S100A6 In Microenvirenment Directly And Indirectly Affects Human Colorectal Carcinoma Cells And Their Mechinisms Research

Backgrounds and objectivesColorectal carcinoma(CRC)is one of the three most common malignancies in the world.It has strong invasiveness and rapid development.In particular,as people’s lifestyle changes,CRC’s incidence rate has been increasing year by year,and young people’s illness accounts for 2%-8% of the entire CRC.The study of the mechanism of CRC progression can provide new ideas for clinical treatment.The S100 protein family is closely related to the occurrence and development of tumors.Some S100 proteins can directly affect process of tumor,and can also affect development of tumor indirectly through the tumor microenvironment.For example,S100 B and S100A7 can directly promote tumor progression and indirectly promote tumor development by promoting the infiltration of tumor associated macrophages(TAMs).Our previous study found that S100A6 has a direct role in promoting the development of CRC,and its mechanism remains to be further elucidated.At the same time,whether S100A6 has the TAMs-mediated indirect effect on CRC has not yet been reported.Phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway regulates many biological processes such as tumor growth and metastasis.Our previous study found that S100A6 can promote the activation of PI3K/Akt signaling pathway in cervical cancer and osteosarcoma cells.In addition,this signaling pathway is also involved in the inflammatory process,up-regulating the expression of M-CSF,and M-CSF can recruit and induce macrophage polarization into M2 type.This study is to explore the direct role and mechanism of S100A6 in CRC cells and the indirect effect of S100A6 on CRC cells via TAMs and its mechanism.We hope to accumulate experimental evidence for clarifying the role and mechanism of S100A6 in the development of CRC and provide new ideas for clinical treatment.Methods1.Preparation and identification of recombinant protein GST-human S100A6(GST-hS100A6): GST-hS100A6 and GST were prepared by prokaryotic expression and identified by SDS-PAGE,Coomassie blue staining and Western blot,and stored at-80°C.2.The direct effect of GST-hS100A6 on human CRC cell lines LoVo and SW480 and its mechanism research2-1 After treating LoVo and SW480 cells by GST-h S100A6(30 μg/ml):1)MTT assay and scratch healing assay were used to detect cells proliferation and migration,respectively.2)The Akt and p-Akt in cells were detected by Western blot.2-2 LoVo and SW480 cells were treated with Inhibitors of PI3K/Akt signaling pathway LY294002 and GST-hS100A6 alone or in combination for 24 hours.Wound healing assay was used to examine the cell migration ability.3.Indirect effect of GST-hS100A6 on human CRC cell lines LoVo and SW480 via TAMs and its mechanism research3-1 Preparation of macrophages: After treating THP-1 cells with phorbol 12-myristate 13-acetate(PMA)for 24 h,the cells stopped proliferating;suspension state of most cells was turned into adherence state,and extended pseudopods.It means that the cells have been differentiated into macrophages and for subsequent experiments.3-2 Preparation of conditioned medium: 1)preparation of CM-A6-CRC: human CRC cells were treated with GST-hS100A6 and the culture supernatant was collected by centrifugation and stored,this was the conditional medium(CM-A6-CRC);1)preparation of CM-A6-CRC-Mφ: macrophages were treated with CM-A6-CRC and the culture supernatant was collected by centrifugation and stored.This was CM-A6-CRC-Mφ.3-3 MTT assay was used to detect proliferation of LoVo cells treated with CM-A6-CRC-Mφ.3-4 THP-1 cells were seeded into upper of Transwell chamber,induced for 24 h by PMA,then treated with CM-A6-CRC for 48 h.After that,these cells were co-cultured with LoVo and SW480 cells at scratches of 0 h,respectively.The migration ability of CRC cells was examined.3-5 The mechanism of S100A6 inducing migration of LoVo and SW480 cells via TAMs1)Effect of CM-A6-CRC on macrophage polarization: after macrophages were treated with CM-A6-CRC for 48 h,q RT-PCR was used to detect the expression of M2 marker CD206,M2 related molecule IL-10 and M1 phenotype marker iNOS in the macrophages.2)qRT-PCR was used to detect the expression of M-CSF and CCL2,which can promote macrophage polarization into M2 phenotype,in LoVo and SW480 cells treated with GST-hS100A6 for 48 h.3)To investigate the mechanism of S100A6 promoting the up-regulation of M-CSF expression in CRC cells: Lo Vo cells were treated with LY294002 and GST-hS100A6 alone or in combination for 48 h.qRT-PCR and Western blot were used to detect the expression of M-CSF in CRC cells,respectively.Results1.GST-hS100A6 and GST were successfully prepared.2.Direct effect and mechanism of S100A6 on human CRC cell lines LoVo and SW4802-1 GST-hS100A6 had no significant effect on the proliferation of LoVo and SW480 cells(P>0.05).2-2 GST-h S100A6 promoted the migration of LoVo and SW480 cells(P<0.01).2-3 PI3K/Akt signaling pathway was involved in S100A6-promoted migration of human CRC cells(P<0.01).3.Indirect effects and mechanisms of S100A6 via TAMs on human CRC cells3-1 No indirect effect of GST-h S100A6 on the proliferation of LoVo cells via TAMs was found(P>0.05).3-2 GST-hS100A6 had an indirect effect on the migration of LoVo and SW480 cells via TAMs(P<0.05).3-3 CM-A6-CRC up-regulated the expression of M2-type marker CD206 and M2-type macrophage-associated factor IL-10(P<0.05),while the expression of M1-type marker iNOS had no significant changes in macrophages(P>0.05).3-4 The expression of M-CSF and CCL2 were up-regulated in Lo Vo and SW480 cells treated with GST-hS100A6(P<0.05).,3-5 The expression of M-CSF of LoVo cells in GST-hS100A6 and LY294002 combination group was significantly lower than that of in GST-hS100A6 alone group(P<0.05).The above results suggest that S100A6 can indirectly promote the migration of CRC cells via TAMs.The mechanism may include that S100A6 up-regulates the expression of M-CSF and CCL2 in CRC cells and the latter two factors induce macrophages to M2 phenotype.At the same time,PI3K/Akt signaling pathway is involved in mediating S100A6 up-regulation of M-CSF expression in CRC cellsConclusion1.S100A6 promotes the migration of human CRC cells and the activation of the PI3K/Akt signaling pathway is involved in mediating this procession.2.S100A6 in the CRC microenvironment can indirectly promote the migration of CRC cells via TAMs,and its mechanism may be related to S100A6 up-regulating the expression of M-CSF and CCL2 in CRC cells and the polarization of macrophages to M2 type inducing by M-CSF and CCL2.3.Activation of PI3K/Akt signaling pathway is involved in the M-CSF up-regulation by S100A6 in CRC cells.