Figure 4.

Metastasis‐associated lung adenocarcinoma transcript 1 (MALAT1) interacts with enhancer of zeste homolog 2 (EZH2) in hepatocellular carcinoma. A, RNA pull‐down assays were carried out with biotinylated MALAT1 probe followed by western blot assay using anti‐EZH2 Ab. B, Schematic illustration of MALAT1 regions used for truncated RNA pull‐down assays. C, RNA pull‐down assays using biotinylated truncated MALAT1 confirmed that MALAT1 bound EZH2 mainly at its 3′‐end. D, Enrichment of the MALAT1 fractions in cross‐link and sonication RNA immunoprecipitation (RIP) using anti‐EZH2 Ab further validated EZH2 interacted with the 3′‐end of MALAT1. E, Anti‐EZH2 RIP was carried out after transiently overexpressing microRNA (miR)‐22 followed by detecting MALAT1 with quantitative PCR. miR‐22 did not compete with EZH2 for binding MALAT1. F, G, ChIP assays were undertaken with anti‐EZH2 and anti‐H3K27me3 Abs after knockdown of MALAT1. Knockdown of MALAT1 attenuated the enrichment of EZH2 at miR‐22 (F) and E‐cadherin (G) promoter. *P < .05, **P < .01, compared with respective control