OBJECTIVETo investigate the effect of blocking polypyrimidine complex binding to DNA site by using peptide nucleic acid (PNA) on γ-globin gene expression.

METHODSPYR-PNA, β-PNA and RS-PNA (random sequence-PNA) were designed and synthesized, then were transfected into K562 cells with the cationic liposome lipofectamine 2000 used as vector. The expression of γ-globin gene at both the transcriptional and translational level was detected by RT-PCR and the Western blot respectively at 24 h, 48 h and 72 h after transfection with PNAs.

RESULTSCompared with RS-PNA and control groups, the expression of &gamma;-globin gene at mRNA and protein levels in PYR-PNA group was significantly up-regulated(P<0.05), especially at 48 h after tranfection, the levels of mRNA and protein in PYR-PNA group were increased by 2.0 and 2.5 times than those in control group, respectively.

CONCLUSIONPYR-PNA can significantly up-regulate the expression of &gamma;-globin gene in K562 cells, this study may provide a new research idea for gene therapy of β-thalassemia.

DNA ; Gene Expression ; Humans ; Peptide Nucleic Acids ; Transfection ; gamma-Globins

β-thalassemia is a chronic hemolytic anemia characterized by the reduction or absence of synthesis of β-globin chains because of the β-globin gene mutations. β-thalassemia belongs to the inherited hemoglobin disease, and occurs in some provinces of China, such as in Guangdong, Guangxi, Fujian, its prevalence is about 2%. The treatment of this disease include transfusion, iron chelating agent, hematopoietic stem cell transplantation, splenectomy, induced expression of Fetal Hemoglobin (HbF) and gene therapies. However, the mortality rate of this disease is still higher, thus some new treatments are urgently needed. In recent years, the study was mainly concentrated in 2 aspects: the normal β-globin gene transfer and endogenous &gamma;-globin re-activation. Some studies showed that the expression of miRNAs was dysregulated in β-thalassemia. Some miRNAs could regulate &gamma;-globin at posttranscriptional level, thus, the clarification of relationship between miRNAs and β-thalassemia is expected to provide experimental bases to β-thalassemia therapy. In this review, the induced therapy of &gamma;-globin for β-thalassemia and its relationship with the miRNA are summarized.
China ; Fetal Hemoglobin ; metabolism ; Genetic Therapy ; Humans ; MicroRNAs ; metabolism ; beta-Globins ; genetics ; beta-Thalassemia ; therapy ; gamma-Globins ; therapeutic use

OBJECTIVETo explore the effects of TBLR1-RARα on the differentiation induction of leukemia cell line K562 cells into erythroid lineage and to investigate its related mechanisms.

METHODSTet-Off inducible system was used to construct the conditional expression vector of TBLR1-RARα fusion gene by cloning the TBLR1-RARα fragment into lentivirus vector pLVX-Tight-Puro, the expression of TBLR1-RARα fusion gene was induced by doxycycline (Dox). Then, K562 cells were transfected with lentivirus pLVX-Tight-Puro-TBLR1-RARα-flag, and the expression of fusion proteins was verified by Western blot. After treatment of K562 with all-trans retinoid acid (ATRA), real time RT-PCR was performed to test the expression of erythroid differentiation-related CD71 and α, ε, &gamma;-globins gene. Flow cytometry was used also to analyze the expression of erythroid differentiation markers CD71 and CD235a. Benzidine staining was used to detect the production of hemoglobin in K562 cells.

RESULTSqRT-RCR showed that ATRA could increase the expression level of CD71 and α, ε, &gamma;-globin genes when TBLR1-RARα was expressed. After treatment of ATRA, the proportion of CD71(+) cells detected by the flow cytometry also increased. Benzidine staining showed that ATRA could induce hemoglobin production in K562 cells with TBLR1-RARα fusion gene expression.

CONCLUSIONThe expression of TBLR1-RARα fusion gene contribute to ATRA-inducing differentiation of K562 cells into erythroid lineage.

Cell Differentiation ; Erythrocytes ; Hemoglobins ; Humans ; K562 Cells ; Nuclear Proteins ; Receptors, Cytoplasmic and Nuclear ; Receptors, Retinoic Acid ; Repressor Proteins ; Retinoic Acid Receptor alpha ; gamma-Globins

The role of ROS in hydroquinone-induced inhibition of K562 cell erythroid differentiation was investigated. After K562 cells were treated with hydroquinone for 24 h, and hemin was later added to induce erythroid differentiation for 48 h, hydroquinone inhibited hemin-induced hemoglobin synthesis and mRNA expression of &gamma;-globin in K562 cells in a concentration-dependent manner. The 24-h exposure to hydroquinone also caused a concentration-dependent increase at an intracellular ROS level, while the presence of N- acetyl-L-cysteine prevented hydroquinone- induced ROS production in K562 cells. The presence of N-acetyl-L-cysteine also prevented hydroquinone inhibiting hemin-induced hemoglobin synthesis and mRNA expression of &gamma;-globin in K562 cells. These evidences indicated that ROS production played a role in hydroquinone-induced inhibition of erythroid differentiation.
Acetylcysteine ; pharmacology ; Cell Differentiation ; drug effects ; Dose-Response Relationship, Drug ; Hemin ; pharmacology ; Humans ; Hydroquinones ; pharmacology ; K562 Cells ; drug effects ; Reactive Oxygen Species ; metabolism ; gamma-Globins ; genetics

This study was aimed to explore the effect of BCL11A gene on transcription of &gamma;-globin gene in K562 cells. B-cell lymphoma/leukemia 11A (BCL11A) gene was silenced by small interfering RNA (siRNA) expression vectors in K562 cells (human erythroblastic leukemia cell line). Gamma-globin mRNA level in K562 cells was determined by RT-PCR. Association between the BCL11A gene and &gamma;-globin gene transcription was explored by comparison of mRNA levels. The results indicated that the silence rate of the BCL11A gene in K562 cells by 4 siRNA expression vectors was 49.7%, 55.4%, 78.2%, and 84.1%, respectively. The siRNA expression vector with 84.1% silence rate was transfected into K562 cells, transcription level of &gamma;-globin mRNA in K562 cells transfected with siRNA expression vector increased 2.4 times as compared with control K562 cells. It is concluded that level of &gamma;-globin mRNA increases when the BCL11A gene is silenced. It indicates that the BCL11A gene may be a negative regulator for &gamma;-globin gene expression.
Carrier Proteins ; genetics ; Gene Expression Regulation, Leukemic ; Genes, Regulator ; Genetic Vectors ; Humans ; K562 Cells ; Nuclear Proteins ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Transcription, Genetic ; Transfection ; gamma-Globins ; genetics

We studied the function and mechanism of miR-24 in regulating beta-like globin gene expression. We first detected the expression of miR-24 during erythroid differentiation and also detected the globin gene expression in miR-24 overexpressing K562 cells through q-PCR. Dual-luciferase reporter assay and Western blotting were used to identify target genes of miR-24. "Rescue experiment" was further used to investigate the regulation of miR-24 on globin gene expression whether depending on targeting Sp1 or not. We found that miR-24 increased during hemin-induced K562 cells and EPO-induced HPCs (hematopoietic progenitor cells) erythroid differentiation. Overexpression of miR-24 in K562 cells promoted the epsilon- and gamma-globin gene expression during hemin-induced erythroid differentiation through targeting the negative globin regulator Sp1. These results suggested that miR-24 can improve the expression of beta-like globin gene through targeting Sp1.
Cell Differentiation ; Gene Expression Regulation ; Hematopoietic Stem Cells ; metabolism ; Humans ; K562 Cells ; MicroRNAs ; genetics ; Sp1 Transcription Factor ; genetics ; epsilon-Globins ; genetics ; gamma-Globins ; genetics

This study was aimed to detect and identify the promoter CpG island methylation of &gamma;-globin gene in peripheral blood mononuclear cells from patients with β-thalassemia major and healthy adult in Guangxi province, as well as to analyze the difference of promoter methylation rate of each CpG sites between them, and then to screen the promoter CpG island main methylation sites which maybe influence &gamma;-globin expression. The template DNA was modified by bisulfite genomic sequencing PCR; the promoter sequences of &gamma;-globin gene were amplified by technique Touchdown PCR, and then the PCR products were cloned and sequenced for obtaining methylation status of each CpG sites in target fragments, and then the accurate methylation sites and levels were detected quantitatively. The results indicated that the 4 CpG methylation sites locating at 28, 122, 231 and 234 bp in sequences were hypermethylated. As compared with healthy adults, the DNA methylation rate of 122 and 231 bp CpG sites in patients with β-thalassemia major was obviously lower, however, methylation rates of 28 and 234 bp sites were not significantly different between patients and healthy adults. It is concluded that the methylation sites 28, 122, 231 and 234 bp of &gamma;-globin gene promoter are found both in patients with β-thalassemia major and healthy adults. The 122 and 231 bp sites are identified preliminarily to be involved in the regulation of &gamma;-globin expression. This study provides the experimental evidence for alleviating the clinical symptoms of β-thalassemia major and targeting gene treatment through the regulation of &gamma;-globin.
Adolescent ; Adult ; Case-Control Studies ; Child ; CpG Islands ; DNA Methylation ; Female ; Humans ; Male ; Promoter Regions, Genetic ; Young Adult ; beta-Thalassemia ; genetics ; gamma-Globins ; genetics

OBJECTIVETo develop a real-time PCR-based chromatin immunoprecipitation (ChIP) assay for determining the effect of sodium butyrate on acetylation of histone in gamma-globin gene promoter regions in K562 cells.

METHODSK562 cells were grown in the presence or absence of 0.5 mmol/L sodium butyrate for 48 h, and 1=10(7) cells per group were used for real-time PCR-based ChIP with anti-acetylated histone H3 or H4 antibodies. The levels of acetylated histone H3 and H4 (acH3 and acH4) in Ggamma- and Agamma-globin gene promoter regions were measured.

RESULTSIn the K562 cells with sodium butyrate treatment or without any treatment, the levels of acH3 or acH4 in Ggamma- or Agamma-globin gene promoter were higher than that in the necdin gene (negative control). Compared with the untreated K562 cells, the cells treated with 0.5 mmol/L sodium butyrate showed a 3.1-fold or 2.6-fold increase in acH3 or acH4 in Ggamma-globin gene promoter region, with also a 3.7-fold or 3.2-fold increase in acH3 or acH4 in Agamma-globin gene promoter region, respectively (P<0.01).

CONCLUSIONWe have successfully developed a real-time PCR-based ChIP assay for analyzing the acetylation of histone H3 and H4 in gamma-globin gene promoter regions. Our results support the role of sodium butyrate in increasing the level of acetylated histone in gamma-globin gene promoter regions.

Acetylation ; Butyrates ; pharmacology ; Chromatin Immunoprecipitation ; methods ; Histones ; chemistry ; Humans ; K562 Cells ; Promoter Regions, Genetic ; genetics ; Real-Time Polymerase Chain Reaction ; methods ; gamma-Globins ; genetics

Selenoprotein S (SelS) is widely expressed in diverse tissues where it localizes in the plasma membrane and endoplasmic reticulum. We studied the potential function of SelS in erythrocyte differentiation using K562 cells stably overexpressing SelS wild-type (WT) or one of two SelS point mutants, U188S or U188C. We found that in the K562 cells treated with 1 microM Ara-C, SelS gradually declined over five days of treatment. On day 4, intracellular ROS levels were higher in cells expressing SelS-WT than in those expressing a SelS mutant. Moreover, the cell cycle patterns in cells expressing SelS-WT or U188C were similar to the controls. The expression and activation of SIRT1 were also reduced during K562 differentiation. Cells expressing SelS-WT showed elevated SIRT1 expression and activation (phosphorylation), as well as higher levels of FoxO3a expression. SIRT1 activation was diminished slightly in cells expressing SelS-WT after treatment with the ROS scavenger NAC (12mM), but not in those expressing a SelS mutant. After four days of Ara-C treatment, SelS-WT-expressing cells showed elevated transcription of beta-globin, gamma-globin, epsilon-globin, GATA-1 and zfpm-1, whereas cells expressing a SelS mutant did not. These results suggest that the suppression of SelS acts as a trigger for proerythrocyte differentiation via the ROS-mediated downregulation of SIRT1.
beta-Globins ; Cell Cycle ; Cell Membrane ; Cytarabine ; Down-Regulation ; Endoplasmic Reticulum ; epsilon-Globins ; Erythrocytes ; gamma-Globins ; K562 Cells ; Selenoproteins

OBJECTIVETo investigate the effects of combined use of low-dose hydroxyurea (HU) and sodium butyrate (NaB) on the expression of 7 globin genes (zeta, alpha, epsilon, Ggamma, Agamma, delta, and beta) in human erythroid progenitor cells.

METHODSHuman erythroid progenitor cells were cultured using a two-step liquid culture system and treated with HU and NaB either alone or in combination. The inhibitory effects of the agents on the cell growth were monitored with trypan blue exclusion assay, and the changes in the mRNA of the 7 globin genes were detected using RT-PCR.

RESULTSLow-dose HU combined with NaB resulted in significantly lower inhibition rate of the erythroid progenitor cells than routine dose HU and NaB used alone (28.56% and 38.80%, respectively, P<0.05). Compared with untreated cells (0.653-/+0.092 and 0.515-/+0.048), HU combined with NaB significantly increased the expression of Ggamma-and Agamma- mRNA (1.203-/+0.018 and 0.915-/+0.088, respectively, P<0.05), and HU and NaB used alone produced similar effects (1.305-/+0.016 and 0.956-/+0.029 for HU, and 1.193-/+0.070 and 0.883-/+0.012 for NaB, P>0.05). HU and NaB, either used alone or in combination or at different doses, caused no significant changes in the other globin genes (zeta, alpha, epsilon, delta and beta) (P>0.05).

CONCLUSIONLow-dose HU combined with NaB can up-regulate gamma globin gene expression, especially Ggamma-mRNA expression, to decrease the growth inhibition on human erythroid progenitor cells in vitro, but produces no significant effect on the expressions of zeta, alpha, epsilon, delta and beta genes.

Anemia, Sickle Cell ; genetics ; Butyrates ; administration & dosage ; pharmacology ; therapeutic use ; Cells, Cultured ; Drug Therapy, Combination ; Erythroid Precursor Cells ; cytology ; drug effects ; physiology ; Erythropoiesis ; drug effects ; Humans ; Hydroxyurea ; administration & dosage ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism ; gamma-Globins ; genetics ; metabolism