微小RNA-223-3p通过调节MYH10基因促进巨核细胞多倍体化

摘要

目的 探讨微小RNA-223-3p(miR-223-3p)对巨核细胞分化和成熟的影响,并初步探索其中可能的机制.方法 通过实时定量PCR检测巨核细胞分化过程中miR-223-3p的内源性表达变化趋势,而后外源调节miR-223-3p在细胞系中的表达量,并通过流式细胞术检测其对于巨核细胞分化和成熟的影响,运用生物信息学分析,找到其发挥相关生物学作用的靶基因MYH10,并通过实时定量PCR、荧光素酶、流式细胞术验证MYH10是miR-223-3p的靶基因.结果 内源性miR-223-3p随着巨核细胞的分化成熟表达量增加,在K562和Meg-01细胞系中转染miR-223-3p mimics后可升高巨核细胞相关表面标志CD41和CD61的阳性率,同时显著促进多倍体的形成,MYH10的基因表达随miR-223-3p表达升高而下降,通过双荧光素酶报告基因技术验证MYH10基因是miR-223-3p的靶基因,进一步抑制MYH10基因表达后,可促进巨核细胞多倍体化.结论 miR-223-3p可通过调控MYH10的表达调节巨核细胞的成熟.%Objective To investigate the effect of microRNA-223-3p (miR-223-3p) on megakaryocytic differentiation and maturation, and explore the potential mechanism .Methods The endogenous expression of miR-223-3p during megakaryocyte ( MK) differentiation was detected by real-time PCR.Flow cytometry further indicated that alteration of miR-223-3p in human cell lines exerted effects on MK differentiation and maturation .By performing integrative bioinformatic analysis, the potential miR-223-3p target gene, MYH10,was identified.Real-time PCR, luciferase reporter assay and flow cytometry revealed that MYH10 was a direct target of miR-223-3p.Results Endogenous expression of miR-223-3p was in-creased with the differentiation and maturation of MK .The expression of megakaryocytic surface markers CD41 and CD61 and the ploidy were significantly increased in K562 and Meg-01 cells after transfection with miR-223-3p mimics.The expression of MYH10 decreased with the increase in miR-223-3p.Using a luciferase reporter assay ,we demonstrated that MYH10 was a direct target of MiR-223-3p.Furthermore, direct downregulation of MYH10 promoted MK polyploidization . Conclusion MiR-223-3p might regulate the polyploidization of MK by targeting MYH10.