TRIM21 Promotes Differentiation Of Hypopharyngeal Squamous Cell Carcinoma Through Improving Protein Stability By Ubiquitination Of Cytoskeleton Protein KRT10

Background & Objective: Hypopharyngeal squamous cell carcinoma(HPSCC)is one of the tumors with low survival rate in head and neck cancers.Patients can be treated with surgery or nonsurgical method.There is no significant difference in survival rate between two therapeutic ways.However,non-surgical treatment can significantly improve the living quality of patients.Therefore,it is of great clinical significance to choose possible target for non-surgical treatment.As an E3 ubiquitin ligase,TRIM21 is primarily studied in the field of immunity.In addition,it has been reported that TRIM21 played roles in the occurrence and development of breast cancer,liver cancer and glioma.But it is still unknown that the function of TRIM21 in HPSCC.Therefore,we explored the role of TRIM21 in the differentiation of hypopharyngeal carcinoma and its molecular mechanism.This may provide possible target for future non-surgical therapy for HPSCC.Methods: 1.Analysis of TRIM21 expression level in hypopharyngeal carcinoma with low and moderate-high differentiation The tissue samples were divided into two groups according to pathological grade: low differentiation(6 pairs)and medium and high differentiation(6 pairs).RT-q PCR and Western Blotting were used to detect the expression of TRIM21 in low and moderate-highly differentiated hypopharyngeal carcinoma tissues at gene and protein levels.2.Effects of TRIM21 overexpression and knockdown on cell biological function Western Blotting was used to detect the effects of overexpression or knockdown of TRIM21 on the expression levels of KRT10,IVL and TGM1 in FaDu cells.CCK-8 and scratch assay were used to detect the effects of TRIM21 overexpression and knockdown on proliferation and migration of FaDu cells,respectively.3.Screening of TRIM21 interacting proteins and molecular mechanism analysis Protein immunoprecipitation and mass spectrometry were used to identify TRIM21 interacting proteins.The protein synthesis inhibitor cycloheximide(CHX)was used to treat TRIM21 overexpression FaDu cells.The expression of KRT10 was detected by Western blotting.FaDu cells were transfected with HA-UbΔGG and HAUb plasmids,and the ubiquitination level of KRT10 was detected by Western blotting.Results: 1.The RNA and protein expression of TRIM21 in moderate and high-differentiated HPSCC was significantly higher than that in low-differentiated HPSCC.2.Overexpression TRIM21 in FaDu cells could increase the expression level of differentiation related protein markers IVL,TGM1 and KRT10,and inhibit cell proliferation and migration as well.On the contrary,TRIM21 knockdown decreased the expression of the above proteins,and promote cell proliferation and migration.3.The data of immunoprecipitation and mass spectrometry analysis showed that there is an interaction between TRIM21 and KRT10.CHX induction result indicated that TRIM21 could inhibit KRT10 degradation.Overexpression of TRIM21 could upregulate KRT10 ubiquitination.Conclusion: TRIM21 may be involved in HPSCC differentiation.One of the possible mechanism maybe that TRIM21 could improve the protein stability of KRT10 by ubiquitination of KRT10 through non-protein degradation,thereby promote cell differentiation.