Role And Mechanism Of Ubiquitin E3 Ligase Deltex3 In Invasion And Metastasis Of Papillary Thyroid Carcinoma

Objective: Papillary thyroid carcinoma(PTC)is the most common endocrine malignancy.The incidence rate has gradually increased in recent years.Although most PTC patients have good prognosis,some patients are prone to lymph node metastasis or distant metastasis,which seriously affects the prognosis of patients.Therefore,it is very important to study the molecular mechanism of invasion and metastasis in PTC.Ubiquitin proteasome pathway is an important molecular mechanism for intracellular protein degradation.Among them,ubiquitin E3 ligase has the specificity of recognizing substrate and plays an important role in the whole ubiquitin proteasome pathway.The expressional and functional changes of ubiquitin E3 ligase can affect the occurrence and development of a variety of malignant tumors.Deltex(DTX)family proteins(DTX1,DTX2,DTX3,DTX3 L,and DTX4),as important members of ubiquitin E3 ligase,regulate ubiquitin proteasome pathway by specifically recognizing substrates and interacting with them,thus participating in a variety of cell biological processes.Up to now,the abnormal expression of DTX family proteins has been found in a variety of human malignant tumors,which indicates that there are inevitable relationships between DTX family proteins and the occurrence and development of malignant tumors.It is likely to become potential biological markers and provide beneficial help for diagnosis,treatment,and prognosis evaluation of tumors.However,the roles and molecular mechanisms of DTX3 in invasion and metastasis of PTC are still unclear.In this project,the specific function of DTX3 in invasion and metastasis of PTC and the downstream molecular regulation mechanisms were deeply studied.Methods: 1.The data in the cancer genome atlas(TCGA)were analyzed to compare the expression of DTX3 in various cancers and corresponding adjacent tissues.Quantitative real-time polymerase chain reaction(q RT-PCR)and western blot were performed in 114 PTC patients and paired normal thyroid tissues(2 cm away from the tumor)to detect the m RNA and protein expression of DTX3.The relationship between the abnormal expression of DTX3 in PTC and the clinicopathological characteristics of PTC patients was analyzed.2.The protein and m RNA expression of DTX3 in Nthy-ori3-1,K1 and TPC-1 cells was detected by western blot and q RT-PCR.The nuclear and cytoplasmic protein of K1 and TPC-1 cells were separated and extracted,and the localization of DTX3 protein were detected by western blot.The gene expression of DTX3 in K1 and TPC-1 cells was down-regulated by plasmid transfection or up-regulated by lentivirus transfection,to construct stable transfected cell lines.The effects of DTX3 knockdown or overexpression on the proliferation,migration,invasion,apoptosis,and cell cycle of PTC cells were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assay,wound healing assay,Transwell invasion assay,Annexin VPE / 7-amino-actinomycin D(7-AAD)double staining assay,and Propidium Iodide(PI)staining assay.3.To evaluate the effect of DTX3 on epithelial mesenchymal transition(EMT)related proteins and the AKT signaling pathway,western blot was used to detect the expression of E-cadherin,vimentin,N-cadherin,phosphorylated AKT(p-AKT)and AKT protein.Next,the interacting proteins of DTX3 were screened by immunoprecipitation(IP)combined with mass spectrometry(MS).The possible ubiquitination substrate of DTX3 was identified by immunofluorescence,in vivo ubiquitination assay and western blot.Results: 1.The results of searching and comparing TCGA database showed that the expression of DTX3 was lower in a variety of human primary cancers,especially in thyroid cancer(p < 0.05).Western blot and q RT-PCR results showed that the relative protein and m RNA expression levels of DTX3 in PTC tissues were significantly lower than those in adjacent tissues(p < 0.05),and the lower expression of DTX3 in PTC was only closely related to cervical lymph node metastasis(p < 0.05).Western blot and q RT-PCR results showed that compared with Nthy-ori3-1,the relative protein and m RNA expression levels of DTX3 in K1 and TPC-1 cells were significantly lower(p < 0.05).The results of nuclear and cytoplasmic protein isolation and western blot showed that DTX3 was mainly expressed in the nucleus of K1 and TPC-1 cells.Western blot and q RT-PCR results showed that the relative protein and m RNA expression levels of DTX3 in the down-regulated group(DTX3-sh RNA)were significantly lower than those in the no transfection group(con)and knockdown empty vector group(neg-sh RNA)(p < 0.05),but there were no significant differences between con and neg-sh RNA groups(p > 0.05).Compared with con and overexpression empty vector(neg-vector)groups,the relative protein and m RNA expression levels of DTX3 in overexpression group(DTX3-c DNA)were significantly higher(p < 0.05),but there were no significant differences between con and neg-vector groups(p > 0.05).The results of wound healing test showed that DTX3 could inhibit the migration of PTC cells: the average percentage of cell migration area in K1 and TPC-1DTX3-c DNA groups were significantly lower than those of neg-vector and con groups respectively(p < 0.05);However,there were no significant differences between con and neg-vector groups(p > 0.05);The average percentage of cell migration area in K1 and TPC-1 DTX3-sh RNA groups were significantly higher than those in neg-sh RNA and con groups respectively(p < 0.05);There were no significant differences between con and negsh RNA groups(p > 0.05).The results of Transwell invasion assay showed that DTX3 decreased the invasion ability of PTC cells: the average number of invasive cells in K1 and TPC-1 DTX3-c DNA groups were significantly less than those in neg-vector and con groups(p < 0.05);There were no significant differences between con and neg-vector groups(p > 0.05);The average number of invasive cells in K1 and TPC-1 DTX3-sh RNA groups were significantly higher than those of neg-sh RNA and con groups(p < 0.05);There were no significant differences between con and neg-sh RNA groups(p > 0.05).The results of MTS assay,Annexin V-PE / 7-AAD double staining assay and PI staining assay showed that DTX3 had no significant effects on the proliferation,apoptosis,and cell cycle of PTC cells(p > 0.05).3.Western blot results showed that DTX3 interfered with EMT in PTC cells: the relative expression levels of vimentin in K1 and TPC-1 DTX3-c DNA groups were significantly lower than those in neg-vector and con groups respectively(p <0.05);The relative expression levels of E-cadherin protein in K1 and TPC-1 DTX3-c DNA groups were significantly higher than those in neg-vector and con groups respectively(p <0.05);The expression levels of N-cadherin protein in K1 and TPC-1 con,neg-vector and DTX3-c DNA groups were no significant differences respectively(p > 0.05);In K1 and TPC-1 cells,the relative expression levels of vimentin in DTX3-sh RNA groups were significantly higher than those in neg-sh RNA and con groups respectively(p < 0.05);In K1 and TPC-1 cells,the relative expression levels of E-cadherin protein in DTX3-sh RNA groups were significantly lower than those in neg-sh RNA and con groups respectively(p< 0.05);In K1 and TPC-1 cells,the relative expression levels of N-cadherin protein in con,neg-sh RNA and DTX3-sh RNA groups were no significant differences respectively(p >0.05).In addition,western blot results showed that DTX3 regulated the AKT signaling pathway: the relative expression levels of p-AKT protein in DTX3-c DNA groups of K1 and TPC-1 cells were significantly lower than those in neg-vector and con groups respectively(p < 0.05);However,the relative expression levels of AKT in con,neg-vector and DTX3-c DNA groups were no significant differences respectively(p > 0.05);The relative expression levels of p-AKT in K1 and TPC-1 DTX3-sh RNA cells were significantly higher than those in neg-sh RNA and con groups(p < 0.05),while AKT protein expression levels in con,neg-sh RNA and DTX3-sh RNA groups were no significant differences respectively(p > 0.05).A total of 46 proteins that might interact with DTX3 were identified by IP-MS.After further Co-IP assays,X-ray repair cross complementing protein 5(XRCC5)and NADH:ubiquinone oxidoreductase complex assembly factor 5(NDUFAF5)were identified as the interacting proteins of DTX3.Immunofluorescence assay showed that DTX3 and XRCC5 were co-localized in the nucleus of K1 and TPC-1.The results of ubiquitination experiment in vivo showed that the ubiquitinated XRCC5 in K1 and TPC-1 DTX3-c DNA groups were significantly more than those in neg-vector groups,indicating that DTX3 could promote the ubiquitination of XRCC5(p < 0.05).Conclusion: 1.The protein and m RNA expression of DTX3 in PTC tissues was significantly lower than that in adjacent tissues,and closely related to cervical lymph node metastasis of PTC.2.The overexpression of DTX3 could significantly inhibit the migration and invasion of PTC cells,and the down-regulation of DTX3 could enhance the migration and invasion of PTC cells,indicating that DTX3 played an important role in inhibiting the invasion and metastasis of PTC cells.3.DTX3 might limit the migration and invasion of PTC by promoting the ubiquitination of XRCC5,reducing the AKT signaling pathway and inhibiting the EMT process.