The Role And Mechanism Of MiR-340 In Regulating Anti-tumor Immunity Of Pancreatic Cancer

Background:The avoidance of immune destruction is a hallmark of cancer cells,and immune checkpoint blockade has emerged as a potential cancer immunotherapy with great promise.The“don’t eat me”signal CD47 in cancer cells binds signal regulatory protein-αon macrophages and prevents their phagocytosis.Thus,inhibition of CD47may offer a novel strategy for the treatment of cancer.MicroRNAs(miRNAs)are important negative regulators of gene expression in the immune system.However,the function of miRNAs in tumor immunity remains largely unknown.miR-340 has a significantly negative relationship with CD47 in pancreatic adenocarcinoma(PAAD).However,the current studies on miR-340 are very limited,especially the role of miR-340 in tumor immunity has not been reported.Objective:On one hand,the study explored the potential mechanism of miR-340 in tumor immunity through in vitro studies.On the other hand,in vivo experiments were conducted to further verify the results of in vitro experiments,to explore whether miR-340 could regulate the growth of PAAD and tumor immune microenvironment,and whether miR-340 could regulate the function of specific immune cells,and ultimately inhibit the development of PAAD.Methods:By using the Oncomine database,we analyzed the expression of CD47 in PAAD tissues and normal pancreatic tissues,and then we evaluated the expression of CD47 on the surface of human pancreatic cancer cell lines PANC1,BxPC3,and mouse pancreatic cancer cell lines Panc02 and H7 by flow cytometry.The relationship between CD47 and survival probability in patients with PAAD was analyzed using LinkedOmics and TCGA.The candidate miRNAs targeted to CD47were derived from the miRanda,DIANA-microT and Targetscan.Furthermore,by using bioinformatics analysis,we screened miR-340,which was significantly negatively correlated with the expression of CD47 in tumor tissues in a variety of cancers.In addition,we assessed the relationship between miR-340 and the survival rate of PAAD,and the relationship with radiation therapy.The regulation of miR-340to the expression of CD47 was explored through bioinformatic,luciferase reporter assays,qRT-PCR and western blot.Next,we evaluated the effect of miR-340 on the proliferation,migration and apoptosis of Panc02 and PANC1 by CCK-8,scratch test and flow cytometry.Moreover,we co-cultured miR-NC or miR-340 stable overexpression Panc02 cells with mouse peritoneal derived macrophages or bone marrow derived macrophages,respectively,and analyzed the effect of miR-340 on macrophage phagocytosis of tumor cells by flow cytometry and immunofluorescence.For in vivo studies,we used mouse miR-NC and miR-340 stable overexpression Panc02 cells to construct subcutaneous transplanted tumor models and in situ tumor models of PAAD in wild-type C57BL/6 mice.For the subcutaneous transplanted tumor model,we observed and measured the tumor size of tumor-bearing mice every other day.The differences in tumor volume and tumor weight between the two groups were compared.For in situ model,we detected the expression of miR-340 and CD47in tumor tissues of the two groups of tumor-bearing mice,and used flow cytometry analysis to detect the percentage changes of critical immune cells in peripheral blood,peripheral immune organs spleen and tumor immune microenvironment.Finally,the role of macrophages in the inhibition of miR-340 in tumor development of PAAD was determined by depleting macrophages using chlorophosphate liposomes in vivo.Results:The expression of CD47 in PAAD tissues was significantly higher than normal pancreatic tissue,and it was highly expressed on the surface of both human and mouse pancreatic cancer cells.MiR-340 regulated and directly inhibited the expression of CD47 by binding its 3’UTR,it is inversely correlated with the expression of CD47 and indicated the prognosis of survival in PAAD.The expression of miR-340 increased after radiotherapy.Functional studies further showed that the high expression of miR-340 in Panc02 cells or PANC1 cells had no significant effect on cell proliferation,migration and apoptosis,but the recovery of miR-340 in cancer cells was sufficient to down-regulate CD47 and further promote macrophages to engulf tumor cells.In vivo studies found that the growth of subcutaneous transplanted and in situ tumors of miR-340 overexpression Panc02 cells were significantly inhibited.In tumors of in situ miR-340 overexpression Panc02 cells-bearing mice,the expression of miR-340 was increased,and CD47 was decreased.In the peripheral blood,peripheral immune organ spleen,especially the tumor immune microenvironment of miR-340 overexpression Panc02 cells-bearing mice,the proportion of cytotoxic CD8~+T lymphocytes increased.In addition,in miR-340overexpression Panc02 cells-bearing mice,the proportion of M1-type macrophages and the ratio of M1-type to M2-type macrophages were significantly increased in the spleen and tumor tissues,showing enhanced polarization toward M1-type.Moreover,the depletion of macrophages could restore the inhibitory effect of miR-340 and promote the growth of PAAD,indicating that macrophages were contributed to the anti-tumor effect of miR-340.Conclusion:The miR-340 in tumor cells significantly inhibited the progression of PAAD in mice by affecting the tumor immune microenvironment.The regulatory mechanisms were as follows:the high expression of miR-340 in tumor cells could inhibit the expression of CD47,further promote the phagocytosis of macrophages to tumor cells by blocking the CD47-SIRPαaxis,and promote the polarization of macrophages to M1-type,increase the proportion of tumor killer CD8~+T cells,ultimately activating the anti-tumor immune response.Therefore,miR-340 is a key regulatory factor that enables macrophages to exert phagocytosis and anti-tumor immunity.It can be used as an effective potential target for clinical treatment of PAAD and even other solid tumors,and has a promising application prospect in tumor immunotherapy.