| 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. Hypoxia inducible factor 1α(hypoxia-inducible factor 1α, HIF-1α) is the main regulatory subunit of oxygen of hypoxia inducible factor 1. It participated broadly in transcriptional activation induced by hypoxia in mammalian cells and plays an important role in angiogenesis. Vascular endothelial cadherin (VE-cadherin/VE-cad) is a specific transmembrane adhesion protein in endothelial cell surface. It mediates adhesion between adjacent endothelial cells connected, and plays an important role in maintaining vascular integrity, regulating the permeability of endothelial cells and intracellular signal transduction. In recent years, some scholars have found that many highly invasive tumor cells also have high expression of VE-cad, and participate in the imitation of tumor cells in angiogenesis, which is called vasculogenic mimicry. Vasculogenic mimicry (vasculogenic mimicry, VM) is a new model of tumor microcirculation which completely different from the classic tumor angiogenesis and do not rely on endothelial cells. VM is closely correlated with tumor invasion and metastasis and prognosis of patients. Overexpression of HIF-1αgene may be related to the VM formation. VE-cad as an important molecular regulator in vascular mimicry of tumor angiogenesis showed a potential anti-tumor effect.Objective1.To investigate the effect of silencing of HIF-1αon growth and vasculogenic mimicry formation in human esophageal squamous cancer cell Eca109 in vivo and further screen out the target genes in VM formation of HIF-1α.2.To observe and discussed the effect of silencing Vascular endothelial cadherin (VE-cad) gene by RNA interference on the formation of vasculogenic mimicry, as well as cell proliferation and apoptosis of esophageal cancer cells.Methods1.Six-week-old BALB/c nude mice were divided into three groups (n=10 per group). Group A were injected subcutaneously with Eca109 cells, group B were injected Eca109 cells transfected with empty expression vector(Eca109/Neo), and group C were injected Eca109/shRNA cells (stably transfected with small hairpin RNA eukaryotic expression vector targeting HIF-1αgene). Tumor growth and volume were measured, tumor volume = (lenth×width2)/2. All nude mice were killed after 28 days, weighed the tumors, drew the tumors growth curves.2.Periodic acid schiff (PAS) and CD31 double staining was used to detect the existence of vasculogenic mimicry (VM) in transplanted tumor tissue and the differences of distribution between the various groups.Immunohistochemistry was used to observe the protein distribution of HIF-1αand epithelial kinase (EphA2), vascular endothelial cadherin (VE-cad), matrix metalloproteinase -2 (MMP2) in tumor tissue; RT-PCR and western blot were adopted to detect the mRNA and protein expression of HIF-1αand EphA2, VE-cad , MMP2 in tumor tissue. 3.Then ESCC cell lines Eca109 and TE13 were transfected by plasmid targeting VE-cad which had been constructed and screened out. After selection by culturing with Blasticidin, inverted fluorescent microscopy was used to observe whether the green fluorescent protein (GFP) of monoclones was expressed. And the expression of VE-cad protein of stablely transfected cell was detected by western blot.4.The proliferation abilities of Eca109,Eca109/neo,Eca109/miVE-cad and TE13,TE13/Neo,TE13/miVE-cad cells were determined by MTT colorimetric assay. Flow cytometry (FCM) was used to detect the apoptosis rate of the cells after 24h. The apoptosis rate was assessed by AnnexinⅤ/7-AAD double labeling. The capillary tube like structures (VM) were observed by three dimensional culture in these cells. RT-PCR and Western blot were used to detect the mRNA and protein expression of VE-cad and HIF-1α, EphA2, Laminin 5γ2 (LN5γ2) in different cells.Results1.Xenograft tumors were observed after cells inoculation about 5-7 days in group A and B, while about 7-10 days in group C. Tumor formation rate is 100%. Xenograft tumors in group C grew delay and slow. Compared the volumes and weights with two control after 28 days, group C had statistical significance(P< 0.05). The inhibitory rate reached to 48%.2.The CD31 negative and PAS positive staining of the pipeline structure with the red blood cells can be found in the vicinity of necrosis and the tumor edge in control groups, as well as individual tissue sections of group C. This structure is called vasculogenic mimicry (VM). Statistics showed that the density of VM in group C was obviously decreased compared with control groups (P<0.05), while no significantly difference between the two control groups. HIF-1αlocated in the nucleus and the cytoplasm and concentrated in the necrotic area around the tumor cells; VE-cad located in the cytoplasm and membrane; EphA2 and MMP2 located in the cytoplasm. In group C, no HIF-1αprotein expressed, EphA2 and VE-cad protein expression was lower than the two control groups, while the expression of MMP2 was no significant difference with the control groups. According to RT-PCR and Western bolt, the protein expression of HIF-1αwas silenced in group C while the mRNA levels of HIF-1αdecreased slightly. The mRNA and protein expression of EphA2, VE-cad were decreased significantly, but the expression of MMP2 did not change obviously.3.The Eca109 and TE13 which were transfected with the plasmid were selected in Blasticidin (3μg/ml).Out of all monoclones, a strong green fluorescence can be observed in clones by inverted fluorescence microscope. The monoclonal cells of fluorescent protein expression rate of about 100% were dentified by western blot. The results showed that VE-cad protein expression of Eca109-32 (later named Eca109/miVE-cad) and TE13-16 (later named TE13/miVE-cad) were inhibited with higher transfection efficiency.4.MTT colorimetric assay showed the proliferation ability of Eca109/miVE-cad and TE13/miVE-cad significantly lowered than non–transfec- -ted and empty vector groups. Flow cytometry showed that the apoptosis rate of Eca109/miVE-cad and TE13/miVE-cad significantly increased than control groups. Eca109 and TE13 cells could form capillary tube like structures in vitro. The number of lumen-like structure of Eca109/miVE-cad and TE13/miVE-cad were significantly decreased. RT-PCR and Western blot showed the mRNA and protein expression of VE-cad in Eca109/miVE-cad and TE13/miVE-cad were suppressed, accordingly, the expression of EphA2, LN5γ2 were also significantly reduced comparing to non-transfected and empty vector groups.The expression of HIF-1αin stably transfected Groups showed no significant differences with non-transfected groups and empty vector groups. Conclusions1.Inhibition of HIF-1αgene expression can inhibit the growth of esophageal squamous cell carcinoma in vivo, which delayed tumor formation and slowed down growth rate.2.The cell Eca109 can form vasculogenic mimicry-like structure in vivo. HIF-1αgene silencing can inhibit the formation of vasculogenic mimicry in esophageal squamous cell carcinoma in vivo.VE-cad, EphA2 genes are all downstream gene of HIF-1α, which regulate the development of vasculogenic mimicry in esophageal squamous cancer.3.The recombinant plasmid pcDNA?6.2-GW/EmGFPmiVE-cad can suppress the protein expression of VE-cad in Eca109 and TE13 cells. VE-cad has significant impact on the biological function of Eca109 and TE13 cells.4.Esophageal squamous cancer cell lines Eca109 and TE13 are all capable of forming VM structures in vitro. Silencing VE-cad gene can suppress their vasculogenic mimicry formation efficiently. VE-cad could inhibit the formation of vasculogenic mimicry by suppressing the expression of EphA2 or LN5γ2. It may be a key site of HIF-1αto regulate the VM formation in esophageal squamous cell carcinoma. |
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