| Objective:Pancreatic cancer(PC)is a solid tumor of the digestive system with a very poor prognosis,and its morbidity and mortality rates remain high,posing a serious threat to human health and quality of life,and its pathogenesis is still not fully understood.Ubiquitination is an important post-translational modification of proteins that plays a crucial role in tumorigenesis and progression,and MINDY2,a member of the motif interacting with Ub-containing novel DUB family(MINDYs),is a newly discovered deubiquitinating enzyme(MINDY).The role of MINDY2,a newly discovered deubiquitinating enzymes(DUBs),in pancreatic cancer is still unclear.The group investigated the potential value of MINDY2 in pancreatic cancer through bioinformatics analysis,and further explored the effect of MINDY2 on the proliferation,invasion and migration ability of pancreatic cancer cells,and investigated the molecular mechanism of MINDY2 playing a pro-cancer role in pancreatic cancer to provide new ideas for the diagnosis and treatment of pancreatic cancer.Methods:Bioinformatics analysis was performed through GEO,UALCAN,TCGA and GTEx databases to understand the expression of MINDY2 in pancreatic cancer and its correlation with prognosis,RNAseq data of pancreatic cancer and corresponding clinical information were obtained from TCGA database to analyze the correlation between MINDY2 and T-stage,N-stage,pathological grading and cancer stage of pancreatic cancer patients,ROC curve to analyze the diagnostic value of MINDY2 in pancreatic cancer,TIDE algorithm to predict the potential immunotherapeutic response of MINDY2 in pancreatic cancer,and TCGA database to analyze the correlation between MINDY2 in pancreatic cancer and EMT(epithelial-mesenchymal transition),inflammatory response,ECM-related genes,angiogenesis,TGFB and tumor inflammatory features,and immune correlation analysis was performed to understand the correlation of MINDY2 with immune cells and immune checkpoint-related gene expression.The expression of MINDY2 in human pancreatic cancer tissues and adjacent normal tissues and pancreatic cancer cell lines(6 strains)and normal pancreatic ductal epithelial cells was detected by q RT-PCR and Western blot assays.Immunohistochemical analysis was performed in tissue microarrays(TMA)containing90 pancreatic cancer and corresponding adjacent normal pancreatic tissue samples to understand MINDY2 expression and to analyze the relationship between MINDY2 expression and clinicopathological parameters in pancreatic cancer patients.MINDY2 was silenced by small molecule interfering RNA.q RT-PCR and Western blot assays were performed to verify the silencing efficiency,and stable lentiviral strains were constructed for overexpression,down-regulation and corresponding negative control groups,and the infection efficiency was verified by CCK-8 assay,clonogenic plate assay,Ed U assay,flow cytometry,Transwell assay,wound healing assay,Western blot assay was performed to detect the effect of MINDY2 on the proliferation,invasion and migration of pancreatic cancer cells in vivo and in vitro.The effect of MINDY2 on EMT-related proteins and cell cycle-related proteins.Co-IP assay,protein profiling and bioinformatics analysis screened ACTN4 as a target protein of MINDY2,and verified the regulatory effect of MINDY2 on ACTN4 by q RT-PCR,Western blot and ubiquitination assay,downregulated ACTN4 by CCK-8 assay,clone plate assay,Ed U assay,flow cytometry,Transwell assay,wound healing assay,bioinformatics analysis to screen PI3K/AKT/m TOR as a downstream target pathway,and Western blot to verify the effect of MINDY2 and ACTN4 on PI3K/AKT/m TOR signaling pathway by Western blot.Results:The results of bioinformatics analysis based on GEO and TCGA databases suggested that MINDY2 expression was higher in pancreatic cancer than in adjacent normal tissues and was associated with poor prognosis,and the ROC curve suggested that MINDY2 had a high diagnostic value in pancreatic cancer,and the TIDE algorithm predicted that the higher the expression of MINDY2,the worse the response of pancreatic cancer to immune checkpoint inhibitors.Combined with the RNAseq data of pancreatic cancer in TCGA database and corresponding clinical information analysis,it was found that MINDY2 in pancreatic cancer was positively correlated with EMT,inflammatory response,ECM-related genes,angiogenesis,TGFB and tumor inflammatory features,and immune correlation analysis suggested that MINDY2 was deeply involved in immune cell infiltration in pancreatic cancer and associated with immune checkpoint-related gene expression.The results of q RT-PCR,Western blot and tissue microarray indicated that the expression of MINDY2 was higher in both pancreatic cancer tissues and pancreatic cancer cell lines than in adjacent normal tissues or normal pancreatic ductal epithelial cells,and the analysis of patients’ clinicopathological data revealed that the expression of MINDY2 correlated with poor patient prognosis.After constructing overexpression and low expression of MINDY2 lentivirus stable transfection strains,MINDY2 was confirmed to promote proliferation,invasion and migration of pancreatic cancer cells in vivo by CCK-8 assay,clonogenic plate assay,Ed U assay,flow cytometry,Transwell assay and wound healing assay.Western blot assay revealed that MINDY2 could promote EMT and correlated with cell cycle-related protein expression.The results of subcutaneous tumorigenesis assay in nude mice and liver metastasis assay in spleen envelope of nude mice suggested that MINDY2 could promote the tumorigenic ability and metastatic ability of pancreatic cancer cells in nude mice,and the immunohistochemical results suggested that MINDY2 was positively correlated with Ki-67 and PCNA expression,and HE staining of dissected liver in liver metastasis model revealed that the size and number of liver metastases were significantly stronger after MINDY2 upregulation than that of the control group.Co-IP and protein profiling combined with bioinformatics analysis identified ACTN4 as the target protein of MINDY2.q RT-PCR and Western blot results found that MINDY2 only affected ACTN4 at the protein level,and ubiquitination experiments suggested that MINDY2 modified ACTN4 through deubiquitination and then stabilized ACTN4 at the protein level.The replies by CCK-8 assay,clonogenic plate assay,Ed U assay,flow cytometry,Transwell assay,and wound healing assay revealed that downregulation of ACTN4 reduced the ability of MINDY2 to promote proliferation,invasion and migration of pancreatic cancer cells.Further bioinformatics analysis revealed that both MINDY2 and ACTN4 were associated with PI3K/AKT/m TOR signaling pathway,and the phosphorylation level of PI3K/AKT/m TOR pathway was significantly increased after overexpression of MINDY2 in Western blot assay.The effect of this MINDY2 on this signaling pathway was attenuated by the addition of PI3 K inhibitor(LY294002),and a similar effect was achieved after downregulation of ACTN4.Conclusions:The Deubiquitinating Enzyme MINDY2 Promotes Pancreatic Cancer Proliferation,Invasion and Metastasis by Stabilizing ACTN4 Expression and Activating the PI3K/AKT/m TOR Signaling Pathway... |
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