Effect Of Lentiviral Vector Of RNA Interference Targeting Against Skp2 On Cell Cycle, Apoptosis And Proliferation Of Endometrial Carcinoma Cell HEC-1-A

Objective: To construct lentiviral vectors of RNA interference (RNAi) specific to S-phase kinase associated protein 2 (Skp2) and to inhibit the expression of Skp2 gene in endometrial carcinoma cell HEC-1-A, and to investigate the effects on the expression of p27, cyclinD1 and caspase-3, as well as cell cycle, apoptosis and proliferation of endometrial carcinoma cell HEC-1-A.Methods: Lentiviral vectors (LV-shRNA), containing U6 promoter and green fluorescent protein (GFP), were constructed to deliver small hairpin RNA (shRNA) targeting Skp2 into HEC-1-A cells. The control group was the HEC-1-A cells transfected with negative vector. The transfection efficiency was determined by detecting the GFP expression with the fluorescent microscopy. The expression level of Skp2 mRNA was examined by reversed transcript polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction (RT-qPCR). The expression level of Skp2 protein was finally examined by Western blotting for identifing the inhibitory efficiency of RNAi. In order to analyze the effects of silencing Skp2 gene on the expression of p27,cyclinD1 and caspase-3 ,as well as cell cycle, apoptosis and proliferation, the methods mentioned above was applied to detect the changes in expression of p27,cyclinD1 and caspase-3 on mRNA and/or protein level respectively. For the same purpose, cell counting, CCK-8 method and flow cytometry were performed to examine the changes in cell cycle, apoptosis and proliferation after RNAi targeting Skp2.Results: 1. Four RNAi lentivirus expression vectors targeting to four various sites of Skp2 gene were produced, and the sequence and correct site of ds oligos inserted were confirmed by PCR and sequencing assay; 2. The lentivirus were packaged in 293T cells with high titer; 3. Small hairpin RNA (shRNA) targeting Skp2 was transfected into endometrial carcinoma cells HEC-1-A by recombinants of lentiviral vectors and the transfection efficiency could reach more than 70%; 4.The expression of Skp2 gene was inhibited by lentiviral recombinants of LV-Skp2-1,LV-Skp2-2,LV-Skp2-3 and LV-Skp2-4. The inhibitory efficiency of LV-Skp2-3 and LV-Skp2-4 could reach over 50%, higher than the other two; 5. Silencing Skp2 gene by RNAi did not influence the expression of p27 and cyclinD1 mRNA; however, it could up-regulate the expression of P27 protein and down-regulate the expression of CyclinD1 protein; 6.Inhibiting the expression of Skp2 gene had the influence on the processes of HEC-1-A cell cycle thus induced G2/M arrest; 7.Down-regulating the expression of Skp2 gene inhibited proliferation of HEC-1-A cell and slowed down its growth velocity; 8. No obvious effect on the apoptosis of HEC-1-A cell was observed by inhibiting the expression of Skp2 gene.Conclusions: Effective down-regulating Skp2 expression by RNAi helped to up-regulate the expression of P27 protein and down-regulate the expression of CyclinD1 protein; however, its impact on the expression of p27 and cyclinD1 mRNA was not observed. Silencing Skp2 gene by RNAi influenced the processes of HEC-1-A cell cycle, induced G2/M arrest and further inhibited the proliferation of cells and slowed down their growth velocities with insignificant influence on apoptosis. Skp2 might be a potential adjuvant gene therapeutic target for human endometrial carcinoma.