MMP12 Knockout Up-regulates M2 Macrophage To Accelerate Intestinal Tumor Growth In Apc^Min/+ Mice

Objective:MMP12?Matrix Metalloproteinases 12?is mainly an endo-protease actively secreted by inflammatory cells such as macrophages and monocytes,which can decompose most of the extracellular matrix and vascular components.In recent years,it has been reported that MMP12 is involved in the occurrence,development,invasion and metastasis of tumors.This study is to investigate the effect of MMP12 deletion on intestinal growth in Apc^Min/+mice and further explore its molecular mechanism.Methods:First,Apc^Min/+mice were crossed with MMP12 knockout mice to establish Apc^Min/+;MMP12^-/-spontaneous intestinal adenoma model,Apc^Min/+;MMP12^-/-mice were used as experimental group,Apc^Min/+mice were control group.Counting the number and volume of intestinal tumors at different ages representing different pathological processes at 9 weeks,15 weeks,and 24 weeks.The pathological morphology and extent of intestinal lesions in Apc^Min/+mice and Apc^Min/+;MMP12^-/-mice were observed by hematoxylin-eosin staining.Immunohistochemistry was used to detect the proliferation,angiogenesis,leukocyte and macrophage infiltration of intestinal tissues in Apc^Min/+mice and Apc^Min/+;MMP12^-/-mice;Further,Western blotting and immunohistochemistry were used to detect the intestinal cancerization in mice at different ages.Finally,Western blotting,immunofluorescence and flow cytometry were used to detect the changes of macrophage subtypes.The relevant cytokines were screened by Mouse Cytokine Array,and real-time qPCR which detect the changes of relevant cytokines secreted by M2 macrophages in Apc^Min/+mice and Apc^Min/+;MMP12^-/-mice.Results:1.Identification of Apc^Min/+;MMP12^-/-gene mice:Apc^Min/+;MMP12^-/-micehave clear Apc^Min/+bands at 600 bp and 340 bp,as well as MMP12^-/-clear strip at 1400 bp.2.Tumor statistical results showed that the number of intestinal tumor in Apc^Min/+;MMP12^-/-mice was significantly higher than that in Apc^Min/+mice,and the tumor volume was larger.3.Hematoxylin-eosin staining results showed that knockout of MMP12could accelerate the pathological process in Apc^Min/+mice,andApc^Min/+;MMP12^-/-mice had a higher degree of malignancy.4.The results of immunohistochemistry showed that the positive expression of Ki67 and CD34 in Apc^Min/+;MMP12^-/-mice was higherthan that in Apc^Min/+mice?*P<0.05?.The positive expression rates ofCD45 and CD68 in tumors were also significantly higher than those in Apc^Min/+mice?**P<0.01 and***P<0.001?.5.The results of immunohistochemistry and Western blotting showed thatthe expression of CA19-9 and CEA in Apc^Min/+;MMP12^-/-miceintestinal tumor was earlier and higher than that in Apc^Min/+mice.Theexpression of?-catenin in Apc^Min/+;MMP12^-/-mice was higher than thatin Apc^Min/+mice?*P<0.05?,and the amount that went inside thenucleus was higher.6.Flow cytometry results showed that the proportion of M2 macrophages in spleen,intestinal tumors and peritoneal macrophages wassignificantly up-regulated in Apc^Min/+;MMP12^-/-mice;The results ofimmunofluorescence and Western blotting also indicated that the expression of M2 macrophage marker?Ym1,Arg1?of intestinal tumor in Apc^Min/+;MMP12^-/-mice was significantly increased.7.The results of Mouse Cytokine Array showed that the levels of inflammatory factors IL-4 and TNF-?in Apc^Min/+;MMP12^-/-mice M2macrophage culture supernatant were significantly increased.Thisresult was also verified by real-time qPCR.Conclusion:1.Knockout of MMP12 promotes the growth and canceration of intestinal tumors in Apc^Min/+mice.2.Knockout of MMP12 promoted the intestinal macrophage infiltration in Apc^Min/+mice;at the same time,the macrophage polarized into M2 typeunder the stimulation of tumor microenvironment.3.Knockout of MMP12 promoted the secretion of IL-4 and TNF-?by M2macrophages in Apc^Min/+mice,and decreased the secretion of CCL2and IL-23,which led to the growth of intestinal tumors.