Study On The Effect Of Skp2 Gene Silencing On The Biological Characteristics Of Esophageal Cancer Cells Eca109

Objective: To investigate the expression level of Skp2 in esophageal cancer and its relation to clinicopathological features.And through adenoviral vector expressing Skp2 short hairpin RNA to transduce Eca109 cells,we explore the influence of the biological behavior about Eca109 cells and the inhibition effect of Skp2 on esophageal cancer in vitro and vivo.Methods:(1)Twenty-four cases of esophageal cancer tissues and corresponding adjacent normal tissuess were studied by immunohistochemical to explore the expressing of Skp2.(2)We used the saved Ad-shSkp2 to transfect 293 cells to amplify the adenovirus and then through the CsCl density gradient to separation and purification.(3)After infected by the Ad-shSkp2,flourescence microcope and Western blot were used to detect transfecting efficiency,Cell viability was assessed by CCK-8 assay,the apoptosis and cell cycle were measured by flow cytometry,the expression of Bcl-2,Bax,PARP eand cleaved-PARP were explored by western blot.(4)The application of Ad-GFP-RFP-LC3 were conducted to detect the autophagy of Eca109 cells,the expression of LC3Ⅱ/Ⅰ and Beclin1 were explored by western blot.(5)Eca109 cells were injected into nude mice.To test the growth inhibitory effect of Ad-Skp2-shRNA on Eca109 xenografts in the nude mice,intratumoral injection of PBS,NC adenovirus,and Ad-Skp2-shRNA were performed every 3 days four times in total.The tumor of this animals were measured and recorded every 3 days and research finished 4 weeks later,the growth curves of tumor were drawed and the growth inhibition rate of tumor calculated.The expression of Skp2 and PCNA were studied by immunohistochemistry(IHC)staining.Results:(1)Skp2 were found in both esophageal cancer tissues and corresponding adjacent normal tissues.But esophageal carcinoma cells were strongly expressed Skp2.(2)Numerous Ad-sh Skp2 and Ad-NC were successfully obtained.(3)Adenoviral vector expressing Skp2-shRNA(Ad-shSkp2)and Ad-NC were transduced into Eca109 cells.The Ad-shSkp2 not only inhibited the expression of Skp2,but also suppressed Eca109 cell proliferation.Skp2-shRNA treatment leaded to cell cycle stagnation at G2/M phase,and a obvisouly apoptotic sub-G1 peak was found.The Skp2-shRNA decreased the expression of Bcl-2 and increased the expression of Bax and PARP cleavage.(4)After infection with AdmRFP-GFP-LC3,Skp2-shRNA treated cells showed a punctate pattern of LC3 fluorescence,representing recruitment of LC3-II to autophagosomes and the formation of autophagic vacuoles.Western blots showed that the LC3-Ⅱ/Ⅰrates and the expression of Beclin-1 were significantly elevated by Skp2-shRNA treatment.(5)Primarily,hematoxylin and eosin staining of the resected tumor specimens were used to prove the success of model.The data of tumor growth curve and dissected tumor suggested that Skp2-shRNA reduced tumor volume and growth rate of Eca109 cells in vivo(P<0.05).The Skp2-shRNA model had clearly proved that the presence of the Skp2-shRNA in the nude-mouse.In the same way,to silence Skp2 had decreased the PCNA-positive cells.Conclusion: The expression level of Skp2 in esophageal cancer is high.Skp2 silencing induce apoptosis and trigger autophagy in esophageal cancer cells.In vivo,Skp2 silencing has meaningful inhibitory effect on esophageal cancer.