| ObjectivesCopines,are a calcium-dependent phospholipid binding family with highly conserved properties in biological evolution.Nine members of the family have been identified.Copine 1(CPNE1),a member of this family,is located in the 20q 11.21 region of human chromosome and is commonly expressed in various tissues and organs.CPNE1 expression is elevated in non-small cell lung cancer and is associated with poor prognosis.Currently,the mechanism of CPNE1 ubiquitination and its specific ubiquitin ligase have not been reported.This study aims to further investigate the degradation mechanism of CPNE1 in NSCLC,and provide new insights for targeting CPNE1 to regulate the occurrence and development of NSCLC.Methods(1)The expression of CPNE1 in a variety of cancers was detected through the database,and the relationship between CPNE1 and prognosis of patients with non-small cell lung cancer was studied.(2)The stability and half-life of CPNE1 were detected by using the protein synthesis inhibitor CHX,proteasomal inhibitor MG 132 and lysosomal inhibitor 3-MA.(3)The interaction between CPNE1 and Ub was detected by immunoprecipitation.CPNE1 lysine residue mutant plasmids were constructed to determine the major residue in the ubiquitination process of CPNE1..(4)The ubiquitin ligase of CPNE1 was predicted by UbiBrowser website,and NEDD4L was screened.The expression of NEDD4L in a variety of cancers was detected by using database.The expression of NEDD4L in cancer was detected by immunohistochemistry.(5)Through database analysis,there was no correlation between NEDD4L and CPNE1 at mRNA level.Tissues collected from patients with non-small cell lung cancer(NSCLC)were detected for the protein levels of CPNE1 and NEDD4L,which were found to be negatively correlated.(6)The interaction between CPNE1 and NEDD4L was detected by immunoprecipitation.Confocal microscopy was used to observe the distribution of CPNE1 and NEDD4L in cells to verify the existence of co-localization.(7)In non-small cell lung cancer cell lines,the expression of CPNE1 protein was detected by NEDD4L interference.(8)Nedd4L knockout lenti virus was prepared and infected with lung cancer cell lines,and the effects of Nedd4L knockout on endogenous CPNE1 protein expression and proliferation and metastasis of non-small cell lung cancer cells were detected.Results(1)CPNE1 was up-regulated in a variety of cancers.In non-small cell lung cancer,high expression of CPNE1 is associated with poor prognosis.(2)CPNE1 was degraded in 2h,and it could be degraded by ubiquitin-proteasome and autophagy-lysosome pathways.(3)CPNE1 can interact with Ub,and the K157 locus of CPNE1 is conserved in many species,which plays a major role in the ubiquitination process of CPNE1.(4)The expression of NEDD4L was low in cancer,and there was no correlation between NEDD4L and CPNE1 at mRNA level.In patient tissues,CPNE1 was negatively correlated with NEDD4L protein levels.(5)NEDD4L and CPNE1 had co-localization in the cytoplasm,and they could interact with each other.Interrupting NEDD4L can up-regulate the protein level of CPNE1,and this up-regulation can be inhibited by proteasome inhibitor MG132.(6)NEDD4L can regulate the proliferation and metastasis of NSCLC by regulating CPNE1.ConclusionCPNE1 is highly expressed in a variety of cancers,and this high expression is associated with poor prognosis in patients with non-small cell lung cancer.In our study,we reported a new mechanism of CPNE 1 degradation,and found that CPNE 1 can be degraded by ubiquitinproteasome and lysosome pathways.These results indicated that NEDD4L promoted the degradation of CPNE1 through the ubiquitin-proteasome pathway.Nedd4L knockout can stabilize CPNE1 protein and promote the proliferation and metastasis of NSCLC cells.In addition,we found that CPNE1 interacts with UB through its K157 site,revealing the specific mechanism of CPNE1 in NSCLC canceration and providing new insights for NSCLC treatment strategies. |
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