Knockdown of DGKZ induces apoptosis and G2/M phase arrest in human acute myeloid leukemia HL-60 cells

Diacylglycerol kinase zeta (DGKZ) is associated with the pathogenesis of a variety of malignant diseases, but its biological function on acute myeloid leukemia (AML) has not been explored. This study was aimed to analyze apoptosis induced by Knockdown of DGKZ and its mechanism in human acute myeloid leukemia HL-60 cells.In the present study qRT-PCR was used to detect the expression of DGKZ in HL-60, THP-1, K562, H9, Jurkat and CD34 cell lines. DGKZ-shRNA lentiviral vector was established and used to infect acute myeloid leukemia HL-60 cells. Cell Counting Kit-8 (CCK-8) assay was used to determine the viability of HL-60 cells DGKZ knocked down. Apoptosis and cell cycle phase of HL-60 cells after DGKZ knockdown were evaluated by flow cytometry. Western blot analysis was performed to investigated the protein expression related to apoptosis and cell cycle. Results showed DGKZ expression were stable and higher in Jurkat, HL-60, THP-1,K562 leukemia cells than those of H9 and CD34 cells. Compared with cells of the shCtrl group, DGKZ was markedly knocked down in cells which were transfected with lentivirus encoding shRNA of DGKZ in HL-60 cells. DGKZ knockdown significantly inhibited the proliferation and induced cycle arrest at the G2/M phase in HL-60 cells. Western blot results indicated the expressions of caspase-3, caspase-8, and survivin markedly increased in HL-60 cells after knockdown of DGKZ. The results suggest Knockdown of DGKZ can inhibit proliferation of acute myeloid leukemia HL-60 cell caused cell cycle arrest at the G2/M phase through caspases pathway.


Introduction
Acute myeloid leukemia (AML) is a malignant disease group with an idiosyncratic nature and its incidence tends to increase year by year. Leukemia poses a major threat to the survival of patients, and researchers have been working on understanding the pathogenesis of the disease [1][2][3] . However, the mechanism remains unclear and a number of studies have suggested that there is a significant correlation between disease and multiple genes [4][5][6] .
Several studies have suggested that diacylglycerol kinase zeta (DGKZ) is associated with the pathogenesis of a variety of malignant diseases, such as acquired aplastic anemia, gastric cancer, and neuroblastoma [7][8][9] . However, its biological function in AML has not been studied.
DGKZ, which is encoded by DGKZ, is a subtype of DGK-IV 10 . It is thought to act in cell cycle regulation, where diacylglycerol (DGA) acts as an intracellular second messenger 11 . It can specifically activate protein kinase C and protein tyrosine kinase, which further activate signal transduction pathways through protein phosphorylation and ultimately affect cell proliferation as well as differentiation 12 . DGKZ is involved in transient ischemic attack, liver function, and myocardial pathological damage repair, showing its protective effects in different cells. Research shows that DGKZ has a role in the regulation of glioma, gastric cancer, liver cancer, and the pathological process of colon cancer [13][14][15][16] .
The purpose of this study is to evaluate the effect of DGKZ knockdown on cell proliferation and apoptosis in leukemia HL-60 cells, which is helpful for exploring therapeutic strategies targeting leukemia.

Identification of DGKZ expression by qRT-PCR
Total RNA was extracted using the TRIzol reagent (Invitrogen, USA). Samples were collected for Trizol cleavage by centrifuging the samples for 5 min at 2,000 rpm and adding 1 ml of Trizol to the cell supernatant. Samples precipitated after being allowed to mix for 5 min at room temperature, subsequently, samples were transferred to a new 1.5 mL tube. Then, cDNA was obtained by reverse transcription using the Promega M-MLV Kit. RNA reverse transcription was performed and quantitative real-time PCR (qRT-PCR) was used to detect expression.The primer sequences for DGKZ detection were as follows: The primer sequences for reference gene(GAPDH) were as follows: and 5′-CACCCTGTTGCTGTAGCCAAA -3′(Reverse).

Western blot
HL-60 cells were lysed after lentivirus infection and centrifuged 12,000 ×g for 15 min at 4 °C. Then, total protein was extracted from the resulting supernatant and the concentration was quantified by the bicinchoninic acid assay (BCA assay). Equal amounts (30 μg) of protein were separated by 10% SDS-polyacrylamide gel, followed by transfer onto PVDF membranes. After blocking, the membranes were treated overnight at 4 °C with rabbit monoclonal anti-human GAPDH, and Flag primary antibodies(1:2000 dilution) . This was followed by incubation with appropriate horseradish Peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1h and detection was achieved with an enhanced chemiluminescence (ECL) kit (GE Healthcare).
After culture for 1, 2, 3, 4, and 5 days, 10 μl CCK-8 was added and cells were cultured 6 for another 4 h. The cell suspensions were vortexed for 5 min, after which the absorbance was read at 450 nm and cell proliferation rate was determined.

Apoptosis assessment
Apoptosis of HL-60 cells with DGKZ knockdown and control were evaluated with the AnnexinV-FITC kit (BD Co. Ltd, USA) according to the manufacturer's instructions.
Analysis was performed by flow cytometry (BD Bioscience, USA) with the CellQuest software (BDIS).

Cell cycle detection
HL-60 cells with DGKZ knockdown (1×10 6 /well) in 6-well plates were incubated in a humidified incubator at 37 °C with 30% humidity and 5% CO 2 for 24 hr. Then, the cells were fixed with 70% ethanol overnight, and incubated with 1 ml of Propidium (PI) solution (20 μg/ ml in PBS with 1% Triton X-100) containing RNaseA at 37°C in the dark for 30 min. Samples were assessed by flow cytometry (BD Bioscience, U.S.A.) with the CellQuest software (BDIS).
The cell suspension was incubated at room temperature for 2 hs, absorbance was obtained, and activity of caspase 3 was determined.

Intracellular signaling array
Cell lysates were prepared as mentioned above and total proteins were isolated. Intracellular signaling molecules were detected using a PathScan intracellular signaling array kit (Cell Signaling Technology) according to the manufacturer's procedure.

Statistical analysis
Data were presented as mean ± standard deviations (SD) from at least three independent experiments. Statistical analysis was performed by Student's t-test. p <0.05 was considered statistically significant.

Expression of DGKZ in different leukemia cell lines
The infected with LV-shDGKZ were significantly suppressed as compared with LV-shCtrl group (Fig. 2 b,c,d). The data confirmed that DGKZ expression was successfully knocked down in HL-60 cells by LV-shDGKZ infection.

DGKZ knockdown inhibited HL-60 cell proliferation
After shRNA-lentivirus infection, 2,000 cells were plated on 96-well plates and cultured in a humidified incubator at 37 °C with 30% humidity and 5% CO 2 for 5 days. The absorbance of the wavelength of 450nm of HL-60 cell shIARS2 group and control group (shCtrl) were evaluated with the microplate reader every day and proliferation rates were calculated. Results showed that the proliferation rate of HL-60 cell infected with LV-shDGKZ was significantly lower than control since the 3th day. The result suggested that DGKZ was significantly associated with the proliferation of HL-60 cells (Fig. 3). 8

Apoptosis of HL-60 cells infected with LV-shDGKZ and LV-shCtrl was evaluated with
the Annexin V-FITC kit and flow cytometry. As shown in Fig. 4, apoptotic rates were 3.37±0.14 in control group and 14.86±0.52 in HL-60 cells after DGKZ knockdown respectively.These findings indicated DGKZ knockdown can induce apoptosis in HL-60 cells.

DGKZ knockdown arrests HL-60 cells at the G2/M phase
To reveal the mechanism of growth suppression effect following DGKZ knockdown, the

DGKZ knockdown promotion the activities of Caspase3 in HL-60 cells
To investigate the apoptotic effect of DGKZ knockdown , relative caspase-3 activity in HL-60 infected with LV-shDGKZ and LV-shCtrl was measured. The results showed the activity of caspase3 in HL-60 infected with LV-shDGKZ was significantly higher than that of LV-shCtrl group (p<0.05) (Fig. 6).

Regulation of signaling molecules in DGKZ knockdown cells
To further illuminate the molecular mechanisms by which DGKZ affects HL-60 cells, a PathScan ® Antibody Array Kit was used to detect changes in signaling molecules in HL-60 cells before and after DGKZ knockdown. The results showed that expression of ERK1/2, HSP27, Smad2, p53, p38 MAPK, and SAPK/JNK were significantly down-regulated in HL-60 cells after RNAi against DGKZ. Meanwhile, expression levels of PARP, caspase-3, and Survivin were up-regulated in HL-60 cells after RNAi against DGKZ.
The data indicated that DGKZ knockdown could significantly inhibit the growth of HL-60 cells via blockade of anti-apoptotic genes and promotion of apoptosis (Fig.7).

Expression of Caspase-3, Caspase-8 and Survivin
To further unveil the mechanism of DGKZ knockdown-induced apoptosis, various key effector of programmed cell death were quantified by western blot. The results showed caspase-3, caspase-8, and survivin protein levels in HL-60 infected with 9 LV-shDGKZ were much higher than those of LV-shCtrl group.

Discussions
The pathogenesis of AML has been unclear and every effort to increase our understanding of AML pathogenesis is of great significance [17][18] . DGKZ is associated with a variety of malignant diseases in humans [19][20] and is also related to proliferation and apoptosis of T cells in the blood system [21][22][23] .With the popularity of precision medical advancement, stem cell technology, cell therapy technology, and the progress of new drugs, survival will greatly enhance in cancer patients. Furthermore, certain cancers may be avoided through preventive treatment, while some may even be completely cured.
Whether cancer can be completely overcome remains a question that can only be