Effect Of HIF-1α-EphA2 Pathway On Vasculogenic Mimicry In Esophageal Squamous Cell Carcinoma

BackgroundEsophageal carcinoma (EC) is the sixth commonest cause of tumour-related death around the world, which mainly includes esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Esophageal carcinoma mortality in China ranks first in the world, where esophageal squamous cell carcinoma accounts for approximately 95% of all esophageal carcinoma. The pathogenesis of esophageal squamous cell carcinoma has been studied extensively. Especially, research on gene level has been the hotspot of cancer research. As a nuclear factor existing in mammals, hypoxia inducible factor-1αmaintains the body's ability to adapt to hypoxia by regulating the hypoxia-inducible genes, which are related to angiogenesis, cell differentiation and apoptosis, etc. Vasculogenic mimicry (VM) is a capillary structure formed by aggressive tumor cells instead of endothelial cells. It is a novel pathway to generate tumor microcirculation. VM is closely correlated with tumor invasion and metastasis and prognosis of patients. It brings bad effect on the traditional antiangiogenic therapy of tumors. High EphA2 expression in cancer cells is correlated with a poor prognosis associated with recurrence due to enhanced metastasis. Interaction of the EphA2 receptor with its ligand (e.g., ephrinA1) triggers events that are deregulated and implicated in carcinogenesis. EphrinA1-independent oncogenic activity and ephrinA1-dependent tumor suppressor roles for EphA2 are described. Molecular interactions of EphA2 with signaling proteins are associated with the modulation of cytoskeleton dynamics, cell adhesion, proliferation, differentiation and metastasis. The deregulated signaling by EphA2 and its involvement in oncogenesis provide multiple avenues for the rational design of intervention approaches.Our former researchs reflect that EphA2 is regulated directly or indirectly by HIF-1αto influence the forming of VM in esophageal squamous cell carcinoma.ObjectiveTo investigate the effect and the correlation of expression of HIF-1αand EphA2 under normoxia and hypoxia on vasculogenic mimicry in esophageal squamous cell carcinoma .Methods1.Two esophageal cancer cell lines Eca109 and TE13 were incubated under normoxia(19%O2) and hypoxia(1% O2), and the expression of HIF-1αand EphA2 was then measured by RT-PCR and western blot.2.The formation of tutular networks of Eca109, TE13 was analyzed by three-dimension culture in vitro under normoxia and hypoxia.3.Eca109 and TE13 were transfected by plasmid harboring interfering RNA targeting EphA2, and the expression of EphA2 was then measured by RT-PCR and western blot.4.The expression of HIF-1αand EphA2 was measured by RT-PCR and western blot under normoxia and hypoxia.5.The formation of tutular networks of Eca109, TE13 transfected miRNA of HIF-1αand EphA2 was analyzed by three-dimension culture in vitro under normoxia and hypoxia. Results1. The expression of HIF-1αand EphA2 in Eca109 and TE13 was obviously enhanced under hypoxia and the numbers of tubular networks were both remarkably increased in cells Eca109 and TE13 under hypoxia.2. The mRNA expression of EphA2 was markedly inhibited in cells Eca109 and TE13 with silencing EphA2 gene.3. The expression of EphA2 and the formation of tutular networks of Eca109, TE13 transfected miRNA of HIF-1αwere obviously decreased both under hypoxia and normoxia and the number of the latter was more than the former.4. The expression of HIF-1αin Eca109 and TE13 were not obviously inhibited with silencing EphA2 both under hypoxia and normoxia, but the tubule-forming ability was significantly inhibited, much more extreme under normoxia.5. The influence of silencing HIF-1αis more intensive than silencing EphA2 in forming tubule under hypoxia; Under normoxia, silencing EphA2's effect is more obvious.Conclusions1. The expressions of HIF-1αand EphA2 exit in two esophageal cancer cell lines Eca109 and TE13.The enhanced expression of HIF-1αand EphA2 under hypoxia can increase the numbers of tubular networks in Eca109 and TE13.2. The environmental oxygen density plays an important role in forming tubule in esophageal cancer cell.HIF-1αand EphA2 may participate in this process.3. The expression of EphA2 in Eca109 and TE13 was inhibited with silencing HIF-1α. On the contrary, the expression of HIF-1αin Eca109 and TE13 was not obviously inhibited with silencing EphA2. EphA2 may be regulated by HIF-1αn the process.4. EphA2 may be one of the important factors regulated by HIF-1αunder hypoxia. The influence of silencing HIF-1αis more intensive than silencing EphA2 in forming tubule under hypoxia; Under normoxia, silencing EphA2's effect is more obvious.