The Effects Of Let-7a On The Ewing’s Sarcoma Malignant Progression And Its Mechanisms

Background: Ewing’s sarcoma (ES), characterized by the proliferation of smallround cells, is the second most common primary bone and soft malignant tumors inchildren and adolescents. Approximately80%of ES cases occur in patients youngerthan20years of age. Although, some advances have been developed in the treatmentstrategies for ES, very little improvement was achieved in the5-year survival rates.The disease-free survival of patients with over metastases is less than30%, and thisrate has not experienced any improvements over the last30years. Thus, a betterunderstanding of the pathological mechanisms about this malignancy is critical to thedevelopment of prognostic biomarkers and novel therapies. MicroRNAs (miRNAs)are a class of small non-coding RNA molecules, which regulate gene expressionpost-transcriptionally. By binding to the complementary sequences in the3’untranslated regions (3’UTR) of their targets, miRNAs induce mRNA degradationor translational repression. Multiple lines of evidence place miRNAs at a critical nodein the progression, development, and maintenance of many diseases, includingcancers. Let-7is one of the most frequently studied miRNA families that have beenreported to fuction as tumor suppressors or oncogenes in tumorigenesis. However, thedetailed mechanisms of let-7in tumorigenesis still need to be elucidated.Objective: Herein, we focus on the functions and mechanisms of let-7a in Ewingsarcoma and we also explore their potential in ES therapy.Methods: Firstly, we explored the expression of let-7a in ES tissues and cell linesusing the stem-loop RT-PCR assays. Secondly, we explored the effects of let-7a onthe cell proliferation, cell cycle progression, apoptosis, migration and invasion ofA673and SK-ES-1cells through the CCK-8assays, flow cytometry technology,wound healing assays and transwell assays, respectively. To further identify themechanisms involved in let-7a-mediated biological behavior, let-7a putative targetswere searched using target prediction programs, and then verified usingdual-luciferase reporter and western-blotting assays; Then, we explored the rescue assays to explore the role of putative target gene in let-7a-mediated tumor suppression.Finnaly, to analysis the putative theraptic role of let-7a, we built the NOD/SCIDimmunodeficiency mouse burdening ES tumor and explored the effects of let-7a onthe growth of ES cells in vivo.Results: The expression of let-7a is downregulated in ES tissues. Consistently, themiRNA level of let-7a is reduced in ES cells relative to the human mesenchymal stemcells (MSCs). These results suggest that let-7a may function as a tumor suppressor inES; Then, we restored the let-7a expression in A673and SK-ES-1cells throughtransfecting with miRNA mimic. Overexpression of let-7a significantly suppressionproliferation and invasion, induced cell apoptosis and arrested cell cycle progreassionof both cell lines, which indicated the tumor suppressor role of let-7a in ES cells; Tofurther identify the mechanisms involved in let-7a-mediated biological behavior,let-7a putative targets were searched using target prediction programs, whichidentified that cyclin dependent kinase-6(CDK6) to be a putative target gene.Dual-luciferase reporter and western-blotting assays demonstrate that let-7asignificantly suppressed that transcription of CDK6. The mRNA and protein levels ofCDK6in ES cells transfected with let-7a were significantly reduced. Sincephosphor-retinoblastoma (p-Rb) plays a key role in CDK6-mediated G1/S cell cycletransition, we explored the levels of Rb and p-Rb upon transfection with let-7a. Theresults showed that overexpression of let-7a significantly suppressed the expressionof p-Rb, while did not influence the level of total Rb, which is consistent withknocking down CDK6. These results demonstrant that let-7a regulates the malignantphenotype of ES cells might be through regulating the expression of CDK6/p-Rb;Further rescue assays showed that retored the expression of CDK6in ES cells thathas been transfected with let-7a mimic before, could partially abolished thesuppressive effects of let-7a on ES cells, which demonstrate that let-7a functions as atumor suppressor directly through targeting CDK6; Finnaly, re-introduction of let-7asignificantly inhibited tumor formation of ES cells in the NOD/SCIDimmunodeficiency mouse, and suppressed the expression CDK6in the xenograftmice.Conclucion: Collectively, our finding characterized the expression properities of let-7a, elucidated the function and molecular mechanism of let-7a in ES and impliedthat let-7a might be emplyed as a noval prognostic markers and therapeutic targets ofES.