Functional Study Of Progranulin’s Role In B16 Melanoma Tumor Growth And Macrophage Polarization

Progranulin（PGRN）is expressed in a variety of tissues,particularly in epithelial and hematopoietic cells.Abnormalities in PGRN are associated with a variety of diseases,including cancers in which overexpression of PGRN has been detected in a variety of clinical specimens（such as breast cancer and ovarian cancers）.However,little is known presently about the function of PGRN in the progression of melanoma.So,first we explored the functional role of progranulin in a melanoma model。On the other hand,PGRN has been shown to be an effective anti-inflammatory factor.PGRN knockout（KO）macrophages are more likely to polarize to M1macrophages that produce more proinflammatory cytokines when stimulated by LPS,but littlel is known about PGRN’s role in M2 macrophage polarization,therefore,in part II,we explored the functional role of PGRN in macrophage polarization.In Part Ⅰ,PGRN in the melanoma cell line B16 was knocked out by transiently transfecting B16 cells with a pair of CRISPR-Cas9 plasmid targeting PGRN for nullification.Single cell clones with PGRN annulment were obtained by serial dilution.The expression level of PGRN in Pgrn-targeted B16 cells was significantly decreased as verified by real-time PCR and Western blot.The effect of PGRN KO on cellular proliferation in vitro was assessed by the MTS assay,which revealed little change compared to wild type B16 cells.Brd U staining also showed little influence on cell cycle.However,PGRN KO B16 cells displayed significant growth deficiency when injected into syngeneic C57BL/6 mice.Using PGRN KO mice,we established that tumor-originated not host-derived PGRN was crucial for tumor growth.Furthermore,flow cytometric analysis of CD4⁺and CD8⁺T cells in the spleen and tumor-draining lymph nodes revealed increased percentages of T cells in mice carrying PGRN KO B16tumor,implying that tumor-derived PGRN may have a paracrine effect on host immune responses to the malignancy.Detection of pulmonary metastasis by tail vein injection of B16 and PGRN KO B16 cells showed that PGRN KO cells significantly reduced lung metastasis,suggesting that tumor-derived PGRN not only affect tumor growth but also affect lung migration.This original study provides insights about PGRN’s role in tumorigenesis and suggests that PGRN may be a tangible therapeutic cancer target.In Part Ⅱ,we mainly explored the role of PGRN in macrophage polarization and macrophage polarization related pulmonary fibrosis disease model.We hypothesized that PGRN can regulate macrophage polarization,furthermore,we hypothesized that these PGRN-dependent changes in macrophage polarization would alter fibrotic responses in the lung.We examined the gene expression changes in WT and PGRN^-/-BMDMs stimulated by LPS or IL-4/IL-13,and found that defficiency of PGRN significantly promoted the M1 macrophages polarizaton while inhibiting the M2macrophages polarization.Then we co-cultured M2-BMDM and CD4⁺T cells in vitro and found that the impared M2 polarization by PGRN deletion led to increased IFN-γsecretion of CD4⁺T cell.Cell signaling pathway studies further revealed that the effect of PGRN on M2 macrophage polarization was involved in Akt and Stat6 pathway.Finally,in the bleomycin-induced pulmonary fibrosis model,we found that the effect of PGRN on macrophage polarization-mediated pulmonary fibrosis is not very obvious.But basically we established the promoting role of PGRN on M2 macrophage polarization.