The Experiment Research On Human Retinal Vascular Endothelial Cells With RNAi Targeting Integrin A_vβ₃

Retinal vascularization is a common complication of hegmato genous hypoxia of retia,which is caused by the proliferation of vascular cells.In recent years,there has been a sustained interest in vascularization processes.Much,if not all,of the work has included the concept of new vessel morphogenesis. Surprisingly,most of the work has not addressed developmental mechanisms directly,but rather as an offshoot of a disease process,wound healing process, or from the perspective of inducing vessels in an ischemic site.One theme has dominated the various studies on capillary or endothelial tube morphogenesis—integrin-mediated cell behavior.Integrin biology impacts virtually every known step of nascent vessel formation.Because the molecular mecharism of retinal vascular ization is very comlex,the optimal therapy for patients with these disease has yet to be defined.Integrins are a family of noncovalently associated hetero dimeric cell surface receptors composed of an[alpha]-and[beta]-subunit that mediate cell-ECM and cell-cell adhesions.There are currently 18[alpha]-and 8 [beta]-subunits,not including splice variants,that combine to form more than 24 different integrins.Integrins are most widely known for their role as adhesion receptors for a variety of ECM proteins,such as FN,vitronectin, collagen,laminin,von Willebrand factor,fibrinogen,thrombospondin,and osteopontin.Most integrins recognize several ECM proteins and conversely, most matrix proteins bind more than one integrin.Integrins participate in many aspects of vasculogenesis,such as angioblast repositioning,extension/ protrusive activity,lumen formation,and vascular fusion.Similarly,integrins are important for endothelial cell behavior during angiogenesis,endothelial cell vacuole formation and coalescence into lumenal structures are arginineglycine-aspartic acid(RGD)-dependent and involve[alpha]_v[beta]₃ and [alpha]₅[beta]₁.Expression of antisense to the[beta]₃-integrin subunit or addition of anti-[alpha]_v[beta]₃ and anti-[alpha]₅ antibodies inhibited vacuole and lumen formation.RNA interference(RNAi)is a phenomenon displayed by most eukaryotic cells to get rid of foreign double-stranded RNA mole cules from themselves. RNAi was discovered in the color test of morning glory,and in anmimal field,It was first studied in animal cells by Fire and colleagues in the nematode, Caenorhabditis elegans.RNAI has now been demonstrated that it can close specific gene's expression in mRNA level,post transcription through double-stranded RNA(dsRNA).the target gene'mRNA can be degra dated efficiently and specifically when the double-stranded RNA(dsRNA)which has homologous sequences with the target gene's mRNA is transfected into cells, and the gene's expression is inhibit ted.Knowledge of RNAi mechanism in mammalian cell in 2001 brought a storm in the field of drug discovery.During the past few years scientists all over the world are focusing on exploiting the therapeutic potential of RNAi for identifying a new class of therapeutics.The applications of RNAi in medicine are unlimited because all cells possess RNAi machinery and hence all genes can be potential targets for therapy.RNAi can be developed as an endogenous host defense mechanism against many infections and diseases.Several studies have demonstrated therapeutic benefits of small interfering RNAs and micro RNAs in animal models.This has led to the rapid advancement of the technique from research discovery to clinical trials.The goal of the experiment is to design and construct u6 promoter-based small interfering RNA(siRNA)expression vectors specific to integrin a_vβ₃ and determine their expression effects on the human retinal vascular endothelial cells for the treatment of retinal neovascularization.[objective]To determine the role of Integrin a_vβ₃ in retinal neovascula rization and discuss the mechanism of down-regulated Integrin a_vβ₃ expression by RNA interference(RNAi)technology,in HRCECs cell line.[Materials and methods]1.The expression of Integrin a_vβ₃ in HRCECs cell line in different condition.2.Synthesis of dsRNA and Construction of shRNA-Expression Vector.2.1 Selection and synthesis of RNAi targets2.2 Construction of hpRNA-expression vector:To construct vector capable of producing hairpin siRNA molecule for Integrin a_vβ₃,we use the mammalian expression plasmid vector pGPU6/GFP/ Neo.Two pairs of Integrin a_vβ₃ oligonucleotides were synthesized.We named them PGCU6-si-Integrin a_v and PGCU6-si-Integrinβ₃ respectively.Oligos were started with Hindâ…¢site and terminated with BamHI site.These dsDNA molecules were ligated to the correspondence sites of pGPU6/GFP/Neo plasmid vector,resulting in the formation of a plasmid containing inverted repeats for Integrin a_vβ₃.3 Transfection of Integrin a_vβ₃ RNAi eukaryotic expression vectors into HRCECs cell line after building retinal vascularization model.3.1 Using Suohua transfectant,the HRCECs cell line expressing highly Integrin a_vβ₃ was transfected with RNAi eukaryotic expression vectors PGCU6-si-Integrin a_vβ₃.3.2 RT-PCR was used to testify the mRNA level in the transfected HRCECs cells.3.3 Western Blot was used to testify the protein level in the plasmid transfected HRCECs cells.3.4 Immunohistochemistry was used to testify the protein level in the plasmid transfected PVR animal model.[Results]1.The expression of Integrin a_vβ₃ in HRCECs cell line.The expression of Integrin a_vβ₃ in HRCECs cell line is very high under hypoxic condition.2.Construction of shRNA-Expression VectorThe oligonucleotides were designed and synthesized which encoding short hairpin transcripts directed against three different parts on Integrin a_vβ₃ mRNA. The oligonucleotides were then ligated into the Linearized plasmid pGCU6/GFP/Neo.The constructed vectors were confirmed by enzyme cutting:restriction endonuclease BamHâ… and Hindâ…¢were employed to cutting the recombinant plasmid,then the cutting product were electrophoresis in 1%agarose gel,and identified by DNA sequencing at last:the plasmid were sequenced by shanghai ShengGong biotechnology limited company.3.Cell transfection and identificationTransfection of Integrin a_vβ3 RNAi eukaryotic expression vectors into HRCECs cell line or Retinal vascularization animal model.3.1 The HRCECs cell line expressing highly Integrin a_vβ₃ was transfected with RNAi eukaryotic expression vectors pGPU6/GFP/Neo using Suohua transfectant. 3.2 RT-PCR and Western Blot suggested that Integrin a_vβ₃ expression in mRNA and protein level was decreased in the transfected cells4.Effects of Integrin a_vβ₃ downregulation on the proliferation of the HRCECs cell lineAdhesion,migration,MTT and FCM method were used to draw the inhibition of proliferative curves of the transfected cells in vitro,showing that the biological function was inhibited in transfected cell.5.Experiment on small mice in vivo testified that Integrin a_vβ₃ SiRNA can down regulation can inhibit the development of retinal neovascularization.6.Statistical AnalysisData statistical analysis was performed using the SPSS 10.0 statistics software package.All results were expressed as Mean±SD,and p＜0.05 was used for significance.[Conclusions]1.The shRNA-expression vector of Integrin a_vβ₃ was constructed successfully.2.RT-PCR and Western Blot results showed that Integrin a_vβ₃ expression was significantly inhibited by siRNA transfectants in HRCECs cells at mRNA and protein levels.3.The results of functional experiments showed that downregulation of Integrin a_vβ₃ expression via RNA interference could influence the biological behaviour of HRCECs cells.It could be presented in the following aspects:a.The expressions of Integrin a_vβ₃ in the transfected cells were inhibited significantly compared with those in control by RT-PCR and western-blot assay. It mainly focused on the decrease of Integrin a_vβ₃ in quantity.b.The suppression of Integrin a_vβ₃ expression in HRCECs cells could reduce the potential proliferation of cells in vitro.Taken together,downregulation of Integrin a_vβ₃ expression via RNA interference could inhibit significantly the expressions of Integrin a_vβ₃ in the transfected cells.It could decrease the proliferative capability of HRCECs.It was demonstrated that targeting Integrin a_vβ₃ could potentially be a new experimental approach for retinal vascularization gene therapy.Alteration the biological behavior of HRCECs cells by RNAi technology is a trend for the treatment of retinal vascularization.