The Role And Mechanism Of ECT2 In Growth And Metastasis Of Laryngeal Squamous Carcinoma

Objective:To investigate the relationship between the expression of ECT2 and the clinicopathological features of laryngeal squamous carcinoma.To investigate the effects of siRNA interference on the expression of mRNA and protein of ECT2 in Hep-2 cells of laryngeal squamous carcinoma in vitro,and to investigate the effects of siRNA interference on apoptosis,proliferation,metastasis and invasion of Hep-2 cells in vitro and its mechanism.To investigate the effect of ECT2 inhibition by ECT2siRNA on growth of laryngeal carcinoma cells in vivo and its mechanism.To study clone informing ability of Hep-2 by lentivirus-mediated ECT2shRNA in vitro.To study the effects of lentivirus-mediated ECT2shRNA targeting on the growth of Hep-2 cells in vivo and its mechanismMethods:The patients with laryngeal squamous carcinoma who taken first treatment in our hospital from September 2013 to March 2015 were retrospectively studied.All patients had complete clinicopathological data and were diagnosed as laryngeal squamous carcinoma by the Pathology Department.The correlation between expression of ECT2 and the clinicopathological features of laryngeal carcinoma was statisticlly analyzed in 81 cases of laryngeal squamous cell carcinoma.The effects of ECT2 siRNA interference on downregulation of ECT2 in Hep-2 cells were detected by Western blot(WB)and reverse transcription polymerase chain reaction(QPCR).The effect of downregulation of ECT2 on the proliferation of Hep-2 cells was detected by using CCK-8 method.The effect of downregulation of ECT2 on the cell cycle of Hep-2 was detected by flow cytometry.The expression levels of cycline D1,cycline E and p27 were measured by using QPCR and Western blot.The effect of downregulation of ECT2 on the apoptosis of Hep-2 cells was detected by using Anexin V-FITC/PI double-dye flow cytometer.The effect of ECT2 siRNA on the invasion and migration of Hep-2 cells in laryngeal carcinoma was detected by invasive and migration experiments.The effect of downregulation of ECT2 on the expression of RhoA,Cdc42,Racl was detected by Quantitative reverse transcription polymerase chain reaction(QPCR)and Western blotting(WB).Hep-2 cells stably transfected with lentivirus-mediated ECT2shRNA cell line were established.The effect of downregulation of ECT2 by lentivirus-mediated ECT2shRNA on clone informing ability of Hep-2 cell was detected by soft agar assay protocol.ECT2 expression in laryngocarcinoma xenograft growth in nude mice was detected by Western blot,and ki67 expression in tumor tissue was verified by immunohistochemistry.Results:The expression of ECT2 was statisticly analyzed higher in laryngeal carcinoma than in pericancerous tissues(P<0.01).The higher expression of ECT2 had significant relationship with different level of pathological differentiation,clinical stage,T stage and lymph node metastasis in laryngeal carcinoma(P<0.05).The high expression of ECT2 had no significant relationship with age,sex,history of smoking,or primary tumor location(P>0.05).After ECT2 was targeted by ECT2siRNA in Hep-2 cells,the levels of mRNA and protein expression of ECT2 were decreased significantly.the proliferation of Hep-2 cells was inhabited by ECT2siRNA.It was found that downregulation of ECT2 inhibited Hep-2 cell division,leading to arrest the cell cycle at G1,decrease the expression of cycline D1 and cycline E,and increase the expression of P27.It was found that downregulation of ECT2 promoted Hep-2 apoptosis,leading to an increase in the expression of cleaved caspase-3 protein.It was found that downregulation of ECT2 inhibited the ability of Hep-2 cells to attack and migrate,leading to a decrease in the expression of Cdc42,Rac1.Hep-2 cell line transfected with lentivirus-mediated ECT2shRNA was successfully constructed.Downregulation of ECT2 decreased the growth of Hep-2 cells in vitro,and in vivo.The decreased expression of cell proliferation marker ki67 was detected by immunohistochemistry.Conclusion:The expression of ECT2 was statisticly analyzed higher in laryngeal carcinoma than in pericancerous tissues.The higher expression of ECT2 had significant relationship with different levels of pathological differentiation,clinical stage,T stage and lymph node metastasis in laryngeal carcinoma.Down-regulation of ECT2 can inhibit the proliferation of Hep-2 cells by arresting cell cycle at G1 phase with down-regulation of cyclinD1 and cyclinE1,up-regulation of p27.Down-regulation of ECT2 can induce apoptosis of Hep-2 cells with up-regulation of cleavedcaspase-3.Down-regulation of ECT2 can inhibit the metastasis and invasion abilities of Hep-2 cells with down-regulation of CDC42 and RAC1.Down-regulation of ECT2 can inhabit tumorigenesis of Hep-2 cells in vivio.