The Mechanism Of MiR-128 Targeting ZEB1/CD47 Axis In Regulating Pancreatic Cancer Immunity

Background:Pancreatic cancer(PDAC)is one of the most malignant tumors in the digestive tract.The early symptoms of pancreatic cancer are insidious and the disease progresses rapidly,but the effective rate of surgical resection and chemotherapy is low,and the prognosis of patients is very poor.It is urgent to study the pathogenesis of pancreatic cancer and develop new immunotherapy methods.MicroRNAs(mi RNAs)are no protein-coding RNAs that regulate tumor growth invasion and immune evasion.However,little is known about the regulatory role of mirnas in pancreatic cancer immunity.Therefore,miRNA is of great significance in regulating the mechanism of action of PDAC and the treatment of other tumors.In this study,we explored the role and mechanism of miR-128 in PDAC.Objective:In this paper,in vitro and in vivo experiments were conducted to investigate the role of miR-128 in regulating pancreatic cancer immunity and its cellular and molecular mechanisms.mi R-128 regulates the infiltration of anti-tumor immune cells including DCs,CD8~+T cells and NKT cells in the immune microenvironment through ZEB1/CD47 axis,inhibits EMT process through ZEB1,and ultimately inhibits tumor growth and metastasis.It provides a new way of understanding tumor immune escape and tumor immunotherapy.Methods:1.Bioinformatics methods were used to analyze the expression changes ofCD47 in pancreatic cancer tissues and normal pancreatic tissues.miRNAsthat might target CD47 were analyzed using miRanda Diana-Microt andTargetScan database.2.The effect of mi R-128 on the growth,migration and invasion of pancreaticcancer cells was investigated by CCK8 scratch migration flow cytometry andluciferase reporter gene in vitro.3.Flow cytometry was used to detect the phagocytosis of macrophages,theproportion of immune cells and the activation of dendritic cells in the tumorimmune microenvironment were measured.4.Panc02 cell lines of pancreatic cancer with stable and high expression ofmiR-NC or miR-128 were constructed,and the effects of miR-128 on thegrowth and metastasis of Panc02 cells and the immune microenvironmentwere studied by constructing orthotopic tumor and subcutaneous tumormodels of pancreatic cancer with C57BL/6 mice.5.Western bloting and qRT-PCR were used to investigate the mechanism ofmiR-128 and the regulation of CD47 ZEB1 in pancreatic cancer.Results:1.Bioinformatics analysis revealed that miR-128 is the miRNA targeting CD47and ZEB1.CD47 ZEB1 was negatively correlated with survival prognosis ofpancreatic cancer patients,and CD47 was positively correlated with ZEB1expression.Low expression of miR-128 was associated with poor overallsurvival of PDAC.2.In vitro experiments showed that miR-128 could inhibit the proliferation,clone formation,migration and invasion of Panc02 cells,and enhance thephagocytosis ability of macrophages and the activation of DCs.3.In vivo experiments,miR-128 was found to inhibit the growth and metastasisof PDAC.Immunomicroenvironment analysis showed that the overexpressionof miR-128 in tumor cells increased the proportion of DCs,CD8~+Tlymphocytes and natural killer T cells(NKT)in tumor and spleen,thusenhancing anti-tumor immunity.4.Western bloting and qRT-PCR confirmed that miR-128 can regulate ZEB1 inpancreatic cancer cells,thereby inhibiting CD47,EMT progression,increasing e-cadherin expression,and down-regulating n-cadherin andVimentin expression.miR-128 can be regulated by ZEB1 by targeting ZEB1to play an anti-cancer role,thus affecting CD47 and EMT.Conclusions:1.Bioinformatics analysis showed that the expression of miR-128 wassignificantly negatively correlated with the survival rate of patients withpancreatic cancer,and the high expression of ZEB1 and CD47 in pancreaticcancer was associated with poor prognosis of patients,and the expression ofZEB1 and CD47 was positively correlated.2.Restoration of miR-128 in Panc02 cells attenuated cell proliferation,migration and invasion,inhibited EMT progression,increased e-cadherinexpression,and down-regulated n-cadherin and Vimentin expression.3.miR-128 can be regulated by ZEB1 to play an anti-cancer role affectingCD47 and EMT.4.In vivo experiments,overexpression of miR-128 significantly inhibitedtumor growth and metastasis of PDAC.mi R-128 regulated invasion ofanti-tumor immune cells including DC CD8+T cells NKT cells in theimmune microenvironment through ZEB1/CD47 axis,and inhibited EMTprocess through ZEB1,ultimately inhibiting tumor growth and metastasis.5.We co-cultured Panc02 cells stabilized with miR-NC or miR-128overexpressed with macrophages or DCs,and found that the panc128overexpressed Panc02 cells enhanced macrophage phagocytosis and DCsactivation.6.miR-128 can act as an important regulator of tumor immunity throughZEB1/CD47 axis and EMT in PDAC.These results provide a theoreticalbasis for innovative preclinical combination immunotherapy based on CD47regulation and EMT inhibitors.