Effects Of RNA Interference For ORC1 Gene On The Phenotype Of Rat Vascular Smooth Muscle Cells

Background :Coronary heart disease,stroke and other vascular complications caused by atherosclerosis are a key cause of death and disability in most countries.The modern cell and molecular biology techniques have shown that atherosclerotic lesions are characterized by macrophages migration,smooth muscle cell proliferation,lipid accumulation,formation of a large number of collagen fibers,elastic fibers and proteoglycans.Vascular smooth muscle cells (VSMCs) are well known to be one of very important components of the vessel wall.Excessive proliferation of VSMCs is common pathologic basis of artherosclerosis,hypertension and restenosis.VSMCs have two phenotypes:contractile phenotype (differentiated phenotype) and synthetic phenotype (non-differentiated or dedifferentiated phenotype).VSMCs of contractile phenotype are highly differentiated and that of synthetic phenotype are immature with low degree of differentiation.In the cardiovascular diseases such as high blood pressure,atherosclerosis and restenosis after angioplasty VSMCs will be transformed from contractile phenotype into synthetic phenotype,which is a key step in its proliferation and migration.The main markers of Contractile phenotype concludesα-smooth muscle actin (α-SM actin),smooth muscle myosin heavy chain2 (SM-2),calponin,vimentin and so on. Matrix Gla protein and osteopontin are main markers of synthetic phenotype.Origin recognition complex (ORC) is composed of six subunits,which can specially bind to the replication origin in the genomes.Cell can't process the DNA replication without ORC.After binding to the replication origin,ORC provide a pad for the other initiation factors (such as cdc6,Cdt1,MCM2-7) to conform the pre-replicative complex (pre-RC).Activated by various kinase,pre-RC will promote the cell from G1-phase into S-phase and commence DNA replication.The initiation of DNA replication is regulated by ORC activity in eucaryotic cell.All of ORC subunits bind to the DNA throughout the cell cycle in yeast,while in mammalian cells,the ORC2-6 subunits bind to the DNA,ORC1 is removed from the chromatin as cells progressed into S phase and rebound to chromatin only as cells progressed into late telophase.RNA interference is a new method for researching the function of gene and committing gene therapy,which can inhibit the expression of target gene specially and effectively.ORC1 is closely related to cell proliferation and strictly control the cell cycle process through rapid combination with or separation from the ORC2-5 complex.It is widely accepted that ORC1 is the key to regulate the activity of ORC. According to this understanding,In previous studies we interfered with ORC1 gene with liposome-mediated siRNA transfection into rat VSMCs,and observe its effect on the cell cycle and proliferation of rat VSMCs,then we have been confirmed that RNA interference with ORC1 gene can inhibit VSMCs'proliferation stimulated by serum.However,It has not yet been studied how the phenotype markers change (do they increase or decrease) and whether VSMCs change from synthetic phenotype to contractile phenotype.It will be answered in this study.Objective:The purpose of present study is as follows:(1)To prove that transfection of specific positive siRNA can inhibit the expression of ORC1 effectively.(2)To explore the change of proliferative ability when VSMCs have been transfected with siRNA targeting ORC1 gene. (3) To explore the change of cell phenotype when the VSMCs have been transfected with siRNA targeting ORC1 gene.Methods:1) VSMCs were obtained by the culture of adherent tissue of rat aortic;Morphological characteristics were observed under the inverted microscope;detection ofα-SM-actin to identify VSMCs was performed by immunofluorescence.2) Lipofectamine2000 was used to transfected siRNA into VSMCs.Inhibitory effect to gene ORC1 was detected by western blot.3) We detected the influence of specific inhibition of the ORC1 gene on the proliferation of rat VSMCs through the MTT test.4) Cell morphology and structure were observed by confocal laser scanning microscope after immunofluorescence;Flow cytometry and Western blot was applied to detection of changes in cell phenotype markers'expression. Results:1) VSMCs in rats was successfully cultured in vitro,then was confirmed by light microscope and Immunofluorescence cytochemistry to establish a platform for further experiment.2) It was confirmed by Western blot that specific positive siRNA transfection can significantly inhibit the ORC1 gene expression in VSMCs,which make further experiment to be possible.3) In VSMCs transfected with siRNA targeting ORC1 gene,the optical density (OD) of MTT (0.1065±0.0257) decreased significantly,Compared with that in the non-transfection group and negative siRNA group(0.1787±0.0412,0.1819±0.0386).4) ORC1 gene silencing increased the expression of VSMC contractile markersα-SM actin and SM-2 and decreased the expression of the synthetic marker osteopontin,in concert with VSMCs differentiated morphology.Conclusion:1) ORC1 gene is closely related to proliferation of VSMCs.When the expression of ORC1 gene was inhibited by RNA interference the proliferation of VSMCs was also significantly suppressed.2) ORC1 gene silenced by RNA intereference can mediate the transition of VSMCs from synthetic phenotype to contractile phenotype.