ZC3H7A And ZC3H15 Regulate The Malignant Phenotype And Mechanism Of Pancreatic Ductal Adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma,the most common type of pancreatic cancer,is known as rapid tumor progression,high mortality,short expected survival,and resist to radiotherapy,chemotherapy,and existing immunotherapies.Our previous study suggested that ZC3H7A was involved in the metastasis of pancreatic cancer.As another candidate protein,ZC3H15,which has the same structure domain,plays a tumor-promoting role by combining with TRAF2 protein in different tumor diseases.The biological roles and mechanisms of ZC3H7A and ZC3H15 in the development of pancreatic cancer are still unclearObjective1.To study the expression levels of ZC3H7A and ZC3H15 in pancreatic ductal adenocarcinoma tissue,and to analyze the relationships among the expression level,the clinicopathological features and prognosis of patients with pancreatic ductal adenocarcinoma.2.To explore the effects of different ZC3H7A and ZC3H15 protein expression levels on malignant phenotypes such as proliferation,apoptosis,invasion,migration and drug resistance of pancreatic cancer cells.And to explore the mechanism of regulating the malignant phenotype of pancreatic cancer cells.Methods1.Expression of ZC3H7A and ZC3H15 in tumor tissues,matched tumor-adjacent pancreas tissues were investigated by immunohistochemistry method,and the association among ZC3H7A/ZC3H15,clinicopathological characteristics and clinical prognosis of patients was analyzed.2.Protein expression levels of ZC3H15 or ZC3H7A in cells were regulated by siRNA interference or transfection with overexpressing plasmids.The stable cell lines of ZC3H7A gene knockout were constructed by CRISPR-Cas9 gene editing technique.Cell proliferation,apoptosis,invasion and migration were observed by CCK8,flow cytometry,plate cloning and transwell test.The effect of gemcitabine was verified in vitro and in vivo.ZC3H7A downstream target genes were explored by mRNA sequencing and microRNA sequencing.The mechanism of ZC3H15 was investigated by immunoprecipitation,mass spectrometry and Western blot.Results1.ZC3H15 protein is over-expressed in pancreatic cancer tissues,and its high expression is significantly correlated with lymph node metastasis and poor differentiation.High ZC3H15 expression level appeared to be an independent risk factor affecting the prognosis of pancreatic cancer patients.The significant positive correlation between ZC3H15 and ZC3H7A could be observed.2.Experimental results of malignant phenotype:(1)The results suggested that ZC3H15 could promote the proliferation of pancreatic cancer cells by interfering with the transformation from G0/G1 to S phase.ZC3H15 can protect pancreatic cancer cells form apoptosis.(2)ZC3H7A gene knockout significantly inhibited proliferation both in vitro and in vivo.The loss of ZC3H7A protein can improve gemcitabine effect both in vivo and in vitro.(3)ZC3H15 and ZC3H7A had no significant effect on the invasion,migration and the ability of single cell cloning in human pancreatic cancer cells.3.The activation state of ERK1/2,a key signaling protein in the MAPK signaling pathway,is significantly positively correlated with ZC3H15.Inhibition of ZC3H15 significantly inhibited the activation of p65,a key signaling protein in the NF-B signaling pathway.The results of protein spectrum analysis after immunoprecipitation and the results of public database showed that ZC3H15 was combined with DRG1.Western blot results after immunoprecipitation showed that ZC3H15 was bound to TRAF2.4.mRNA sequencing and microRNA sequencing results obtained a number of potential downstream targets.CD40 and VASH1 may be the key downstream targets of ZC3H7A.ConclusionsZC3H15 is highly expressed in pancreatic cancer tissues,and its high expression is a risk factor for the prognosis of pancreatic cancer.ZC3H15 activates the MAPK and NF-B signaling pathways by binding to TRAF2 to promote proliferation and inhibit apoptosis of pancreatic cancer cells.The expressions of ZC3H15 and ZC3H7A were positively correlated.After the knockout of ZC3H7A gene,the cell proliferation was inhibited and the gemcitabine effect was improved.CD40 and VASH1 may be important downstream targets of ZC3H7A;DRG1 is another potential target of ZC3H15。...