KLF6 Regulates β-like Globin Expression And Is Modulated By MiR-2355-5p During Erythroid Cells Differentiation

BackgroundThe β-thalassemia is one of the common monogenic genetic diseases.The main pathogenic mechanism is that β-globin gene cluster mutation leads to β-globin synthesis disorder,which makes the α/β-globin chain ratio unbalanced and cannot be synthesized with normal hemoglobin tetramers,causing anemia.Different types of hemoglobin are expressed in different developmental stages of the blood system,embryonic hemoglobin Gower-1（ζ2ε2）is expressed in the early yolk sac of the embryo,fetal hemoglobin HbF（α2γ2）is expressed in the fetal liver,and adult hemoglobin is expressed in the bone marrow of the baby after birth（HbA,α2β2）,this process is called hemoglobin switching.For patients with β-thalassemia,an increase expression of HbF can alleviates the patient’s clinical symptoms to some extent.The regulation of HbF expression is influenced by multiple family members of Kriippel-like factors.So far,KLFs family member KLF6 has been reported to be involved in the process of promoting erythroid differentiation and maturation,but there is no related research can prove that KLF6 participate in regulation of β-globin or y-globin,so the focus of this study is the regulation of β-like globin by the transcription factor KLF6,and its mechanism of action.Our results suggest that KLF6 as a repressor on γ-globin,so we looked for microRNAs（miR-2355-5p）that can target the 3’UTR region of KLF6 to investigate whether miR-2355-5p has down-regulation for KLF6 in transcriptional level.The investigation of the mechanism of microRNA-target gene-globin gene regulatory network,provides a new target for improving HbF levels and improving the severity of clinical symptoms in patients with β-thalassemia.MethodsThe endogenously expressed of KLF6 in HUDEP-2 cells and human peripheral blood hematopoietic progenitor cells（CD34+cells）was knocked down by shRNA lentivirus,and knockdown was proved by RT-qPCR/Western Blot in mRNA level and protein level.To verify whether the transcription factor KLF6 has a regulatory effect on the β/γ-globin gene,the RT-qPCR/Western Blot/HPLC assay was used to detect the mRNA level and protein content of β/γ-globin.Thereafter,experiments such as ChIP/EMSA/Luciferase assay was perforemed to prove that KLF6 whether can bind in the DNAse I high-sensitivity site（Hypersensitivity Sites,HS）in the LCR region of the β-globin gene cluster,and the promoter region of HBB/HBG genes.In combination,the luciferase assay co-transformed with microRNA mimics and plasmid with KLF6 3’UTR region indicated that miR-2355-5p binds to KLF6 3’UTR.To further verify the relationship between miR-2355-5p and KLF6,the lentivirus of overexpression miR-2355-5p was transfected to HUDEP-2 cells and CD34+cells,RT-qPCR/WB was preformed to detect the level of KLF6,and the same detection ofβ/γ-globin gene content,to verify whether miR-2355-5p can regulates β/γ-globin expression by affecting the expression of KLF6.Rescue experiment was performed that the lentivirus of overexpression target gene KLF6 was transfected to HUDEP-2 cells that stably overexpressed mature miR-235 5-5p to observe expression changes about KLF6 and β/γ-globin.ResultsIt was found from the experimental results that the successful knockdown of the transcription factor KLF6 in HUDEP-2 cells and CD34+cells resulted in a decrease inβ-globin content and an increase in the expression of the γ-globin gene.The mRNA and protein levels of β-globin decreased significantly,the mRNA level of γ-globin gene increased,and the content of fetal hemoglobin HbF increased significantly.The results of ChIP/EMSA/Luciferase assay showed that KLF6 was significantly enriched at the HS2 site in the LCR region and the HBB promoter region.Specially KLF6 was significantly enriched in the medium term during differention of HUDEP-2 and CD34+cells,suggesting that KLF6 may promote LCR region and the HBB promoter form a physical structure looping,which regulates the expression of β-like globin gene,which is characterized by down-regulating HBG gene expression and promoting HBB gene expression.miR-2355-5p can bind to the 3’UTR region of KLF6 and overexpression of miR-2355-5p decreases the mRNA level and protein level of KLF6,which proves that miR-2355-5p has a post-transcriptional silencing effect on KLF6,and decreases the expression of β-globin.Meanwhile the γ-globin gene is up-regulated,indicating that miR-2355-5p can up-regulate γ-globin gene expression and increase HbF content by inhibiting the production of KLF6 protein.ConclusionThis study demonstrates that KLF6 regulates the expression of β-like globin gene,which promotes the expression of β-globin and inhibits the expression ofγ-globin.The main mechanism of action is KLF6 binding to the β-globin gene cluster through CACCC box of the HS2 site in the LCR region and the the promoter region of the HBB gene to activate β-globin expression by the enhancer action of the LCR region.However,miR-2355-5p can bind to the 3’UTR region of KLF6,causing post-transcriptional silencing of KLF6,and releasing KLF6 to promote the expression of β-globin gene,which leads an up-regulation in γ-globin gene expression.This study was the first time to explore the inhibitory effect of the transcription factor KLF6 on γ-globin,and its expression is regulated by miR-2355-5p,which provides a new perspective for the KLF family in regulation about β-like globin expression and possible pathway in increasing fetal hemoglobin HbF content.