ZMAT1 Acts As A Tumor Suppressor In Pancreatic Ductal Adenocarcinoma By Inducing SIRT3/p53 Signaling Pathway

Objective:Pancreatic ductal adenocarcinoma(PDAC)is one of the deadliest cancer due to its highly aggressive phenotype and lack of effective biomarkers or treatment strategies,with a 5-year survival of approximately 10%.Although advancements in chemotherapy including the FOLFIRINOX regimen and gemcitabine plus nab-paclitaxel has improved the prognosis of patients,limited breakthroughs in the recurrence rate or long-term outcomes have emerged.Recently,such associations have been reported between the zinc-finger proteins and cellular stress response pathways including those involved in DNA damage,cell cycle arrest and apoptosis.However,the biological function of zinc finger matrin-type 1(ZMAT1)in the context of tumorigenicity and tumor progression is unknown.The present study aimed to investigate the properties of expression level,clinical significance and potential biological function of ZMAT1 in PDAC in vitro and in vivo.Methods:1.Analyses of the expression level of ZMAT1 and its associations with clinicopathological characteristics and survival of PDAC.Bio-informatics analyses were performed to explore the expression of ZMAT1 across PDAC patients cohorts in TCGA,GEO and Oncomine databases.122 pairs PDAC and pancreas tissues from Guangdong Provincial People’s Hospital were collected and used to validate the protein levels of ZMAT1 and evaluate its correlations with clinicopathological characteristics and survival of PDAC patients.2.Evaluation of the effect of ZMAT1 on tumor phenotype in vitro and in vivo;The EGFP-tagged h ZMAT1-CMV Puro vector and three si RNAs targeting ZMAT1 were transfected into cells to over-express and silence ZMAT1 for the generation of cell lines with ZMAT1 over-expression(SW1990/OV)or knockdown(BXPC-3/KD,Canpan-2/KD).Cell counting Kit-8(CCK-8)and colony formation assays were used to determine cell viability.Transwell and wound-healing assays were used to evaluate cell migration.Flow cytometry was performed to detected cell cycle and apoptosis.SW1990/OV cells and control cells were subcutaneously injected to BALB/c nude mice to establish the xenograft mouse models.The tumor sizes and weights of mice were recorded and the tumors were subjected to IHC analysis with the ZMAT1,p53,Ki67 and CD34 antibodies.3.Exploration of potential downstream regulatory mechanism of ZMAT1 in PDAC;RNA sequencing was performed by Illumina on SW1990/OV and control cells groups,subsequently functional and pathway enrichment analyses were conducted on sequencing and TCGA data.RT-q PCR and Western Blot were used to detect the changes in downstream gene expressions after ZMAT1 overexpression and knockdown.Immunofluorescence was carried out to investigated the intracellular localization of ZMAT1 in PDAC cells.Chromatin immunoprecipitation(Ch IP)assays and Ch IP-based sequencing were performed to find the direct downstream effector of ZMAT1 and the potential binding sites.Then Ch IP-q PCR and Luciferase assays were used to verify binding efficiency of the found binding sites.Finally the rescue experiments were conducted for the validation.Results:1.As shown in TCGA,GEO and Oncomine databases,ZMAT1 expression was down-regulated in PDAC compared to adjacent normal tissues.As expected,the results of IHC on 122 pairs of PDAC and pancreas tissues validated the down-regulation of ZMAT1 in tumor tissues.Reduced ZMAT1 expression in PDAC correlated with poor tumor differentiation,advanced TNM stage,high CA19-9 index and positive lymph nodes metastasis.Kaplan-Meier analyses results indicated that PDAC patients with low ZMAT1 expression had inferior OS and DFS than those with high expression.2.Results of CCK-8,colony formation,transwell and wound healing assays showed that up-regulation of ZMAT1 significantly inhibited SW1990/OV cell proliferation and migration.In contrast,down-regulation of ZMAT1 enhanced the proliferative and migratory ability of BXPC-3/KD and Capan-2/KD cell lines.Flow cytometry suggested the cell cycles of SW1990/OV cells was partly arrested at S/G2 stage,while the silencing of ZMAT1 decreased the percentage of cells in the S phase.It’s also indicated up-regulation of ZMAT1 increased the percentage of apoptotic SW1990cells,while ZMAT1 depletion reduced the proportion of apoptotic BXPC-3 and Capan-2 cells.The established xenograft mouse models showed that ZMAT1over-expression significantly inhibited tumor growth and IHC assays on tumors revealed up-regulation of ZMAT1 and p53,low levels of Ki-67 and CD34 in the ZMAT1-OV group.3.Functional and pathway enrichment analyses were performed and the significantly enriched biological processes included “Nucleus,” “P53 Signaling Pathway,”“Apoptotic process,” and “Cell Cycle”.Results of RT-q PCR and western blot showed that over-expression of ZMAT1 significantly upregulated p53,BAD and BAX levels and inhibited CDK2,CCNA2 and Bcl-2 expression in SW1990 cells;while the reverse results were observed in knock-down cell lines.Since ZMAT1 is mainly localized in nucleus,Ch IP-sequencing was performed and it’s suggested ZMAT1 physically interacted with some DNA fragments but not those of P53.As shown in Ch IP-sequencing,SIRT3 gene contained a ZMAT1-binding region which included three potential binding sites in its promoter region,which was verified via Ch IP-q PCR analysis.Luciferase assays showed increased luciferase activity upon SIRT3 binding in cells transfected with any of the three segments.Rescue experiments indicated the increased expression of p53 and enhanced cell growth in ZMAT1-OV cells were reversed by SIRT3 depletion.Collectively,the results suggested that ZMAT1 promoted the transcription of SIRT3 by binding to three sites in its promoter,and subsequently up-regulated p53 and inhibited tumor cell growth.Conclusion: The study identified a novel regulatory model in which ZMAT1 promoted the transcription of SIRT3,which subsequently up-regulated the expression of p53 and contributed to modulate cell cycle progression and apoptosis in PDAC.The findings indicated a role for ZMAT1-SIRT3-p53 signaling during tumor growth,highlighting that ZMAT1 is a novel prognostic and therapeutic biomarker of PDAC.