| Backgrounds and objectivesColorectal carcinoma(CRC),a gastrointestinal malignant tumor of the colon or rectum,is the second most common tumor in women and the third most common tumor in men.It severely threatens human life.Although CRC-related deaths have been relatively reduced by using certain screening methods,there is a shortage of quicker and more effective diagnosis methods are lacking,and the five-year survival rate of patients with advanced and intermediate-stage CRC is still low.Therefore,in-depth study of the molecular mechanism of the occurrence and development of CRC and the search for more effective prevention and diagnosis methods have become important for global medicine and health.The etiology and pathogenesis of CRC are extremely complex,and the changes of gene and environment may affect its process.Advances in microbial detection technology and human microbiome research provide new perspectives on the relationship between pathogenesis and development of CRC.More and more reports have shown that the imbalance of intestinal flora is significantly related to a variety of intestinal diseases,including inflammatory bowel disease(IBD)and colorectal tumors such as CRC.In recent years,studies have found that Fusobacterium nucleatum(Fn),a Gram-negative anaerobic bacteria that is more common in oral inflammatory diseases,is closely related to the occurrence,metastasis,recurrence and survival of CRC.A study from North America suggests that Fn has great potential value in the diagnosis and prognosis of CRC patients,and can be used as a new risk factor for the development of disease from adenoma to cancer.However,the specific role and its molecular mechanism of Fn during the progress of CRC remains to be further explored.The inflammatory microenvironment of tumor is critical to its progress.Macrophage(Mφ),which is differentiated from monocyte infiltrating into tumor microenvironment(TME),is important immune cell,providing a key link between inflammation and tumor.Under the stimulation of various components in TME,Mcp further differentiates into pro-inflammatory and anti-tumor M1-Mφ or pro-tumor and anti-inflammatory M2-Mcp.It has been reported that M2-Mcp promotes the growth and metastasis of CRC and is significantly associated with a high recurrence rate in CRC patients.Fn can create an inflammatory microenvironment for Mcp infiltration by promoting the secretion of inflammatory factors in TME and then resulted in further progress of CRC.However,in the Fn-related CRC microenvironment(Fn-CRC-TME),the factors which are involved in Mcp polarization and the underlying molecular mechanisms have not been fully elucidated.The inflammatory molecule S100A9 belongs to the S100 family and is expressed in many cells,mainly in myeloid cells,so it is also called myeloid-related protein.S100A9 is known to play a key role in the inflammatory response and is highly expressed in a variety of tumors.In serum and tumor tissues of CRC patients,S100A9 levels were significantly increased and also is closely related to the clinical stage and distant metastasis of CRC.Our previous researches have found that S100A9 can not only directly promote the proliferation and migration of CRC cells,but also play a role in immune suppression by regulating myeloid-derived suppressor cells(MDSCs),which further promotes the development of CRC,suggesting that S100A9 is involved in regulating the CRC immune microenvironment and promoting disease progress.However,the specific mechanism remains to be elucidated.Based on above,Fn,Mcp and human CRC cells(HCT116 and SW480)were used to explore the effect and mechanism of Fn on Mcp polarization,the direct and indirect effects(via Mφ)of Fn on the proliferation and migration of CRC cells and their molecular mechanisms,and to explore whether S100A9 is involved in all these processes.The results will aim to clarify the complex interactions among Fn,S100A9,Mcp and CRC cells in Fn-CRC-TME,and their effects on CRC progression.The study also provides some new clues to improve CRC prevention,clinical diagnosis and treatment strategy.Methods1 The effect and mechanism of Fn on Mcp polarization1.1 Preparation and identification of recombinant proteinGST-human S100A9(rS100A9)and GST protein.1.2 The effect of Fn on Mcp polarization1)The markers of M1-Mφ detected were the mRNA of iNOS and TNF-α,and CD86 protein.The markers of M2-Mφ detected were the mRNA of IL-10 and CD206,and CD206 protein.They are commonly known as M1 and M2 markers;2)Mcp was treated with Fn and then the changes of M1 and M2 markers were detected by qPCR after 24 h and detected by Western blot,immunofluorescence test,and flow cytometry after 48 h;1.3 The role of S100A9 in Fn-mediated Mφ polarization1)qPCR,Western blot and enzyme-linked immunosorbent assay(ELISA)were used to detect S100A9 mRNA and protein levels in Mφ and CRC cells treated with Fn;2)Mcp was transfected with S100A9 small fragment interfering RNA(siS100A9)for 6h and then treated with Fn:The changes of M1 and M2 markers were detected by qPCR after 24 h and detected by Western blot,immunofluorescence test,and flow cytometry after 48 h;3)After CRC cells were transfected with siS 100A9 for 6 h and then treated with Fn for 48 h,The supernatant was collected as conditioned media[(CRC+siS100A9+Fn)-CM];qPCR and Western blot were used to detect the changes of M1 and M2 marker molecules after Mcp were treated with this CM;4)Mcp was treated with rS100A9 and then qPCR and Western blot were used to detect the changes of M1 and M2 markers;1.4 The molecular mechanism by which Fn can regulate S100A9 expression of Mcp and CRC cells and induce Mcp polarization to M2 in Fn-CRC-TME1)qPCR was used to detect TLR4 mRNA after Mcp and CRC cells were treated with Fn for 24 h;2)Western blot was used to detect p-p65 protein of Mcp and CRC cells,which were collected at different time points after cells were treated with Fn;3)Mcp and CRC cells were pretreated with TLR4 signal pathway inhibitor TAK-242 for 60 min and then treated with Fn for 2 h,and Western blot was used to detect p-p65 protein of these cells;4)Mcp and CRC cells were pretreated with TAK-242 or Bay 11-7082 for 60 min and then treated with Fn for 48 h,and Western blot and ELISA were used to detect S100A9 expression of these cells;5)Mcp was pretreated with TAK-242 or Bay 11-7082 for 60min and then treated with Fn for 24 h,and qPCR was used to detect the mRNA of M1 and M2 markers;6)After CRC cells were pretreated with TAK-242 or Bay 11-7082 for 60 min and then treated with Fn for 48 h,the supernatant was collected as CM[(Fn+TAK-242+CRC)-CM or(Fn+CRC+BAY 11-7082)-CM],respectively.Mcp was treated with the two diffenent CMs for 24 h and then qPCR was used to detect M1 and M2 markers;1.5 The relationships among the degree of Fn enrichment,Mcp phenotype and S100A9 expression in tumor tissue of CRC patients1)Fuorescence in situ hybridization(FISH)was used to detect and divide Fn(-)and Fn(+)specimens of CRC patients;2)IHC was used to detect the expression of Mcp marker CD68,M1 marker CD86,M2 marker CD206 and S100A9 in the Fn(-)or Fn(+)samples in order to determine the relationships between the degree of Fn enrichment,Mcp phenotype and S100A9;2.The direct and indirect effects of Fn on the progression of CRC and their mechanisms2.1 The direct effect of Fn on the proliferation and migration of CRC cells its mechanism1)In this study,CCK8 and Trans well were used to detect the proliferation and migration ability of CRC cells,respectively;2)After the CRC cells were treated with Fn,the cellular abilities of proliferation and migration were detected;3)After the CRC cells were transfected with siS100A9 for 6 h and then treated with Fn,the cellular abilities of proliferation and migration were detected;2.2 The indirect effects of Fn on CRC cell proliferation and migration through Mcp and their mechanisms1)The indirect effects of Fn:After Mcp were treated with Fn for 48 h,the supernatant was collected as CM[(Mcp+Fn)-CM].CRC cells were incubated with the CM,and then the cellular abilities of proliferation and migration were detected;2)The mechanism:(1)The influence of suppressing the polarization-promoting of Fn on this indirect effect:After CRC cells were transfected with siS100A9 for 6 h and then treated with Fn for 48 h,the supernatant was collected as CM[(Mcp+siS100A9+Fn)-CM].CRC cells were incubated with the CM,and then the cellular abilities of proliferation and migration were detected;(2)The effect of M2-Mcp on CRC:Mcp was treated with rS100A9 for 24 h to induce its polarization and then cultured for another 48 h with new medium.The supernatant was collected as CM[(Mcp+rS100A9)-CM].CRC cells were incubated with the CM,and then the cellular abilities of proliferation and migration were detected;The proliferation and migration abilities of CRC cells were detected after cells were incubated with CM above.2.3 Effect of Fn-treated Mcp on subcutaneous tumor growth in miceA mouse subcutaneous xenograft model was constructed by co-injecting Mcp and CRC into mice,verifying the indirect effect of Fn on CRC via Mcp.Mice were grouped according to the different treatments for Mcp,as follows:Control E.coli group,Fn group,(Fn+siNC)group and(Fn+siS100A9)group.The tumor growth was observed and its length and width were detected by the vernier caliper every three days.The tumor volume was calculated according to the following formula:volume=(width)2xlength/2.The mice were sacrificed after 21 days and their tumor tissues were taken out and weighed one by one.Part of the tissue was excised to extract total protein for Western blot analysis;the remaining were fixed with 4%formaldehyde buffer and embedded in paraffin,and then sectioned for IHC analysis.IHC and Western blot were used to detect the proliferating cell nuclear antigen(PCNA),EMT-related proteins E-cadherin and N-cadherin.Results1.The effect of Fn on Mcp polarization and its mechanism1.1 The rS100A9 and GST proteins were successfully prepared and identified.1.2 Fn promotes M2 polarization of McpThe changes of Mcp treated with Fn:1)The mRNA levels of the M1 marker genes iNOS and TNF-α were decreased(P<0.05 or 0.01),while M2 marker genes IL-10 and CD206 were increased(P<0.05 or 0.01);2)CD86 protein was decreased,while CD206 protein was increased.It suggested that Fn could promote Mcp to M2 polarization.We called it M2-Mcp.1.3 S100A9 mediated in the promotive effect of Fn on Mcp polarization in Fn-CRC-TME1)The levels of S100A9 mRNA and protein in Fn-treated Mcp and CRC cells were higher than those of their control groups(P<0.05 or 0.01),and the contents of S100A9 in their supernatants were also higher(P<0.05 or 0.01),suggesting that Fn could up-regulate the expression and secretion of S100A9 of Mcp and CRC cells in TME.2)S100A9 in Mcp mediated in the promotive effect of Fn on Mcp polarization.Experimental results:The mRNA of M1 marker genes iNOS,TNF-α and CD86 protein in(Fn+siS100A9)group were higher than those of their control groups(P<0.05),while the mRNA of M2 marker genes IL-10,CD206 and CD206 protein were lower(P<0.01 or 0.001),suggesting down-regulation of M1 markers and up-regulation of M2 markers induced by Fn were reversed after disturbing S100A9 expression of Mcp,that is,the M2 polarization of Mcp induced by Fn was suppressed.3)Incubating Mcp with(CRC+siS100A9+Fn)-CM inhibited the Fn-induced Mcp polarization,as shown:(1)Compared with the control group(CRC+siNC+Fn),S100A9 in the supernatant of(CRC+siS100A9+Fn)group was significantly decreased(P<0.01);(2)Incubating Mcp with the CM made from this supernatant,the mRNA of the Ml marker genes iNOS and TNF-α were higher than those of the control group(P<0.05),while M2 marker genes IL-10 and CD206 were lower(P<0.05 or 0.01);Consistent with the changes,CD86 protein was increased while CD206 protein was decreased,suggesting again that S100A9 can mediate Mφ polarization to M2 in Fn-CRC-TME.4)After Mcp were treated with rS100A9,the mRNA of M1 marker genes iNOS and TNF-α were decreased(both P<0.05)while M2 marker genes IL-10 and CD206 were increased(both P<0.05);Meanwhile,CD86 protein was decreased while CD206 protein was increased.It was further that S100A9 in the microenvironment can promote Mcp polarization to M2.The results in this section(1.3)consistently suggested that S100A9 plays a key role in Fn-induced Mcp polarization to M2 in Fn-CRC-TME.1.4 Fn up-regulated the expression and secretion of S100A9 of Mcp and CRC cells in TME through the TLR4/NF-κB signaling pathway,and thereby promoted Mcp polarization to M2.1)TLR4 mRNA in Fn-treated Mcp and CRC cells were higher than those of their control groups(P<0.01),and p-p65 protein were also increased in time-dependent manner;However,when Mcp and CRC cells were pretreated with TLR4 signaling pathway inhibitors TAK-242 for 60 min,the p-p65 up-regulation induced by Fn was inhibited.All results suggested that Fn can activate TLR4/NF-κB signaling pathway in Mφ and CRC cells.2)After Mcp and CRC cells were pretreated with TAK-242 or for 60 min,the S100A9 up-regulation induced by Fn was also significantly inhibited,suggesting that the mechanism of S100A9 up-regulation induced by Fn is involved in activation of TLR4/NF-κB signaling pathway in Mcp and CRC cells.3)After Mcp was pre-treated with TAK-242 or Bay 11-7082 for 60 min,Fn-induced M2 formation was significantly inhibited.The mRNA of M1 marker genes iNOS and TNF-α of(Fn+TAK-242)and(Fn+Bay 11-7082)group were higher than those of the control group(P<0.05),while the M2 marker genes IL-10 and CD206 were significantly lower(P<0.05 or 0.01).The results above suggested that Fn could promote Mcp polarization to M2 and its mechanism is involved the activation of the TLR4/NF-κB/S100A9 signaling pathway in Mφ.4)CRC cells were pretreated with TAK-242 or Bay 11-7082 for 60 min and treated with Fn,and then(Fn+TAK-242+CRC)-CM or(Fn+CRC+BAY 11-7082)-CM were prepared.Mcp was incubated with the two different CMs,and then the down-regulation of M1 marker genes iNOS and TNF-α(P<0.05 or 0.01)and the up-regulation of M2 marker genes IL-10 and CD206 were significantly suppressed(P<0.05 or 0.01),suggesting that the activation of the TLR4/NF-κB/S100A9 signaling pathway in CRC cells is one of the mechanisms by which Fn induces Mcp polarization to M2.The results in this section(1.4)suggested that S100A9 from CRC and Mcp was a key factor for Fn-induced Mcp polarization to M2 in Fn-CRC-TME.TLR4/NF-κB signaling pathway is involved in Fn-induced up-regulation of S100A9.1.5 The degree of Fn enrichment in tumor tissue of CRC patients was positively correlated with M2-Mcp phenotype and S100A9The number of Fn in each field in the case of<5,5～20 and>20 was defined as negative,weak,and positive,respectively.Compared with Fn(-)tissue specimens,S100A9 was higher and there was more infiltration of Mcp(mostly M2-Mcp)in Fn(+)specimens,suggesting that the degree of Fn enrichment was positively correlated with M2-Mcp phenotype and S100A9 level in CRC.Those results were consistent with the previous experiment.Comprehensive analysis showed that Fn can promote the expression and secretion of S100A9 of Mcp and CRC cells by activating the TLR4/NF-κB signaling pathway,therefor increasing S100A9 level in the microenvironment;The increased S100A9 in Fn-CRC-TME can take part in mediating the promotive effect of Fn on Mcp polarization to M2.2.The direct and indirect effects of Fn on CRC cells and their mechanismsThe proliferation capacity at 24 h and 48 h and migration capacity at 24 h of HCT116 and SW480 cells treated with Fn were significantly increased(P<0.001 or 0.01);However,the cellular abilities of proliferation and migration induced by Fn were significantly inhibited after S100A9 expression of CRC cells was reduced(all P<0.001),suggesting that S100A9 is involved in the indirectly promotive role of Fn on the proliferation and migration of CRC cells.2.2 Fn promoted the proliferation and migration of CRC cells and tumor growth through Mcp,and the mechanism was involved the formation of M2-Mcp1)Compared with the control group,the proliferation ability of HCT116 and SW480 cells in the(Mcp+Fn)-CM group was significantly enhanced at 24 h and 48 h(both P<0.001),while the proliferation capacity of both cells at the two time points was significantly lower in(Mcp+siS100A9+Fn)-CM group than that in the(Mcp+siNC+Fn)-CM control group(P<0.05,0.01,or 0.001).At the same time,the volume and weight of subcutaneous tumors in the Fn group were significantly larger than those in the Control E.coli control group(P<0.001)and PCNA proteinwas also higher.And the volume and weight in the(Fn+siS100A9)group were significantly smaller than those in the(Fn+siNC)control group(P<0.001),and PCNA protein was also significantly lower.The above results in vitro and in vivo were consistent,all suggesting that Fn could indirectly promote CRC cells proliferation through Fn-induced Mcp polarization to M2.2)Compared with the control group,the migration ability of HCT116 and SW480 cells at 24 h was significantly enhanced in the(Mφ+Fn)-CM group(all P<0.001).And the migration ability of cells in the(Mφ+siS100A9+Fn)-CM group was significantly lower than that in(Mcp+siNC+Fn)-CM control group(all P<0.001).At the same time,We found that EMT-related protein E-cadherin in the Fn group was lower than that in the control group,while N-cadherin was higher;However,the protein levels above were significantly reversed in the(Fn+siS100A9)group.In summary,the results in vitro and in vivo suggested that Fn could indirectly promote CRC cells migration and EMT through Fn-induced Mcp polarization to M2.3)M2-Mcp promoted the proliferation and migration of CRC cells:rS100A9 was used to induce Mcp polarization to M2.The proliferation and migration ability of HCT116 and SW480 cells in the(Mcp+rS100A9)-CM group were higher than those in the(Mφ+GST)-CM control group(all P<0.001).Combining the results of the first part,Fn promoted Mcp polarization to M2,the results of this part(2.2)proved that Fn can promote CRC cell proliferation and migration through Mcp,and the mechanism is involved the induction and formation of M2-Mφ.Conclusiond1.Fn promotes S100A9 expression of Mcp and CRC cells in Fn-CRC-TME,the mechanism involves the activation of TLR4/NF-κB signaling pathway.2.Fn promotes Mcp polarization to M2,its mechanism involves the activation of TLR4/NF-κB/S100A9 signaling pathway.3.Fn can directly promote the proliferation and migration of CRC cells,the mechanism is related to Fn-upregulated S100A9 expression of CRC cells.4.Fn has the effect of indirectly promotive effect on the cell proliferation,migration,and tumor growth of CRC via Mcp,the mechanism involves the formation of M2-Mφ. |
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