Study On Expression, Function And Mechanism Of EGFL7 Gene In Human Gliomas

BACKGROUND: Gliomas, especially the glioblastomas, are the most common brain malignancy tumor and are marked by an extremely poor prognosis, despite advances in surgical and clinical neuro-oncology. That is why central nervous system gliomas is quite a challenging neoplasm requiring much further research to understand the molecular and cellular clinical basis.Angiogenesis is required for several physiological processes as well as pathological conditions. Growth of solid tumors is highly dependent on angiogenesis. In recent years, the involvement of angiogenesis in the development of malignant tumors and metastasis has been extensively studied. Therapeutic strategies to suppress angiogenesis have been examined on experimental and clinic tumor models with successful outcomes.Gliomas, most of which are solid neoplasm, are among the best vascularized human tumors. The proliferation and invasion of them are depend on the angiogenesis so as to acquire O₂ and nutritive material. So angiogenesis is crucial to the growth of malignant gliomas too. Therefore, antiangiogenic therapy may be a promising therapeutic modality for malignant gliomas. But little is known about how angiogenic mechanism in glioms is regulated in vivo. To investigate and identify a key regulator in this process may be an efficient path and be of far reaching importance.EGF-like domain 7 (EGFL7) is a new endothelial cell-derived secreted factor. It is expressed at high levels in the vasculature associated with tissue proliferation, and is downregulated in most of the mature vessels in normal adult tissues. The documented evidences demonstrated that formation of cord-like structures process fails to take place in EGFL7 knockdown embryos, leading to the failure of vascular tube formation. EGFL7 may be a hopeful target for suppressing angiogenesis of gliomas.But, functioned as a key regulator that controls a specific and important step in vasculogenesis, the expression, function and mechanism of EGFL7 in human gliomas are also remained unknown to date. EGFL7 represents a unique mechanism for embryogenesis and recovery of vascular injury. It is conceived that EGFL7 may play a key role in the angiogenesis of gliomas. However, this hypothesis has not been rigorously tested in gliomas models in vivo or in vitro.OBJECTIVE: The present study was carried out to investigate the expression of EGFL7 in human glioma tissues. According to knockdown the expression of EGFL7 gene by RNAi in U251 and HUVEC cell lines, we also explored the potential role and mechanism of EGFL7 in the process of glioma angiogenesis.METHODS: Fresh tissues of 36 patients with glioma, including low malignance 21 (grade I～II) and high malignance 15 (grade III～IV), were used in the present study. The expression of EGFL7 mRNA by these glioma tissues was examined by RT-PCR. Another 45 cases of formalin-fixed, paraffin-embedded specimens of glioma, including low malignance 24 (grade I～II) and high malignance 21 (grade III～IV), were used to assess the expression status of EGFL7 protein, Factor VIII and Ki-67 by immunohistochemical staining. The Ki-67 labeling index (LI) and microvessel density (MVD) were calculated. To analyze whether or not EGFL7 could be used as an index of the malignancy and a predictor of poor prognostic for the patients with glioma, the correlation between EGFL7 expression and the Ki-67 LI or the MVD was investigated.Small interfering RNA (siRNA) targeted to human EGFL7 was constructed to determine the influence of EGFL7 on the angiogenesis of gliomas: Four pairs of complementary Oligo DNA targeted to human EGFL7 gene sequence and a universal negative control sequence were designed, respectively. A hairpin structure was inserted between the sense and antisense sequence. Then the complementary Oligo DNAs were synthesized and cloned into the pGCL-GFP vector. The resulting lentiviral expression vector containing EGFL7 short hairpin RNA (shRNA) and universal negative control sequence was confirmed by PCR and DNA sequencing. To screen the effective shRNA sequence, a recombinant plasmid vector containing transgenes encoding for human EGFL7-flag recombinant fusion protein and enhanced green fluorescent protein (EGFP), was used to induce overexpression of EGFL7. The recombinant plasmid pGCL-GFP and pEGFP-C1 were co-transfected into 293T cells by lipofectamin method. The gene silencing efficiency of shRNAs on EGFL7 expression by 293T cells was identified with Western Blot. The highest knockdown efficiency plasmid, named pGCL-GFP-vshEGFL7, was packaged by lentivirus packaging vector system. The titer of lentivirus was tested according to the expression level of GFP.The expression of EGFL7 mRNA and protein on U251 cells and HUVECs was examined by real-time quantitative PCR and immunohistochemical staining. And then, U251 cells and HUVECs were infected with. pGCL-GFP-vshEGFL7. The knockdown efficiency of shRNAs on EGFL7 expression by U251 cells and HUVECs was assayed by real-time quantitative PCR. A co-culture model with U251 cells and HUVECs was established by Transwell method in vitro to mimic the effect of tumor cell on endothelial cell in microecological environment of glioma in vivo. Cell proliferative activity of U251 and HUVECs was measured by MTT assay. Cell cycle and apoptosis of HUVECs were analyzed by flow cytometry. Adhesion and migration capability of HUVECs were evaluated by Methyl Violet staining. In vitro angiogenesis of HUVECs was assessed using a capillary-like network formation assay.RESULTS:â‘ The expression of EGFL7mRNA and protein by human gliomas was detected. EGFL7 protein located in plasm of tumor cells and endothelial cells, and its expression correlated directly with the degree of malignancy(t=4.399, P<0.01), KI-67 LI(r=0.793, P<0. 01) and MVD(r=0.684, P<0. 01) of human gliomas. However, it was not detected in normal brain.â‘¡Both the lentiviral expression vector pGCL-GFP containing EGFL7 shRNA and the eukaryotic expression vector pEGFP-C1 containing human EGFL7 were in agreement with expected results confirmed by PCR and sequencing analysis, and the strong expression of reported gene EGFP was observed under inverted fluorescent microscope in 293T cells. After co-transfected with recombinant plasmid pGCL-GFP and pEGFP-C1 into 293T cells by lipofectamin method, Western Blot analysis suggested that the highest knockdown efficiency of shRNA sequence targeted to EGFL7 was 5'-CCGAACCATCTATAGGACC-3' (512～530). The titers of concentrated virus of pGCL-GFP-vshEGFL7 and universal negative control sequence were above 4.8×10⁷ TU/ml and 5.0×10⁷ TU/ml, respectively.â‘¢The enhanced expression of EGFL7mRNA and protein on U251 cells and HUVECs was detected by real-time quantitative PCR and immunohistochemical staining. EGFL7-like immunoreactivity located in plasm of U251 cells and HUVECs. The EGFL7 mRNA expression level of U251 cells and HUVECs was suppressed significantly in EGFL7 knockdown group compared with that in universal negative control. group and normal negative control group (P<0.01), and there was no difference between the two control groups (P>0.05).â‘£Cell proliferation activity of U251 was inhibited significantly in EGFL7 knockdown group compared with that in universal negative control group and normal negative control group (P<0.01) at the same time after 48h, and there was no difference between the two control groups (P> 0.05); Proliferative activity, migration capability, cell cycle and apoptosis rate of HUVECs were no difference between the three groups (P>0.05) in solo-culture system; Migration capability of HUVECs was no difference between the three groups (P>0.05) in co-culture system too; Proliferation activity of HUVECs was decreased significantly in EGFL7 knockdown group compared with that in universal negative control group and normal negative control group (P<0.01) at the same time from 48h to 4d in co-culture system, however, there was no difference between the three groups at 5d(P>0.05); Whether solo-cultured HUVECs or co-cultured HUVECs, the capillary-like network formation was completely inhibited by EGFL7siRNA; On the contrary, it was normal in universal negative control group and normal negative control group (P<0.01); The obvious depression of adhesion capability of HUVECs was also found in EGFL7 knockdown group compared with that in universal negative control group and normal negative control group (P<0.01) both in co-culture system and in solo-culture system. There was no difference between the two control groups (P>0.05).CONCLUSION:1. Tumor cells and endothelial cells in human glioma tissues can express EGFL7 gene. HUVEC or U251 cells can also synthesis and secrete EGFL7 protein respectively. The level of EGFL7 expression directly correlates with the degree of malignancy, KI-67 LI and MVD of human gliomas and may contribute to their rapid growth and invasion.2. The effective sequence of siRNA targeted to human EGFL7 gene is screened out, and the lentivirus RNAi vector producing EGFL7 shRNA is constructed successfully in this study. It is confirmed that the shRNA can significantly knockdown the expression of EGFL7 on HUVEC and U251 cells.3. Lentivirus-mediated EGFL7 gene silencing can inhibit the adhesion and tube formation of HUVECs and depress the proliferation activity of U251 cells. These findings suggest that EGFL7 plays a key role for capillary-like network formation of endothelial cells in the process of glioma angiogenesis and may directly contribute to malignant proliferation of gliomas. EGFL7 may become an attractive target for gene therapy of gliomas and RNAi mediated by shRNA targeted to EGFL7 may be a new promising strategy for it.