Pancreatic tumor cell induction of macrophage polarization through sonic hedgehog regulation of interferon regulatory factors
Objective To explore the specific role mechanism of sonic hedgehog(Shh)regulating macrophage pol-arization in pancreatic tumor cells.Methods Clinically isolated pancreatic tumor tissues and adjacent tissues were collected,and the expression level of Shh was detected by immunohistochemistry.The relationship between Shh ex-pression difference in TCGA database and prognosis in patients with pancreatic cancer was analyzed.Pancreatic tum-or cells and macrophages were co-cultured in vitro.The macrophage polarization was identified by flow cytometry and the level of Shh protein was detected by Western blot.RT-qPCR assay was used to detect the mRNA relative ex-pression levels of Shh,nitric oxide synthase(iNOS)and arginase-1(Arg-1).The co-cultured cells were divided into four groups,including control group,NC group,Shh group and Shh-RNAi group.The Shh expression was regulated by lentivirus vector,and the same items were detected.The expression levels of interferon regulatory factor(IRF)4 and IRF5 were detected by Western blot and RT-qPCR respectively.Results Immunohistochemistry showed that the Shh positive cell rate in pancreatic cancer tissues was significantly higher than that in adjacent tissues(P＜0.05).TCGA data analysis revealed that the expression of Shh in patients with pancreatic cancer was significantly higher than that in normal controls(P＜0.05),and the low expression of Shh was correlated with the improvement of long-term survival rate(P＜0.05).Compared with normal RAW264.7 cells,the expression of Shh protein,mRNA relative expressions of Shh,iNOS and Arg-1 and M2/M1 ratio in co-cultured RAW264.7 cells in pancreatic tumors were incre-ased(allP＜0.05).Compared with control group and negative control group,the protein and mRNA relative expression levels of Shh and IRF4 and mRNA relative expression level of Arg-1 in Shh group were up-regulated,the macrophage M2/M1 ratio was increased,and the protein and mRNA relative expression levels of IRF5 and mRNA relative expression level of iNOS were down-regulated(allP＜0.05).Compared with Shh group,the protein and mRNA relative expression levels of Shh and IRF4 and mRNA relative expression level of Arg-1 in Shh-RNAi group were down-regulated(all P＜0.05),the M2/M1 ratio was decreased(P＜0.05),and the relative expression levels of IRF5 protein and mRNA and relative expression level of iNOS mRNA were up-regulated(all P＜0.05).Conclusions Shh is highly expressed in pancreatic tumor cells.Over-expression of Shh can promote the M2 macrophage polarization and inhibit the M1 macrop-hage polarization,which may be achieved by regulating the expressions of IRF4 and IRF5 proteins and genes.
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