Macrophage Migration Inhibitory Factor Promotes Tumour Growth And Lung Metastasis Through Activating RAS/MAPK Pathway In Human Osteosarcoma

Osteosarcoma(OS)is the most common type of primary bone cancer diagnosed in children and adolescents.The incidence of osteosarcoma counts for little part of all cancers,but 20% of patients present detectable metastases after first clinical diagnosis.OS a leading cause of cancer death in adolescents.Emerging evidence suggests that the tumor microenvironment plays a critical role in driving cancer development.As a pleiotropic cytokine,macrophage migration inhibitory factor(MIF)is confirmed to be overexpressed in many types of cancer and is demonstrated to promote cell proliferation,angiogenesis and metastasis.Highly expressed MIF in cancer is an additional predictor for poor patient outcome.However,the mechanism of MIF in osteosarcoma remains unclear.In this study,the level of MIF was proved to be significantly increased in the tissues and serum samples of OS patients and the MIF expression was found to be associated with tumour size,pulmonary metastasis and the survival rate of OS patients.A positive correlation between the levels of MIF and p-ERK1/2 was found in OS patient tissues.Furthermore,MIF could activate RAS/MAPK pathway by a time-and dose-dependent way in 143 B cells and MNNG/HOS cells,thereby promoting the cell proliferation and migration.We established shRNA-MIF-143 B and shRNA-MIF-MNNG/HOS cell lines by MIF-shRNA lentivirus.CCK-8 kit,flow cytometry,transwell and qRT-PCR were performaned to find that cell proliferation,migration,anti-apoptosis are inhibited by MIF-shRNA.And then the down-regulation of MIF could significantly inhibit the growth and the lung metastasis of osteosarcoma in mouse xenograft and orthotopic models.Our findings demonstrated that MIF could be an important cytokine during OS progression and the activation of RAS/MAPK pathway triggered by MIF could be a possible mechanism for OS tumor growth and lung metastasis.MIF may represent a promising target for osteosarcoma diagnosis and therapy.Part ?: Study of expression and significance of MIF in tissues and serum of patients with osteosarcomaObjective: To study the expression of MIF in tumor tissues and serum of osteosarcoma patients,and to explore the relationship between the expression of MIF and the tumor size,lung metastasis and the survival rate of osteosarcoma patients.Methods: Osteosarcoma(OS)tissues and paired normal adjacent tissues(NAT)were obtained from OS patients who underwent radical resection at Jinling hospital(Nanjing,P.R.China)from 2008 to 2012.Surgically removed tissues were quickly frozen in liquid nitrogen until analysis.Serum samples were obtained from a subset of 35 patients at the time of diagnosis.Thirty-five volunteers showing no evidence of disease were selected as the healthy controls.All protocols concerning the use of patient samples in this study were approved by the Medical Ethics Committee of the affiliated Jinling hospital of Nanjing University(Nanjing,China).A signed informed consent form was obtained from all subjects.To investigate the relevance of MIF expression with survival situation,we followed up the 60 OS patients for three years since diagnosis.Results: Sixty paired OS and NAT tissues were preliminary examined by HE staining,IHC staining was then performed using specific anti-MIF antibody to detect the level of MIF in tissues of 60 patients.We found that 38 cases(63.3%)showed the high expression of MIF(with more than 20% MIF-positive cells)and 22 cases(36.7%)showed the low expression of MIF(with less than 20% MIF-positive cells).Additionally,8 paired tissues were randomly selected from 38 cases with high expression of MIF and were detected by western blot.The results indicated the significant increase of MIF in the OS tissues compared with the normal adjacent tissues,which was in accordance with the results of IHC staining.the MIF expression was associated with tumour size(p=0.009)and pulmonary metastasis(p=0.008),but not gender,age,location,histologic subtype and enneking stage analyzed by ?2 test.The serum MIF level of 1.524�0.482 ng/ml in OS patients were significantly higher than 1.146�0.274 ng/ml in healthy people(p<0.01).the results of ?2 test showed that the serum MIF level were associated with the clinicopathological parameters of enneking stage(p=0.014),tumour size(p=0.003)and pulmonary metastasis(p=0.001).we found that the OS patients with high MIF expression had a lower overall survival rate(68.42%)than those with low MIF expression(95.45%;p=0.014),which was analyzed by the Kaplan-Meier method.Conclusions: The up regulation of MIF occurs frequently in OS tissue and serum samples,and may be associated with tumor size and lung metastasis in patients with OS.Part ?: MIF promotes tumour growth and lung metastasis through activating RAS/MAPK pathway in human osteosarcomaObjective: Detecting the ERK1 / 2 phosphorylation level and the changes of RAS /MAPK pathways downstream cell proliferation markers(KI-67 and PC 200 NA)andmetastasis related genes(MMP-9,MMP-13 and VEGF)through the stimulation of MIF osteosarcoma cells to investigate the mechanism of MIF promoting the growth and metastasis of osteosarcoma.Methods: MNNG/HOS and 143 B cells(2�105)were seeded in 6-well plates and cultured in MEM medium with 10% FBS for 12 hours.Cells were washed with PBS and changed with serum-free MEM for 12 h and then incubated with various concentrations(0,50,100 ng/mL)of rMIF for different times(0 min,10 min,30 min,2 h,6 h).After that,cells were harvested for western blot analysis of total-ERK1/2 and p-ERK1/2.Additionally,cells were incubated with rMIF at the concentration of 100 ng/m L for 2 h in presence or absence of 10 mM U0126(MEK1/2 inhibitor,Sigma-Aldrich)for the following protein and RNA analysis.Results: MIF induced the phosphorylation of ERK1/2 in both time-and dose-dependent fashions.After MIF stimulation,the enhanced p-ERK1/2 was detected as early as 30 min at the concentration of 100 ng/mL and the most significant phosphorylation of ERK1/2 was observed in 143 B and MNNG/HOS cells after MIF treatment for 2 h.Therefore,the treatment condition of 100 ng/mL for 2 h was used in the following assays.The expressions of p-SRC,total SRC,RAS-GTP,total RAS,p-MEK1/2,total MEK1/2,p-ERK1/2 and total ERK1/2 were detected using western blot assay to study the possible mechanism of ERK1/2 activated by MIF.The levels of total SRC,RAS,MEK1/2 in 143 B and MNNG/HOS cells were not significantly changed compared with the controls after stimulation with MIF.However,MIF remarkably enhanced the protein phosphorylation of SRC,MEK1/2 and ERK1/2 and strengthened the ability of RAS to bind to GTP.In addition,the protein level of p-ERK1/2 in the eight OS samples had a significant increase compared with the paired NAT samples.The result of spearman rank correlation test(R=0.6176)suggested a positive correlation between the protein levels of MIF and p-ERK1/2 in the OS patients.Moreover,cell proliferation markers(KI-67 and PCNA)and the metastasis associated genes(MMP-9,MMP-13 and VEGF)in the downstream of RAS/MAPK pathway were found to significantly increase after MIF stimulation.Thereafter,interference experiments with a MEK1/2 inhibitor were performed,the fold changes of the genes of KI-67,PCNA,MMP-9,MMP-13 and VEGF significantly decreased after treatment with MIF in presence of 10 mM U0126 when compared with those cells with MIF stimulation in absence of 10 mM U0126.Taken together,these resultssuggested that MIF might promote the osteosarcoma progression through activating RAS/MAPK pathway.Conclusions: MIF may promote the progression of osteosarcoma by activating the RAS / MAPK pathway.Part ?: Downregulation of MIF inhibits proliferation and metastasis of osteosarcoma cellsObjective: To establish a stable transfected RNA cell line to inhibit the expression of MIF,and to investigate the mechanism of MIF affecting the development and metastasis of osteosarcoma.Methods: Establish a stable inhibition of MIF expression of MNNG / HOS and 143 B cells(shRNA-MIF-LV-143 B and shRNA-MIF-LV-MNNG / HOS)and control cells(shRNA-NC-LV-143 B and shRNA-NC-MNNG / HOS),4 groups of cells were observed in MIF,PCNA,KI-67,MMP-9,MMP-13 and VEGF levels in mRNA.Results: The mRNA expression of MIF was reduced by 75.5% in 143 B cells and66.4% in MNNG/HOS cells after shRNA-MIF-LV infection.While,the protein expression of MIF was reduced by 84.1% in 143 B cells and 71.9% in MNNG/HOS cells,and the expression of p-ERK was reduced by 56.2% in 143 B cells and 41.1% in MNNG/HOS cells after shRNA-MIF-LV infection.Cell proliferation was inhibited in a time-dependent manner in 143 B or MNNG/HOS cells stably suppressing MIF expression,when compared with the control cells.The m RNA levels of PCNA,KI-67,MMP-9,MMP-13 and VEGF markedly decreased in shRNA-MIF-LV-MNNG/HOS cells or 143 B cells compared with the controls.Additionally,the migrating and invading cell number respectively decreased approximately 55.4% and 65.1% in shRNA-MIF-LV-143 B cells.Similarly,in shRNA-MIF-LV-MNNG/HOS cells,the migrating and invading cell number were 46.9%and 53.1% less than the control cells.Conclusions: MIF interference can significantly inhibit the proliferation and metastasis of osteosarcoma cells.Part ?: Downregulation of MIF inhibits osteosarcoma growth and metastasis in vivoObjective: To study the effect of down-regulation of MIF on the growth and metastasis of osteosarcoma through a mouse xenograft model for the observation of osteosarcoma growth and an orthotopic mouse model for monitoring spontaneous lung metastasis of osteosarcoma.Methods: Construct the mouse xenograft model to observe the growth of osteosarcomaThe animals were divided equally into 4 groups(7 mice per group)and injected subcutaneously into their right flanks with 5�106 viable shRNA-MIF-LV-143 B cells,shRNA-MIF-LV-MNNG/HOS cells or their control cells.After subcutaneous implantation of cells,animals were daily observed for tumour growth.The tumours were measured on days 9,12,15,18,21,24,27 and 30.The mice were sacrificed and photographed at 30 days post-implantation.The mice were sacrificed and photographed at 30 days post-implantation.Construct an orthotopic mouse model of spontaneous lung metastasis in osteosarcoma.The 143 B cells with the stable inhibition of MIF expression and a luciferase reporter,or the control cells,were grown to near confluence.Cell suspension(20 ?l;1�106cells/mL PBS)was then injected percutaneously into the tibia of anesthetized nude mice.Each group contains 8 mice.Five weeks later,the animals were sacrificed.The lung tissues around nodules were harvested and fixed in 10% formalin solution.Microscopic lung metastases were visualized on 5 ?m paraffin-embedded sections with HE staining.Results: At 1 week after inoculation with cells,the signal from luciferase bioluminescence was detected merely at the primary lesion in each group,but no signal was detected in the pulmonary area.At 3 weeks,2 of the 8 mice from shRNA-NC-LV-143 B group showed the signals in the pulmonary area,suggesting the metastatic lesion.But no similar signal was detected in the shRNA-MIF-LV group.At days27,1 of the 8 mice from the shRNA-NC-LV group died and the lung metastasis was confirmed by autopsy.At 5 weeks,the lung metastasis was detected by IVIS in all the 7alive mice from the shRNA-NC-LV group.Whereas,only 2 of the 8 mice from the shRNA-MIF-LV group showed the lung metastasis.Accordingly,the mice from shRNA-MIF-LV-143 B group appeared the significantly inhibited tumour growth.Theorthotopic tumour weight of shRNA-MIF-LV-143 B group reduced by 63.6% compared with the control group.Moreover,much less micrometastatic nodules were observed from the resected lungs of shRNA-MIF-LV group compared with the shRNA-NC-LV group and the metastatic lesions were confirmed using HE staining.The mRNA levels of MIF,PCNA,KI-67,MMP-9,MMP-13 and VEGF from orthotopic tumours in nude mice significantly declined after down-regulation of MIF in shRNA-MIF-LV group.Conclusions: The downregulation of MIF can significantly inhibit the growth of osteosarcoma in vivo and weaken the lung metastasis of OS,suggesting that MIF may be a valuable target for the treatment of OS...