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Full-Text (PDF) Original Article Iran J Ped Hematol Oncol. 2019, Vol9, No4, 49-56 Effect of Estradiol on miR-21& miR-155 Expression in promyelocytic leukemia-derived cell line NB4 Robab Naghibzadeh MSc1, Narges Obeidi PhD1,2,*, Alireza Farsinejd PhD3,Gholamreza Khamisipour PhD1, Masoud TohidFar PhD4 1. Department of Hematology, School of Para Medicine, Bushehr University of Medical Sciences, Bushehr, Iran 2. Blood Transfusion Organization, Bushehr, Iran 3. Department of Hematology and Medical Laboratory Sciences, Faculty of Allied Medical Sciences, Kerman University of Medical Sciences, Kerman, Iran 4. Department of Hematology and Medical Laboratory Sciences, Faculty of Allied Medical Sciences, ShahidBeheshti University of Medical Sciences, Tehran, Iran *Corresponding author:Dr Narges Obeidi, Department of Hematology, School of Para Medicine, Bushehr University of Medical Sciences, Bushehr, Iran. E-mail: [email protected]. Orchid ID:0000-0001-8087-2850 Received: 25 March 2019 Accepted: 10 November 2019 Abstract Background: Due to the estrogen participation in modulating the proliferation and commitment of stem cells and the effects of miR-21 and miR-155 expression on reduced proliferation and colony formation of acute myeloid leukemia (AML), the aim of the present study was to evaluate the effect of estradiol on expression of miR-21 and miR-155 in the NB4 cell line, as an acute promyelocytic leukemia (APL). Materials and Methods: In the present experiment, NB4 cells were treated with different quantities of estradiol (5, 25, 50, 75, 100, 150, 200, 250 μg/ml) and vehicle control for 24 and 48 hours. Viability, apoptosis, and cellular proliferation were estimated by trypan blue exclusion, flow cytometry, and MTT assays, respectively. The level of miR-155 and miR-21 expression was studied using absolute quantitative real-time PCR. Results: Results showed that estradiol in the effective dose (200 μg/ml) led to decreased cellular viability (in a dose dependent manner, P = 0.004) and apoptosis of NB4 cells. In addition, the expressions of miR-155 and miR-21 were significantly and dose-dependently decreased (p<0.05). Conclusion: Estradiol at the effective dose caused apoptosis in NB4 cell line. This substance can be used as a drug for the treatment of APL. However, further assessments are needed to support the effectiveness of estradiol in the treatment of APL. Keyword: Acute Myeloid Leukemia (AML), Estradiol, MiR-155, MiR-21 Introduction (2). Based on the morphology of Leukemia as a most common hematologic promyelocytic cells, the acute malignancy is known by atypical promyelocytic leukemia (APL) is proliferation of stem cells and identified as a M3 subtype of AML. The Downloaded from ijpho.ssu.ac.ir at 20:09 IRST on Sunday October 3rd 2021 [ DOI: 10.18502/ijpho.v10i1.2169 ] development of leukocytes and their characteristic features of this abnormality precursors in blood and bone marrow (1). are hemorrhagic episodes, differentiation Acute myeloid leukemia (AML), a group arrest at the promyelocytic stage, and of heterogeneous clonal disorders of sensitivity to the differentiation response hematopoietic progenitors, is driven by to all-trans-retinoic acid (ATRA). numerous genetic perturbations that affect Cytogenetically, a balanced reciprocal cell proliferation, differentiation, and translocation between chromosomes 15 apoptosis. As a result, immature blasts of and 17 and production of PML/RARA myeloid lineage stack up in the bone fusion protein are responsible of APL. marrow, the generation of mature end Interestingly, current experiments reported stage cells is blocked and finally fatal that microRNAs are contributed to the infections and hemorrhages are occurred pathogenesis of APL. Mature miRNAs are Predictors of Caregiver Burden among Parents of Children with Cancer non-protein encoding RNAs that range metastasis (8). MiR-21 is frequently from 19-25 nucleotides in size, regulate overexpressed in AML blasts, and its gene expression post-transcriptionally (3), expression is particularly increased in and play important roles in a diversity of NPM1 mutant AMLs (9). biologic processes and differentiation of Estradiol, namely 17β-Estradiol (E2), is stem cells into RBC lineage (4). They are the most potent mammalian estrogenic critical regulators of cellular activities such steroids that circulates endogenously as differentiation, development, throughout the body and acts as the major proliferation, and apoptosis. Dysregulation female sex hormone. Its effects are of miRNAs can cause cancer and miRNA mediated through Estrogen Receptor (ER) expression profile has been used to classify that is found in varied tissues such as in cancers. MiR-155, an important miRNAs breasts, uterus, ovaries, bone, skin, fat, involved in hematopoiesis (control both prostate, and brain. This hormone can bind myelopoiesis and erythropoiesis), is to two types of Estrogen Receptors, Alpha expressed in high and low levels in normal (ERα) and Beta (ERβ) (10). Studies have hematopoietic stem-progenitor cells indicated that Ets-1/miR-126 possibly (HSPCs) and mature hematopoietic cells, mediated the protective effect of estradiol respectively. However, the exact against atherogenesis (10). Increasing regulation mechanisms of normal myeloid evidence indicates that estrogen has role in commitment have not been obviously modulation of stem cell niche (11) as well realized until now. Negative regulation of as proliferation and lineage commitment of apoptosis, increase of the proliferation rate adult and pluripotent stem cells. In the of myeloid progenitors, and promotion of current study, the effect of estradiol on HSPCs commitment to the common miR-21 and miR-155 expression in AML myeloid progenitors are probable functions cell line (NB4) was investigated by the of this microRNA. MiR-155 was researchers. frequently overexpressed in solid tumors as well as in hematological malignancies, Materials and Methods such as diffuse large B-cell lymphoma Cell cultivation (DLBCL), follicular lymphoma, Hodgkins NB4 cells (purchased from Pasteur lymphoma, primary mediastinal B-cell Institute, Tehran, Iran) were cultured at ° lymphoma, chronic lymphoid leukemia, 37 C along with 5% CO2 in RPMI 1640 and acute myeloid leukemia (AML) (5). enriched with fetal bovine serum (FBS, Depending on the tissue and malignancy 10%), penicillin (100 units/ml), and type, it can be either oncogene or tumor- streptomycin (100 μg/ml). All components suppressor. of culture medium were provided from In addition, it was found to be presented in Gibco Life Technologies, USA. Dimethyl CD34+ human HSPCS, and guess that sulfoxide (DMSO; Merck, Germany) was Downloaded from ijpho.ssu.ac.ir at 20:09 IRST on Sunday October 3rd 2021 [ DOI: 10.18502/ijpho.v10i1.2169 ] may hold these cells at an early stem- used for preparation of NB4-stock progenitor stage, inhibiting their suspension. transformation into more mature cells (6). Hormones High expression of miR-155 in AML cell 17β-Estradiol Hormone (Sigma,USA) was lines causes reduced proliferation and purchased from Sigma. Using methanol, as colony formation (7). On the other hand, diluents, hormone was formulated as 10-3 MiR-21 functions as an oncogene and M stock suspensions and stored at -20°C. often over-expressed in various In vitro treatment with estradiol malignancies and has been participated in Treatment was done in three groups. NB4 various malignancy-related processes such cells were cultured for 1 to 3 days under as uncontrolled proliferation, inhibition of the following conditions: i. Blank: NB4 apoptosis, and promotion of invasion and did not receive any treatment (untreated 50IranJ Ped Hematol Oncol. 2020, Vol10.No1, 49-56 Ahmadi et al group), ii. Estradiol: NB4 cells were apoptosis was performed with Annexin V- treated with Estradiol and iii. Methanol: FITC/Propidium iodide kit (B.D, USA). NB4 cells were treated with methanol After 1 day, the cells were centrifuged, the (negative control group). Each groups had resulting pellet was washed with cold PBS three samples. Two-five×105 cells/ml were (2×) and then resuspended in binding seeded in culture flasks containing buffer (1×), 5 µl of FITC-Annexin V and 5 antibiotic-supplemented RPMI enriched µlof PI. Upon 15minutes incubation at with FBS. Next, NB4 cells were treated room temperature in dark, the cells were with 5, 25, 50, 75, 100, 150, 200, 250 analyzed by flow cytometer (FloMax μg/ml of estradiol, and control groups software V2.0). were treated with methanol as diluents. RNA Extraction After 24, 48, and 72 hours of treatment, After treatment with Estradiol, total RNAs enumeration of viable and dead cells was from NB4 cells were isolated by Trizol performed with Neubauerhemocytometry RNA extraction kit (Invitrogen, USA) and Trypan blue stain. based on the manufacturer protocol. Cell viability measured by Trypan Blue Agarose gel electrophoresis and optical Exclusion Assay density measurement (A260/A280 ratio, As described above, to investigate the Nano Drop 1000 Spectrophotometer) were estradiol effect on the cell viability, cells applied for evaluation of quality and purity were seeded into a 12-well culture plate of extracted RNA, respectively. (2×105 cells/well) and treated with cDNA synthesis and Real-time PCR concentrations of estradiol.After mixing Aftersynthesis of cDNA using (1:1 ratio) of cellsuspension with 0.4% corresponding synthesis kit (Thermo, trypan blue solution (Sigma-Aldrich, USA), the expression levels of miR-155 USA) and incubation at room temperature and miR-21 was investigated by
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