Plasma microRNA-451 as a novel hemolytic marker for β0-thalassemia/HbE disease

Abstract

In Southeast Asia, particularly in Thailand, β0-thalassemia/hemoglobin E (HbE) disease is a common hereditary hematological disease. It is associated with pathophysiological processes, such as the intramedullary destruction of immature erythroid cells and peripheral hemolysis of mature red blood cells. MicroRNA (miR) sequences, which are short non-coding RNA that regulate gene expression in a suppressive manner, serve a crucial role in human erythropoiesis. In the present study, the plasma levels of the erythroid-expressed miRNAs, miR‑451 and miR‑155, were analyzed in 23 patients with β0-thalassemia/HbE and 16 control subjects. Reverse transcription‑quantitative polymerase chain reaction analysis revealed significantly higher levels of plasma miR‑451 and miR‑155 in β0‑thalassemia/HbE patients when compared to the control subjects. Notably, among the β0‑thalassemia/HbE patients, a significant increase in miR‑451 levels was detected in severe cases when compared with mild cases. The levels of plasma miR‑451 correlated with reticulocyte and platelet counts. The results suggest that increased plasma miR‑451 levels may be associated with the degree of hemolysis and accelerated erythropoiesis in β0‑thalassemia/HbE patients. In conclusion, miR‑451 may represent a relevant biomarker for pathological erythropoiesis associated with β0-thalassemia/HbE.

Figure 1.

Increased levels of plasma miR-451 and miR-155 in patients with β⁰-thalassemia/HbE. ∆C_q values of miRNA-451 and miRNA-155 were calculated using the C_q values of the internal control, cel-miR-39. (A) Patients with β⁰-thalassemia/HbE exhibited significantly higher plasma miR-451 and miR-155 levels when compared with normal control subjects. (B) The association between plasma miR-451, miR-155 levels, and disease severity was analyzed using the Student's t-test (black triangles, ∆C_q values of patients without splenectomy; white triangles, ∆C_q values of the patients that had undergone splenectomy). Plasma miR-451 and miR-155 levels were significantly associated with the degree of β⁰-thalassemia/HbE disease severity. The severe cases exhibited significantly higher levels of miR-451 and miR-155 when compared with the control subjects and mild cases. HbE, hemoglobin E; C_q, quantification cycle; miR, microRNA.

Figure 2.

ROC analysis of plasma miR-451 and miR-155 levels. ROC analysis of plasma (A) miR-451 and (B) miR-155 levels in normal control subjects (n=16), β⁰-thalassemia/HbE patients (n=23) and those with mild (n=10) and severe disease (n=13). The sensitivity and specificity values were defined as the maximized AUC with 95% CI value. ROC, receiver operating characteristic; miR, microRNA; HbE, hemoglobin E; AUC, area under the ROC curve; CI, confidence interval.

Figure 3.

Correlation between the level of miR-451 and miR-155 and clinical features. Levels of miR-451 and miR-155 correlated with (A) reticulocyte and (B) platelet counts as depicted by plots of miR-451 and miR-155 ∆C_q values against the absolute number of reticulocytes and platelets. Data were analyzed using a Pearson's correlation test. miRNA, microRNA; C_q, quantification cycle.

Figure 4.

miR-451 levels in PRCs. (A) The levels of miR-451 in PRCs from 9 control subjects and 18 patients with β⁰-thalassemia/HbE. (B) The levels of miR-451 between normal control, and patients with mild and severe cases of β⁰-thalassemia/HbE. ∆C_q values of miRNA-451 in PRCs were calculated using the following formula: ∆C_q=C_q(miR-451)-C_q(miR-let7a). miR, microRNA; PRC, packed red cells; HbE, hemoglobin E; C_q, quantification cycle.