【摘要】 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。更多还原

【Abstract】 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.更多还原