| Objectives Colorectal cancer(CRC) is one of the most malignant gastrointestinal carcinomas worldwide and ranks one of the eight common cancers in China,and the incidence rate of CRC increases 4.2% every year which higher than the global average. With the progression of colorectal cancer, cancer tissue can gradually break through intestinal, and invade adjacent tissues, such as enterocoelia and ovary, even be transferred to the remote argan(liver and lung). It indicates that CRC has became a major health problem in our country nowadays. The mechanisms of CRC to improve the level of early diagnosis and the prevention of CRC have became the important scientific issues for health care system in China.Chronic inflammation and carcinogens are two important carcinogenic agents. In 2011, two famous Cancer scholar, Hanahan and Weinberg,recruited "inflammation" as one of the ten characteristics of cancer. Epidemiological and molecular biological researches indicate that inflammation is closely tied to the development of CRC, namely, CRC belongs to inflammation-related tumors. Although the relationship between inflammation and CRC is definited, its specific process and molecular mechanisms are still limited. It’s necessary to perform in-depth interpretation of the relationship between them.After the concept of tumor microenvironment(TME) was raised, it opened a new view to clarify the relationship between inflammation and cancer. Therefore, macrophage is an important component of the tumor microenvironment. Affected by the tumor microenvironment, macrophages is named as tumor-associated macrophages(TAMs) for the transformation in phenotype and function and influenced the development of tumors.Moreover, studies have shown that the number and density of infiltrated macrophages are tightly related to ulcerative colitis(UC) and CRC. The apical surface of the intestinal epithelium is exposed to lipopolysaccharide(LPS) from the lumen because of bacterial translocation and intestinal permeability changes. Therefore, LPS is often used to mimick an CRC inflammatory microenvironment for the study of the relationship between the tumor microenvironment macrophages and CRC.S100A8(Mrp8, Calgranulin A), an important inflammatory molecule,is one of the S100 protein family. Gene Ontology analysis revealed that S100A8 is involved in many biological process, such as acute and chronic inflammation, chemotaxis and LPS-induced reactions. It is reported that increased level of S100A8 is measured in virous tumor cells and infiltrated inflammatory cells in peritumoral regions, which is correlated with tumor stage, differentiation, angiogenesis and prognosis. Furthermore, S100A8 is not only involved in inflammatory disease processes by directly stimulating the inflammatory cells under certain conditions to release inflammatory factors, but also by playing an important role in chronic arthritis by upregulating Fcγ receptor of macrophages through the activation of TLR4.Studies have shown that TLR4 is the key receptor for the regulation of macrophages by S100A8, and the NF-κB signaling pathway is an important signaling cascade in the downstream of the TLR4 pathway, which plays an important role in the inflammatory response.MiR-155, a typical multi-functional miRNA, is involved in a variety of pathophysiological processes such as inflammation, immune response and tumor. Abnormal expression of mi R-155 is correlated with activation of the NF-κB signaling pathway. Overexpression of mi R-155 boosts the immune cells to secrete a variety of inflammatory cytokines, amplifies the inflammation effects, increases the tissue damage, precancerous lesions and tumor formation.As an important member of inflammatory microenvironment,whether S100A8 is involved in the regulation of macrophages in the inflammatory microenvironment? mechanism? What the effect of S100A8 on the progress of CRC through regulating macrophages in the inflammatory microenvironment is?Based on this, in the study, we mimicked the CRC inflammatory microenvironment by LPS and used macrophages in the inflammatory microenvironment as subject(in this paper, we named them "the inflammatory tumor associated macrophages", i TAMs) to investigate the role of S100A8 in the regulation of the i TAMs and the effects and the underlying molecular mechanism of S100A8 on proliferation and migration of CRC cells. The aim is to elucidate the role of S100A8 in the CRC process and accumulate experimental evidence for promoting the development of CRC by inflammation, and ultimately offering new strategies and new intervening targets for the prevention and treatment for CRC.Method1 Effect and molecular mechanism of S100A8 on regulation of the i TAMs1.1 Preparation of recombinant GST-S100A8 and control GST proteinThe recombinant plasmid p GST-moluc-S100A8 and p GST-moluc were transformed into competent BL21 cells, isopropyl-β-Dthiogalactoside(IPTG) was added to induce the expression of recombinant proteins. After the bacteria were sonicated, the recombinant proteins were isolated and purified by glutathione-Sepharose 4B beads. At last, the recombinant proteins were filtered with a bacteria filter and stored at-80℃.1.2 Differentiation of THP-1 cells to macrophages by PMA Human monocyte/macrophage(THP-1) cells were cultured in RPMI-1640 medium, and induced by different concentrations of PMA and differentiated to macrophages. Optimal induction concentration was selected according to cell morphology and viability.After the differentiated THP-1 macrophages were treated with LPS,the cells were defined as the inflammatory tumor associated macrophages(i TAMs).1.3 Effect of LPS and recombinant protein GST-S100A8 on the viability of macrophages by MTT assayTHP-1 cells were cultured in complete RPMI-1640 medium, adjusted to 4×104/ml and seeded in 96-well plates 100 μl/well with appropriate concentration of PMA. After the cell were incubated for 24 h, old medium was removed and the cells were washed with PBS three times to eliminate PMA. The cells were treated with different concentrations of LPS(0-400ng/ml) and GST-S100A8(0-40 μg/ml) and incubated in a controlled atmosphere of 5% CO2 and 37℃. It is recorded as 0 h and conducted an MTT assay for the following five days for selection of optimum concentration of intervention.1.4 Effect of S100A8 on regulation of the iTAMs1) Detection of mi R-155 expression in the i TAMs after S100A8 treatment by real-time PCRAfter the i TAMs were treated with GST-h S100A8, the cells were collected and the total RNA was extracted. Then reverse transcription was performed with specific primers and mi R-155 expression was detected by real-time PCR.2) Detection of the expression of inflammatory cytokines IL-1β and TNF-αin the i TAMs after S100A8 treatment by RT-PCRAfter the i TAMs were treated with GST-h S100A8 for 6-48 h, the cells were collected and the total RNA was extracted. Then reverse transcription was performed with random primers and m RNA expression of IL-1β and TNF-α was detected by RT-PCR.After the iTAMs were treated with GST-hS100A8 for 24 h, the supernatant was collected, centrifuged and the secretion of IL-1β and TNF-α was measured by ELISA according to the manufacturer’s instructions.1.5 Molecular mechanisms of S100A8 on regulation of the iTAMs1) Detection of TLR4 expression and NF-κB nuclear translocation in the i TAMs after S100A8 treatment by immunofluorescence stainingThe i TAMs were seeded on sterile cell coverslips in 24-well culture plates and treated without and with GST and GST-S100A8 in serum-free RPMI-1640 medim for indicating time. Then TLR4 expression and NF-κB nuclear translocation were measured by immunofluorescence staining.2) Detection of NF-κB nuclear translocation in the i TAMs after S100A8 treatment by western bolt analysisAfter the i TAMs were treated without and with GST and GST-h S100A8 for the indicated time, or pretreated with the NF-κB signaling pathway inhibitor(Bay 11-7082) for 30 min prior to the treatment of GST-h S100A8 for 1 h, total protein and nucleoprotein of the i TAMs were extracted and NF-κB nuclear translocation was measured by western bolt analysis.3) Detection of activation of NF-κB in the i TAMs after S100A8 treatmentSignaling pathway plasmid p NF-κB-luc was transfected into the i TAMs. After pretreatment of the i TAMs with Bay 11-7082 for 30 min, the cells were treated with GST-h S100 A for 24 h, luciferase activity was detected.4) Relationship among the NF-κB signaling pathway, miR-155 and inflammatory cytokines IL-1β and TNF-α in the i TAMs after S100A8treatment(1) Whether S100A8-induced upregulation of mi R-155 is involved in activation of the NF-κB signaling pathway:After pretreatment of the iTAMs with Bay 11-7082 for 30 min, the cells were treated with GST-h S100 A for 24 h, the expression of mi R-155 was measured by real-time PCR.(2) Whether S100A8-induced upregulation of L-1β and TNF-α is involved in activation of the NF-κB signaling pathway:After pretreatment of Bay 11-7082 in the i TAMs for 30 min, the cells were treated with GST-h S100A8 for 24 h, the m RNA expression and secretion of IL-1β and TNF-α were measured by RT-PCR and ELISA.(3) Whether S100A8-induced upregulation of mi R-155 is involved in upregulation of mi R-155:After transfected mi R-155 inhibitor and mi R-155 NC into the i TAMs,the cells were treated with GST-h S100 A for 24 h, the expression of mi R-155 was measured by real-time PCR, the m RNA expression and the secretion of IL-1β and TNF-α were measured by RT-PCR and ELISA.2 Effect and molecular mechanisms of S100A8 on the viability and migration of CRC cells through the regulation of the i TAMs2.1 Preparation of the i TAMs conditioned medium(i TAMs-CM):The i TAMs in 100 mm dishes were treated without and with GST and GST-h S100A8 for 24 h, the medium was collected, filtered and used as the i TAMs-CM(stored at 4 ℃ for 2 weeks).2.2 Effect of S100A8 on the viability of CRC cells through regulating the i TAMs(1)Detection of the proliferation of CRC cells after treatment of the i TAMs-CM by MTT assay.(2)The iTAMs were co-cultured with CRC cells and treated with GST-h S100A8 for 24 h, cell cycle of CRC cells was detected by flow cytometric analysis.(3)The i TAMs were co-cultured with CRC cells and treated with GST-h S100A8 for 24 h, cell apoptosis of CRC cells was detected by Hoechest staining assay.2.3 Effect of S100A8 on the migration of CRC cells through the regulation of the i TAMsCRC cells were seeded into 6-well culture plates or 24-well Transwell cell culture chambers and co-cultured with CRC cells. After pretreatment of Bay 11-7082 in the i TAMs for 30 min, the cells were treated with GST-h S100A8 for indicated time, the migration of CRC cells was detected by wound healing assay and Transwell migration assay.Results1 Effect and molecular mechanism of S100A8 on regulation of the i TAMs1.1 Identification of recombinant proteins GST-S100A8 and control GSTPrepared recombinant proteins GST and GST-S100A8 were identified by SDS-PAGE, Coomassie Brilliant Blue staining and western blot analysis.Their purities were confirmed to be >90% by Quantity One software.1.2 Differentiation of THP-1 cells to macrophages by PMAAfter treatment of 0, 25, 50, 100, 200 and 400 ng/ml PMA in THP-1cells for 24 h, we found that the cells were characterized by reduced proliferation, increased adherence and extended pseudopodia. The effect of induction after treatment of 50 ng/ml PMA was more significant compared to others. Therefore, 50 ng/ml of PMA was selected as an inducer in subsequent experiments.1.3 Effect of LPS and S100A8 on viability of macrophagesAfter macrophages were treated with 0, 50, 100, 200 and 400 ng/ml of LPS, there was no significant effect on the cell viability compared to each group(p>0.05). Therefore, 100 ng/ml of LPS was used to treat macrophages and defined as the inflammatory tumor associated macrophages(i TAMs) for subsequent experiments.After macrophages were treated with 0, 5, 10, 20 and 40 μg/ml of GST-h S100A8, there was no significant difference on the cell viability compared with each group(p>0.05).1.4 Effect of S100A8 on regulation of the i TAMs1) S100A8 promotes the expression of mi R-155 in the i TAMsCompared to the control group, GST-h S100A8 significantly promoted the expression of mi R-155 in the i TAMs(P<0.05). After treatment of 10μg/ml GST-h S100A8 in the i TAMs, the expression of mi R-155 was more significantly increased than other concentration groups. Therefore, 10μg/ml of GST-h S100A8 was selected as the treatment in subsequent experiments.In addition, after the i TAMs were treated with GST-h S100A8 for 6, 12, 24 and 48 h, we found that the expression of mi R-155 were increased with time(P<0.05). It should be noted that S100A8 upregulated the expression of mi R-155 in a time-dependent manner.2) S100A8 facilitated the mRNA expression of IL-1β and TNF-α in the i TAMsThe expression of IL-1β and TNF-α m RNA in the i TAMs was increased more significantly than the control group(P<0.05) after treatment of GST-S100A8, and reaching a peak value at 24 h.3) S100A8 facilitated the secretion of IL-1β and TNF-α in the supernatant of the i TAMsCompared to control group, the secretion of IL-1β and TNF-α in the supernatant of the i TAMs were significantly increased after treatment of GST-S100A8(P<0.01).1.5 Molecular mechanisms of S100A8 on the regulation of the iTAMs1) S100A8 promoted the expression of TLR4 in the i TAMsThe expression of TLR4 was more obvious in macrophages treated with LPS or GST-S100A8. After the i TAMs were treated with GST-S100A8, the expression of TLR4 was more significantly increased(P<0.05). It is indicated that S100A8 promoted the expression of TLR4 in the i TAMs.2) S100A8 promoted nuclear translocation of NF-κB in the i TAMsAfter the treatment of GST-S100A8 in the i TAMs, nuclear translocation of p-NF-κB p65 was significantly promoted than other groups(P<0.05) and reached a peak value 1 h after treatment(P<0.05).In line with the results of immunofluorescence staining, the results of western blot showed that after the treatment of GST-S100A8 in the i TAMs for 1 h, the level of p-NF-κB p65 in nucleus was much higher than other groups(P<0.05), but the total p-NF-κB p65 wasn’t significantly altered(P>0.05). In addition, after treatment of GST-S100A8 in the i TAMs for different times, the levels of p-NF-κB p65 in nucleus were all increased,but it wasn’t in a time-dependent manner and the total p-NF-κB p65 wasn’t significantly changed(P>0.05). After the i TAMs pretreated with Bay11-7082 for 30 min, the S100A8-induced upregulation of p-NF-κB p65 in nucleus was partially inhibited(P<0.05).Furthermore, luciferase activity assay was used to analyze the activation of the NF-κB pathway. After the treatment of GST-S100A8 in the i TAMs for 24 h, the luciferase activity of NF-κB in the supernatant was much higher than other groups(P<0.05), but after the i TAMs were pretreated with Bay 11-7082 for 30 min, the luciferase activity was significantly inhibited(P<0.05). These findings indicated that GST-S100A8 increased activity of NF-κB transcription factor in the i TAMs.3) S100A8 upregulated the expression of mi R-155 in the i TAMs through the activation of the NF-κB pathwayAfter the iTAMs were pretreated with Bay 11-7082 for 30 min, the S100A8-induced upregulation of the expression of mi R-155 in the i TAMs was also significantly suppressed(P<0.05). It’s indicated that S100A8 upregulates the expression of mi R-155 in the i TAMs through activating the NF-κB pathway.4) S100A8-induced upregulation of mi R-155 enhanced the expression of inflammatory cytokines IL-1β and TNF-αAfter the i TAMs were transfected with mi R-155 inhibitor,S100A8-induced upregulation of the expression of mi R-155 in the i TAMs was conspicuously inhibited(P<0.05).To determine whether mi R-155 expression is involved in the secretion of inflammatory cytokines in the i TAMs, we detected the m RNA of IL-1βand TNF-α and their protein levels in the supernatant by RT-PCR and ELISA respectively. The results showed that the levels of IL-1β and TNF-αm RNA and the secretion of IL-1β and TNF-α in the supernatant were markedly reduced after transfected with mi R-155 inhibitor or pretreated with Bay 11-7082 for 30 min in the S100A8-treated i TAMs(P<0.05).2 Effect and Molecular mechanisms of S100A8 on the viability and migration of CRC cells through regulation of the i TAMs2.1 Effect of S100A8 on the viability of CRC cells through regulation of the i TAMs1) Effect of S100A8 on the proliferation of CRC cells through regulating the i TAMsCompared to other groups, the proliferation of HCT116 and SW480 cells after treated with the S100A8-treated i TAMs-CM for 24 h wasn’t significantly changed(P>0.05).2) Effect of S100A8 on the cell cycle of CRC cells through regulation of the i TAMsThere was no significant difference in the cell cycle of CRC cells which were co-cultured with the S100A8-treated i TAMs for 24 h Compared to other groups(P>0.05).3) Effect of S100A8 on the apoptosis of CRC cells through regulation of the i TAMsCompared to other groups, there was no significant difference in cell apoptosis of CRC cells which were co-cultured with the S100A8-treated i TAMs for 24 h(P>0.05).2.2 Effect and Molecular mechanisms of S100A8 on the migration of CRC cells through regulation of the i TAMsA wound healing assay was used to detect the migration of CRC cells co-cultured with the i TAMs after different treatments. There was a significant increase in wound closure rate after co-cultured with the i TAMs which treated with GST-S100A8 compared to that of the other groups(P<0.05), and the effect could be impaired by Bay 11-7082(P<0.05).The effect of cell migration was further confirmed by Transwell migration assay, which showed that the number of trans-membrane migrated CRC cells co-cultured with the i TAMs after the treatment of GST-S100A8 was markedly increased compared to that of the other groups(P<0.05), but the effect could be impaired by Bay 11-7082(P<0.05).These results suggested that S100A8 facilitated the migration of CRC cells co-cultured with the i TAMs through the activation of the NF-κB signaling pathway.Conclusion(1) S100A8 could significantly promote the expression of mi R-155 and inflammatory cytokines IL-1β and TNF-α in the i TAMs.(2) Activation of TLR4/NF-κB/miR-155 signaling pathways was involved in the cellular effects of the i TAMs mediated by S100A8.(3) S100A8 could significantly facilitate the migration but not the viability of colorectal cancer cells through the regulation of the i TAMs. |
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