The Mechanism Of HID1 Regulated By Snail And The Effect Of HID1 On Pancreatic Cancer Cell Migration

Background:Our study illustrated the mechanism of HID1 repression by Snail in pancreatic ductal adenocarcinoma(PDAC)cell lines and the repression of HID1 on cancer cell migration.PDAC is currently one of the deadliest solid malignancies due to its aggressive nature,late detection,and resistance to chemotherapy.Recent studies have shown that Snail plays an important role in cancer metastasis and progression.The zinc finger transcription factor Snail contains a C-terminus DNA binding domain and an N-terminus SNAG domain.The DNA binding domain can bind E-box sequence(CANNTG)of target genes.The SNAG domain can recruit several co-repressor complexes to its target promoters to exert its repressive function and to induce epithelial-mesenchymal transition(EMT).In EMT progression,Snail enhances cell motility by down-regulating epithelial markers,such as E-cadherin and up-regulating mesenchymal markers,such as N-cadherin,fibronectin and vimentin.HID1 was found in our Chromatin immunoprecipitation-sequence(Ch IP-seq)assay by using H2AK119Ub1 antibody in PDAC tissues.Caenorhabditis elegans hid-1 gene was first identified in a screen for mutants with a high-temperature-induced dauer formation(hid)phenotype,and it acts in the early steps of DCV exocytosis by controlling the correct sorting of DCV cargoes.A research showed loss of expression of HID1 in multiple cancer cell lines derived from tumors that arose in various tissues.However,there is no report show HID1 plays an important role in cancer metastasis.The TFSEARCH program predicts there are two E-boxes in HID1 promoter,which indicates HID1 is a potential target gene of Snail.Methods:We overexpressed and knocked down HID1 in multiple cancer cell lines and tested the mRNA levels of HID1 by using Real-time PCR.At the same time,we knocked down the components of polycombrepressive complexes Ring1 A and EZH2 and examined the expression of HID1.Ring1 A and EZH2 are reported have an effect on H2AK119Ub1.In order to investigate the mechanism of HID1 repression by Snail,we cloned HID1 wild type promoter and generated several mutants of promoter-luciferase constructs based on the location of E-boxes.Snail was co-transfected with HID1 promoter into HEK293 T cells and HID1 promoter activity was tested by luciferase assay.To examine whether Snail directly binds to HID1 promoter,we performed chromatin immunoprecipitation(Ch IP).To examine whether HID1 has a role in Snail induced EMT progression,we established stable clones to silence or induce the expression of HID1 in PANC-1 cell line.Moreover,we co-expressed HID1 with exogenous Snail in PATU8988 cell lines.The EMT markers and cell motility were checked by Western blotting and transwell assay.Results: In multiple cancer cell lines,ectopic Snail expression can inhibit the transcription of HID1 and knock-down of Snail can increase the expression of HID1.Moreover,the components of polycomb repressive complex Ring1 A and EZH2 can also repress the expression of HID1.Luciferase and ChIP assay show that Snail can directly bind to the E-box1 and E-box2 and repress HID1 transcription.Western blot and transwell assay showed that ectopic HID1 expression blocked the upregulation of vimentin,inhibited the ability of cell migration.However,ectopic HID1 can not completely reverse the promotion of Snail on cell migration.In addition,in 19 cases of paired tumor and peri-tumor specimens,we found HID1 is down-regulated in tumor tissues compared to the peri-tumor tissues.Conclusion: Our results showed HID1 is a direct target of Snail and HID1 has an important role in cancer cell migration.