Study On Expression, Function And Mechanism Of Fibulin-5 Gene In Human Gliomas

BACKGROUND:Gliomas,especially the high-grade gliomas,are the most common primary brain malignant tumor whose characteristic is quickly recurrence and an extremely poor prognosis,despite advances in surgical and clinical neuro-oncology.The microvessel density is an independent index in judging the grade of gliomas as to the conclusive evidence.Angiogenesis maybe the most important factor for the proliferation and metastasis of gliomas,and the anti-angiogenesis treatment maybe the most worth expectation for the better prognosis of the patients who had got the gliomas.fibulin-5(FBLN-5,DANCE or EVEC),which is a 448 amino acid glycoprotein,is the member of the fibulin family of extracellular matrix (ECM) proteins,and is mapped in chromosome 14q32.1.It contains six EGF-like repeats,all containing consensus sequences for Ca²⁺-binding(CB)-EGF-like repeats.The first CB-EGF-like repeat of fibulin-5 also contains a RGD sequence,which mediates interaction with cell surface integrins,includingαvβ3,αvβ5 andα9β1.fibulin-5 is a multifunctional extracellular matrix protein that mediates cell-cell and cell-matrix communication.It regulates the formation and assembly of elastic fibers primarily by binding tropoelastin, and secondarily by binding lysyl oxidase-like 1(Loxl-1) and EMILIN-1 (elastin microfibril interface-located protein).It can enhanced wound closure and healing by increasing tissue granulation and type I collagen expression.Its expression is normally low in adult vasculature,but it is remarkably elevated in developing or injured blood vessels,and in uterine myometrial arteries undergoing cyclic angiogenesis.Also,it takes an important role in the angiosclerosis and cutis laxa.The expression of fibulin-5 also augments the tumorigenicity of human HT1080 fibrosarcoma cells by increasing their DNA synthesis, migration toward fibronectin,and invasion through synthetic basement membranes.In stark contrast,Its expression is down-regulated in the majority of metastatic human malignancies,particularly in cancers of the kidney,breast,ovary,and colon.It also can slow the growth of cancer tumors in mice by preventing blood vessels from sprouting,fibulin-5 may play an important part in the tumor procession.In our research,we will discuss the role that fibulin-5 plays in the process of gliomas.Part 1 Expression and function of fibulin-5 gene in the gliomasObjective:The present study was carried out to investigate the expression of fibulin-5 in human glioma tissues.Methods:40 cases of formalin-fixed,paraffin-embedded specimens of tissue,including low-grade glioma of 21 patients(gradeⅠ～Ⅱ) and high-grade glioma of 19 patients(gradeⅢ～Ⅳ),and another normal brain tissues of 7 patients, were used to assess the expression status of fibulin-5 protein,Ki-67, Factorâ…§and HIF-1αby immunohistochemical staining.The Ki-67 labeling index(LI) and microvessel density(MVD) were calculated. Then we also analyzed the correlation between fibulin-5's expression and the Ki-67 LI,MVD or the expression of HIF-1α.Another 44 cases of the fresh tissue,including low-grade glioma of 22 patients(gradeⅠ～Ⅱ), high-grade glioma of 15 patients(gradeⅢ～Ⅳ) and the normal brain tissues of 7 patients,were used to assess the expression status of fibulin-5 mRNA by RT-PCR.Results:The expression of the fibulin-5's protein usually lay between cell and cell or cell and matrix.In the normal brain tissue,it always could be found;in the low grade gliomas tissue,there were 13 sample could be found;in the high grade gliomas,there were only 4 sample could be found.The expression of the fibulin-5's protein in the normal brain tissues and the low grade gliomas were higher than its expression in the high grade gliomas(P＞0.05).The expression of fibulin-5's protein in the vessel which was in the normal brain tissue usually lay in the great vessel,but in the low grade tissue,it usually lay in the capillary vessel.The expression of the fibulin-5's protein inverse correlated with the degree of malignancy,Ki-67 LI,MVD and the expression of HIF-1αof human gliomas(r=-0.455,-0.779,-0.320;P =0.003,0.000,0.044 ).Then we found that fibulin-5's mRNA always expressed in the normal brain tissue;there were 14 sample could be found in the low grade gliomas tissue;there were only 3 sample could be found in the high grade gliomas tissue.The expression of the fibulin-5's mRNA in the normal brain tissues and the low grade gliomas were higher than its expression in the high grade gliomas(P＞0.05).Conclusion:fibulin-5 could expressed in the normal brain tissue.The expression of fibulin-5 may correlated with the process and the angiogenesis of gliomas.Part 2 To construct the cell strain(HUVEC-2C) for expressing the fibulin-5 gene through the stable transfectionObjective:To construct an lentiviral vector carrying fibulin-5 gene and then stable transfected to the huaman umbilical vein endothelial cells -2C(HUVEC-2C).Methods:The expression of fibulin-5 in the HUVEC-2C and the U251 was checked by RT-PCR first.Then fibulin-5 was amplified by PCR and subcloned fibulin-5 into the lentiviral vector(pGC-FU),to generate the lentiviral expression vector, (pGC-FU-FIBULIN-5).Recombinant lentiviruses were produced by 293T cells following the co-transfection of pGC-FU-FIBULIN-5,with the packaging plasmids pHelper1.0 and pHelp2.0.So,we got the lentiveruses called KL425-LV.Last,we use the KL425-LV to stable transfect into HUVEC-2C and got the cell strain called Fibulin-5-GFP-LV. Results:There was no expression in the HUVEC-2C and the U251.The cell strain(Fibulin-5-GFP-LV) was expressing the fibulin-5 gene through the check by Western blot and Real-time PCR.Conclusion:The cell strain which was expressing the fibulin-5 gene was successed constructing.Part 3 Effect on the bionomics of HUVEC-2C by the fibulin-5 gene expressingObjective:The present study was carried out to investigate the effect on the bionomics of HUVEC-2C by the fibulin-5 gene expressing. Then we could figure out the function of the fibulin-5 during the angiogenesis.Methods:There were three groups in the experiment:CON group(used the normal HUVEC-2C),NC group(used the HUVEC-2C which was transfected the negative vector) and KD group(used the cell strain called Fibulin-5-GFP-LV,which was contructed in the Part 2).So we could exam the change of the ability of the proliferation,adhesion, invasion,apoptosis and vessel-sprouting of the HUVEC-2C during the fibulin-5 gene expressing.Results:By the MTT test,we found that the OD value in the KD group was lower than that in the CON group and the NC group(P＜0.05).In the adhesion's test,the OD value in the KD group was higher than that in the CON group and the NC group(P＜0.05).In the invasion's test,we found that the OD value in the KD group was lower than that in the CON group and the NC group(P＜0.05).By the flow cytometry's test,we found that the cell cycle in the KD group was prolonged(P＜0.05).In the vessel-sprouting abiltity test,we found that the number of the vessel was decreased in the KD group(P＜0.05). Conclusion:The expression of fibulin-5 gene could prolong the period of the cell cycle,inhibit the ability of the proliferation,invasion and the vessel-sprouting of HUVEC-2C.But it could enhance the ability of the adhesion,fibulin-5 gene expressing could inhibit the angiogenesis and make the vessel more stable.Part 4 Effect on the bionomics of HUVEC-2C and U251 by fibulin-5 gene expressing in the gliomas's microenvironmentObjective:The present study was carried out to investigate the effect on the bionomics of HUVEC-2C or U251 by the fibulin-5 gene expressing in the gliomas's microenvironment.Then we could figure out the function of the fibulin-5 during the process of gliomas.Methods:We first constructed the gliomas's microenvironment by HUVEC-2C and U251 co-cultured.There were three groups in the experiment:CON group(used the normal HUVEC-2C),NC group(used the HUVEC-2C which was transfected the negative vector) and KD group(used the cell strain called Fibulin-5-GFP-LV,which was contructed in the Part 2).So we could exam the change of the ability of the proliferation,adhesion, invasion and vessel-sprouting of the HUVEC-2C during the fibulin-5 gene expressing in the gliomas's microenvironment.We also examed the change of ability of the proliferation and invasion of the U251 by the same way.Results:â‘ Testing the change of the ability of HUVEC-2C's bionomics in the gliomas's microenvironment:By the MTT test,we found that the OD value in the KD group was lower than that in the CON group and the NC group(P＜0.05).In the adhesion's test,the OD value in the KD group was higher than that in the CON group and the NC group (P＜0.05).In the invasion's test,we found that the OD value in the KD group was lower than that in the CON group and the NC group(P＜0.05). In the vessel-sprouting ability test,we found that the number of the vessel was decreased in the KD group(P＜0.05).â‘¡Testing the change of the ability of U251's bionomics in the gliomas's microenvironment:By the MTT test,we found that the OD value in the KD group was lower than that in the CON group and the NC group(P＜0.05).In the invasion's test, we found that there were no difference in the KD group,CON group and NC group(P＞0.05).Conclusion:The expression of fibulin-5 gene in the gliomas's microenvironment could inhibit the ability of the proliferation, invasion and the vessel-sprouting of HUVEC-2C.It could enhanced the ability of the adhesion of HUVEC-2C.It could also inhibit the U251 's reproductive activity,fibulin-5 gene expressing could inhibit the angiogenesis and make the vessel more stable in the gliomas's microenvironment.It mightbe make the tumor vessel normalization. Mightbe,it inhibit the gliomas's ability of the proliferation and the invasion directly.