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SOX6 Downregulation Induces y-Globin in Human [beta]-Thalassemia Major Erythroid Cells.

1. Introduction

Beta-thalassemia syndromes are a group of hereditary blood disorders characterized by reduced or absent [beta]-globin chain synthesis, resulting in anemia [1]. Clinical evidence indicates that elevated fetal hemoglobin (HbF; [[alpha].sub.2][[gamma].sub.2]) production mitigates the severity of [beta]-thalassemia [2]. Patients with two [beta]-thalassemia alleles combined with hereditary persistence of fetal hemoglobin (HPFH) allele have milder clinical presentations [3,4]. Therefore, HbF is a major modifier of the severity in [beta]-thalassemia.

The predominant globin in normal infants is [gamma]-globin, which switches to [beta]-globin few months after birth, by a process known as the fetal-to-adult hemoglobin switch [5]. Thus, understanding the molecular mechanisms responsible for [gamma]- to [beta]-globin switching and increasing [gamma]-globin gene expression as a potential therapy for [beta]-hemoglobinopathy is critical. Recent studies on [gamma]-globin expression have shed new light on this complex regulatory process.

Sry type HMG box (SOX6) is a chromatin-associated protein (member of the SRY-related high mobility group (HMG) box transcription factors) that binds and induces a marked bending of DNA [6]. Recently, SOX6 was shown to stimulate erythroid cell survival, proliferation, and terminal maturation during definitive murine erythropoiesis [7, 8]. In 2006, SOX6 was first identified as a novel and crucial silencing factor of eyglobin in mice. SOX6 acts as a repressor by directly binding to the ey promoter [9]. SOX6 and BCL11A, the latter being a master repressor of [gamma]-globin [10-12], cooccupy the human globin cluster along with GATA1 and cooperate in silencing [gamma]-globin transcription in adult human erythroid progenitors [13]. SOX6 could be a potentially promising target for [gamma]-globin induction. There is no report yet on downregulation of SOX6 to induce [gamma]-globin expression in human erythroblasts derived from [beta]-thalassemia major.

Taken together, SOX6 was chosen as target mediator to induce [gamma]-globin expression in human erythroblasts derived from [beta]-thalassemia major.

2. Materials and Methods

2.1. Cell Culture. Mononuclear cells (MNC) were isolated from normal human peripheral blood (PB) and thalassemia major patients bone marrow (BM) by density gradient centrifugation with Ficoll-Hypaque solution (Solarbio life sciences). The cells were incubated in a one-stage liquid culture system (Table 1).

Three normal donors and three [beta]-thalassemia major patients were analyzed. Each donor's cells were separately cultured. Cells were incubated at 37[degrees]C in a humidified atmosphere of 5% C[O.sub.2] and maintained at a density of 5 x [10.sup.5] cells/ml by supplementing cultures with fresh media every four days. Cell numbers and viability were determined by trypan blue exclusion. Cell morphology was assessed by phase-contrast microscopy (Olympus CKX41) and Wright-Giemsa staining of cytocentrifuge preparations.

The information on the cultured cells is shown in Supplementary Figures 1-5.

We also tried to purify [CD34.sup.+] cells from MNCs by immunomagnetic beads separation technique. The harvested [CD34.sup.+] cells were cultured till the end stage but did not reach enough numbers to extract mRNA or protein. So, MNC was chosen for culture.

K562 cells (gift from Institute of Hematology, Chinese Academy of Medical Sciences) were incubated in DMEM (Invitrogen) with 10% fetal calf serum (Invitrogen).

There were three groups of cells: [1] control group (C), untreated; [2] control vector group (CV), treated with control lentiviruses; and [3] sh-RNA group (SH), treated with lentiviruses containing target shRNA.

The research protocol was approved by the ethics committee of Guangxi Medical University in China.

2.2. Lentiviral shRNA and Lentivirus Production and Transduction. Lentiviral shRNA constructs in the pLKO.1-PURO vector were purchased from Sigma[R] Life Science. The pLenti4/BLOCK-IT[TM]-DEST vector (gift from Tsinghua University in China) was used to produce control lentiviruses. We selected two different shRNA sequences for SOX6 and then chose the sequence with the most obvious inhibitory effect. The shRNA sequence is listed in Table 2.

293FT cells were transduced with 10 [micro]g pLKO.1PURO/pLenti4/BLOCK-IT-DEST vector, 15 [micro]g VSVG 1.0 vector, and 15 [micro]g [DELTA]8.9 vector. The cells were incubated with 20 ml Opti-MEM (Gibco) and 120 [micro]l Lipofectamine 2000 (Invitrogen) solutions for six hours, after which the media was replaced. After 72 hours, media containing virus was filtered and collected. The virus titer was determined by crystal violet staining method and was found to be 5 x 106 TU/ml.

For transduction, MNCs were grown for 0 and 24 hours at a concentration of 5 x [10.sup.5] cells/ml in media incubated overnight with vector particles (multiplicity of infection [MOI], 10). Cells at end stage of differentiation (the 15th day) were harvested to extract mRNA.

K562 cells were incubated with the viruses at a concentration of 5 x [10.sup.5] cells/ml (MOI, 10) overnight. Transduced cells were selected by puromycin. On the 12th day of culture, the cells were harvested to extract mRNA and protein.

2.3. Western Blot. Whole cell extracts were obtained by lysis in RIPA buffer (Table 3). A total of 60 [micro]g of protein was diluted with 6x SDS-loading buffer, denatured by boiling for five minutes, and resolved on a 10% or 12% SDS-PAGE gel. Proteins were then transferred to a PVDF membrane by wet transfer procedure. The membrane was incubated with relevant primary antibodies overnight at 4[degrees]C. This was followed by incubation with anti-rabbit or anti-goat horseradish peroxidase- (HRP-) conjugated secondary antibodies (1: 3000 dilutions) in 5% milk in 1x TBS and 0.1% Tween-20 for one hour.

Antibodies against [gamma]-hemoglobin (ab137096) was purchased from Abcam. Antibody against SOX6 (A303-553A) was purchased from Bethyl. Antibody against GAPDH (60004-I-Ig) was purchased from Proteintech. Molecular weights and dilution ratios of primary antibodies are listed in Table 4.

2.4. Flow Cytometry. Cells (50-100,000) at the end stage of differentiation (the 15th day) were rendered into single-cell suspensions for flow cytometric analysis. Cells were tested for expression of cell surface receptors with antibodies specific for CD235a (BD Biosciences) conjugated to fluorescein isothiocyanate on a FACS Calibur System (BD Biosciences) using CellQuest analysis software (BD Biosciences). Isotype control was also used. CD235a is a type I transmembrane sialoglycoprotein that is expressed on human erythrocytes and is useful for their identification and characterization.

2.5. RNA Isolation and qPCR. RNA was isolated using the RNeasy mini kit (Qiagen) according to the manufacturer's protocol. cDNA was synthesized with the iScript cDNA synthesis kit (Bio-Rad). Quantitative real-time PCR (qPCR) was performed using the Faststart Universal SYBR Green (Roche). Amplification reactions were performed with an Optical IQ Thermal Cycler (Bio-Rad) with the following conditions: 95[degrees]C for 10 minutes, followed by 40 cycles of 95[degrees]C for 15 seconds and 60[degrees]C for 60 seconds. All reactions were performed in triplicate. Relative gene expression was quantified using the [DELTA][DELTA]Ct method. Target gene expression was normalized to GAPDH expression, unless indicated otherwise. The relative values of [gamma]-globin and [beta]-globin mRNA for each sample were calculated using the Ct method using GAPDH transcript signal as an internal control. Primers used in this study are listed in Table 5.

2.6. Statistical Analysis. Statistical analysis of gene expression data obtained from qPCR was performed with the one-way ANOVA test and SPSS16.0 statistical software. All results are means [+ or -] SD from three independent experiments. A value of two-sided p less than 0.05 was considered statistically significant.

3. Results

K562 cell line is an erythroleukemic human "fetal" erythroid cell line, expressing [gamma]-globin gene dominantly and a little [beta]-globin gene. First, we selected this cell line to observe the effect of downregulation of SOX6 on [gamma]-globin expression.

We then repeated the procedure in primary adult erythroid cells from normal donors or patients with [beta]-thalassemia major. Human cytokine-mobilized adult PB MNCs from 3 normal donors and human [beta]-thalassemia major bone marrow MNCs from 3 patients were transduced with vector. Three of the thalassemia major patients were compound heterozygous for [[beta].sup.0/] [[beta].sup.+] mutation. Each donor's cells were separately cultured. MNCs were placed in liquid cultures that promote unilineage erythroid differentiation. Erythroid cell differentiation was analyzed by cell morphology (Wright-Giemsa staining), CD235a expression (qPCR and flow cytometry), and hemoglobin gene expression (qPCR). At the end of the culture period, about 90% of the cultured cells were erythrocytes (the information on the cultured cells is shown in Supplementary Figures 1-5). Taken together, these data show effective erythropoiesis in vitro.

Downregulation of SOX6 expression in K562 cells and primary adult erythroid cells from normal donors or patients with [beta]-thalassemia major led to elevated [gamma]-globin expression.

In K562 cells, the effects of SOX6 knockdown on altering the expression of [gamma]-globin were analyzed by qPCR and WB. As shown in Figure 1(a), [gamma]-globin mRNA expression showed 2.15-fold increase in shRNA group compared to control group. WB analysis also demonstrated elevated yglobin protein level in shRNA group, as shown in Figure 1(b).

In primary adult erythroid cells, the effects of SOX6 knockdown on altering the expression of [gamma]-globin were analyzed only by qPCR on day 15 of erythroid differentiation due to the reasons mentioned above. As shown in Figure 1(c), [gamma]-globin mRNA expression showed 3.35-fold increase in shRNA group compared to control group in primary adult erythroid cells from normal donors. And in erythroid cells from patients with [beta]-thalassemia major, [gamma]-globin mRNA expression showed 1.97 fold increase in shRNA group compared to control group, as shown in Figure 1(d).

In shRNA group, there were significant differences in yglobin expression as compared to the control group or control vector group (p < 0.01) both in K562 cells and in primary adult erythroid cells from normal donors or patients with [beta]-thalassemia major (Figures 1(a), 1(c), and 1(d)).

Erythroid differentiation was not affected by SOX6 knockdown as all cultures showed similar levels of CD235 positivity, as shown in Figures 1(e) and 1(f). In primary adult erythroid cells transduced with SOX6 shRNA, there were no significant differences in CD235a expression as compared to the control group or control vector group (Figure 1(f)).

Each normal donor and [beta]-thalassemia major patient data were listed in Table 6.

4. Discussion

[beta]-thalassemia remains a major global health challenge, particularly in the developing countries. Its clinical management includes supportive care and iron chelation. Bone marrow transplantation provides a genetic cure but cannot be widely applied [14]. Several important natural observations demonstrated that the severity of [beta]-thalassemia could be ameliorated via increased production of [gamma]-globin [2-4]. Modifier genes of [gamma]-globin and hemoglobin switching have been long studied due to their potential usage in developing targeted therapeutic approaches for [beta]-thalassemia [5].

SOX6 is a member of the SOX (Sry-type HMG box) family of transcription factors, characterized by the presence of an HMG domain that recognizes the minor groove on DNA. The binding of SOX proteins to DNA forces it to bend at about 75[degrees], introducing local conformational changes. The ability of SOX proteins to bind in close proximity to other transcription factors and to distort DNA suggests that they can act as "architectural proteins," possibly by promoting the assembly of biologically active multiprotein complexes. These complexes, in turn, mediate the interactions between distant chromatin domains, bringing together promoter/enhancer regions, finally assembling the "chromatin hubs" that control gene expression regulation [15-17]. The SOX6 transcription factor plays critical roles in various cell types, including erythroid cells [18]. A potential role for SOX6 in globin gene regulation was first recognized by analysis of the SOX6-deficient mouse. At the fetal liver stage, expression of mouse embryonic [beta]-like globins ([epsilon]y and [beta]h1) was dramatically elevated in the SOX6-deficient p100H mouse [9]. BCL11A is a critical mediator of [gamma]- to [beta] globin switching in mammals. Induction of [gamma]-globin by downregulation of BCL11A was demonstrated in primary human adult erythroid progenitors and mice [10-12]. A recent study suggests that transcriptional silencing of [gamma]-globin by BCL11A involves long-range interactions and cooperation with SOX6. BCL11A mediates silencing of [gamma]-globin genes through both long-range interaction within the human [gamma]-globin cluster and local interactions with the chromatin-associated SOX6 proteins at the proximal promoters of the [gamma]-globin genes [13]. SOX6, by interacting with y-proximal promoters, may help recruit BCL11A to the proximal regions of the y-genes during hemoglobin switching [13]. SCF-mediated [gamma]-globin gene expression in adult human erythroid cells is also associated with SOX6 downregulation [19]. Taken together, SOX6 may also be a potentially promising target for HbF induction.

Our data showed that downregulation of SOX6 induced production of [gamma]-globin in K562 cells and primary adult erythroid cells from normal donors or patients with thalassemia major. Downregulation of SOX6 did not impair differentiation of erythroblast, which is consistent with previous report [13]. This is the first report on the induction of yglobin by downregulation of SOX6 in primary adult erythroid cells from patients with [beta]-thalassemia major.

Besides its role in hemoglobin regulation, SOX6 also plays an important role in the development of the central nervous system [20], cartilage [21], and muscle [22]. Murine SOX6 null mutants (p100H) show delayed growth, myopathy, and atrioventricular heart block and die within 2 weeks following birth [23]. Previous studies on the role of SOX6 in hemoglobin regulation have not revealed any impact of its downregulation on other systems.

5. Conclusions

So, SOX6 is a critical regulator of [gamma]-globin. Our data and previous studies suggest that this transcription factor is promising targets to influence [gamma]-globin expression for alleviating anemia in [beta]-thalassemia. Given that SOX6 is not erythroid specific, its safety and exact roles in [gamma]-globin regulation need to be intensively explored.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (Grant no. 81360085), Research Fund for the Doctoral Program of Higher Education of China (no. 20124503110009), and Foundation of Department for Science and Technology of Guangxi Zhuang Autonomous Region (no. Gui Ke Gong 1598011-1).

Supplementary Materials

Supplementary 1. Supplementary Figure 1: Representative cytospin images of proerythroblasts (Pro-E), basophilic erythroblasts (Baso-E), polychromatic erythroblasts (Poly-E), orthochromatic erythroblasts (Ortho-E), and mature erythrocytes.

Supplementary 2. Supplementary Figure 2: Proportion of erythrocytes in the differentiated cultured cells on day 5, day 10, and day 15, verified by cell count based on Wright-Giemsastained morphology (counts of cells include Pro-E, Baso-E, Poly-E, Ortho-E, and mature erythrocytes).

Supplementary 3. Supplementary Figure 3: Flow cytometry analysis showing CD235a expression in cultured cells after 15 days.

Supplementary 4. Supplementary Figure 4: qPCR showing CD235a expression in differentiated cultured cells on day 5, day 10, and day 15.

Supplementary 5. Supplementary Figure 5: qPCR showing [gamma]/[beta]-globin mRNA ratios in differentiated cultured cells on day 5, day 10, and day 15.

https://doi.org/10.1155/2017/9496058

References

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Jing Li, Yongrong Lai, Jun Luo, Lin Luo, Rongrong Liu, Zhenfang Liu, and Weihua Zhao

Department of Hematology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, China

Correspondence should be addressed to Yongrong Lai; laiyongrong@263.net

Received 2 June 2017; Revised 30 October 2017; Accepted 2 November 2017; Published 28 November 2017

Academic Editor: Wen-Hwa Lee

Caption: Figure 1: (a) qPCR analysis showed that downregulation of SOX6 expression in K562 cells induces [gamma]-globin production. (b) Western blot analysis showed that downregulation of SOX6 expression in K562 cells induces [gamma]-globin production. (c) qPCR analysis showed that downregulation of SOX6 expression in primary adult erythroid cells from normal donors induces [gamma]-globin production. (d) qPCR analysis showed that downregulation of SOX6 expression in primary adult erythroid cells from patients with [beta]-thalassemia major induces [gamma]-globin production. (e) Flow cytometry analysis showed that in cells from patients with [beta]-thalassemia major transduced with SOX6 shRNA, there are no obvious changes in CD235a expression as compared to the control group or control vector group. (f) qPCR analysis showed that, in primary adult erythroid cells transduced with SOX6 shRNA, there are no significant differences in CD235a expression as compared to the control group or control vector group. C group: control; CV group: control vector; SH group: shRNA. * p < 0.01.
Table 1: One-stage liquid culture system.

Reagent                Company                       Concentration

RPMI1640               Hyclone
Fetal calf serum       Invitrogen                    30%
Erythropoietin         Kyowa Hakko Kirin Co., Ltd.   3 units/ml
Dexamethasone          Sigma                         10-6 M
[beta]-Estradiol       Solarbio life sciences        10-4 M

Table 2: shRNA sequence.

shRNA targeting gene   shRNA sequence

SOX6                   CCGGCGTGAGATAATGACCAGTGTTCTCGAGAACACTGGTCATTA
                       TCTCACGTTTTT

Table 3: RIPA buffer.

50 mM Tris             0.6057 g                      1.51 g
150 mM NaCl            0.8766 g                      2.19 g
0.5% NaDeoxycholate    0.5 g                         1.25 g
1% NP40                1ml                           2.5ml
0.1% SDS               0.1 g                         0.25 g
pHto 8                 dH2O to 100 ml                dH2O to 250 ml

Table 4: Dilution ratio of primary antibody.

Antibody name          Molecular weight              Dilution ratio

SOX6                   96kDa                         1:1000
[gamma]-Hemoglobin     16 kDa                        1:1000
GAPDH                  36kDa                         1:3000

Table 5: Primer sequence.

Name of locus          Primes (5'-3')              Product size

SOX6                   CTCCTGCAGCAACAGATCCA        115 bp
                       AGAGGAATCCCTGTTGGGCA
[gamma]-Hemoglobin     TCGCTTCTGGAACGTCTGAG        157 bp
                       GTAGACAACCAGGAGCCTTCC
[beta]-Hemoglobin      GTCTACCCTTGGACCCAGAGGTTC    131 bp
                       TGAGCCAGGCCATCACTAAAG
GAPDH                  GTCAGCCGCATCTTCTTT          99 bp
                       CGCCCAATACGACCAAAT

Table 6: Information of each donor or patient (SOX6).

             [gamma]-Globin        SOX6                  HbF
             (fold increase)    (percentage           (baseline)
                                  knock down)

Donor 1      3.85                  48.7%                 1.1%
Donor 2      4.07                  55.2%                 0.8%
Donor 3      2.08                  35.1%                 2.3%
Patient 1    2.63                  29.9%                31.3%
Patient 2    2.08                  36.0%                45.7%
Patient 3    1.16                  41.7%                65.6%

             Genotype

Donor 1      /
Donor 2      /
Donor 3      /
Patient 1    [[beta].sup.0][[beta].sup.+]
Patient 2    [[beta].sup.0][[beta].sup.+]
Patient 3    [[beta].sup.0][[beta].sup.+]

Donor: health donor; Patient: [beta]-thalassemia major patient;
[gamma]-globin (fold increase): fold increase in [gamma]-globin
gene expression in shRNA group compared to control group; SOX6
(percentage knockdown): percentage knockdown of SOX6; HbF
(baseline): inherent baseline HbF percentage of each donor and
patient; genotype: genotype of each [beta]-thalassemia major
patient.
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Title Annotation:Research Article
Author:Li, Jing; Lai, Yongrong; Luo, Jun; Luo, Lin; Liu, Rongrong; Liu, Zhenfang; Zhao, Weihua
Publication:BioMed Research International
Article Type:Report
Date:Jan 1, 2017
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