The Effect Of Macrophage Educated By S100A6 On Proliferation Of Human Crc Cells And Its Mechanism

Backgrounds and objectivesColorectal carcinoma(CRC)is one of the most common malignance of digestive system neoplasm,and its morbidity and mortality rank third and fourth respectively in the world.In China,morbidity of CRC is rising year by year;the 5-year survival rate of advanced or advanced CRC is low.Therefore,there is much significance to explore the pathogenesis of CRC and search for innovative prevention and treatment targets.Tumor microenvironment(TME)provides an appropriate environment for tumor progression,which mainly includes two types of components: one is non-cellular components,including cytokines and secreted proteins(such as S100A6);another component is cells,including tumor cells,immune cells(such as macrophages)and other stroma cells.These components interact with each other in TME to promote tumor progression.Calcium cyclin S100A6 is a member of S100 family and regulates cell proliferation,apoptosis,cytoskeleton remodeling and stress response.S100A6 is overexpressed in CRC tissues and cell lines and closely related to poor prognosis of CRC patients.S100A6 promotes the proliferation,migration and invasion of CRC cells.As a secretory protein,S100A6 can bind to the receptor for advanced glycation end products(RAGE)and toll-like receptors(TLRs)on the surface of target cells in a paracrine/autocrine manner.Macrophages is the largest number of immune cells infiltrating into the TME,accounting for 30% to 50% of tumor stroma cells.It can promote the occurrence and development of cancers through a variety of ways,such as immune escape,cell activation and proliferation,tumor invasion and metastasis,etc.Due to the cytokine balance or other factors in TME,bone marrow-derived macrophages mainly differentiate into two types: M1,inflammation related macrophages,and M2,tumor related macrophages.In tumor tissues of CRC patients,the infiltration of M2 significantly increased,which promote the growth and invasion of CRC,and are closely related to the treatment failure and poor prognosis of patients.However,how the secretory proteins in TME affect on macrophages has not been fully elucidated as well as the effects and molecular mechanisms of the affected macrophages on CRC cells.As is well-known,S100A6 in CRC microenvironment can bind to target cells by RAGE or TLRs,while macrophages in TME has both RAGE expression and corresponding signal transduction mechanism.Therefore,we speculate that S100A6 in the CRC microenvironment can affect the functions of macrophages by binding to its RAGE or TLRs,and therefore promotes the development of CRC.In this study,human mononuclear cell line THP-1,human CRC cells LoVo and HCT116 were selected as subjects to explore the effect of S100A6 on macrophages and the indirect effect of S100A6 in TME on the proliferation of human CRC cells via educating macrophages and theinvolved mechanisms.It is hoped to accumulate experiment evidence for clarifying the complex interactions among S100A6,macrophages and CRC cells in the CRC microenvironment,as well as to provide new ideas for clinical treatment of CRC patients.Methods1 Preparation and identification of recombinant human protein GST-human S100A6(rhS100A6)and GST.2 The treatment of rhS100A6 on macrophages(Mφ)2.1 THP-1 was induced to macrophagesAfter THP-1 was induced by 100 ng/mL PMA(phorbol 12-myristate13-acetate)for 24 hours,the cells stopped proliferation and changed from the suspension state into adherent state,from round to flat oval,polygon;and some of them extended pseudopod.It means that the cells have differentiated into macrophages and can be used for subsequent experiments.2.2 Treating macrophages by recombinant protein: A6-Mφ or GSTMφ were the macrophages treated with rhS100A6(100μg/mL)or GST(100μg/mL)for 24 h,respectively.3 The effect of S100A6 on macrophages and its mechanism3.1 The influence of S100A6 on differentiation of macrophages and its mechanism:3.1.1 qPCR was used to test expression of M1 marker genes(TNF-a、iNOS),M2 marker genes(CD206、CD163)and M2 related genes(CD206、CD163)in A6-Mφ and GST-Mφ.3.1.2 Western blot was used to detect the levels of M2 related protein CD206,PD-1,MMP7,MMP9,and VEGF in A6-Mφ and GST-Mφ.3.1.3 The mRNA and protein levels of RAGE and TLR4 in A6-Mφwere examined by qPCR and Western blot,respectively.3.1.4 Macrophages were pre-treated by RAGE inhibitor FBS-ZM1 or TLR4 inhibitor TAM-242,and then treated by rhS100A6 for 24 h,then M1 marker genes(TNF-a、iNOS)and M2(CD206、CD163)marker genes in Mφwere detected by qPCR.3.2 Effect of S100A6 on macrophage proliferation and its mechanism.3.2.1 CCK8 and MTT assay both were used to test the survival of macrophages treated by various concentration of rhS100A6(A6-Mφ)and diverse treatment time.3.2.2 qPCR and Western blot were used to detect mRNA and protein levels of RAGE and TLR4 in A6-Mφ.3.2.3 Macrophages were pre-treated by RAGE inhibitor FBS-ZM1 or TLR4 inhibitor TAM-242,and then treated by various concentration of rhS100A6 and diverse treatment time,then survival of macrophages was tested by CCK8.3.3 Influence of S100A6 on S100A6 expression in Macrophages.qPCR and Western blot were used to test the mRNA and protein levels of S100A6 in A6-Mφ.4 Effect of macrophages educated by S100A6(A6-Mφ)on proliferation of CRC cells.4.1 LoVo and HCT116 cells were co-cultrued with A6-Mφ for 24h:4.1.1 Typan blue staining,MTT,CCK8,crystal violet staining and FCM were used to detect the survival,proliferation and apoptosis incidence of CRC cells,respectively.4.1.2 Western blot was used to detect protein levels of proliferation related proteins,c-Myc and Proliferating Cell Nuclear Antigen(PCNA),and apoptosis related proteins,cleaved-caspase3 and cleaved-caspase8 inHCT116 cells.4.2 Mechanism that A6-Mφ affects on proliferation of CRC cells.4.2.1 qPCR and Western blot were used to detect the mRNA and protein levels of IL-6 in A6-Mφ.4.2.2 HCT116 cells were pretreated by IL-6R blocking peptide for30 min,and then co-cultured with A6-Mφ for 24h;survival,proliferation and apoptosis rate of HCT116 cells were tested by Typan blue staining,MTT,CCK8,crystal violet staining and FCM,respectively.4.2.3 HCT116 cells were co-cultured with A6-Mφ for 24 h,and then Western blot was used to test the levels of t-JAK2,p-JAK2,t-STAT3 and p-STAT3 in HCT116 cells.Results1.rhS100A6 and GST were successfully prepared.2 Effect of S100A6 on macrophages and its mechanisms2.1 S100A6 promoted polarization of macrophages to M2;one of the mechanisms was activating RAGE pathway.2.1.1 rhS100A6 downregulated M1 marker genes,iNOS and TNF-a(P<0.05),but upregulated M2 marker genes,CD206 and CD163(P<0.05),which indicated that S100A6 can promote macrophages polarization to M2.2.1.2 The macrophage polarization to M2 promoted by rhS100A6 was rescued by RAGE inhibitor FBS-ZM1(P<0.05),not TLR4 inhibitor TAM-242(P>0.05).2.2 S100A6 promoted macrophage proliferation,the mechanisms included RAGE and TLR4 pathway activation.rhS100A6 enhanced survival of macrophages(P<0.05),which could be rescued by RAGE inhibitor FBS-ZM1 or TLR4 inhibitor TAM-242(P<0.05).2.3 S100A6 upregulated S100A6 expression: the mRNA and proteinlevels of S100A6 in A6-Mφ were 1.6 times and 2.1 times as much as those in GST-Mφ,respectively(P<0.05).3 Macrophages educated by S100A6(A6-Mφ)promoted HCT116 cell proliferation,and one of the mechanisms was up-regulating IL-6 of macrophages and then activating IL-6/JAK2/STAT3 signaling pathway in HCT116 cells.3.1 A6-Mφ promoted and inhibited proliferation and apoptosis of CRC cells,respectively(P<0.05),upregulated the levels of c-Myc and PCNA(P<0.05),and downregulated the levels of cleaved-caspase3 and cleaved-caspase8 in HCT116 cells(P<0.05).All suggested that the macrophages educated by S100A6 can enhance the vitality of human CRC cells,and that the mechanisms that it promotes CRC cells proliferation include inhibiting cell apoptosis.3.2 The mRNA and protein levels of IL-6 in A6-Mφ were increased(P<0.05).A6-Mφ upregulated the levels of p-JAK2 and p-STAT3 in HCT116 cells(P<0.05),but had no significant effect on the expression of t-JAK 2 and t-STAT3(P>0.05).Meanwhile,the activity of A6-Mφ in promoting HCT116 cell proliferation and inhibiting apoptosis was partially reversed by IL-6R blocking peptide(P<0.05).It suggested that macrophages educated by S100A6 can promote CRC cell proliferation through the activating IL-6/JAK2/STAT3 signaling pathway.Conclusions1 S100A6 can educate macrophages in CRC mcrioenvironment,which includes promoting macrophage differentiation into M2 via binding to RAGE,improving macrophage viability through interaction with RAGE or TLR4,and upregulating the expression of S100A6 in macrophages.2 The macrophages educated by S100A6 can promote the proliferation of CRC cells through the activation of IL-6/JAK2/STAT3 signalingpathway.