MiR-566 Modulates VEGF Via Targeting VHL In Human Glioblastoma In Vitro And Vivo

Glioblastoma is a highly vascularized and common tumor in central nervous system,and the medium survival is less than a year.Although the advances in the understanding of cancer biology and therapeutic advances in other tumors, but we only know a little pathogenesis of this high malignant tumor,the current therapeutic approaches, such as surgical resection, postoperative radiotherapy, chemotherapy and immunotherapy, and so on,GBM has poor prognosis and high recurrence rate.So,it is crucial to explore the pathogenesis of glioblastoma.micro RNAs(mi RN As) are a class of short non-coding RNAs that control the expression of a large number of genes in eukaryotes,the size is about 20~25 nucleotides(1), they are involved in various regulation ways, such as the development, hematopoiesis, cell proliferation and apoptosis,and so on(2,3).Previous study showed mi RN As are associated with initiation and development of glioma,they function as tumor suppressor or carcinogenic in initiation and progression of glioma through regulating the pathway which involved with their target genes(1).The mi RN As could be a novel breakthrough on glioma molecular therapy in future.VHL gene(Von Hippel Lindau) is first found by Von Hippel(4) and Linda(5), VHL function as tumor suppressor gene that located on chromosome 3p25~26(6,7), encoding 213 amino acid protein, called p VHL, the mutation of VHL contributes to tumorigenesis of various tumors,such as renal cell carcinoma, pancreatic cancer, central nervous system hemangioma, pheochromocytoma, and so on(8).VHL is also involved in initiation and progression of glioma,HIF-1α is a distinguished target of the VHL as an E3 ligase for degradation(9,10),the expression of VEGF is regulated by HIF-1a,and the VEGF is the most important vascular growth factor in initiation and progression of glioma(11).Except regulating HIF-1a level and anti-angiogenesis, VHL also can regulate apoptosis(12), cell cycle(13)and the effect of extracellular matrix(14).Our previous research suggest that VHL gene is down-regulated in glioblastoma, and negatively correlated with patient survival(unpublished data), in research of glioma,we found mi R- 566 is associated with initiation,malignant and prognosis of glioblastoma(9),bioinformatics predicted that VHL gene is regulated by mi R-566. Therefore, there may be a relationship among mi R- 566,VHL and VEGF play important roles in glioma development.This study will explore mi R-566 modulates VEGF via targeting VHL in human glioblastoma, to find a novel target of glioma therapy and provide a new and more effective method and thought of comprehensive treatment of glioma.The present study was divided into two parts:The first part of our study is mi R-566 modulates VEGF via targeting VHL in human glioblastoma in vitro.We first infected U87 GBM cells by lenti-ASmi R-566,Real time PCR was conducted to detect the expression of mi RNA-566 and VEGF m RNA after transfection,Western blot assay was used to detect protein(VHL, HIF-1α, VEGF,MMP-2,MMP-9) expression.Then transfected U87 GBM cells by VHL expression plasmid,detecting protein(VHL,HIF-1α,VEGF) expression by western blot.Luciferase reporter gene assay was conducted to detect the relationship between mi R-566 and VHL.The results showed that in U87 cells infected by lenti-AS- mi R-566, the expression of mi RNA-566,VEGF m RNA and HIF-1α, VEGF,MMP-2 and MMP-9 protein were decreased obviously,the expression of VHL protein was increased,in U87 cells transfected by VHL expression plasmid,the expression of VHL protein was increased,the expression of HIF-1α and VEGF protein were decreased; luciferase reporter gene assay was proved that mi R-566 could modulate VHL directly.Taken together, these results demonstrated that mi R-566 can modulate VEGF via targeting VHL in vitro and can affect the invasion ability of glioblastoma.The second part of this study is construction of the intracranial model,to further demonstrate mi R-566 can modulate VEGF via targeting VHL in vivo,we constructed intracranial model by U87 cells which infected by lenti-AS- mi R-566, immunehistochemical staining was used to measure protein expression and FISH was used to detect the expression of mi R-566 in tissue section. FISH that the expression of mi R-566 was decreased after transfection, immunohistochemical staining proved that the expression of VEGF,MMP-2 and MMP-9 protein was decreased and VHL protein was increased after transfection. These results demonstrated that mi R-566 can modulate VEGF via targeting VHL in vivo.Conclusion:Through fluorescent reporter assay and western blot, we demonstrated that VHL is a target of mi RN A-566,both in vitro and vivo,the inhibition of mi RNA-566 can down-regulate the expression of HIF-1α, VEGF,MMP-2 and MMP-9,oppositely up-regulate the expression of VHL;and the increase of VHL expression can inhibit the expression of HIF-1α and VEGF. These data showed that mi R-566 can modulates VEGF via targeting VHL both in vitro and vivo, and the inhibition of mi R-566 expression can inhibit the ability of invasion in glioblastoma.