Investigation On The Roles And Underlying Mechanisms Of CDK5 In Regulating The Proliferation And Invasion Of Gastric Cancer

Objective: Gastric Cancer(GC)is one of the most common malignancies with poor outcome in China,but the molecular mechanisms underlying this cancer is largely unclear.Cyclindependent kinase 5(CDK5)is a proline-directed serine/threonine kinase.Previous studies indicated that CDK5 participates in a variety of neurodegenerative diseases associated with its karyoplasm localization.Recently,increasing evidence suggested a function of CDK5 in tumorigenesis and progression.However,the function and underlying mechanism of CDK5 in GC remains unknown.In this study,we examined the expression of CDK5 in GC tissues and analyzed its clinical value.We investigated the biological function of CDK5 with different karyoplasm localization in the GC cells in vitro.We explored binding proteins of CDK5 and the potential downstream genes.This study not only would further reveal the molecular mechanisms of proliferation and metastasis of GC,but also to provide new intervention targets for the treatment of GC.Methods:(1).Immunohistochemistry was used to detect the expression of CDk5 in 244 GC patient tissues.The ?2 test(or the Fisher exact test),univariate survival analysis(Kaplan-Meier method)and multivariate survival analysis(Cox proportional hazards regression model)were used to analyze the relationship between CDK5 expression and clinicopathological parameters together with overall survival in GC patients.(2).Different karyoplasm localization and the knockdown lentivirus systems of CDK5 were constructed,and HGC-27 GC cells infected to generate the stable cell models.(3).In vitro experiments,CCK8 and SRB assay were used to measure the GC cell proliferation,plate clone formation assay was applied to measure the GC cell cloning ability and transwell migration assay was performed to measure the GC cell invasion and metastasis.(4).The binding protein and the candidate target genes for CDK5 were identified by immunoprecipitation and mass-spectrometry.Results:(1).The expression of CDK5 protein was scored as low in 93(38.11%)samples and high in 151(61.89%)samples.GC patients with low expression of CDK5 had a poorer prognosis than those with high CDK5 expression(P < 0.05).The 3-and 5-year cumulative survival rates were 50.5% and 40.6% for patients with low CDK5 expression,and 62.7% and 52.7% for those with high CDK5 expression(P < 0.05).The mean survival time for patients with low and high expression of CDK5 was 40 and 75 months respectively(P < 0.05).Mmultivariate analysis showed CDK5 expression status was an independent prognostic factor for patients with GC(P < 0.05).(2).In in vitro experiments,compared with control group,upregulation of CDK5-WT and CDK5-NLS dramatically suppressed the proliferation,cloning formation and metastasis of GC cells,where upregulation CDK5-NES only inhibited GC cells metastasis.Similarly,downregulation CDK5 could promote metastasis but not affect on proliferation and clone formation of GC cells.(3).Seven CDK5-interacting protein was discovered by immunoprecipitation and mass-spectrometric peptide sequencing(4).PP2 A was identified as a novel CDK5-interacting protein,and the PP2 A inhibitor Okadaic Acid reversed the migration and invasion but no proliferation and clone formation induced by CDK5 overexpression in GC cells.Conclusions:(1).GC patients with low expression of CDK5 had a poorer prognosis,and CDK5 expression status was an independent prognostic factor for GC patients.(2).Knockdown of endogenous CDK5 expression significantly enhanced migration and invasion of GC cells,but had no impact on proliferation or clone formation.3.CDK5-PP2 A protein complex was critical for the ability of CDK5 to suppress metastasis of GC.