Figure 3.

ETV5 activates MMPs through regulating SKA1 and further regulating TRPV2 in ESCC. (A)(B) Volcano Plot and Clustering analysis showed significantly upregulated and downregulated gene signature in ECA109, KYSE150, or TE1 siETV5 groups normalized to NC siRNA groups by RNA-seq. (C-F) QPCR indicated that the knockdown of ETV5 could downregulate SKA1 and TRPV2 mRNA expression in ECA109 and KYSE150 cells. (G)(H) Western blot showed that knockdown of ETV5 could downregulate SKA1 and TRPV2 protein expression in ECA109 and KYSE150 cells. (I) Chip assay revealed that a positive signal was detected via primers targeting SKA1. (J) Luciferase reporter gene assays showed that mutant at the predicted binding site of SKA1 significantly decreased the luciferase activity in ECA109 cells. (K) Chip assay revealed that a positive signal was detected via primers targeting TRPV2. (L) Luciferase reporter gene assays showed that mutant at the predicted binding site of TRPV2 significantly decreased the luciferase activity in ECA109 cells. (M)Western blot indicated that protein SKA1 and TRPV2 were higher in selected ESCC tissues than in paired normal esophageal mucosa tissues. (N) Correlation analysis indicated that ETV5 was in positive correlation with SKA1 and TRPV2 respectively. (O)(P) Western blot indicated that SKA1 knockdown markedly downregulated protein expression of MMPs in EAC109 and KYSE150 cells. (Q)(R) Western blot indicated that TRPV2 knockdown markedly downregulated protein expression of MMPs in ECA109 and KYSE150 cells. (S)(T) Western blot indicated that SKA1 and TRPV2 could significantly rescue the effects of ETV5 overexpression on MMPs in ECA109 and KYSE150 cells. *P<0.05; **P<0.01; ***P<0.001.