Effects Of PNUTS On Proliferation And Invasion Of Laryngeal Squamous Cell Carcinomas Hep-2 Cells And Its Mechanism

ObjectiveTo explore the effects of Adv vector-mediated siRNA targeting PNUTS on proliferation,invasion,migration and epithelial-mesenchymal transition of laryngeal squamous cell carcinomas Hep-2 and its probable regulatory mechanism,hoping to find a new treatment target of linlaryngeal carcinomas.Methods1.Construction of PNUTS siRNA Recombinant adenovirus;screening of the best MOI and identification of its effects(1)Hep-2 cells was infection with different MOI,and the best MOI was the one whose transfection efficiency was more than 80%.(2)Levels of PNUTS mRNA and protein were detected by RT-PCR and Western blotting respectively.2.Effects of Ad-siPNUTS on biological function of Hep-2 cells(1)MTT assay was used to detect the effect of Ad-siPNUTS on the proliferation of Hep-2 cells.(2)Transwell assays were used to detect the effect of Ad-siPNUTS on the invasion and migration of Hep-2 cells.(3)Western Blotting was used to detect the effect of Ad-siPNUTS on the expressions of E-cadherin and N-cadherin proteins.3.The regulation mechanisms of PNUTS on Hep-2 cells Western Blotting was used to detect the effect of Ad-siPNUTS on the expressions of PI3K?P-AKT?total Rb?p-Rb?E2FI and ZEB1 proteins.Results1.PNUTS siRNA Recombinant adenovirus was constructed successfully and the best MOI was 100;RT-PCR and Western Blotting results showed that the expression of PNUTS in Hep-2 cells could be silenced by Ad-siPNUTS specifically and efficiently.2.MTT assay showed that compared with the group Ad-GFP,the proliferation of Hep-2 cells in group Ad-siPNUTS were inhibited on the second day(P=0.004),the third day(P=0.001)and the fourth day(P=0.000).There was no significant difference between the group Ad-GFP and group PBS(P>0.05).3.Transwell migration assy showed that compared with group Ad-GFP,the migration ability of Hep-2 cells in group Ad-siPNUTS were decreased significantly(P=0.000),and there was no significant difference between group Ad-GFP and group PBS(P=0.372).4.Transwell invasion assy showed that compared with group Ad-GFP,the invasion ability of Hep-2 cells in group Ad-siPNUTS were decreased significantly(P=0.000),and there was no significant difference between group Ad-GFP and group PBS(P=0.420).5.Western Blotting results showed tha compared with group Ad-GFP,protein expressions of total Rb(P=0.000),p-Rb(P=0.000),PI3K(P=0.023),p-AKT(P=0.000),E2F1(P=0.000),N-cadherin(P=0.005),ZEB1(P=0.000)were decreased while the E-cadherin(P=0.003)was increased in groupAd-siPNUTS.There was no significant difference between group Ad-GFP and group PBS(P>0.05).Conclusion1.Expressions of PNUTS in Hep-2 cells could be silence by PNUTS siRNA Recombinant adenovirus specifically and efficiently.2.Ad-siPNUTS could inhibit the proliferation,invasion,metastasis and EMT of Hep-2 cells.3.The regulation of PNUTS on Hep-2 cells may be related to PI3K/AKT,Rb/E2F1 and Rb/ZEB1 signaling pathways.4.PNUTS may be a new target for gene therapy of larynx cancer.