Research On Cell Penetrating Peptides VP22 Affecting The Antitumor Activity Of The Tumor Suppressor Protein PTEN In Vitro

Objectives:1.By constructing a PTEN/VP22 fusion protein eukaryotic expression vector pc DNA3-PTEN-VP22, verify whether VP22 can mediated PTEN transport in esophageal cancer cell line Eca109 cells.2.Study on whether PTEN/VP22 can play a role in anti-tumor in vitro than PTEN significantly, and lay the foundation for the development of novel PTEN/VP22 double gene antitumor drugs.Methods:Using the esophageal carcinoma cell line Eca109 as the experimental object, the experiment was divided into four groups: pc DNA3-PTEN group, pc DNA3-PTEN-VP22 group, pc DNA3-VP22 group, and pc DNA3(the control group).1. The expression of PTEN protein were detected by the method of cell immunofluorescence.2. Western blot assay was used to determine PTEN and p-Akt level.3. CCK-8 method was used to detect the cell proliferation activity.4. Flow cytometry method was performed to measure the cell cycle and apoptosis rate.Results:1.Cell immunofluorescence showed that the recombinant plasmid pc DNA3-PTEN,pc DNA3-PTEN-VP22 can express green fluorescent protein PTEN in Eca109 cells, while in group pc DNA3 and group pc DNA3-VP22,there were not observed the green fluorescent protein, PTEN protein expression in group pc DNA3-PTEN-VP22 was higher than that in group pc DNA3-PTEN(P < 0.05).2.Western-blot showed that there were detected PTEN protein and PTEN-VP22 fusion protein in group pc DNA3-PTEN and group pc DNA3-PTEN-VP22 separately,while group pc DNA3 and group pc DNA3-VP22 were not detected PTEN protein.3. CCK8 results showed that compared with pc DNA3 control group, pc DNA3-PTEN and group pc DNA3-PTEN-VP22 had an inhibitory effect on the growth of Eca109 cells, the inhibition was a time- and concentration dependent manner(P < 0.05), but the group pc DNA3-VP22 on the growth of Eca109 cells had no inhibitory effect(P > 0.05), meanwhile the inhibition of group pc DNA3-PTEN-VP22 on the growth of Eca109 cells was more stronger than that of group pc DNA3-PTEN at the concentration of 0.5μg/ml(P < 0.05),and the the inhibitory rate was close to 50% after 48 h.4. Cell cycle results show that, compared with the control group, the G1 phase percentage of cells in group pc DNA3-PTEN and group pc DNA3-PTEN-VP22 increased(P < 0.05),while the G1 phase percentage of cells in group pc DNA3-VP22 had no difference(P > 0.05). Compared with group pc DNA3-PTEN, group pc DNA3-PTEN-VP22 had a significantly higher proportion of cells in the G1 phase(P < 0.05).5. The apoptosis results show that, compared with control group pc DNA3,the apoptosis rate was(7.65±0.68)%,and that in group pc DNA3-PTEN-VP22 and group pc DNA3-PTEN were(30.66 ±0.99)% and(69.55±0.38)% separately, which were increased(P < 0.05),while in group pc DNA3-VP22, the apoptotic rate was(7.86±0.13)%(P > 0.05),meanwhile the apoptosis rate in pc DNA3-PTEN-VP22 group was higher than that of pc DNA3-PTEN group(P < 0.05).6. Western-blot assay showed that, compared with pc DNA3 group, the p-Akt expression level in pc DNA3-PTEN and pc DNA3-PTEN-VP22 group were significantly lower(P < 0.05),meanwhile the expression of p-Akt in pc DNA3-PTEN-VP22 group was significantly lower than that in pc DNA3-PTEN group(P < 0.05), while in pc DNA3-VP22 group, p-Akt protein expression was no difference(P > 0.05).Conclusion:1. The eukaryotic expression vector pc DNA3-PTEN-vp22 can express PTEN/VP22 fusion protein in Eca109 cells.2. VP22 promotes the expression and distribution of PTEN in more Eca109 cells in vitro, and ultimately play a more significant antitumor effect.3. PTEN may inhibit cell signaling molecules Akt phosphorylation and decrease the expression of p-Akt,thereby inhibiting proliferation and inducing apoptosis.