Study Of Hypoxia-inducible Factor-1α On Carcinogenesis Of Esophageal Squamous Cell Cancer

Background:Hypoxia-inducible factors(HIFs) is a kind of transcription factors inducedby hypoxic condition, play key roles in the pleiotropic response observedunder hypoxic conditions. Hypoxia-inducible factor-1 is one subtype of HIFs,as a heterodimer includingαandβsubunits, andαsubunit is function oneregulated by hypoxia, which overexpressed in various cancer tissues andmay play critical roles in tumorigenesis and progression by inducingvascular endothelial growth factor (VEGF) to involve in tumor angiogenesis.Aims:1. To investigate the expression of HIF-1α, VEGF in the carcinogenesis ofESCC and the relationship between HIF-1α, VEGF expression andclinicopathologic features2. To study the expression and significance of HIF-1αand VEGF in humanesophageal squamous cancer cell line Eca-109 in vitro. 3. To explore the effect of silencing HIF-1αgene by RNA interference invitro on esophageal squamous cancer cell line Eca-109.4. To evaluate the growth influence of grafted tumor in nude mice throughsilencing HIF-1αgene by RNA interference in vivo.Methods:1. The expression of HIF-1αmRNA, VEGF mRNA were examined byhybridization in situ and the expression of HIF-1α, VEGF, CD31 proteinwere examined by immunohistochemical assay, In 67 surgically resectedspecimens from paitents with ESCC, 42 with normal esoghageal mucosaand 40 biopsy specimens from patients with esophageal dysplasia. Weinvestigated the changes of HIF-1α, VEGF and CD31 expression in threegroups, and the correlation of its genes expression between MVD andclinicopathologic features were also explored.2. The expressions of HIF-1α,VEGF mRNA and HIF-1αprotein inesophageal squamous cancerous cell line Eca-109 were detected byRT-PCR and Western blot in hypoxia or normal oxygen pressurerespectively.3. Human esophageal squamous cancer cell Eca-109 was transfected withthree eukaryotic expression vectors with short hairpin RNA (shRNA)homologous to HIF-1αgene. We examined expression levels of HIF-1,VEGF, HO-1, MMP-2 and PTEN by western blotting or RT-PCRrespectivly to determine the relationship between its genes and HIF-1α. Moreover, the changes of cell cycle after silencing HIF-1αgene weredetected by flow cytometry. The proliferating activities of three cellgroups were investigated using cell proliferating assay.4. Silencing HIF-1αgene Eca-109 Cells and nomal Eca-109 cells wereimplanted to nude mice respectively, and observing the growth of graftedtumors. The expressions of HIF-1α, VEGF protein in the grafted tumorswere exemined by Western blot.Results:1. The expression rate of HIF-1α, VEGF and CD₃₁ in normal esoghagealmucosa, esophageal dysplasia and ESCC were: HIF-1αmRNA (16.67ï¼…, 35.0ï¼…, 65.67ï¼…); HIF-1αprotein (0ï¼…, 20.0ï¼…, 58.21ï¼…); VEGFmRNA (26.19ï¼…, 34.29ï¼…, 62.29ï¼…);VEGF protein (11.9ï¼…, 27.5ï¼…,58.21ï¼…); MVD (12.71±4.571, 16.95±5.486, 33.39±6.688 )respectively. In ESCC, the overexpression of HIF-1α, VEGF weresignificantly associated with high MVD, and high HIF-1αexpressioncorrelated with VEGF protein expression. HIF-1α, VEGF proteinexpression correlated with TNM classification, depth of tumour invasionand lymph node metastasis.2. Semiquantitive RT-PCR showed that HIF-1αand VEGF over-expressedin oxygen-deprived esophageal squamous cancerous cell line Eca-109,and reached the peak at 24h; The result of Western blot showed thatHIF-1αprotein has no significant up-regulation in hypoxia vs normal oxygen pressure.3. The transcription of HIF-1αgene and the expression level of HIF-1αwere down-regulated by RNA interference. The expression level ofHIF-1αgene in the cell groups with HIF-1αgene silencing was lowerthan control group. The expressions of VEGF, HO-1, MMP-2, PTENgenes were also decreased at various levels. The results of flowcytometry showed that cells stayed in G0/G1 stage in HIF-1αgenesilenced cell groups were much more than in control groups. Theproportion of cells in S stage was decreased after RNAi. There was littledifference in proportion of G2/M stage. Moreover, we found that theproliferating activity ofEca-109 cells was also decreased after RNAi.4. Study in nude mice showed that transplanted tumors from esophagealsquamous cancerous cell line Eca-109 with HIF-1αRNAi grew slowerand smaller than that without HIF-1αRNAi. Western blot showed thatHIF-1αprotein has significantly down-regulated in hypoxia.Conclusions:1. The expressions of HIF-1α, VEGF and MVD increased gradually as thecarcinogenesis of ESCC progressed. HIF-1αmay play an important rolein tumorigenesis of ESCC by inducing VEGF expression in tumorangiogenesis. Overexpression of HIF-1αand VEGF protein may beused as biological markers for evaluating the behavior of ESCC.2. HIF-1αmRNA and VEGF mRNA were over-expressed in esophageal squamous cancerous cell line Eca-109 in hypoxia condition, but HIF-1αprotein has no significant up-regulation in hypoxia. HIF-1αmay play akey role in esophageal squamous cancerous cell line Eca-109 adapting tohypoxia. The regulation of HIF-1αin esophageal squamous cancerouscell line Eca-109 may be at transcription level.3. The recombinant plasmids pGCsi-H1-shHIF transfected can suppress theexpression of HIF-1αefficiently in esophageal squamous cancerous cellsEca-109, and the cloned esophageal squamous cell carcinoma cell strainthat silenced HIF-1αgene stablly were cultivated successfully afterscreening with antibiotics and monoclonize. The results of cellproliferation assay indicated that HIF-1αhas positive correlation withproliferate activity of esophageal squamous cancer cells. HIF-1αalsoplays a role in DNA replication of squamous cancer cells.4. RNA inference targeting HIF-1αcan inhibit growth of transplantedtumor significantly. HIF-1αmay play an important role in carcinogenesisof ESCC.