inhibitor counteracts SOX2-OT knockdown-induced downregulation of SOX2 and SOX2-OT. D: Expressions of SOX2 and SOX2-OT are positively correlated based on data from cBioPortal database. E: SOX2 overexpression attenuated SOX2-OT knockdown-induced inhibition of GLI1 expression. **P < 0.01.