Let-7a Suppresses Macrophage Infiltrations And Malignant Phenotype Of Ewing Sarcoma And Its Mechanisms

Background: The present study that interaction between tumors cells,tumor-derived humoral factors and the bone marrow in the bone niches has been shown to be essential for bone tumor initiation and promotion.Among the tumor stromal cells,tumor-associated macrophages(TAMs)are usually the most abundant immune population.In our previous researches shown there are a number of infiltrating-TAMs in the ewing sarcoma(ES)tissue.Moreover,some studies demosrared let-7a inhibited ES cells proliferation which act as a tumor suppressor gene,but,it has not been reported whether the lower-expression of let-7a relates to TAMs infiltration in ES.In order to solve this problem,we has carried on the related research.Objective: To explore the effects of let-7a regulated the macrophage infiltration,ES cells malignant phenotype and its mechanism in ES.Methods: Firstly,we explored the expression of let-7a in 16 cases of ES tissues and cell lines using the Real-time-PCR assays.To investigate the association between let-7a expression and tumormacrophage infiltration,ES specimens were characterized for the number of TAMs by immunohistochemistry(IHC)using anti-CD68 antibody.Secondly,we explored the effects of let-7a on the cell proliferation,migration,invasion and monocyte chemotaxis of A673 and SK-ES-1 cells through the CCK-8 assays,wound healing assays,transwell assays and monocyte chemotaxis,respectively.To further identify the mechanisms involved in let-7a-mediated biological behavior,let-7a putative targets were searched using target prediction programs,and then verified using dual-luciferase reporter,rescue assays and western-blotting assays;Finnaly,to analysis the putative theraptic role of let-7a,we built the NOD/SCID immunodeficiency mouse burdening ES tumor and explored the effects of let-7a on the growth of ES cells and mediated TAMs infiltration in vivo.Results: The expression of let-7a is downregulated in ES tissues.Consistently,the mi RNA level of let-7a is reduced in ES cells relative to the human mesenchymal stem cells(MSCs).These results suggest that let-7a may function as a tumor suppressor in ES.Immunohistochemical results showed that high expression of CD68-positive TAMs in ES,we demonstrated that the let-7a expression is negatively related to macrophage infiltration in ES and mediated macrophage infiltration.Overexpression of let-7a significantly suppression proliferation,migration,invasion,and monocyte chemotaxis induced of ES cells.To further identify the mechanisms involved in let-7a-mediated biological behavior,let-7a putative targets were searched using target prediction programs,which identified that signal transducer and activator of transcription 3(STAT3)to be a putative target gene.Dual-luciferase reporter,western blotting assays demonstrate that let-7a significantly suppressed that transcription of STAT3 directly.Rescue assays showed that let-7a functions as a tumor suppressor directly through targeting STAT3.Finnaly,reintroduction of let-7a significantly inhibited tumor formation and macrophages infiltration of ES cells in the NOD/SCID immunodeficiency mouse,and suppressed the expression STAT3 in the xenograft mice.Conclucion: In summary,let-7a/STAT3 axis performs suppressive effects not only limited at suppressing the malignant phenotypes of tumor cells but also on the regulating of macrophage infiltration in ES.These provide theoretical basis for the future molecular therapeutics for ES patients.