Research On The Mechanism Of RIOK3 Regulating Macrophage Function And Related Role In Tumor Invasion/Metastasis

Macrophages are multifunctional immune cells that play important roles in both specific and non-specific immune responses.In addition to systemic effects such as phagocytosis of foreign bodies,antigen presentation,and inflammatory responses,the infiltration of macrophages in local tissues is closely related to the development of various diseases.In particular,macrophages infiltrated in tumor tissue can affect the prognosis of patients by regulating tumor invasion and metastasis,but the relevant mechanisms are not clear.Studying the molecular mechanisms that regulate the proliferation,differentiation and functional polarization of macrophages will help to elucidate its correlation with tumorigenesis and development,and provide new targets for tumor immunotherapy targeting tumor macrophages.RIOK3(RIO Kinase 3)is an atypical serine/threonine protein kinase that belongs to the RIO(right open reading frame)family along with RIOK1 and RIOK2.Studies have shown that RIOK3 is involved in the production of type ? interferon,NFkB signal transduction pathway,Hedgehog signal transduction pathway and Akt/mTOR pathway,and might throw impacts on cellular immune function and cell proliferation.The results of mRNA expression profiling showed that RIOK3 was highly expressed in myeloid cells,suggesting that this protein may be involved in the related fimctions of macrophages.In this study,using our established macrophage RIOK3-specific knockout mice(RIOK3-/-),malignant melanoma in situ and lung tissue metastasis models along with RNA-seq-based transcriptome studies and other research methods,the regulation of RIOK3 on macrophage function and tumor invasion and metastasis was comprehensively analyzed at the level of molecular,cells and model animals,:1)the effect of RIOK3 on macrophage proliferation,differentiation and polarization;2)The regulatory effect of RIOK3 on the key pathway M-CSF/PI3KjAkt during macrophage activation;3)RIOK3 regulates tumor invasion and metastasis by affecting the proliferation,differentiation and polarization of macrophages in tumor tissues.The main research results are as follows:1)RIOK3 was found to inhibit the proliferation of macrophages,and promote its apoptosis;RIOK3 exerts an inhibition effect on M2 type polarization of macrophages,but not affect the differentiation and maturation of macrophages;2)RIOK3 can inhibit M-CSF/PI3K/Akt pathway activation and thereby reduce macrophage proliferation;3)RIOK3 inhibits the infiltration of tumor-associated macrophages in malignant melanoma tissue and inhibits tumor growth and metastasis.For the first time,this study found that RIOK3 can inhibit macrophage proliferation and M2-type polarization by regulating M-CSF/PI3K/Akt pathway,thereby reducing macrophage infiltration in tumor tissues,and restrain the invasion and metastasis of malignant melanoma.The results provide a scientific basis for using RIOK3 as a new target for immunotherapy of tumor-associated macrophages.Part ?.Effects of RIOK3 on macrophage proliferation,differentiation and polarizationOBJECTIVE:To explore the effects of serine/threonine kinase RIOK3 on the function of macrophages.METHODS:Bone marrow nucleated cells were isolated from 4-6 weeks old female mouse(C57 wild type and RIOK3-/-)and differentiated into primary macrophages with M-CSF cultivation for 7 days.Then,in order to overview the effects of RIOK3 on macrophage proliferation,differentiation and polarization,BMDMs were stimulated with macrophage colony-stimulating factor(M-CSF,MO),interferon-?/lipopolysaccharide combination(IFN-?+LPS,M1 type polarization),and interleukin-4(IL-4,M2 type polarization)respectively.Cell RNA was extracted for genomic transcriptome RNA sequencing(RNA-seq),followed by functional enrichment analysis on significantly differentially expressed genes(DEGs)and validation of gene expression by RT-qPCR.Moreove,during macrophages cultivation with M-CSF,CCK8 was used to detect cell proliferation,CFSE staining was combined with flow cytometry to detect the dynamic process of cell division and division,cell cycle assay was used to calculate the proportion of cells in active mitosis state,and apoptosis assay was used to detect cell apoptosis rate after M-CSF starvation treatment.The differentiation index stained on macrophages and assayed by flow cytometry was used to evaluate cell differentiation.RESULTS:RNA-seq results identified differentially expressed genes(DEGs)between the transcriptome of RIOK3 knockout and wild-type macrophages.The functional enrichment analysis of DEGs in M-CSF(MO)stimulation group showed that Cell cycle is the top term,following by other related terms such as cell division,apoptosis,and cell proliferation.Wild-type cells and RIOK3 knockout cells stimulated by M-CSF were collected for RT-qPCR assay and the results confirmed that the expression of cell cycle-related gene Ccnd1 was significantly increased.The results of CCK8 showed that the cell proliferation signal was enhanced,and the cell count was confirmed with crystal violet staining which showed that cell quantity is significantly increased.The cell division was dynamically deteeted by flow cytometry.The results showed that the average number of divisions and the proportion of dividing cells in RIOK3 knockout cells were higher than those in the control group.The results of cell cycle experiments showed that the proportion of cells in the active phase of cell division(S phase and G2 phase)in RIOK3 knockout group was higher than that in the control group.The results of apoptosis assay showed that after M-CSF starvation treatment,wild-type cells showed obvious apoptosis,while RIOK3 knockout macrophages responded to M-CSF starvation without significant apoptosis.Analysis of RNA-seq data under polarized conditions(Ml or M2)indicated that RIOK3 knockout macrophages were prone to be transformed into repairing macrophages(M2);RT-qPCR confirmed its M2 signature.When stimulated by IL-45 the expression of cytokine Arg1,Retnla,and 1110 genes was up-regulated,and when IFN??/LPS was combined cytokines such as 1112 were down-regulated.The results of whole blood cell count in mouse showed that there was no significant difference between the proportion of whole blood cells in RIOK3-/-mice and wild type mouse.Fluorescence staining of bone marrow nucleated cells combined with flow cytometry showed that there was no significant difference in the proportion of mononuclear dendritic precursor cells(MDP),monocyte progenitor cells(cMoP),and monocytes between RIOK3-/-and wild-type group.In the process of M-CSF-induced monocyte maturation and differentiation,the marker of macrophage maturation was detected by flow cytometry.The results showed that the ratio of total cells to mature cells and the average fluorescence intensity of each index have no significant differences in the two groups.CONCLUSION:RIOK3 can inhibit macrophage proliferation,promote its apoptosis,suppress M2 type polarization,and reduce the expression of related genes Argl,Retnla and 1110.RIOK3 does not affect the differentiation and maturation of macrophages.Part ?.The mechanism of RIOK3 in regulating macrophage proliferationOBJECTIVE:To analyze the molecular mechanism of RIOK3 in regulating macrophage proliferation.METHODS:BMDMs were harvested and stimulated by M-CSF in vitro for a corresponding period of time,and collected for protein extraction to detect the activation of CSFIR downstream pathway by Western blot,including PI3K/Akt signaling pathway,MAPE/Erk signaling pathway and NF-kB signaling pathway.Regulatory effects of RIOK3 on related pathways were venfied by corresponding cell pathway inhibitors.The interaction between RIOK3 and the target protein was explored by fluorescence staining combined with laser confocal microscopy and venfied by immunoprecipitation experiments.RESULTS:The results of WB assay on the M-CSF activation pathway showed that phosphorylation levels of PI3K and Akt were increased in RIOK3 knockout macrophages,and Erk phosphorylation level was unchanged.Immunofluorescence confocal experiments showed that there was no significant co-localization between RIOK3 and CSFIR.The results of CSF1R staining assayed by flow cytometry showed that RIOK3 knockout did not affect the total expression,endocytosis and degradation of CSF1R on macrophage surface.There is a cytoplasmic co-localization between RIOK3 and PI3K(p85 subunit).Using PI3K(p85 subunit)protein as a bait,WB can detect RIOK3 protein,which means RIOK3 co-precipitates with PI3K.CONCLUSION:RIOK3 inhibits PI3K phosphorylation during M-CSF stimulation by binding to PI3K(p85 subunit),thereby suppressing the proliferation of macrophages;RIOK3 does not affect the expression,endocytosis and degradation of CSF1R.Part ?.Effect of RIOK3 deficient macrophage on melanoma developmentOBJECTIVE:To further explore the effect of RIOK3 expressed in macrophage on tumor prognosis through melanoma bearing mouse model.METHODS:Several pairs of 6-8 weeks old female RIOK3-/-and wild-type mouse were enrolled.B16-F10 cells were injected subcutaneously into the dorsal,and the tumor size of melanoma was recorded every 2-4 days.In situ tumors were harvested 14 days later,followed by collagenase digesting to prepare single suspension cells.Different types of immune cells were labeled with fluorescent antibodies,and were assayed by flow cytometry to detect the proportion of lymphocytes,macrophages,etc.in tumors.Morever,immunohistochemical staining was performed to further validate the proportion of macrophages in the tumor.The melanoma cells labeled with HMB45+MART1+were flow-selected.And cell RNA was extracted for RNA-seq to identify differentially expressed genes(DEGs)between the two groups of melanoma cells.Functional analysis was managed to predict the gene clusters classification that cause tumor progression.o To establish a model of lung metastasis in melanoma,B16-F10 cells was injected into the tail vein and the mouse were sacrificed to observe tumor metastasis after 12 days.RESULTS:The size of in situ melanoma in RIOK3-/-group was significantly larger than that in the control group,and the proportion of macrophages in the tumor tissues was significantly increased.The flow cytometry assay indicated that there was no significant difference in the proportion of M1 and M2 types TMAs between two groups.The RNA-seq results of melanoma cells suggest that melanoma cells in RIOK3-/-mice have higher invasiveness.The lung metastasis model in mice suggests that the distant metastasis lesions in the lung of RIOK3-/-mice are significantly increased,the body weight is significantly shrunken,and the prognosis is poor.CONCLUSION:Deficient expression of RIOK3 in macrophages can promote disease progression on melanoma-bearing mice.RIOK3 may suppress tumor growth and metastasis by inhibiting tumor-associated macrophages infiltrating in malignant melanoma tissue.