The Effects Of RNA Interference Targeting Geminin Gene On The Proliferation Of Vascular Smooth Muscle Cells In Rats

Background：The proliferation, migration and synthesis of extracellular matrix of vascular smoothmuscle cells (VSMCs) is the common pathologic basis of cardiovascular diseases such asatherosclerosis and restenosis of angioplasty, but the mechanism of pathogenesis is stillunclear. The recent reports have found that the phenotype transformation of VSMCs was akey link which led to the above pathological process. Geminin is now regarded as amuliti-functional small-moleculear protein, and an important synergy role during theproliferation-differentiation process in the eukaryotic cells, suggesting that Geminin geneinvolved in the regulation of the phenotype transformation of VSMCs. However, it is notknown how Geminin gene affects on the proliferation of VSMCs, and whether it is involvedin the regulation of cells phenotype. In our study, the effects of geminin gene on theproliferation and phenotype of VSMCs in rats was analyzed by using siRNA method. Itmay be useful for providing some data of experiments in the area of prevention in theatherosclerosis and angioplasty restenosis.VSMCs have two phenotypes usually: dedifferentiated phenotype (synthetic phenotype)and differentiated phenotype (contractile phenotype), they can transform with each otherunder certain condition. In the adult artery wall, there are three laminae of blood vessels,including intima, tunica media and adventitia. VSMCs are mainly located in tunica mediaand occured as the differentiated phenotype under physiologic state. When artery injured orcultured in vitro, VSMCs can transform from differentiated phenotype into dedifferentiatedphenotype, and migrate into intima and proliferate. The synthetic phenotype is an immaturestate with low degree of differentiation, meanwhile contractile phenotype is a highlydifferentiated one. The main markers of synthetic phenotype include in vasoactivesubstance and extracellular matrix protein, which could repair the injured arterial wall. In addition, growth factors could stimulate proliferation and the transformation of thephenotype. The initiation complex consists of ORC1and Cdt1, which play an importantphysiological role in the regulation of cell proliferation. It is well known that DNAreplication is a basic feature of cell proliferation. Initiation complex is a kind of initiatorprotein in the eukaryotic replication. Abundance evidence supported that successfulcomposition of the initiation complex is the prerequisite of DNA replication.It is not kown whether Geminin involved in the regulation of VSMCs phenotypictransformation, and what is the relationship between cells proliferation and Geminin gene.We hypothesis that the Geminin which could participate in the regulation of phenotypetransformation and proliferation of VSMCs is a repressor of origin licensing.Objective：To investigate the effects of geminin gene on the proliferation and phenotype ofVSMCs in rats by using the siRNA method, and analyze the mechanism of the Geminingene regulating cells proliferation.Methods：1. VSMCs were transfected with three paris of Interference sequence to knock down ofGeminin gene, and screened out the interference effect of the the most significant sequencewhich was used to build the model of slow virus packages.2. The differentiated phenotype of VSMCs was obtained by serum stimulation, thechange of Geminin expression was measured by Western blotting and RT-PCR.3. The phenotypic markers of VSMCs were detected by Western blotting afterGeminin gene silencing, the proliferation of VSMCs was observed by [3H]-TdR and EdUincorporation.4. The changes of Initiation complex—ORC1,Cdt1protion were analyzed afterGeminin gene silencing, and their interrelationship was examed by immunofluorescenceand co-immunoprecipitation.Results:1. After transfected three pairs of siRNA sequences targeting Geminin generespectively, a significant decrease of Geminin expression was observed in one of them,which acted as the model of building lentiviral interference.2. The differentiated phenotype of VSMCs was obtained successfully after serum stimulation, the expression of differentiation phenotypic marker-α-actin significantlydecreased, while that of dedifferentiation phenotypic marker-OPN significantly increased,indicating the Geminin gene could be involved in the regulation of cells phenotypictransformation.3. After Geminin gene silencing, The [3H]-TdR and EdU incorporation of Positiveinterference group was significantly higher than that of two controls, suggesting thatGeminin gene could inhibit the proliferative of VSMCs and maintain The differentiatedphenotype.4. The expression of ORC1and Cdt1in positive group significantly increased,compared with that of two controls, the result of immunofluorescence andco-immunoprecipitation showed the direct interaction between Cdt1and Geminin gene.Conclusion：Geminin gene was closely related to the proliferative and the phenotypetransformation of VSMCs, speculating that Geminin gene could be involved in regulationof cells proliferation by affecting the formation of initiation complex.