Effects Of RNAi-mediated Endoglin Gene Silencing In Endothelial Cells Of Ovarian Carcinoma

Background:Tumor angiogenesis depends on the activation,proliferation and migration of vascular endothelial cells.Tumor-derived microvascular endothelial cells(TdMECs)are different from those in normal tissues.It's important to isolate and culture TdMECs for the study on the mechanism of tumor angiogenesis and the antiangiogenesis therapy. Endoglin (CD105) is a cell-surface glycoprotein most recently identifiedas an optimal indicator of proliferation of human endothelial cells. It is a component of the receptor complex of Transforming Growth Factor (TGF-β), a pleiotropic cytokine involved in cellular proliferation, differentiation and migration .The inhibition of CD105 expression enhanced the ability of TGF-βto suppress growth, migration and capacity to form capillary tubes of cultured endothelial cells.Several findings suggest for the involvement of CD105 in angiogenesis and vascular development, and in maintaining vessel wall integrity. The expression of endoglin is elevated on the endothelial cells of healing wounds, developing embryos, inflammatory tissues, and solid tumors. The finding has prompted several pre-clinical studies designed to get a deeper understanding on the role of CD105 in angiogenesis, and to evaluate the most appropriate clinical settings to utilize CD105 as a therapeutic target.Objective:Based on our understanding of the heterogeneity of tumor-derived endothelial cells and the occurrence of abundant newly-formed microvessels in ovarian carcinoma,the present study tried to isolate and purify ovarian carcinoma-derived microvascular endothelial cells(ODMECs).To construct a siRNA expression plasmid transcripted by RNA interference (RNAi),using gene endoglin as a clone target.To identify the recombinant plasmid nucleic acid sequence by sequencing on the standard sequencer,and to evaluate the plasmid impact to the endoglin gene expression in transfected ODMECs.These results lay a foundation for the further research on the function of endoglin in the development and therapy of ODMECs.Methods:1. The ovarian carcinoma tissues were digested by combination of trypase,collagenaseⅠand dNaseⅠ.ODMECs were purified using a MACS magnetic cell sorting system with antiCD31 antibody. The obtained ODMECs had a high purification and could be successfully cultured in vitro.cells were identified by Morphological,biochemical and phenotypic characteristics. biological property of cells was investigated.2. Two DNA sequences with small hairpin structure were designed and synthesized.The complement form was obtained by annealing and cloned into Plasmid pGenesil-1.Recombinant plasmid was transformed into strain DH5α,identified by using restriction enzyme and sequencing of nucleic acid.3. The short interfering RNA(siRNA)expression plasmid pGenesil-ENG1 and pGenesil-ENG2 were transfected into ODMECs by lipofectamine.The expression of the endoglin gene Messenger Ribonucleic Acid(mRNA)on the transfected cells was measured by reverse transcription polymerase chainreactio(RT-PCR) . Metromethyl Thiazolyl Blue(MTT) was used to observe the proliferation of ODMECs which transfected and no-transfected with the plasmid. The formation of two-dimensional tubular structure of ODMECs in vitro was observed by light microscopy after transfection pGenesil-ENG1.Results:1. The ODMECs grew as gconfluentmonolayer like cobblestones and kept their features through passages. They expressed CD31, CD105,FⅧ-RAg and the purity was about 99.6 %. They bing of Ulex europaeus lectin I(UEA-I) and uptake of Acetylated-Low-Density Lipoprotein (DiI-ac-LDL). ODMECs could form tubule-like structure(TLS) in medium.2. By restriction endonuclease,eukaryotic expression plasmid of Endoglin proved to be successfully constructed. Sequence analysis of inserted fragment revealed the same sequence as synthesized oligonucleotides.3. The pGenesil-1 Plasmid was cotransfected into ODMECs by lipofectamine with three siRNA expression plasmids pGenesil-ENG1,pGenesil-ENG2 and pGenesil-H (Negative control)respectively.We could find the green fluorescent in ODMECs under the fluorescent microscope. 4. The expression of the Endoglin mRNA in ODMECs transfected with recombinant plasmid decreased was Reduced after 48 hours, and the growth of ODMECs cells was suppressed significantly(P<0.01).ODMECs two-dimensional in vitro formation of TLS are also significantly reduced (P <0.01).Conclusion:this study revealed that the combined digestion with trypase,collagenaseⅠand dNaseⅠfollowed by the purification with the magnetic MicroBeads could effectively improve the quantity and purity of ODMECs. The method can be useful to endothelial study in ovarian carcinoma.The results of the present study is very helpful to our understanding the heterogeneity induced by tumor environment and provide more useful experimental material and in vitro model for the studies on angiogenesis and anti-angiogenesis.The Endoglin siRNA expression vector has been successfully constructed. It effectively downregulated the expression of Endoglin in ODMECs, inhibiting proliferation and TLS formation.These results suggest that a strategy based on siRNA targeting of endoglin may build the foundation to the clinical management of ovarian carcinoma.