The Study On The Effect Of EVI5 In Non-small Cell Lung Cancer Growth And Metastasis And Its Underlying Mechanism

Objectives:Ecotropic viral integration site 5(EVI5),an important protein regulating cell cycle,cytokinesis and cellular membrane traffic,functions as a stabilizing factor maintaining anaphase-promoting complex/cyclosome(APC/C)inhibitor Emil in S/G2 phase.EVI5 has been reported to play a pro-oncogene role in many cancers,however,the exact mechanism by which EVI5 causes malignant transformation of NSCLC remains unknown.In the present study,we addressed the role of EVI5 in NSCLC by regulating tumor growth,migration and invasion.Methods:The expression levels of EVI5 and miR-486-5p in NSCLC tissues and cells were measured by real-time PCR.Meanwhile,EVI5 and its associated protein expression were analyzed by western blot and co-immunoprecipitation assay in NSCLC.Flow cytometry was performed to determine cell proliferation and apoptosis.CCK-8 and clonogenic assays were used to analyze cell growth viability.Transwell assays and wound healing were utilized to assess the invasion and migration ability of NSCLC cells.A dual-luciferase was used to determine the relationship between miR-486-5p and EVI5 in NSCLC cell lines.To investigate the role of EVI5 in vivo,lung carcinoma xenograft mouse model was applied.Results:(1)EVI5 was upregulated in NSCLC tissues and cell lines when compared with that in normal tissues and cell line(P<0.05).(2)In vitro knockdown of EVI5 inhibits growth,migration and invasion of tumor cells in NSCLC cells and induced G1 cell cycle arrest in NSCLC cells(P<0.05).(3)The stable EVI5-knockout cell lines(A549-EVI5-KO vs A549-Cas-9,H226-EVI5-KO vs H226-Cas-9)and the stable EVI5-overexpressed cell lines(A549-PLVX vs A549-EVI5)were successfully established.The lentivirus-mediated EVI5 knockout remarkably suppressed growth,migration and invasion in NSCLC cells(P<0.05).Whereas overexpression of EVI5 promoted in vitro growth,migration and invasion in NSCLC cells.(4)Inoculation of EVI5-deficient tumor cells into nude mice suppressed tumor growth and metastasis compared to control mice inoculated with unmanipulated tumor cells.(5)EVI5 protein level has a positive correlation with Emi1 protein level in human NSCLC tissues(P<0.001).And EVI5 was co-expressed with Emil in NSCLC cells.When EVI5 was knockdown,the expression of Emil,p-Akt and p-Erk,CyclinA2 and CyclinD1 were significantly decreased compared with the control cells in A549 and H226 cell lines.Furthermore,in the cell lines with EVI5 overexpression,the Emil siRNA induced decrease the growth ability induced by EVI5 overexpressing(P<0.001).(6)We examined complexes of EVI5 and TGF-? receptors using co-immunoprecipitation experiments,EVI5 was co-expressed with TGF-? receptors in the A549 cell line.When EVI5 was knockdown,the expression of TGF-? receptor ?,TGF-?receptor ? and its downstream signaling molecules was inhibited were significantly decreased compared with the control cells in A549 and H226 cell lines.Furthermore,the migration and invasion ability of NSCLC cell lines induced by exogenous TGF-?1 was significantly inhibited by EVI5 knockout.(7)A dual-luciferase reporter vector containing the EVI5 3'-UTR seed region specific to miR-486-5p,or the corresponding mutant sequence was used.the luciferase activities of the reporter vector containing the seed region of miR-486-5p in NSCLC cells was significantly suppressed by miR-486-5p transfection.(8)miR-486-5p was downregulated in NSCLC tissues and cell lines when compared with that in normal tissues and cell line.(9)Overexpressing miR-486-5p inhibits growth,migration and invasion of tumor cells in NSCLC cells and induces G1 cell cycle arrest in NSCLC cells(P<0.05).(10)In NSCLC cell lines,ectopic miR-486-5p decreased the growth,migration and invasion of NSCLC cells induced by EVI5 overexpression,the level of EVI5 and its downstream signaling molecules was inhibited.Conclusion:EVI5 is highly elevated in human NSCLC tissue and cell lines.Then,we found that EVI5 affects the efficacy of Emil-targeted cell cycle dysregulation in NSCLC cells.In addition,EVI5 was shown to activate downstream TGF-?/Smad signaling pathways by interacting with TGF-? receptors.Further experiments found that the overexpression of EVI5 was correlated with a decrease in miR-486-5p expression with decreased miR-486-5p in NSCLC.In conclusion,our study may provide a theoretical basis for the determination of new targets for NSCLC therapy.