CXCR4 Promoted Invasion And Metastasis Of Esophageal Squamous Cell Carcinoma As A Downstream Target Of HIF-1α

Aims:The purpose of this study was to explore the effect of hypoxia inducible factor-1α(HIF-1α)regulating C-X-C chemokine receptor 4(CXCR4)expression on the invasion and metastasis of esophageal squamous cell carcinoma(ESCC)cells,and to clarify the molecular regulation mechanism of HIF-1α-induced CXCR4 expression in ESCC cells,so as to provide a theoretical basis for clinical targeted therapy and development of new drugs in patients with ESCC.Methods:1.By analyzing the sequencing data sets(GSE23400 and GSE75241)of ESCC in GEO database,the m RNA level of CXCR4 in ESCC tissue samples and esophageal mucosal tissues was compared.In addition,we also detected the m RNA and protein expression of CXCR4 in paired ESCC tissues and normal tissues by q RT-PCR and Western blot,respectively.The expression of CXCR4 in ESCC was detected by immunohistochemical(IHC)and the relationship between CXCR4 expression and clinicopathological features was analyzed.we also detect the expression of CXCR4 protein in 7 ESCC cell lines and immortalized esophageal normal epithelial cells by Western blot.2.We constructed CXCR4 overexpression and CXCR4 knock-down virus.According to the expression level of CXCR4 in ESCC cell line,we selected KYSE30 and KYSE150 to construct CXCR4 knock-down stable line,KYSE450 and KYSE510 to construct CXCR4 over-expression stable line.The effects of CXCR4 on the migration,invasion and proliferation of ESCC were observed by transwell experiments,CCK8,clone formation experiments and Ed U experiments in vitro,and the effects of CXCR4 on the metastasis and growth of ESCC were detected by constructing subcutaneous tumour model and metastatic tumour model in nude mice.3.The effect of hypoxia on the expression of CXCR4 in ESCC cells was observed in vitro.Transcriptome sequencing was used to find the differentially expressed genes in ESCC cells after the decreased expression of HIF-1α.q RT-PCR and Western blot experiments were used to further verify the change of CXCR4 expression after the change of HIF-1α expression level.Transwell experiment was used to compare the changes of migration and invasion ability of ESCC cells under hypoxia,and the changes of migration and invasion ability of ESCC cells under hypoxia condition by interfering with the expression of CXCR4 or using CXCR4 inhibitor,and the role of CXCR4 in HIF-1α promoting the migration and invasion ability of ESCC cells was confirmed by functional rescue experiment.4.Through the analysis of JASPAR database,we found that there were seven potential HRE sites in the promoter region of CXCR4 gene.We selected 2000 bp upstream of CXCR4 gene promoter and used about 500 bp as a truncated body.Finally,we constructed a full-length luciferase reporter gene plasmid(including HRE1-7 sites),two truncated luciferase reporter gene plasmids(including HRE1 site and HRE1-5 sites),and the mutation luciferase reporter gene plasmid(HRE1-7 site complete mutation).HIF-1α overexpression plasmid and control plasmid were also constructed.Chromatin immunoprecipitation technique and Dual-luciferase assay were used to verify HIF-1α directly bound to HRE sites of CXCR4 promoter in ESCC cells and activates CXCR4 promotor transcription.5.The expression of HIF-1α in ESCC was detected by IHC,the relationship between the expression of HIF-1α and clinicopathological features was analyzed,and the correlation between the expression of HIF-1α and CXCR4 in ESCC tissue was analyzed by Spearman’s coefficient tests.Results:1.GEO database revealed that CXCR4 were found to be highly expressed in ESCC tissues.In addition,q RT-PCR and Western blot revealed that CXCR4 was up-regulated in ESCC.Immunohistochemical revealed that CXCR4 were up-regulated in ESCC,and high expression of CXCR4 was found to be associated with lymph node metastasis and poor prognosis of ESCC.2.Transwell experiments showed that compared with the control group,CXCR4 knockdown significantly reduced the migration and invasion ability of KYSE30 and KYSE150,while CXCR4 overexpression exhibited opposite effects in KYSE450 and KYSE510 cells.The results of animal experiments showed that compared with the control group,the lung metastasis and growth ability of ESCC cells were weakened after the decrease of CXCR4 expression level.MSX-122,a CXCR4 antagonist,could significantly reduce the lung metastatic ability of KYSE30 cells.3.After hypoxia treatment,the expression of CXCR4 increased significantly in KYSE30,KYSE150,KYSE450 and KYSE510 cells.The results of transcriptome sequencing showed that when the expression of HIF-1α decreased,the difference of CXCR4 was particularly significant among the down-regulated genes.HIF-1α knockdown in KYSE30 and KYSE150 cells markedly reduced the expression of both CXCR4 m RNA and protein under normoxia or hypoxia,whereas HIF-1α overexpression up-regulated CXCR4 protein in KYSE450 and KYSE510 cells.Furthermore,in vitro transwell assay demonstrated the increased metastasis ability of KYSE30 and KYSE150 induced by hypoxia or HIF-1α could be weakened by CXCR4 interference or treatment with MSX-122(100n M).4.Dual-luciferase analysis demonstrated that hypoxia significantly enhanced the full-length group of CXCR4 gene promoter activity,while the transcriptional activity of luciferase reporter gene plasmid in two truncated and mutant groups had no significant change.HIF-1α overexpression significantly enhanced the full-length group of CXCR4 gene promoter activity,while the transcriptional activity of luciferase reporter gene plasmid in two truncated and mutant groups had no significant change.Ch IP assay revealed that HIF-1α directly bound to HRE sites 1,4,5 and 7 in the CXCR4 promoter in KYSE30 cells.5.Immunohistochemical results showed that the expression of HIF-1α in ESCC tissue was higher than that in esophageal mucosa,and the expression of HIF-1α was positively correlated with CXCR4 in ESCC.Conclusion:1.CXCR4 is highly expressed in ESCC and the high expression of CXCR4 is closely related to lymph node metastasis and poor prognosis.2.CXCR4 can promote the invasion,metastasis,proliferation and growth of ESCC.3.HIF-1α regulates the expression of CXCR4 in hypoxic environment.Hypoxia treatment or HIF-1α overexpression promotes the migration and invasion of ESCC cells,and the promoting effect of hypoxia/HIF-1α on the migration and invasion of ESCC cells could be weekend by interfering with CXCR4 expression4.HIF-1α binds to the HRE site of CXCR4 gene promoter and regulates its expression.5.The expression of HIF-1α was positively correlated with that of CXCR4 in ESCC.