TRIM21 Promotes The Progression Of Pancreatic Cancer Via SMAD4 Ubiquitination

Background:The incidence rate of pancreatic cancer has shown an upward trend in recent years.It ranks seventh in the inceidence rate of male malignant tumors,11 th of women.And the rank of mortality related to malignant tumors is 6th.The five-year survival rate of PC in China is 5%-10%.However,The mechanism of pancreatic cancer is still unclear.Previous studies have shown that transforming growth factor beta(TGFβ)signaling pathway plays an important role in the progression of pancreatic cancer.The TGFβ-SMADs signaling pathway is an important growth-related pathway in vivo.The key tumor suppressor factor SMAD4 in the pathway is in a state of functional loss in about 50%of pancreatic cancer,which is closely related to the occurrence and development of pancreatic cancer.The sequencing data of a large number of pancreatic cancer cases shows that about 30% of pancreatic cancer patients have a deep deletion of SMAD4 copy number,which directly leads to low expression and non-function of SMAD4.But it is worth noting that 20% of pancreatic cancer patients have no copy number loss of SMAD4 but still have loss of SMAD4 function,which indicates that post translational modification plays an important role in the loss of SMAD4 function.The mechanism of this has not been fully elucidated.The tripartite motif(TRIM)superfamily is a group of E3 ubiquitin ligases contain the RING domain.TRIM21,as an important menber of this family,has been confirmed to be involved in the regulation of biological functions of various solid tumors.Its exact function and role are still debated.At present,it is believed that the tumor-suppressing or tumor-promoting function of TRIM21 is tumor-specific.In pancreatic cancer,TRIM21 was significantly overexpressed and correlated with poor prognosis of patients.More importantly,in patients with non-SMAD4 copy number loss,the overexpression of TRIM21 was significantly correlated with the low expression of SMAD4 protein.Our research group also found that there is protein interaction between SMAD4 and TRIM21 through SMAD4 protein interaction mass spectrometry analysis.So is the high expression of TRIM21 the reason for the loss of SMAD4 function? What function exists in the protein interaction of TRIM21 and SMAD4? This study will explore these questions.Methods:The correlation between TRIM21 m RNA expression level and prognosis in pancreatic cancer patients was found using GEPIA web tool.The relationship between TRIM21 and SMAD4 in case protein chip was analyzed using HPA database.The interaction between TRIM21 and SMAD4 was found by mass spectrometry analysis and immunoprecipitation.Western Blot,real-time quantitative polymerase chain reaction(RT-q PCR),immunocytochemistry and immunofluorescence(ICC/IF)methods were used to identify the functional effects of TRIM21 on SMAD4 at the transcriptional and translational levels;through Construct a truncated body to clarify the interaction domain between TRIM21 and SMAD4;the regulatory effect of NLK on TRIM21 ubiquitination modification of SMAD4 was verified by Western Blot;determine the ubiquitination type of TRIM21 for SMAD4 by denaturing ubiquitination method;verify the effect of TRIM21 on the migration and proliferation of pancreatic cancer cells by Transwell,plate cloning,and scratch experiments.Results:TRIM21 interacts with SMAD4,and binds to the MH1 domain of SMAD4 through the RING-Bbox domain.TRIM21 has a higher expression level in pancreatic cancer than in normal tissues,and is negatively correlated with SMAD4.After overexpression of TRIM21,SMAD4 is inhibited,and it works at the protein level.The inhibitory effect of TRIM21 on SMAD4 depends on its ubiquitination modification of SMAD4.The ubiquitination of SMAD4 by TRIM21 leads to the degradation of SMAD4 through the proteasomal pathway.Phosphorylation of SMAD4 by NLK is critical for TRIM21 ubiquitination of SMAD4.Cell experiments found that overexpression of TRIM21 enhanced the proliferation and invasion of pancreatic cancer cells.Conclusion:In pancreatic cancer cells,TRIM21 interacts with SMAD4,and modifies the MH1 domain of SMAD4 through ubiquitination to cause its degradation through the ubiquitin-proteasome pathway,and NLK is crucial for the regulation of TRIM21 ubiquitination and modification of SMAD4 effect.The invasiveness and proliferation of pancreatic cancer cells increased after TRIM21 was overexpressed,and its expression was correlated with poor prognosis of patients.Further research on its mechanism may provide new ideas and therapeutic strategies for the inactivation mechanism of TGFβ-SMAD4 pathway in non-SMAD4-deficient pancreatic cancer.