Nogo-B Fosters HCC Progression By Enhancing Yap/Taz-mediated Tumor-associated Macrophages M2 Polarization

Objective: Liver cancers are the fourth most common cause of cancer-related death and rank sixth in terms of incident cases.Therefore,the treatment of liver cancer is still a severe test for us.Tumor-associated macrophages(TAMs)and their M2-type extremely promote tumor proliferation,invasion and metastasis.Nogo-B is expressed in most tissues and participates in macrophage polarization.The purpose of this study was to explore the role of Nogo-B in the polarization of tumor-associated macrophages M2 and its potential mechanism,and provide a potential target for the treatment of liver cancer.Methods: Tissue immunofluorescence staining and q-PCR were used to detect the expression level of Nogo-B in cancer and adjacent macrophages in 67 clinical liver cancer specimens.Follow up the relationship between Nogo-B expression level and tumor-free survival rate.Univariate and multivariate regression analysis were used to evaluate the relationship between Nogo-B expression level and patient prognosis.Western blot,q-PCR,and flow cytometry were used to detect the expression levels of Nogo-B and polarization markers in macrophages(TNF-?,IL-1?,iNOS,IL-10,MR,Arg-1,CD163).Subsequently,the expression of Nogo-B in macrophages was knockdown by lentivirus,and the expression of polarization markers of macrophages in the control group and knockdown group was detected by WB.The control group and knockdown group macrophages were co-cultured with mouse HCC cells,CCK-8 and colony formation experiments were used to detect the proliferation of liver cancer cells,and the Transwell assay was used to detect the migration and invasion of HCC cells.Subsequently,a nude mouse subcutaneous tumor model was established and the tumor-bearing growth status was analyzed(HCC cells: macrophages = 4: 1).Ki67 immunohistochemical staining was used to assess the level of cancer cell proliferation,and immunofluorescence staining was used to assess the level of macrophage polarization.Subsequently,immunofluorescence staining and WB were used to detect the localization and expression of Yap and Taz in macrophages.Verteporfin,the Yap inhibitor was used to pretreat macrophages,and the expression of Yap and Taz and the polarization level of macrophages were detected by WB and q-PCR.Results: The expression of Nogo-B in TAMs of HCC patients is significantly increased,which correlated with the poor prognosis of the patients with HCC.Coincidentally,HCC conditioned medium(HCM)facilitated Nogo-B expression and the M2 phenotype of macrophages.Nogo-B knockdown Nogo-B significantly suppressed the M2-type polarization of macrophages and inhibited HCC cells proliferation both in vivo and in vitro(Tumor volume,day= 19,shNC vs shNogo-B 2276.73 ± 361.2 vs 1289.67 ± 321.5,p <0.01,n = 3;colony number,shNC vs shNogo-B 151 ± 37 vs 66 ± 26,p <0.05,n = 3).Furthermore,Nogo-B knockdown inhibited macrophage-mediated tumor cells migration and invasion(migration,shNC vs shNogo-B 50 ± 6 vs 28 ± 5,p <0.001,n = 5;invasion,shNC vs shNogo-B 43 ± 7 vs 24 ± 4,p <0.001,n = 5),and promoted tumor cells apoptosis(shNC vs shNogo-B 4.60 ± 1.67 vs 10.46 ± 2.41,p <0.05,n = 3).Nogo-B meaningfully enhanced IL4-stimulated the alternative activation of macrophages as well as expression of the transcriptional regulators Yes-associated protein(Yap)/transcriptional coactivator with PDZ-binding motif(Taz).An inhibitor of Yap,Verteporfin,could block Nogo-B-Yap/Taz-mediated macrophages M2 polarization.Conclusion: Nogo-B expression in macrophages facilitates tumor-associated macrophages M2 polarization and protumoral effects of TAMs in HCC.Targeting Nogo-B /Yap/Taz in macrophages could provide a new therapeutic strategy in HCC therapy.