| Background:Esophageal 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. A number of studies have shown that HIF-1αis over-expressed in esophageal squamous cell carcinoma. Moreover, the expression of HIF-1αis closely associated with tumor angiogenesis, metabasis and infiltration.Objective: To explore the impact of RNA interference targeting HIF-1αon the biological behavior of esophagus squamous cell carcinoma cell line TE13 and the relationship between HIF-1αand vasculogenic mimicry- related genes in esophageal squamous cell carcinoma. Methods1. To acquire the optimal selecting concentration of G418 for transfection, TE13 cell were plated in the DMEM containing different concentration G418 (0, 100, 200, 300, 400, 500 and 600μg/ml) and observed two weeks. Then esophagus squamous cell carcinoma cell line TE13 was transfected by plasmid targeting HIF-1α. .After selection by culturing with G418, inverted fluorescent microscopy were used to observe whether the green fluorescent protein(GFP) of monoclones were expressed. And the expression of HIF-1αprotein of stable transfected TE13 cell was detected by western blot.2. Flow cytometry (FCM) was used to detect the apoptosis rate of TE13, TE13/Neo and TE13/siHIF cells after 24hr hypoxic exposure. The apoptosis rate was assessed by AnnexinⅤ/7-AAD double labeling.3. The proliferation abilities of TE13, TE13/Neo and TE13/siHIF cells were determined by MTT colorimetric assay.4. The capabilities of moving of TE13, TE13/Neo and TE13/siHIF cells were tested by Transwell method.5. Esophageal Squamous Cancer Cells TE13 and Eca109 were cultured under hypoxia at different time (6, 12, 24, and 48 h). The expressions of HIF-1αprotein were detected by western blot, and the proper culture time under hypoxia for two cell lines were confirmed.6. Eca109, Eca109/Neo, Eca109/siHIF, TE13, TE13/Neo and TE13/siHIF cells were cultured under normoxic and hypoxic conditions for proper time. The expressions of HIF-1α, EphA2/ECK, VE-cadherin, Laminin5γ2 and MMP-2 protein were detected by Western blot. Results:1. Identification of the stable transfected TE13 cellTE13 cells were cultured in DMEM containing different concentrations of G418 for two weeks and the cells in G418 (400, 500, 600μg/ml) all died. So, 400μg/ml was selected as the optimal concentration of G418 for stable transfection. Out of all monoclones, a strong green fluorescence can be observed in clone 12 by inverted fluorescence microscope. HIF-1αprotein can not be detected by Western blot in clone 12. So we named this clone as TE13/siHIF.2. The apoptosis rate assessed by Flow cytometryAfter hypoxia for 24hr, TE13, TE13/Neo and TE13/siHIF cells were double-labeled with AnnexinⅤ/ 7-AAD. The results of Flow cytometry showed that the apoptosis rate of TE13, TE13/Neo and TE13/siHIF cells were (4.19±0.82) %, (3.69±0.67) % and (11.91±0.96) %. The apoptosis rate of TE13/siHIF cell significantly increased than TE13 cell (P< 0.05) and the apoptosis rate of TE13 and TE13/Neo was not different significantly (P>0.05).3. The proliferation determined by MTT colorimetric assayTE13, TE13/Neo and TE13/siHIF cells were cultured for 7 days. The results of MTT colorimetric assay showed the proliferation ability of TE13/siHIF significantly lowered than TE13 (P <0.05) and the difference between TE13 cell and TE13/Neo cell was not significant (P>0.05).4. The capabilities of moving tested by Transwell methodTranswell experiment showed less cells moving through the artificial basement membrane with silencing of HIF-1α(18.2±3.7vs103.8±8.52, P<0.05) and the difference of moving capacity between TE13 cell and TE13/Neo cell was not significant (96.8±9.44 vs103.8±8.52, P >0.05).5. The optimal hypoxia time of two ESCC cells The optimal hypoxia time was disparity in the two ESCC cell lines. The expression of HIF-1αprotein in Eca109 cell reached a peak under hypoxia for 6hrs. But for TE13 cell, the expression of HIF-1αprotein reached the highest level after hypoxia 24hrs. So, 6hrs and 24hrs were selected for optimal hypoxia time for Eca109 and TE13 cell respectively.6. The expression of HIF-1α, EphA2/ECK, VE-cadherin, Laminin5γ2 and MMP-2 protein in the two ESCC cellsThe expression of HIF-1α, EphA2/ECK, VE-cadherin, Laminin5γ2 and MMP-2 protein were markedly inhibited in Eca109/siHIF and TE13/siHIF cell under normoxia (P<0.05) and the expression of MMP2 did not change obviously. The expression of HIF-1α, EphA2/ECK, VE-cadherin and Laminin5γ2 protein of TE13 and TE13/Neo (Eca109 and Eca109/Neo) under hypoxia than those under normoxia (P<0.05) and the expression of MMP2 had an increasing trend but no statistical significance (P>0.05). The expression of HIF-1αand the four vasculogenic mimicry-related genes in TE13/siHIF and Eca109/siHIF under hypoxia did not increase than those under Normoxia (P>0.05).Conclusions:1. The expression of HIF-1αprotein of ESCC cells are closely related to the time of hypoxia. And it appeared to have a decline trend after an initial ascent. But different cell line has different response to hypoxia.2. TE13 cell silencing HIF-1αcan be acquired after TE13 cell stable transfected with pGC-siHIF3. Clone 12 is the most satisfactory monoclone, the expression of HIF-1αof which can not be detected by Western blot.3. Compared with TE13 cell, the apoptosis rate of TE13/siHIF cell increase obviously, the proliferation ability and the moving capacity of TE13/siHIF decreased markedly. These results demonstrate HIF-1αhas significant impact on the biological function of TE13 cell.4. Though the expression of HIF-1αof TE13/siHIF can not be detected by western blot, TE13/siHIF still has the proliferation ability and the moving capacity and the apoptosis rate of TE13/siHIF cell only increase about 10%. This indicates that the development of esophageal squamous cell carcinoma is a complex process involving multiple genes.5. VE-cadherin, EphA2 and LN5γ2 genes are all downstream gene of HIF-1α, which regulate the development of vasculogenic mimicry in esophageal squamous cancerous cell. Though MMP2 gene is related to vasculogenic mimicry, it is not in the signaling pathway of HIF-1αregulating vasculogenic mimicry.
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