Ward Christina M, Li Biaoru, Pace Betty S
Department of Biochemistry and Molecular Biology, Augusta University, Augusta, GA 30912, USA Department of Pediatrics, Augusta University, Augusta, GA 30912, USA.
Department of Pediatrics, Augusta University, Augusta, GA 30912, USA.
Exp Biol Med (Maywood). 2016 Apr;241(7):719-29. doi: 10.1177/1535370216636725. Epub 2016 Mar 2.
Sickle cell anemia is a common genetic disorder caused by a point mutation in the sixth codon of the β-globin gene affecting people of African descent worldwide. A wide variety of clinical phenotypes ranging from mild to severe symptoms and complications occur due to hemoglobin S polymerization, red blood cell sickling, and vaso-occlusion. Research efforts are ongoing to develop strategies of fetal hemoglobin (HbF; α2γ2) induction to inhibit sickle hemoglobin polymerization and improve clinical outcomes. Insights have been gained from investigating mutations in the β-globin locus or transcription factors involved in the mechanisms of hemoglobin switching. Recent efforts to expand molecular targets that modulate γ-globin expression involve microRNAs that work through posttranscriptional gene regulation. Therefore, the goal of our study was to identify novel microRNA genes involved in fetal hemoglobin expression. Using in silico analysis, we identified a miR-34a binding site in the γ-globin mRNA which was tested for functional relevance. Stable expression of the shMIMIC miR-34a lentivirus vector increased fetal hemoglobin levels in single cell K562 clones consistent with silencing of a γ-globin gene repressor. Furthermore, miR-34a promoted cell differentiation supported by increased expression of KLF1, glycophorin A, and the erythropoietin receptor. Western blot analysis of known negative regulators of γ-globin including YY1, histone deacetylase 1, and STAT3, which are regulated by miR-34a showed no change in YY1 and histone deacetylase 1 levels; however, total- and phosphorylated-STAT3 levels were decreased in single cell miR-34a K562 clones. These data support a mechanism of fetal hemoglobin activation by miR-34a involving STAT3 gene silencing.
镰状细胞贫血是一种常见的遗传性疾病，由β-珠蛋白基因第六密码子的点突变引起，影响着全球非洲裔人群。由于血红蛋白S聚合、红细胞镰变和血管阻塞，会出现从轻度到重度症状及并发症的多种临床表型。目前正在进行研究以制定诱导胎儿血红蛋白（HbF；α2γ2）的策略，以抑制镰状血红蛋白聚合并改善临床结果。通过研究β-珠蛋白基因座中的突变或参与血红蛋白转换机制的转录因子，已经获得了一些见解。最近扩大调节γ-珠蛋白表达的分子靶点的努力涉及通过转录后基因调控发挥作用的微小RNA。因此，我们研究的目标是鉴定参与胎儿血红蛋白表达的新型微小RNA基因。通过计算机分析，我们在γ-珠蛋白mRNA中鉴定出一个miR-34a结合位点，并对其功能相关性进行了测试。shMIMIC miR-34a慢病毒载体的稳定表达增加了单细胞K562克隆中的胎儿血红蛋白水平，这与γ-珠蛋白基因阻遏物的沉默一致。此外，miR-34a通过增加KLF1、血型糖蛋白A和促红细胞生成素受体的表达来促进细胞分化。对受miR-34a调控的γ-珠蛋白已知负调节因子包括YY1、组蛋白去乙酰化酶1和STAT3的蛋白质印迹分析显示，YY1和组蛋白去乙酰化酶1水平没有变化；然而，单细胞miR-34a K562克隆中的总STAT3和磷酸化STAT3水平降低。这些数据支持miR-34a通过STAT3基因沉默激活胎儿血红蛋白的机制。
