Modification Of ADP-ribosylation Of LRP6 By PARP-16 Regulates The Invasion And Metastasis Of Oral Squamous Cell Carcinoma

Objective:Oral squamous cell carcinoma(OSCC)is one of the common malignant neoplasms in the head and neck region.Multimodal therapy including surgical resection,with or without radiotherapy,chemotherapy,and immunotherapy,has improved the quality of life as well as the survival of patients.However,the 5-year survival rate is around 50%.Tumor invasion and metastasis is the leading causes of death in patients with OSCC and is an urgent clinical problem to be solved.PARP family proteins are a class of ADP-ribose transferases,and their family members are involved in cellular processes such as DNA damage repair and stress response.However,the mechanism of PARPs on invasion and metastasis of OSCC are rarely studied.However,the mechanism of PARPs on invasion and metastasis of OSCC has been little studied.PARP-16 is a newly discovered PARP family member,and its role in tumors is still unclear.Therefore,in this study,we investigated the biological role of PARP-16 in OSCC and revealed the molecular mechanism of PARP-16 in regulating OSCC invasion and metastasis.Methods:1)RNA sequencing and clinical data of HNSCC patients were obtained from TCGA data portal.Univariate Cox regression analysis was used to estimate the association between the expression of 17 PARP genes and the overall survival(OS)of patients.TCGA and several OSCC datasets obtained from the Gene Expression Omnibus(GEO)database were used to analyze the expression differences of PARP-7 and PARP-16 in tumor tissues and normal tissues,and the relationship with clinical prognosis.2)We constructed OSCC cell lines transfected with PARP-16 overexpression plasmid and sh RNA to study the effect of PARP-16 on the biological behavior of OSCC cells,such as proliferation,invasion and metastasis,in vivo and in vitro.3)RNA-seq was performed in OSCC cell lines transfected with PARP-16 overexpression plasmid and control vector to screen the differentially expressed genes,and functional enrichment analysis of the DEGs were performed.The activation status of relevant signaling pathways in the m RNA and protein levels was verified.We utilized the Bio Plex Interactome database to construct protein-protein interaction(PPI)network of PARP-16 to identify PARP-16 target proteins.The protein interactions and binding region were validated in vitro.The expression relationship between PARP-16 and target proteins was examined in clinical specimens,and the effect of PARP-16 on the expression of target proteins was examined at the cellular level.The regulation of PARP-16 on target proteins was studied from both protein synthesis process and protein degradation process.Further,we explored PARP-16 on the ribosylation and ubiquitination modifications of target proteins.Results:1)Using univariate Cox regression analysis,PARP-7 and PARP-16 were found to be associated with the survival of HNSCC in the TCGA cohort.The expression of PARP-16 in the GEO datasets was lower in OSCC tumor tissues than in normal tissues,and low PARP-16 expression was associated with poor prognosis.In OSCC clinical specimens,the expression of PARP-16 was lower in tumor tissues than in normal tissues and was correlated with lymph node metastasis.Furthermore,PARP-16 expression was lower in metastatic lymph nodes than in situ tumors,and PARP-16 was associated with worse prognosis.2)Silencing PARP-16 promotes OSCC cell invasion,metastasis,tube formation and ectopic proliferation in vitro and in vivo.3)Functional enrichment analysis showed that PARP-16 is involved in the regulation of EMT,Wnt and other signaling pathways,and silencing PARP-16 promotes the activation of Wnt signaling pathway to regulate partial EMT in OSCC cells.LRP6 is a substrate protein of PARP-16,and PARP-16 interacts with LRP6 through its intracellular structural domain.PARP-16 modifies LRP6 by ribosylation to induce its ubiquitinated degradation.Conclusion:1)Low expression of PAPR-16 in OSCC is associated with lymph node metastasis and poor prognosis.2)Silencing PARP-16 regulates partial EMT through Wnt signaling pathway and promotes OSCC cell invasion and metastasis.3)PARP-16 regulates LRP6 protein homeostasis through ribosylation-dependent ubiquitination pathway to inhibit Wnt signaling pathway.