Inhibition of α-globin gene expression by RNAi

Biochemical and Biophysical Research Communications 369 (2008) 935–938

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Inhibition of a-globin gene expression by RNAi Orawan Sarakul a,1, Phantip Vattanaviboon a,*, Prapon Wilairat b, Suthat Fucharoen c, Yasunobu Abe d, Koichiro Muta d,y a

Department of Clinical Microscopy, Faculty of Medical Technology, Mahidol University, Siriraj Campus Bangkoknoi, Bangkok 10700, Thailand Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand c Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Nakornpathom 73170, Thailand d Department of Medicine and Bioregulatory Science, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan b

a r t i c l e

i n f o

Article history: Received 1 February 2008 Available online 5 March 2008 Keywords: RNAi siRNA a-Globin Thalassemia Erythroid precursor cell

a b s t r a c t RNA interference (RNAi), a process by which target messenger RNA (mRNA) is cleaved by small interfering complementary RNA (siRNA), is widely used for investigations of regulation of gene expression in various cells. In this study, siRNA complementary to 50 region of exon II of a-globin mRNA was examined for its role in erythroid colony forming cells (ECFCs) isolated from normal peripheral blood donor. On day 6 of cell culture, 1  106 ECFCs were transfected with lipofectamine-containing a-globin specific siRNA. After 48 h of transfection, a-globin specific siRNA produced significantly reduction of a-globin mRNA level in a dose-dependent manner, but it did not affect the level of b-globin mRNA. Significantly, decreased numbers of hemoglobinized erythroid cells relative to the control were observed supporting the inhibitory effect of this a-globin mRNA specific siRNA. Ó 2008 Elsevier Inc. All rights reserved.

RNA interference (RNAi) has recently emerged as a powerful tool for post-transcriptional gene silencing, which have been applied to various mammalian cells [1–3]. The mechanism of RNAi involves the generation of small interfering RNA (siRNA), which assembles with proteins into an RNA-induced silencing complex (RISC) that targets complementary mRNA for degradation [4,5]. This technique has been developed into an effective tool to specifically down-regulate gene expression in a wide variety of target cells as a means for specific therapeutic intervention in preclinical models of diseases characterized by aberrant gene expression [3]. In Thailand, thalassemia is the most common genetic disorder that poses an important health problem for the country [6,7]. Absence or decreased globin synthesis in thalassemia leads to unmatched globins that results in symptoms ranging from clinically mild anemia to lethality of the patients [6,7]. An excess of unmatched a-globin chains in b-thalassemia gives rise to ineffective erythropoiesis, due to an arrest of b-thalassemic normoblast stages in bone marrow, and also to reduced lifespan of circulating b-thalassemic erythrocytes in peripheral blood [8]. Therefore, restoration of a more balanced globin expression in b-thalassemic erythroid precursor cells should result in improve-

* Corresponding author. Fax: +66 2 412 4110. E-mail address: [email protected] (P. Vattanaviboon). 1 Present address: School of Allied Health Science and Public Health, Walailak University, Nakhonsithammarat, Thailand. y Deceased. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.02.124

ment in cell pathology. Towards achieving this goal, in this study we have examined the ability of a siRNA, complementary to 50 region of exon II of a-globin mRNA, to specifically inhibit a-globin gene expression in normal erythroid precursor cells in vitro. Materials and methods Peripheral blood samples. Peripheral blood buffy coat samples were obtained from normal, healthy Japanese volunteers with informed consent. The experiment was approved by the Institutional Board for Human Research, Kyushu University. All samples were free from hepatitis and HIV. siRNA design. An a-globin specific siRNA was designed using algorithm available at Ambion website (www.ambion.com), following published siRNA design guidelines [9,10]. Analysis revealed three potential target sites of siRNA on a-globin gene located at codons 41–47 (sequence no. 1), codons 97–103 (sequence no. 2) and codons 127–133 (sequence no. 3). Preliminary studies showed that only siRNA from sequence no. 1 (located in the 50 region of exon II) significantly reduced a-globin gene expression (data not shown). Therefore, sequence no. 1 (sense strand 50 -GAC CUACUUCCCGCACUUCTT-30 and antisense strand 50 -GAAGUGCGGGAAGUAGGUC TT-30 ) was used in the subsequent experiments. Isolation and culture of ECFCs. Erythroid colony forming cells (ECFCs) were prepared and cultured as previously described [11,12]. ECFCs were isolated from peripheral blood mononuclear cells by negative cell selection through Vario-MACS column (Miltenyi Biotech, USA) after incubating the cells with anti-CD3, -CD11b, -CD15, and -CD45RA antibodies conjugated to immunomagnetic beads (Miltenyi Biotech, Germany). ECFCs in filtrate were cultured in Isocove’s modified Dulbecco medium (IMDM; Gibco BRL, USA), containing 15% heat-inactivated fetal calf serum (FCS; Commonwealth Serum Laboratories, Australia), 15% pooled human AB serum, 2 U/ml of recombinant human erythropoietin (rhEPO, kindly provided by Chugai Pharmaceutical Co. Ltd., Japan), 20 ng/ml of recombinant human stem cell factor (rhSCF), 10 ng/ml of recombinant human interleukin-3 (rhIL-3) (R&D Systems, Inc., USA, kindly provided by Kirin-Brewery Co. Ltd., Japan), 100 U/ml penicillin

O. Sarakul et al. / Biochemical and Biophysical Research Communications 369 (2008) 935–938

Results Effect of a-globin siRNA on a- and b-globin mRNA level The optimal time for transfection was determined by transfecting ECFCs on day 6 of culture with a-globin specific siRNA–lipofectamine complex (1.0:5.0 lg) and incubated for 24, 48, and 96 h before RNA was extracted and a-globin mRNA level was measured by quantitative reverse-transcription PCR relative to untransfected cells. Level of a-globin mRNA at 24, 48, and 96 h was 81.0%, 55.3%, and 78.4% (average of two independent measurements) of control, respectively. Therefore, mRNA was quantitated after 48 h transfection in all the following experiments. ECFCs were then transfected with 5 lg of lipofectamine-containing varying amounts (0.2, 0.5, and 1.0 lg) of a-globin siRNA. After 48 h, total RNA was extracted, and mRNA of a- and b-globin were reversely transcribed and levels of cDNA were determined by quantitative PCR. As shown in Fig. 1A, a-globin siRNA could spe-

A

120 1

Normalized α-globin mRNA level

α/β globin mRNA ratio 0.83 0.98 0.53

100 80 60 40 20

un Co tra nt ns rol fe ct ed Li po fe c on tam ly in e

0

B

0.44

1.0 0.5 0.2 α-globin siRNA (μg)

un Co tra nt ns rol fe ct ed Li po fe c on tam ly in e

and 100 lg/ml streptomycin (Gibco BRL, USA) in a high-humidity incubator at 37 °C under 5% CO2/95% air (defined as day 0). On day 3, ECFCs were enriched and cultured until day 6 as described above, but without rhIL-3. siRNA-transfection. The 6-day cultured cells were collected and incubated in serum-free medium composed of 50% IMDM/50% F-12 [HAM] medium (Sigma, USA) containing 1% detoxified bovine serum albumin (BSA; Stem Cell Technologies Inc., Canada), 300 lg/ml of iron-saturated transferrin (Boehringer Mannheim, Germany), 2 U/ml of rhEPO, and 20 ng/ml of SCF at 37 °C under high-humidity 5% CO2 and 95% air [13]. siRNA was transfected into cells by lipofectamineTM 2000 solution (Invitrogen, USA) as previously described [13,14]. In brief, 1 ml of ECFCs (1  106 cells in serum-free medium) was placed into each well of 12-well plate. Lipofectamine (5 lg) was diluted in 250 ll of Opti-MEM I reduced serum medium (Gibco BRL, USA) and incubated for 5 min at room temperature. siRNA was diluted in 250 ll of OptiMEM-I to the assigned amount (0, 0.2, 0.5, and 1.0 lg), before being mixed with diluted lipofectamine and incubated for 20 min at room temperature. Lipofectamine–siRNA complex was added to each well of ECFCs and the solution was mixed gently. Transfected cells were incubated at 37 °C in 5% CO2 incubator and at indicated time (24, 48, and 96 h) cells were harvested for determination of mRNA level and hemoglobinized cells. Determination of cell viability, apoptosis, and cell development. On the day of mRNA assay, erythroid cell morphology was determined for cell development using May–Gruenwald’s–Giemsa staining (Merck, Germany) for differential cell count. Two hundred cells were counted and percent cells of each stage were calculated. Cell viability was measured by 1% trypan blue vital staining (Sigma, USA). Percent dye-excluded cells from 500 cells were determined in a hemocytometer under a light microscope. Cell apoptosis was assayed by annexin V-FITC staining for phosphatidylserine membrane externalization and by propidium iodide for nuclei staining following manufacturer’s protocol (Immunotech, France). Then fluorescence positive cells were analyzed by the EPICS Elite ESP flow cytometer (Beckman Coulter, USA). Determination of mRNA level using real time quantitative PCR. Total RNA was extracted from ECFCs using TRIzol reagent according to manufacturer’s instruction (Invitrogen, USA). First-strand cDNA was synthesized from 1 lg of total RNA using a reverse transcriptase-polymerase chain reaction (RT-PCR) kit (Takara, Japan). The reaction was started by incubating at 30 °C for 10 min, followed by 42 °C for 30 min, and finally 99 °C for 5 min. For detection and quantification of mRNA levels of either a- or b-globin, Light Cycler (Roche Diagnostics, Germany) technology was performed according to the manufacturer’s instructions. PCR primers for a-globin and b-globin mRNA determination were as follows: a-globin: 50 -ATGGTGCTG TCTCCTGCCGAC-30 (sense strand), 50 -GGGTCACCAGCAGGCAGTGG-30 (antisense strand); b-globin: 50 -AGGTTCTTTGAGTCCTTTG-30 (sense strand), 50 -AGCCACCACT TTCTGATAG-30 (antisense strand) [15]. The cycling condition was initial heating at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 65 °C for 15 s (for determination of a-globin mRNA level) or 54 °C for 15 s (for determination of b-globin mRNA level), and 72 °C for 13 s. Target quantities were normalized against standard curve constructed from serial dilution of standard a-globin cDNA amplification and plotting the threshold cycle of each standard against the DNA concentration. Determination of benzidine positive cells. Hemoglobinized cells were detected by benzidine staining [11]. Cells (104–105 cells per slide, prepared by cytocentrifugation) were fixed with methanol and stained with 2% 3,30 -dimethoxybenzidine in methanol for 15–20 min, followed by washing in freshly prepared H2O2 solution (30% H2O2:CH3OH:H2O = 1:22.5:16.5). Four hundred cells were counted under a light microscope and percent stained cells calculated based on intensity grade (1 + for pale staining and 2 + for strong staining). Statistical analysis. Statistical analysis was performed using paired t-test. p < 0.05 was considered statistically significant.

Normalized β-globin mRNA level

936

0.2

120 100 80 60 40 20 0 0.5

1.0

α-globin siRNA (μg)

Fig. 1. Determination of the levels of a-globin and b-globin mRNA. The levels of a-globin (A) and b-globin (B) mRNA relative to untransfected cell control were measured. Various amounts of a-globin siRNA (0.2, 0.5, and 1.0 lg) were transfected into the 6-day cultured erythroid colony forming cells by lipofectamide. The mRNA levels were determined after 48 h incubation. The numbers above of the bars represent a/b globin mRNA ratio. Data are means ± SD of three independent experiments.

cifically suppress a-globin mRNA level in a dose-dependent manner (p < 0.05 for all siRNA concentrations compared to both controls), while the level of b-globin mRNA was not affected (Fig. 1B). When the mRNA levels were normalized with respect to untransfected control, a/b-globin mRNA ratio decreased inversely with increasing amounts of a-globin siRNA–lipofectamine complex used. The a/b-globin mRNA ratios were 0.83 ± 0.05, 0.53 ± 0.09, and 0.44 ± 0.08 for 0.2, 0.5, and 1.0 lg of the transfected complex, respectively (Fig. 1). Effect of a-globin siRNA on hemoglobin synthesis ECFCs were collected after 48 and 96 h of transfection with a-globin specific siRNA–lipofectamine complex (1.0:5.0 lg) and stained with benzidine staining to detect hemoglobinized cells (Fig. 2). ECFCs were classified into three groups based on their ben-

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Control

apoptosis analysis. siRNA–lipofectamine transfection had neither effect on cell viability (90 ± 3% viable transfected cells compared to 93 ± 2% of untransfected control, p > 0.05) nor cell apoptosis (2.4 ± 1.7% apoptotic transfected cell compared to 2.7 ± 1.3% of untransfected control, p > 0.05). Non-significant delay of cell development from basophilic normoblast to polychromatic normoblast was observed by differential cell counting of May–Gruenwald’s– Giemsa stained cells (Table 2).

1+

2+

Lipofectamine

α-globinsiRNA

Negative

48 h

A

B

C

D

E

F

96 h

Fig. 2. Determination of hemoglobinized cells. Erythroid colony forming cells were transfected with 1.0 lg a-globin siRNA–lipofectamine complexes and hemoglobinized erythroid cells were detected by benzidine staining after 48 h (C) and 96 h (F) incubation. (A) and (D) are untransfected cell controls while (B) and (E) are cells treated with lipofectamine only and incubated for 48 and 96 h, respectively. The upper panels represent cells giving negative, 1+ and 2+ positive for benzidine staining, respectively.

zidine staining property. Strongly stained, pale stained, and unstained cells were classified as 2+ positive, 1+ positive, and as negative cells, respectively [11]. All groups were counted and calculated in percentages as shown in Table 1. Decrease in numbers of benzidine positive cells was observed at 48 h after transfection and became more obvious at 96 h. Significantly decreased numbers of benzidine positive cells, especially strongly stained cells at 96 h after transfection supported the inhibitory function of a-globin siRNA (p < 0.05). A reduction in the intensity of benzidine staining indicates a lowering in the amount of hemoglobin in transfected ECFC, mimicking the situation in thalassemic erythroblasts. Effect of a-globin siRNA on cell viability, apoptosis and cell development After transfection by siRNA–lipofectamine (1.0:5.0 lg) complex for 48 and 96 h, ECFCs were stained with trypan blue for cell viability measurement and with annexin V-FITC/propidium iodide for

Discussion Transfection of erythroid colony forming cells in vitro with siRNA complementary to a-globin mRNA was able to specifically reduce the level of a-globin mRNA, and not that of the closely related b-globin mRNA. Amount of siRNA used as well as duration of transfection are two additional parameters that need to be considered. Although a dose–response effect was obtained up to 1 lg of siRNA, the suppression function on mRNA level was not increased when 5 lg of the siRNA was applied indicating a saturation phenomenon (data not shown). Reduction of a-globin mRNA level was greatest after 48 h of transfection and rebounded at 96 h. Usually, siRNA inhibits mRNA expression during 2–4 days of lipofectamine transfection [14]. The stage of ECFCs development was mainly pronormoblast (proerythroblast) after day 6 which differentiated to basophilic normoblast and polychromatic normoblast on day 8 and to mostly polychromatic normoblast on day 10 of culture [11,16]. The cells then continue proliferating and differentiating to orthochromatic normoblast, reticulocyte, and finally erythrocyte. Production of globin protein begins in the basophilic normoblast stage and ends in the reticulocyte. The reticulocyte is able to synthesize hemoglobin for approximately 2 days after the cell has been lost its nucleus [17]. This implies the inhibitory effect of a-globin siRNA still remained at rebounding phase. A significant decrease in the numbers and intensity of benzidine positive siRNA-transfected ECFC (Fig. 2, Table 1) strongly suggest that the siRNA successfully lowered the level of hemoglobin in the transfected cells. The reduction of intracellular hemoglobin was likely due to inhibition of a-globin synthesis because b-globin gene expression was not affected by this siRNA (Fig. 1B). Cationic lipid lipofectamine was chosen to introduce siRNA into erythroid precursor cells by transfection. Alternative delivery systems, such as electroporation, delivery via PCR-based amplification of siRNA expression cassette, viral vectors (viz. lentiviral, retroviral and adenoviral vectors), have also been used for transferring siRNA into host cells [18,19]. The efficiency of lipofectamine transfection in our hands using the well-established siRNA suppressed lamin A/ C system (Lamin A/C siRNA-FITC, Greiner Bio-One, Japan) varied from 26% to 56%, depending on the amounts of lipofectamine and siRNA used (data not shown). Under the optimal condition

Table 1 Percentage of hemoglobinized cells at 48 and 96 h after transfection determined by benzidine staining method Conditions

Time(h)

Hemoglobinized cells (mean percentage ± SD)a Benzidine negative

Benzidine positive 2+

Total

Untransfected control

48 96

35 ± 3 21 ± 3

23 ± 3 15 ± 4

42 ± 6 64 ± 1

65 ± 3 79 ± 3

Lipofectamine only

48 96

32 ± 4 23 ± 2

26 ± 4 17 ± 5

42 ± 8 60 ± 3

68 ± 4 77 ± 2

a-Globin siRNA–lipofectamine complex

48 96

40 ± 4* (p = 0.01) 47 ± 5* (p = 0.001)

26 ± 3 11 ± 2

34 ± 7* (p = 0.005) 42 ± 7* (p = 0.004)

60 ± 4* (p = 0.013) 53 ± 5* (p = 0.002)

a *

1+

Data from three independent experiments. Significantly different compared with untransfected control at the same period of time (p < 0.05). Number in parenthesis represents p-value.

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Table 2 Stages of cell development at 96 h after transfection Conditions

Untransfected control Lipofectamine only a-globin siRNA–lipofectamine complex a

Stages of cell development (mean percentage ± SD)a Pronormoblast

Basophilic normoblast

Polychromatic normoblast

Orthochromatic normoblast

5±1 4±2 5±1

18 ± 4 21 ± 1 25 ± 4

76 ± 5 74 ± 1 69 ± 3

1 1 1

Data from three independent experiments.

using 5 lg of lipofectamine and 1.0 lg of siRNA, the transfection efficiency of up to 56% was obtained. Lipofectamine treatment did not affect erythroid colony forming cell viability or caused any increase in apoptosis compared to control untransfected cells. Transfection via liposome is recommended as a routine procedure as it is easy to perform and less harmful to host cells, although it is a transient phenomenon. PCR-based expression cassettes provide longer function of siRNA in cells [18]. Samakoglu et al. [20] have demonstrated the therapeutic potential of coregulating c-globin transgene expression with bS-globin RNAi to achieve c-globin production and reduction of bS-globin transcription in sickle cell anemia. The reduction of b-globin chain synthesis leading to the presence of unmatched unstable a-hemogobin, which binds and causes oxidative damage to red cell membrane, explains the more severe anemia of b-thalassemia syndrome in comparison with a-thalassemia where the unmatched b-hemoglobin monomers can form the relatively stable homotetramer hemoglobin H. Thus, a reduction in a-globin synthesis by this specific a-globin siRNA may ameliorate the severity of b-thalassemia by lowering the level of unmatched a-globin chains. This condition mimics the co-inheritance of both a- and b-thalassemia in which milder clinical manifestations have been observed [21]. In summary, inhibition of a-globin mRNA expression developed in this study can also be used as a convenient model for studying pathophysiology of a-thalassemic erythroid precursor cells in vitro, and may possibly be applied in reducing a-globin levels in severe forms of b-thalassemia in a clinical setting. Acknowledgments This work was supported by Japan Society for the Promotion Science (JSPS) and Japan Science and Technology Agency (JST) to K.M. and P.V., and by the Thailand Research Fund Senior Researcher Fellowship to S.F. References [1] A. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, C.C. Mello, Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature 391 (1998) 806–811.

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