Inhibition of Stat3 expression induces apoptosis and suppresses proliferation in human leukemia HL-60 cells

Abstract

Acute myeloid leukemia (AML) is the most common myeloid leukemia. It is highly malignant, thus, most patients with AML will relapse and die after traditional treatment. Stat3, a member of the signal transducer and activator of transcription (Stat) family, is involved in the development and progression of many tumors. The purpose of the study was to investigate whether the down-regulation of Stat3 expression by RNA interference is effective against human leukemia HL-60 cells. The results indicated that constitutively expressed Stat3 is present in human leukemia HL-60 cells, and the down-regulation of Stat3 expression caused significant induction of apoptosis as well as inhibition of proliferation in HL-60 cells. These data further demonstrated that Stat3 plays a critical role in human leukemia HL-60 cell apoptosis and proliferation. Thus, targeting Stat3 may be a useful adjunctive treatment strategy in AML.

Keywords:

• Acute myeloid leukemia
• Stat3
• RNA interference
• Apoptosis
• Proliferation

Introduction

Acute myeloid leukemia (AML), also known as acute myelogenous leukemia, is a malignant disorder of the blood that is characterized by maturation arrest of a malignant clone of myeloid cells.1 It is reported that AML accounts for 70% of newly diagnosed acute leukemias and its incidence increases with age.2^–4 Despite considerable advances in antileukemic chemotherapy, the relapse and the low survival rate of AML remain significant problems.5^–7 Hence, better approaches for the treatment of AML are needed.

In mammals, the Stat family consists of seven members: Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b, and Stat6.8 Stat3, a member of Stat family, participates in regulating cell apoptosis, proliferation, and differentiation, and is involved in the development and progression of many tumors.9^–13 Constitutive Stat3 activity has been observed in ∼50% of AML cases and patients whose leukemia cells exhibit constitutive Stat3 activity perform shorter disease-free survival than those without such constitutive activity.¹⁴14,15

In this report, we further testified whether constitutively expressed Stat3 is present in human leukemia HL-60 cells and examined whether the down-regulation of Stat3 expression by RNA interference (RNAi) could be able to affect the apoptosis and proliferation of human leukemia HL-60 cells.

Materials and Methods

Cell culture

Human leukemia HL-60 cell line was obtained from Academy of Sciences (Shanghai, China). Cells were grown as suspension culture in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco; Invitrogen; Carlsbad, CA, USA) in a humidified 37°C incubator and aired with 5% CO₂.

Construction of the short hairpin RNA (shRNA) expression plasmid

A double-stranded shRNA oligonucleotide was designed against Stat3 (5′-ACACCGCAGCTGCACCAGCTGTACTTGCTTGAAGTACAGCTGGTGCAGCTGCT-3′); (5′-AAAAAGCAGCTGCACCAGCTGTACTTCAAGCAAGTACAGCTGGTGCAGCTGCG-3′). This oligonucleotide was designed according to the protocol of SilenCircle RNAi transcription kit (Allele Biotechnology, San Diego, CA, USA). After being annealed at 95°C, the products were ligated into the pSilenCircle linearized vector. Then, the Escherichia coli transformation and plasmid DNA preparation were performed according to the standard protocols,16 and the shRNA expression vector sequence was identified.

Cell transfection

Before transfection, about 5×10⁵ cells were seeded into each well of a 24-well plate. FuGENE® HD (Roche Molecular Biochemicals, Indianapolis, IN, USA) was used as the transfection reagent according to the manufacturer’s instructions. In this study, pSilenCircle empty vector was used as a control.

Immunofluorescence staining

HL-60 cells were incubated on the poly-L-lysine pretreated coverslips overnight. After that, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 minutes, washed with PBS, permeabilized, and blocked in PBS/Triton containing 5% goat serum albumin at room temperature for 1 hour. Then, 1∶400 dilution of anti-Stat3 antibody (Cell Signaling Technology, Beverly, MA, USA) was added and the cells were incubated overnight at 4°C. In the following day, the cells were washed with PBS and 1∶200 dilution of fluorescein isothiocyanate (FITC)-conjugated secondary antibody was added. After incubation for 1 hour, the cells were washed with PBS, and then mounted with 50% glycerol in PBS. Fluorescence was observed by a Fluoview FV300 confocal laser scanning biological microscope (Olympus, Essex, UK).

Reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was extracted from cultured HL-60 cells with Trizol reagent. cDNA was synthesized from total RNA by using reverse transcription assay according to the previous protocol.17 Primers used to detect Stat3 expression were 5′-GGGTCTGGCTAGACAATATCATCG-3′ and 5′-CACTACCTGGGTCGGCTTCG-3′, and primers against beta-actin as a positive control were 5′-CCAAGGCCAACCGCGAGAAGATGAC-3′ and 5′-AGGGTACATGGTGGTGCCGCCAGAC-3′. After amplification, polymerase chain reaction products were electrophoresed on a 1% agarose gel and visualized by ethidium bromide staining.

Western blot

Total protein was extracted from cultured HL-60 cells with protein lysis buffer. The lysates were centrifuged at 12 000 rpm at 4°C for 10 minutes. Protein samples were electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to polyvinylidene fluoride membranes. The membranes were then incubated with anti-Stat3 antibody (Cell Signaling Technology) or anti-beta-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoreactive bands were detected by enhanced chemiluminescence detection system.

Cell apoptosis assay

HL-60 cells were cultured in 24-well plates. We used an Annexin V-FITC apoptosis detection kit (Bipec Biopharma Corporation, Cambridge, MA, USA) to quantify. In brief, we harvested 10⁶ cells, washed, and resuspended cells in 400 μl binding buffer containing 5 μl Annexin V-FITC, and subsequently incubated cells for 15 minutes at 4°C in the dark. After that, we observed Annexin V-FITC-positive cells under a fluorescence microscope. The percentage of apoptotic cells was determined for each of the treatments by counting five visual fields and at least 100 cells. The experiment was repeated three times.

Cell proliferation assay

HL-60 cells were cultured in 96-well plates. Cell proliferation ability was determined using MTS assay following the manufacturer’s directions (CellTiter 96 AQueous Non-radioactive proliferation kit; Promega, Son Luis, CA, USA). Absorption value was measured at 490 nm with a 96-well plate reader. The cell proliferation of samples was calculated by dividing the absorption value of samples with that of control. The experiment was repeated three times.

Statistical analysis

Data are expressed as mean±SEM. Statistical analysis of the data was performed by one-way analysis of variance. P<0·05 was considered to be significant.

Results

Constitutively expressed Stat3 proteins are present in HL-60 cells

Previous studies have shown that constitutively activated Stat3 is present in many human cancer cells and tumor tissues.18^–20 In the current study, the immunofluorescence staining method was used to further testify whether Stat3 proteins are constitutively or transiently expressed in HL-60 cells. The result shows no obvious change in the expression level of Stat3 protein with the passage of time (Fig. 1A).

Figure 1. (A) Photomicrograph of Stat3 proteins expressed in HL-60 cells. (B) The control of secondary antibody only. Bar = 50 μm.

RNAi specifically reduces the expression of Stat3 in HL-60 cells

The effect of RNAi was analyzed by RT-PCR, western blot, and immunofluorescence staining. The results of RT-PCR and western blot showed that Stat3 RNAi effectively down-regulated mRNA and protein expression of Stat3 in HL-60 cells. In contrast, the expression of beta-actin remained unaffected after transfection with Stat3 shRNA (Fig. 2A and B). Immunofluorescence staining further confirmed that Stat3 protein expression was markedly reduced in HL-60 cells after RNAi (Fig. 2C).

Figure 2. RT-PCR, western blot, and immunofluorescence staining showed that there was a reduction in the Stat3 expression level in the RNAi treatment group. (A) Results from RT-PCR. (B) Results from western blot. (C) Results from immunofluorescence staining. Bar = 50 μm.

Down-regulation of Stat3 by RNAi induces apoptosis of HL-60 cells

To examine whether down-regulation of Stat3 affects HL-60 cell apoptosis, Annexin V assay was performed. As depicted in Fig. 3, the percentage of apoptotic cells in the Stat3 RNAi treatment group was significantly higher than that in the control group. These results illustrated that the down-regulation of Stat3 resulted in induction of HL-60 cell apoptosis.

Figure 3. The effect of Stat3 RNAi on human leukemia HL-60 cell apoptosis was examined by using Annexin V assay. The statistical analysis showed that the apoptosis rate was increased after transfection with Stat3 shRNA (**P <0·01, ***P<0·001).

Down-regulation of Stat3 by RNAi inhibits proliferation of HL-60 cells

Seventy-two hours after transfection, the MTS assay was used to examine the cell proliferation. As shown in Fig. 4, the proliferation of HL-60 cells was significantly suppressed by the treatment with Stat3 shRNA, compared with control group cells. These results illustrated that the down-regulation of Stat3 resulted in the inhibition of HL-60 cell proliferation.

Figure 4. The effect of Stat3 RNAi on human leukemia HL-60 cell proliferation was examined by using MTS assay. The statistical analysis showed that the proliferation ability of HL-60 cells transfected with Stat3 shRNA was weakened (***P<0·001).

Discussion

Since Stat3 acts as a master regulator in many areas of tumor progression, including cell proliferation, apoptosis, and cell cycle regulation, it has been recognized as an oncoprotein.21 In previous researches, scientists have conducted numerous experiments targeting Stat3 to investigate tumor treatment. These studies showed that the inhibition of Stat3 signaling-induced growth inhibition and apoptosis in prostate, ovarian, and breast cancer cells.22^–24 Our previous studies also demonstrated that the down-regulation of Stat3 induced apoptosis and decreased the invasion activity in glioma cells.²⁵25,26 All of these findings suggest that targeting Stat3 might pave a new way into cancer therapy.

It has been reported that Stat3 activity is most prevalent in AML.27 Our results also testified that constitutively expressed Stat3 is present in human leukemia HL-60 cells. Subsequently, to examine the role of Stat3 in human leukemia cells, we analyzed the cell apoptosis and proliferation events that occurred in HL-60 cells transfected transiently with Stat3 shRNA. In this study, RNAi-mediated gene silencing was utilized to down-regulate the expression of Stat3. An obvious reduction in both the mRNA level and the protein level of Stat3 in the RNAi treatment group can be detected by RT-PCR, western blot, and immunofluorescence staining analyses. Further analysis revealed that the down-regulation of Stat3 expression by RNAi induces apoptosis and inhibits proliferation in human leukemia HL-60 cells. In addition, induction of apoptosis and inhibition of proliferation only occur in some of HL-60 cells in the RNAi treatment group, suggesting that the apoptosis and proliferation changes in HL-60 cells might be partially due to the inhibition of Stat3 and there might exist other signaling molecules involved in this regulatory process. Our future work will focus on the functional analysis of these molecules and further investigating the molecular mechanism of Stat3 in the pathogenesis of AML, which might foster the development of targeting therapies for AML.

In conclusion, constitutively expressed Stat3 is present in human leukemia HL-60 cells. After the down-regulation of Stat3, human leukemia HL-60 cells underwent induction of apoptosis and inhibition of proliferation. The results of this study imply that Stat3 plays an important role in the apoptosis and proliferation of human leukemia HL-60 cells and may be a potential therapeutic target in AML treatment.

This work was supported by the Natural Science Foundation of Shanghai (10ZR1410800), Leading Academic Discipline Project of Shanghai Municipal Education Commission ‘Molecular Physiology’, National Basic Research Program of China (2010CB529806), and Key Discipline Project of Renji Hospital, Shanghai JiaoTong University School of Medicine (RJ 4101307).