Induction of G1 cell cycle arrest and p15^INK4b expression by ECRG1 through interaction with Miz-1

ECRG1 is a novel candidate of tumor suppressor gene identified from human esophagus. To study the biological role of ECRG1 gene, we performed a GAL4-based yeast two-hybrid screen of a human fetal liver cDNA library. Using the ECRG1 cDNA as bait, we identified two putative clones as associated proteins, Miz-1 and FLNA (Filamin A). The interaction of ECRG1 and Miz-1 was confirmed by glutathione-S-transferase (GST)-pull-down assays in vitro and co-immunoprecipitation experiments in vivo. ECRG1 was co-localized with Miz-1 in nucleus, as shown by confocal microscopy. Transfection of ECRG1 gene into the esophageal cancer (EC) cells inhibited cell proliferation and induced G1 phase arrest of cell cycle. In the co-transfection of ECRG1 and Miz-1 assays, we found inhibition of cell proliferation and G1/S phase in EC cells, but the levels of cell proliferation inhibition and G1/S phase arrest were more strongly compared with the transfection of ECRG1 or Miz-1 alone. In addition, the interaction of ECRG1 and Miz-1 could induce expression of p1^INK4b gene in esophageal cancer 9706 (EC9706) cells. However, the transfection of ECRG1 or Miz-1 alone was not revealed the expressions of P15^INK4b gene. When antisense ECRG1 interdicted expression of endogenous ECRG1 in Balb/C-3T3 cells, Transfection of Miz-1 couldn't induce p15^INK4b expression. The results provide evidences that ECRG1 and Miz-1 in EC cells may be acting as a co-functional protein associated with regulation of cell cycle and induction of P15^INK4b expression. It suggests that ECRG1 may inhibit tumor cell growth by affecting cell cycle, and that expression of P15^INK4b may be likely to enhance G1 cell cycle arrest during the interaction of ECRG1 and Miz-1. The physical interaction of ECRG1 and Miz-1 may play an important role in carcinogenesis of EC.