MiR-19b-3p Regulates Fetal Hemoglobin Synthesis By Targeting SOX6 In Beta Thalassemia

Objective The overall carriage prevalence of β thalassemia is 6.66% in Guangxi province.β-TM patients are severely anemic and require lifelong transfusion and iron chelation therapy to survive.Therapies targeting Hb F induction may be a promising approach for treating β-thalassemia.However,the regulatory mechanisms of fetal hemoglobin(Hb F)have not been unclear.The purpose of this study is to screen core micro RNAs related to fetal hemoglobin(Hb F)by performing weighted gene co-expression network analysis(WGCNA)and differential gene expression(DEG)analysis,and investigates the role of core micro RNA in Hb F synthesisMethod The microarray data related to Hb F,was used to construct a co-expression network.Hub micro RNA genes were identified basing on the integrative analysis of WGCNA and DEG.The expression levels of core mi RNA and its potential target genes were further verified by PT-q PCR.Correlations between micro RNA levels and hematological parameters such as Hb F were analyzed.k562 cell lines were transfected with micro RNA mimics or inhibitor,the expression of micro RNA target gene and its downstream gene HBG were verified by RT-q PCR,Western blot and cellular immunofluorescence.Simultaneously,CCK8 was used to examine cell proliferation,Flow cytometry was used to examine cell apoptosis.Dual luciferase reporter assay and AGO2-RIP were applied to verify the relationship among micro RNA and its target gene.Result Pink module was positively related to Hb F was identified through WGCNA analysis,Therefore,we screened hub genes in the pink module and identified mi R-19b-3p as a core micro RNA which was also highly expressed in high-Hb F β-thalassemia carriers.SOX6 was predicted to be the target gene of mi R-19b-3p.Peripheral blood reticulocytes of 20 high-Hb F β-thalassemia carriers and 20 healthy controls were collected to analyze the expression levels of mi R-19b-3p and SOX6 using RT-q PCR.RT-q PCR showed mi R-19b-3p was up-regulated in high-Hb F group comparing with the normal group,and was positivity correlated with Hb F level.The predicted relationships between the mi R-19b-3p and SOX6 were identified using luciferase reporter assay,AGO2-RIP experiments.Furthermore,overexpression of mi R-19b-3p in k562 cell lines inhibited the expression of SOX6,further induced Hb F synthesis.While inhibition of mi R-19b-3p leaded to promote the expression of SOX6 in k562 cells,consequently,reduced Hb F expression.The inhibition of Hb F mediated by mi R-19b-3p inhibitor could be rescued by knockdown the SOX6 activity using si RNA.However,mi R-19b-3p had no effect on cell proliferation and apoptosis in vitro.Conclusion By performing WGCNA,mi R-19b-3p was identified as a novel micro RNA associated with Hb F.mi R-19b-3p was highly expressed in high-Hb Fβ-thalassemia carriers,and was positively related to Hb F level.mi R-19b-3p modified Hb F synthesis by targeting SOX6 in k562 cells.Our study provided a new insight into the regulation of Hb F expression and a novel potential biomarker for further research in future studies.