Both in vivo and in vitro studies revealed that EZR-AS1 promoted cell migration through up-regulation of EZR expression. Mechanistically, antisense lncRNA EZR-AS1 formed a complex with RNA polymerase II to activate the transcription of EZR. Moreover, EZR-AS1 could recruit SMYD3 to a binding site, present in a GC-rich region downstream of the EZR promoter, causing the binding of SMYD3 and local enrichment of H3K4me3. Finally, the interaction of EZR-AS1 with SMYD3 further enhanced EZR transcription and expression. Our findings suggest that antisense lncRNA EZR-AS1, as a member of an RNA polymerase complex and through enhanced SMYD3-dependent H3K4 methylation, plays an important role in enhancing transcription of the EZR gene to promote the mobility and invasiveness of human cancer cells.EZR-AS1 regulates EZR transcriptional activity in ESCC cells by interacting with RNA polymerase II, but not by affecting the binding of SP1 and AP-1 transcription factors. the overexpression of EZR-AS1 accompanied the enhanced expression of EZR (variant 1), both at the mRNA and protein levels, in KYSE150 cells (Figure 1H and I).