Let-7a对尤文肉瘤干细胞恶性表型的影响及其可能的机制

Effect of let-7a on malignant phenotype of Ewing sarcoma stem cells and its possible mechanism

目的:探讨微RNA let-7a对尤文肉瘤中肿瘤干细胞恶性表型的影响及其可能的分子作用机制。方法:采用侧群(side population,SP)细胞分选法分选出尤文肉瘤A673和SK-ES-1细胞中的肿瘤干细胞样细胞,并进行鉴定。采用实时荧光定量PCR法检测SP细胞与非SP(non-SP)细胞中let-7a的表达差异。将人工合成的let-7a模拟物(let-7a mimic)分别转染至A673和SK-ES-1的SP细胞中,随后通过细胞集落形成实验和Transwell小室侵袭实验分别检测let-7a对SP细胞集落形成和侵袭能力的影响。蛋白质印迹法检测SP细胞和non-SP细胞中信号转导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)及其下游的磷酸化STAT3(phospho-STAT3,p-STAT3)、基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)蛋白的表达差异,以及转染let-7a mimic后SP细胞中这些蛋白表达水平的变化。结果:成功分离出尤文肉瘤A673和SK-ES-1细胞中的肿瘤干细胞样SP细胞,并发现SP细胞中干细胞相关蛋白SOX2和CD133的表达水平均明显上调(P值均<0.01)。与non-SP细胞相比,SP细胞中let-7a呈低表达(P<0.01),而STAT3信号通路相关蛋白STAT3、p-STAT3和MMP2明显高表达(P值均<0.01),细胞集落形成能力和侵袭能力均明显升高(P值均<0.05)。let-7a过表达的尤文肉瘤SP细胞中,细胞集落形成能力和侵袭能力均较阴性对照组明显降低(P值均<0.05),而且STAT3及其下游p-STAT3和MMP2蛋白表达水平较阴性对照组均明显下调(P值均<0.01)。结论:尤文肉瘤干细胞中Let-7a低表达。过表达let-7a可能通过STAT3信号通路介导抑制尤文肉瘤干细胞的集落形成和侵袭能力。

Objective:To investigate the effect of microRNA let-7a on the malignant phenotype of cancer stem cells in Ewing sarcoma and its possible molecular mechanism.Methods:The cancer stem cell-like cells of Ewing sarcoma A673 and SK-ES-1 cells were isolated through side population(SP)cell isolation methods and then identified.The expression difference of let-7a in SP cells and non-SP cells was detected by real-time fluorescence quantitative PCR.The artificially synthesized let-7a mimic was transfected into SP cells isolated from A673 and SK-ES-1 cells,respectively.The effects of let-7a on the colony formation and invasion capacities of SP cells were detected by cell colony formation assay and Transwell invasion assay,respectively.The protein expression differences of signal transduction and activator of transcription 3(STAT3)and its downstream phospho-STAT3(p-STAT3)and matrix metalloproteinase 2(MMP2)in SP cells and non-SP cells were detected by Western blotting.The expression changes of these proteins in SP cells after let-7a mimic transfection were also detected.Results:The cancer stem cell-like SP cells were successfully isolated from Ewing sarcoma A673 and SK-ES-1 cells.The expression levels of stem cell-related proteins SOX2 and CD133 in SP cells isolated from A673 and SK-ES-1 cells were significantly up-regulated(both P<0.01).Compared with the non-SP cells,the expression level of let-7a in SP cells was low(P<0.01),while the expressions of STAT3 signaling pathway-related STAT3,p-STAT3 and MMP2 proteins were opposite(all P<0.01),and the capacities of clone formation and invasion were significantly increased(both P<0.05).After the overexpression of let-7a,the colony formation and invasion capacities of SP cells were significantly decreased(both P<0.05),the expression levels of STAT3 and its downstream p-STAT3 and MMP2 were significantly down-regulated as compared with the control group(all P<0.01).Conclusion:The expression of let-7a in cancer stem cells of Ewing sarcoma is low,and the overexpression of let-7a can suppress the colony formation and invasion capacities of cancer stem cells in Ewing sarcoma,which may be mediated through STAT3 signaling pathway.