TRIM29 Promotes Pancreatic Cancer Growth And Progression By Upregulating YAP1 Protein Level

Background and Objective:Pancreatic cancer(PC)is a highly aggressive malignant tumor of the digestive system.It is commonly known as “the king of cancer”.Its morbidity and mortality are increasing year by year worldwide,which seriously endangers human health.The early diagnosis of pancreatic cancer is difficult,the degree of malignancy is high,the resection rate is low,and the overall 5 year survival rate of the patients is only about10%.Therefore,the current situation of diagnosis and treatment of pancreatic cancer is still very serious,and it is of great clinical significance to actively explore the molecular mechanism of the occurrence and development of pancreatic cancer and to seek new effective therapeutic targets.TRIM29,as a member of TRIM protein family,is a protein with various biological functions that has attracted increasing attention in recent years.In recent years,it has been confirmed that the dysregulation of TRIM29 expression is closely related to the occurrence,development,proliferation,invasion and metastasis of tumors.However,the specific biological functions and mechanisms of TRIM29 in pancreatic cancer remain unclear.As an intracellular connexin and transcriptional activator,YAP1 protein is the most important effector of the Hippo signaling pathway.A large number of studies have shown that YAP1 protein affects cancer development in a variety of ways,including promoting malignant phenotype,dry transformation and chemotherapy resistance of tumor cells.However,the specific regulatory mechanism of YAP1 in pancreatic cancer has not been clarified.The purpose of this study was to explore the expression and correlation of TRIM29 and YAP1 in pancreatic cancer,and further study the mechanism of their influence on the growth and progression of pancreatic cancer.Methods:(1)The expression of TRIM29 in pancreatic cancer tissues was analyzed online by GEPIA database.Western blotting(WB),quantitative Real Time Polymerase Chain Reaction(qRT-PCR)and immunohistochemistry(IHC)were used to detect the expression of TRIM29 in pancreatic cancer tissues.The relationship between TRIM29 expression and main clinical features and prognosis of patients with pancreatic cancer was statistically analyzed.(2)The expression of TRIM29 in pancreatic cancer cells was interfered,and the changes of cell growth ability were observed by CCK8 and EdU experiments.The effect of TRIM29 expression on pancreatic cancer cell growth was studied in vivo by subcutaneous xenotransplantation in nude mice.The effects of TRIM29 expression changes on cell cycle and apoptosis were detected by flow cytometry and TUNEL assay,and the expressions of cycle-related proteins and apoptosis-related proteins were further detected by WB.(3)After TRIM29 expression was changed,THE expression of YAP1 in pancreatic cancer cells was detected by WB and qRT-PCR.The expression of YAP1 in pancreatic cancer tissues and corresponding non-cancer tissues was detected by WB,qRT-PCR and IHC,and the correlation between TRIM29 and YAP1 protein expression in tumor tissues was further analyzed.In pancreatic cancer cells with high expression of TRIM29,TRIM29 was knocked down and the expression of YAP1 was upregulated;In contrast,the overexpression of TRIM29 in pancreatic cancer cells with low expression of TRIM29 simultaneously downregulated the expression of YAP1;Then CCK8 and EdU experiments were used to observe the changes of cell growth ability.(4)The direct binding of TRIM29 and YAP1 in pancreatic cancer cells was detected by Co-Immunoprecipitation(Co-IP).On the basis of blocking protein degradation and synthesis of pancreatic cancer cells,WB was used to detect the influence of overexpression or interference of TRIM29 on YAP1 protein expression.Co-IP assay was used to detect the effect of overexpression or knockdown of TRIM29 expression on YAP1 ubiquitin level in pancreatic cancer cells.Results:(1)GEPIA online database showed that the expression of TRIM29 in pancreatic cancer tissues was significantly higher than that in adjacent non-tumor tissues.WB,qRT-PCR and IHC results also confirmed that the expression level of TRIM29 in pancreatic cancer tissues was significantly increased.Statistical analysis suggested that the high expression of TRIM29 was closely related to tumor size(P =0.003)and lymph node metastasis(P =0.001).However,there was no significant correlation with age(P =0.951),gender(P =0.531),pathological grade(P =0.328),vascular invasion(P =0.568)and nerve invasion(P =0.460).In addition,patients with high TRIM29 expression level showed shorter postoperative overall survival and tumor-free survival than patients with low TRIM29 expression level.(2)WB and qRT-PCR results indicated that TRIM29 was highly expressed in pancreatic cancer cells.CCK8 and EdU experiments showed that downregulated expression of TRIM29 in pancreatic cancer cells reduced the cell growth ability.Animal experiments have also confirmed that TRIM29 knockdown can significantly inhibit the growth of pancreatic cancer.The results of flow cytometry showed that downregulating the expression of TRIM29 could arrest the cell cycle in G1 phase and promote the apoptosis of pancreatic cancer cells.WB results showed that downregulated expression of TRIM29 significantly inhibited the expression of cyclin D1 and PCNA.Meanwhile,knockdown of TRIM29 expression up-regulated Bcl-2,and down-regulated Bax and Caspase 3 expression.TUNEL analysis further confirmed that down-regulated TRIM29 expression increased the number of apoptotic cells in xenograft tumors.IHC analysis showed that down-regulation of TRIM29 could inhibit the expression of Ki67.(3)qRT-PCR and WB results showed that TRIM29 could promote the protein expression of YAP1 in pancreatic cancer cells,but had no effect on the mRNA expression level.WB,qRT-PCR and IHC results confirmed that YAP1 was significantly overexpressed in pancreatic cancer tissues,and was positively correlated with TRIM29 expression.Kaplan-Meier survival curve showed that patients with high YAP1 expression had shorter postoperative overall survival and tumor-free survival than patients with low YAP1 expression.Rescue experiments showed that upregulation of YAP1 could promote the proliferation of pancreatic cancer cells,and the decrease in cell proliferation caused by down-regulation of TRIM29 could be partially reversed by up-regulation of YAP.On the contrary,down-regulation of YAP1 inhibited the proliferation of pancreatic cancer cells.Moreover,the increase in cell proliferation caused by up-regulation of TRIM29 could be partially reversed by down-regulation of YAP.(4)Co-IP assay showed that TRIM29 protein could directly bind to YAP1 protein in pancreatic cancer cells.The expression of YAP1 protein accumulates gradually with the increase of time of protease inhibitor MG132.After pancreatic cancer cells were treated with actinomycin(CHX),overexpression of TRIM29 slowed down the degradation rate of YAP1 protein.Further studies confirmed that up-regulation of TRIM29 could significantly inhibit the ubiquitination level of YAP1,while down-regulation of TRIM29 could significantly increase the ubiquitination level of YAP1.Conclusions:TRIM29 is highly expressed in pancreatic cancer tissues,and its expression level is closely related to the malignant progression of pancreatic cancer patients.In addition,TRIM29 can promote the growth and proliferation of pancreatic cancer cells by regulating the protein expression of YAP1.Mechanistically,TRIM29 directly binds to YAP1 and stabilizes the expression of YAP1 by inhibiting its ubiquitination,thus promoting the progression of pancreatic cancer.Our results provide a new theoretical basis for the study of pancreatic cancer growth,and will provide a new theoretical research direction for targeted therapy of pancreatic cancer.