The Study On The Angiogenesis In Esophageal Carcinoma Tissues And Radiosensitivity In Esophageal Squamous Carcinoma Cells Regulated By HIF-1? And Its Downstream Genes About Angiogenesis

Objective:To objective the effect HIF-1?and the genes it regulates played on angiogenesis and prognosis in esophageal carcinoma tissues as well as radiosensitivity in cells.To investigate the effects of 2-methoxyestradiol(2ME2)on the radiosensitivity of EC.Methods:1.A total of six ESCC and para-cancerous tissues(control)were harvested.Microarray based m RNA expression was determined in the ESCC(n=3)and compared with para-cancerous tissues(n=3).Using online software DAVID to analyze biological function and pathway enrichment of significantly differentially expressed genes.Choosing the genes associated with angiogenesis.Microarray data were validated by quantitative real-time reverse transcription polymerase chain reaction(q RT-PCR)in a separate group.2.The expressions of AGGF1?HIF-1?and VEGF in 70 cases of ESCC and 30 cases of normal esophageal tissue were examined using SP immunohistochemical staining and were analyzed according to the clinicopathological features and follow-up data.3.Human esophageal squamous cell carcinoma(ESCC)Eca109 cells were subjected to hypoxia and/or irradiation(IR)in the presence or absence of 2ME2.Cell growth,colony formation,and apoptosis were subsequently evaluated by CCK8 assay,clonogenic survival assay and flow cytometry.Moreover,the m RNA and protein expression levels of HIF-1?,VEGF and AGGF1 in ESCC cells were determined using q RT-PCR and Western blotting.Results:1.A total of 936 significantly differentially expressed genes is confirmed,including 306 up regulation and 630 down regulatiaon.2.GO functional annotation results:the differentially expressed genes of up regulation are mainly related to the cell division,DNA repair,angiogenesis,cell cycle and DNA replication etc.The differentially expressed genes of down regulation are mainly related to muscle contraction,vasoconstriction,signal transduction and acute inflammation etc.3.The pathway enrichment results:the differentially expressed genes of up regulation are mainly focus on the DNA replication,angiogenesis,Fanconi anemia pathway,cell cycle etc.The differentially expressed genes of down regulation are mainly focus on:c GMP-PKG signaling pathway,Vascular smooth muscle contraction,c AMP signaling pathway,MAPK signaling pathway etc.2.The expressions of AGGF1(?2=8.129,P=0.004)?HIF-1?(?2=21.212,P=0.000)and VEGF(?2=4.116,P=0.042)in 70 cases of ESCC were significantly higher than those in 30 cases of normal esophageal tissue.The expressions of AGGF1?HIF-1?and VEGF in ESCC were significantly related to the differention?the depth of invasion?the lymph node metastasis?the M stage?the clinical stage and the radiotherapy response(P all<0.05).The expression of VEGF in ESCC was significantly positively related to the expression of HIF-1?(r=0.333,P=0.003).The expression of AGGF1 was significantly positively related to the expression of VEGF(r=0.290,P=0.015).The radiotherapy curative effect was significantly negatively related to the expression of HIF-1?and VEGF(r=-0.288?-0.241,P=0.016?0.044).The OS of the positive team of HIF-1?was shorter than the negative team significantly(P=0.022).Cox-proportional multivariate analysis showed that positive expression of HIF-1?(P=0.000)?depth of invasion(P=0.049)and chemotherapy(P=0.037)were significant prognostic factors in overall survival.3.With the increasing concentration of Co Cl₂,the proliferation activity of Eca109 cells was becoming slowly(P<0.05).Co Cl₂induced hypoxia could increase cell cycle arrest in G0/G1 phase and decrease arrest in S phase.The G0/G1 phase and S phase cell after hypoxia 24h and 48h was statistically different from that of the control group and after hypoxia 6h and 12h(P<0.05).The decrease of alive Eca109 cells in IR/hypoxia+2ME2 was more than other groups(P<0.05),and they also had more apoptosis(P<0.05).With 2ME2,the increasing expression of HIF-1?,VEGF and AGGF1 m RNA/protein decreased(P<0.001).Conclusions:1.1)A total of 936 significantly differentially expressed genes is confirmed,including 306 up regulation and 630 down regulatiaon.2)Genes associated with angiogenesis are as follows:WNT5A,VEGFC,CHRNA7,ANG,ECM1,VEGFA,HOXB13,AGGF1,HIF1A,VEGFB,HEY1,HIF3A,MMP27 etc.Most of them are up regulation.3)The results of q RT-PCR are the same as microarray.2.AGGF1?HIF-1?and VEGF have high expressions in ESCC,and they are closely related to the clinicopathological features of ESCC.The over expression of them may have influence on the curative effect of radiotherapy and the survival prognosis of ESCC,which can offer new ideals and methods for the treatment of ESCC.3.1)The expression of HIF-1?,VEGF and AGGF1m RNA/protein increased after Co Cl₂ induced hypoxia.2)IR can reduce the Co Cl₂induced the expression of HIF-1?,VEGF and AGGF1 m RNA/protein increased by hypoxia.Cell cycle arrest in G0/G1 phase in hypoxia may associate with decreased radiosensitivity in ESCC cells.3)2ME2 may confer radiosensitivity by inhibiting the expression of HIF-1?.