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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 explore the effect of microRNA-223-3p (miR-223-3p) on the differentiation and maturation of megakaryocytes and to explore the possible mechanism. Methods Real-time quantitative PCR was used to detect the endogenous expression of miR-223-3p during megakaryocyte differentiation. The expression level of miR-223-3p was then exogenously regulated by flow cytometry Megakaryocyte differentiation and maturation, using bioinformatics analysis to find its target gene MYH10 play a relevant biological role, and by real-time quantitative PCR, luciferase, flow cytometry to verify that MYH10 is the target of miR-223-3p gene. Results Endogenous miR-223-3p increased with the maturation of megakaryocytes, and the expression of miR-223-3p mimics in K562 and Meg-01 cell lines increased the expression of macrophage-associated surface markers CD41 and CD61 Positive rate and significantly promote the formation of polyploidy. The gene expression of MYH10 decreased with the increase of miR-223-3p expression. MYH10 gene was identified as the target gene of miR-223-3p by dual luciferase reporter assay. Inhibition of MYH10 gene expression, can promote megakaryocyte polyploidization. Conclusion miR-223-3p regulates the maturation of megakaryocytes by regulating the expression of MYH10.