Objectives Silencing Geminin gene selectively by using RNA interference and investigate the mechanism of the Geminin gene silence regulating VSMCs proliferation.
Methods VSMCs proliferation were determined by [3H]-thymidine incorporation, 5-ethynyl-2’-deoxyuridine (EdU) incorporation and flow cytometry; mRNA and Protein expression of Geminin and Cdt1 were determined by real time-PCR, westernblotting, and immunohistochemistry; the interrelationships of Geminin and Cdt1 were examed by immunofluorescence and co-immunoprecipitation (Co-IP).
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 a stable model of building lentiviral interference. After Geminin gene silencing, the 3H-thymidine and EdU incorporation of positive interference group was significantly higher than that of two controls. The flow cytometry showed Geminin gene silencing contributed to the proliferation of VSMCs by supporting the G0/G1-S cell-cycle progression, and may be no apoptotic exists in this process. The protein expression and mRNA of Cdt1 in positive group significantly increased, compared with that of two controls. The results of immunofluorescence and Co-IP showed the close interaction between Cdt1 and Geminin gene in the proliferation of the VSMCs.
Conclusions Geminin gene silencing contributes to the proliferation of A10 by supporting the G0/G1-S cell-cycle progression has been demonstrated, interestingly, this positive effect is in short term (24-48h) and reversibility. We believed that the results of the study are not caused by our research method, on account of lentivirus-delivered siRNA transfection instead of liposomes. Lentivirus is one kind of reversal viruses that could exhibit the basic feature and structure of reversal. It can integrate into the host genome and demonstrate long-term expression of integrated genes, and causes efficient down-regulation of gene expression, resulting in a functional inactivation of the target genes. In our study, we constructed a lentivirus vector-mediating RNAi targeting of Geminin (RNAi group), and it down-regulated Geminin expression of up to 80-90% efficacy in A10 cells with great specificity. In conclusion, lentivirus-delivered siRNAs are capable of specific, highly stable and functional silencing of Geminin gene expression in our study.