Introduction

Acute lymphoblastic leukemia (ALL) is a malignant disease that arises from various genetic mutations in B or T-lymphoid progenitors, leading to blast cell proliferation, and accumulation of leukemic cells in bone marrow (1). T‑cell ALL (T‑ALL) is a subtype of ALL based on immunophenotype. It accounts for ~12% and ~25% of cases of ALL in children and adult patients, respectively (2). In general, patients with T‑ALL have a poorer prognosis than other ALL patients, including ~15% and ~50% of pediatric and adult cases respectively, which indicates resistance to treatment or the risk of experiencing relapse (2, 3). Therefore, in order to improve the prognosis of patients with ALL and increase the efficacy of current treatments while reducing their side effects, identification of the molecular mechanisms is vital (4).
 Finding new therapeutic targets like microRNAs (miRNAs) is appealing and worth investigating. MiRNAs are 18–25 nucleotide, single-stranded non-coding RNAs that mediate the expression of various genes (5, 6). They regulate gene expression by binding directly to the 3' untranslated region (3'UTR) of protein-coding genes (7, 8). It is estimated that approximately 50% of all protein-coding genes are regulated by miRNAs. Also, these small molecules play critical roles in different types of biological cell processes, including cell proliferation and differentiation, organ development, apoptosis, and metabolism (9).
However, some miRNAs act as oncogenes in cancers by negatively regulating tumor suppressor genes (10). Among these targets, the tumor suppressor genes p53 and p21 are particularly important, since they can be a direct target of miRNAs and regulate the expression and processing of them (11, 12). It has been shown that these tumor suppressors can be inactivated by miRNAs overexpression in malignancies and may result in the development of cancerous phenotypes (12, 13). Therefore, it is important to identify dysregulated miRNAs and their targets for the detection of cancers and finding new therapeutic strategies (10).
In T-ALL like other cancers miRNAs can be dysregulated (14). Sayadi et al. showed that the overexpression of miR-192 in ALL patients upregulated tumor suppressor gene p53 and downregulated prosurvival gene BCL2, which in turn induced apoptosis (15). Mavrakis et al.  identified five miRNAs (miR-19b, miR-20a, miR-26a, miR-92, and miR-223) that could promote  T-ALL development in a mouse model. They found that these miRNAs increased the pathogenesis of T-ALL by targeting tumor suppressor genes (16). Moreover, in another study, it was shown that four miRNAs (miR-101, miR-140-5p, miR-448, and miR-485-5p) were downregulated in T-ALL patient specimens and cell lines (17). However, for better diagnosis and treatment of T-ALL patients, other leukemic oncogenic miRNAs are required to be identified.
MiR-506 is considered as a component of an X chromosome-linked miRNA cluster in the primate testis (18, 19). Depending on the tumor types, miR-506 has reportedly functioned as a tumor-suppressive or an oncogenic miRNA (20). Moreover, previous studies have focused on the role of miR-506 in solid tumors like ovarian cancer (21), pancreatic cancer (22) and cervical cancer (18). However, the biological function of miR‑506 in T‑ALL cells remains unclear.
This study aimed to investigate the effects of miR‑506 on the proliferation and viability of the T‑ALL cell line (Jurkat). In addition, to examine the molecular mechanism of miR-506, the effect of this miRNA on p53 pathway genes like p21, p53, and prosurvival B-cell lymphoma-2 (BCL-2) has been investigated by quantitative real-time polymerase chain reaction (qRT-PCR).

Cell Line and Cell Culture
The human T-ALL cell line Jurkat was obtained from the Iranian Stem Cell Technology Research Center (Tehran, Iran), and cultured in RPMI-1640 medium with L-glutamine (Cell Biotechnology Saba Arna, Fars, Iran), with 10% fetal bovine serum (FBS) (Gibco, USA), 100 U/mL penicillin and 100 mg/mL streptomycin. Then, the cell was incubated at 37°C under a humidified atmosphere consisting of 95% air and 5% CO₂. Finally, suspension-cultured cells were used for transfection at 80% confluence (6).

Transfection
The Jurkat cell lines were divided into control group, scramble group, as well as two treated groups that were transfected by 50 and 100 nM of miR-506 (Eurofins Genomics Germany). The sequence of the mature miR-506 was as follows: (5'-UAAGGCACCCUUCUGAGUAGA‑3').  For Jurkat cells transfection, Lipofectamine 2000 (Invitrogen, USA) was used according to the manufacturer’s manual. Briefly, firstly, one day before the transfection, Jurkat cells were seeded in 24-well plates at a concentration of 2×10⁵ cells/well in a medium with serum but without antibiotics. Secondly, 50 and 100 nM miR-506 were diluted in 50 μL OptiMEM in two tubes (Gibco, USA). Thirdly, in two other tubes, 1.5 μL Lipofectamine was diluted in 50 μL OptiMEM in each. After 5 min of incubation, the diluted miR-506 from stage 2 was combined with the diluted Lipofectamine from stage 3. Then, the compound was incubated at room temperature for 20 min. Finally, it was added directly to each well-containing cells (from stage 1) and was mixed gently. Non-transfected Jurkat cell line was considered as the control group. siRNA conjugated with FITC (Santa Cruz Biotechnology, Inc, Texas, USA) was utilized as a scramble and transfected at a final concentration of 50 nM in a similar manner. Transfection efficiency was detected by scramble and qRT-PCR.

MTT Assay
The day after transfection, fresh RPMI containing 10% FBS replaced the culture medium. The culture plates were incubated at 37°C for the following day in a humidified atmosphere containing 5% CO₂. Then, 10 μL 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT) solution (5 mg/mL) was added to each well and the plates were incubated for a further 4 h. After removal of the medium, dimethyl sulfoxide (DMSO) was added to each well (23). The culture plates were shaken for 15 min and the absorption was measured at the wavelength of 570 nm by an enzyme-labeled analyzer. Three independent experiments were performed.

Flow Cytometry Analysis
Cell apoptosis was evaluated using an Annexin V-FITC Apoptosis Detection kit with 7-AAD (BioLegend, San Diego, CA, USA) (48 h after transfection) according to the following manufacturer’s manual (24): First, cells were washed with cold BioLegend's cell staining buffer and were resuspended in binding buffer. Subsequently, cells were incubated with 5 μL Annexin V/FITC and 5 μL 7-AAD for 15 min at room temperature in the dark. Then, 400 µL of Annexin V binding buffer was added to each tube of the cell. A flow cytometer (BD Biosciences) was used to detect apoptosis in Jurkat cells. Three independent experiments were performed.

RNA Extraction and Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR)  
Total RNA was extracted from cultured cells in the different groups by the Total RNA Isolation Kit (Santa Cruz Biotechnology, Inc., Texas, USA), according to the manufacturer’s instruction, 48 h after the transfection. Nanodrop spectrophotometer (Thermo Fisher Scientific, Inc.) and electrophoresis on 2% agarose gel were used to determine RNA concentration after extraction (25). According to the manufacturer's instructions, cDNA of miRNA and other genes were generated using a highly sensitive cDNA Synthesis kit (Bonbiotech, Inc, Iran) and H Minus First Strand cDNA Synthesis Revert Aid kit (Thermo Fisher Scientifc, Inc., USA) respectively. Specific primers for PCR amplification were synthesized by Bonbiotech Company (Bonbiotech, Inc, Iran) as represented in Table 1.
 The expression level of miR-506 and mRNA in the cells were evaluated on the Step One Plus Real-Time PCR systems (ABI, US). Expression of p53, p21, and BCL-2 mRNA in Jurkat cells were quanti­fied using a 2X Real MOD Green PCR Master Mix kit (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's instructions. Briefly, 2 µL cDNA product was diluted in a final volume of 20 µL, containing 10 pmol of each primer, 10 µL 2× reaction mixture of SYBR Green and 7.4 µL sterile deionized water (12). The cycling program was as follows: An initial denaturation step at 95°C for 10 min, followed by 40 cycles including a denaturation step at 95°C for 10 sec, annealing and extension at 55°C for 40 sec. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and U6 snRNA were selected as internal housekeeping controls. All PCRs were performed in triplicate. The relative expres­sion levels of miR-506 and p53, p21, and BCL-2 mRNA were calculated by the 2^-ΔΔCT method (26).

Statistical Analysis
All statistical analyses were performed using SPSS 21.0 software (SPSS, Inc., Chicago, IL, USA). The data are presented as the mean ± standard deviation (SD) from at least three independent experiments. In order to make a general comparison of the difference between treated cell groups and the control group, the Kruskal-Wallis test was utilized. The difference among the groups (two by two) was analyzed by the Mann-Whitney test. P-value<0.05 was considered to indicate a statistically signifi­cant difference.