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Abstract
Objectives To investigate the effects of geminin gene on the proliferation and phenotype of VSMCs in rats by using the siRNA method, and analyse the mechanism of the Geminin gene regulating cells proliferation.
Methods
VSMCs were transfected with three paris of Interference sequence to knock down of Geminin gene, and screened out the interference effect of the the most significant sequence which was used to build the model of slow virus packages.
The differentiated phenotype of VSMCs was obtained by serum stimulation, the change of Geminin expression was measured by Western blotting and RT-PCR.
The phenotypic markers of VSMCs were detected by Western blotting after Geminin gene silencing, the proliferation of VSMCs was observed by [3H]-TdR and EdU incorporation.
The changes of Initiation complex—ORC1, Cdt1 protion were analysed after Geminin gene silencing, and their interrelationship was examed by immunofluorescence and co-immunoprecipitation.
Results
After transfected three pairs of siRNA sequences targeting Geminin gene respectively, a significant decrease of Geminin expression was observed in one of them, which acted as the model of building lentiviral interference.
The differentiated phenotype of VSMCs was obtained successfully after serum stimulation, the expression of differentiation phenotypic marker-α-actin significantly decreased, while that of dedifferentiation phenotypic marker-OPN significantly increased, indicating the Geminin gene could be involved in the regulation of cells phenotypic transformation.
After Geminin gene silencing, The [3H]-TdR and EdU incorporation of Positive interference group was significantly higher than that of two controls, suggesting that Geminin gene could inhibit the proliferative of VSMCs and maintain the differentiated phenotype.
The expression of ORC1 and Cdt1 in positive group significantly increased, compared with that of two controls, the result of immunofluorescence and co-immunoprecipitation showed the direct interaction between Cdt1 and Geminin gene.
Conclusions Geminin gene was closely related to the proliferative and the phenotype transformation of VSMCs, speculating that Geminin gene could be involved in regulation of cells proliferation by affecting the formation of initiation complex.