MicroRNA-145-3p suppresses the malignant behaviors of T-cell acute lymphoblastic leukemia Jurkat cells via inhibiting the NF-kappaB signaling pathway

: T-cell acute lymphoblastic leukemia (T-ALL) is a hematological tumor caused by the malignant transformation of immature T-cell progenitor cells. Emerging studies have stated that microRNAs (miRNAs) may play key roles in T-ALL progression. This study aimed to investigate the roles of miR-145-3p in T-ALL cell proliferation, invasion, and apoptosis with the involvement of the nuclear factor-kappaB (NF-κB) signaling pathway. T-ALL Jurkat cells were harvested, and the expression of miR-145-3p and NF-κB-p65 was measured. Gain- and loss-of-functions of miR-145-3p and NF-κB-p65 were performed to identify their roles in the biological behaviors of Jurkat cells, including proliferation, apoptosis, and invasion. Consequently, the current study demonstrated that miR-145-3p was downregulated while NF-κB-p65 was up-regulated in Jurkat cells. miR-145-3p directly bound to the 3’ untranslated region of NF-κB-p65. Over-expression of miR-145-3p inhibited Jurkat cell proliferation, invasion, and resistance to apoptosis, while over-expression of NF-κB-p65 presented opposite trends. Co-transfection of miR-145-3p and NF-κB-p65 promoted the malignant behaviors of Jurkat cells compared to miR-145-3p transfection alone, while it reduced these behaviors of Jurkat cells compared to NF-κB-p65 transfection alone. Taken together, this study provided evidence that miR-145-3p could suppress proliferation, invasion, and resistance to the death of T-ALL cells via inactivating the NF-κB signaling pathway.


Introduction
T-cell acute lymphoblastic leukemia (T-ALL) is a fast-growing blood malignancy that is caused by the clonal expansion of transformed T-cell precursors (Evangelisti et al., 2018). It accounts for approximately 12-15% of T-ALL cases in children and 25% in adults with unique clinical and biological features (Raetz and Teachey, 2016;Habiel et al., 2016). T-ALL is correlated with a variety of acquired genetic abnormalities that lead to developmental arrest and aberrant growth of malignant lymphoid progenitors (Bond et al., 2016). Most pediatric T-ALL patients are expected to be cured, and the survival rate of adults younger than 60 years subjected to conventional chemotherapy reached nearly 50%, while older patients have a more unfavorable prognosis (Gianfelici et al., 2016). Despite the advanced therapy protocols, there are still 15-25% of children and 40-50% adult T-ALL patients that relapse and develop drug resistance (Habiel et al., 2016). Moreover, the T-ALL survivors, particularly the children survivors, are at higher risk of acquiring long-run adverse health problems, such as secondary malignancies resulting *Address correspondence to: Xin Yang, Dr___YangXin@163.com from genotoxic drugs (Teepen et al., 2017). Therefore, novel and more effective therapeutic options are urgently needed to improve the outcome and life quality of T-ALL patients.
MicroRNAs (miRNAs) are a class of endogenous short non-coding RNAs that post-transcriptionally regulate protein-coding genes via binding to the 3' untranslated regions (3' UTR) of target mRNAs, contributing to mRNA degradation or translational inhibition (Shioya et al., 2010). miRNAs may play a crucial regulatory role in key cellular processes, including cell growth, cell cycle, or cell apoptosis (Coskun et al., 2011). One single miRNA can mediate thousands of mRNAs, and over 60% of human proteincoding genes are mediated by miRNAs (Lewis et al., 2005). Emerging studies suggest that miRNAs might play significant roles in the pathogenesis of human leukemia (Seca H et al., 2010;Qian Lu et al., 2016). Meanwhile, miR-145-3p has been suggested to act as a tumor suppressor in several cancer types Chen et al., 2018). While the effect of miR-145-3p on T-ALL remains unknown. Importantly, our study identified that miR-145-3p directly bound to the 3'UTR of NF-κB-p65. The NF-κB pathway is a key regulator of apoptosis and plays significant roles in many normal cellular functions (Yu et al., 2017). Aberrant activation of the NF-κB pathway has been suggested to be involved in the pathogenesis of several tumors (Wang et al., 2009;Kuck et al., 2017), as well as in ALL cells (Kordes et al., 2000). Taken together, the current study was designed to explore the role of miR-145-3p in the development of T-ALL with the involvement of the NF-κB signaling pathway.

Cell culture and grouping
Human T-acute lymphoblastic leukemia (T-ALL) Jurkat cell line purchased from American Type Culture Collection (Rockville, MD, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 complete medium containing 10% fetal bovine serum (FBS), 100 μg/mL streptomycin and 100 U/mL penicillin in a 37°C incubator with 5% CO 2 . The medium was refreshed 3-4 times each week until the cell confluence reached 80%, after which the cells began to be passaged. Mononuclear cells (MNCs) collected from human blood from five healthy volunteers (people with major diseases or infectious diseases were excluded). The study was approved by the Ethics committee of the First Affiliated Hospital of Chengdu Medical College, and signed informed consent was obtained from each eligible volunteer.
Jurkat cells in the logarithmic growth phase were seeded into 6-well plates at a density of 5 × 10 5 cells/mL. Next, the cells were assigned into control, mimic negative control (NC), miR-145-3p mimic, inhibitor NC, miR-145-3p inhibitor, over-expression (oe)-NF-κB-p65 and miR-145-3p mimic + oe-NF-κB-p65 groups after corresponding transfections. All transfections were performed following the instructions of the Lipofectamine TM 2000 kit (Invitrogen Inc., Carlsbad, CA, USA), with all the plasmids purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Briefly, 5 μg plasmids and 10 μL Lipofectamine TM 2000 were diluted with 250 μL serum-free Opti-MEM (Gibco Company, Grand Island, NY, USA) and mixed, respectively. Followed by standing at room temperature for 5 min, the two tubes were mixed and allowed to stand for 20 min. Then the mixture was seeded into wells and incubated at 37°C with 5% CO 2 for 8 h, after which the medium was refreshed as complete medium for 72 h of culture, and the cells were collected for following experiments.
Dual luciferase reporter gene assay A computer-based miR target detection program (http:// www.targetscan.org) was performed to predict the binding sites of miR-145-3p and NF-κB-p65. The pMIR-based reporter plasmids (Beijing Huayueyang Biotechnology Inc, Beijing, China) containing wild-type NF-κB-p65 (pMIR-NF-κB-p65-WT) or NF-κB-p65 mutated at the putative miRNA-145-3p binding sites (pMIR-NF-κB-p65-MT) were designed. Well-designed pMIR-based reporter plasmids along with either miRNA-145-3p mimic or mimic NC were cotransfected into HEK293T cells (Shanghai Cell Bank of the Chinese Academy of Sciences, Shanghai, China), with pMIR-Renilla luciferase used as an internal reference. Cells were lysed 48 h after transfections. Relative luciferase activity was normalized to that of Renilla luciferase and determined using the dual-luciferase reporter assay system according to the kit's instructions (E1910, Promega Corp., Madison, Wisconsin, USA). The relative value of firefly luciferase activity/Renilla luciferase activity was evaluated and analyzed.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The RNA concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), and 1 μg of RNA was reversely transcribed into cDNA using a PrimeScript TM RT reagent kit with gDNA Eraser (Takara Holdings Inc., Kyoto, Japan) following the kit's instructions. Briefly, extracted RNA was treated with 200 μL 5× gDNA Eraser Buffer and 100 μL gDNA Eraser at 42°C for 2 min for DNA exclusion. RT was performed at 37°C for 15 min and 85°C for 5 s to produce cDNA. Next, the real time-PCR was performed using a SYBR ® Premix Ex Taq TM (TliRNase H Plus) assay kit (Takara Holdings Inc., Kyoto, Japan) on an ABI7500 qPCR kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The PCR conditions were as follow: predenaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and a final extension at 72°C for 30 s. U6 was set as an internal control for miR-145-3p expression while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was set as an internal control for other genes. The 2 -ΔΔCt method was applied in which 2 -ΔΔCt refers to the ratio of the target gene expression between experimental group and control group, the formula was as follows: ΔΔCt = [Ct (target gene)-Ct (internal control gene)] experimental group -[Ct (target gene)-Ct (internal control gene)] control group . The Ct value was determined by the PCR cycle number at which fluorescence reaches a threshold value, during which the amplification was in logarithmic growth. The primers (Tab. (1)) were provided by Shanghai GenePharma Co., Ltd. (Shanghai, China). Biotechnology Co., Ltd., Shanghai, China). The experiment was repeated three times. Cell cycle measurement: cells were fixed with 1 mL pre-cooled 75% ethanol (-20°C) at 4°C overnight. Thereafter, the cells were washed with cold PBS twice. The PI staining solution was prepared, and each sample was stained with 535 μL mixture containing 500 μL binding buffer, 25 μL PI staining solution (20×), and 10 μL RNase (50×). Cells were treated with 500 μL PI binding buffer, resuspended, bathed at 37°C in the dark for 30 min, and detected using a flow cytometer (C1052, Beyotime Biotechnology Co., Ltd., Shanghai, China) at 4°C in the dark. The experiment was repeated three times.

Transwell assay
Forty-eight hours after transfection, cells were successively cultured in serum-free medium, starved for 24 h, trypsinized, washed twice with PBS, and then resuspended with serumfree medium for following experiments. The 24-well (8 μm) transwell plates (Corning Glass Works, Corning, NY, USA) were used in this study, with three wells set for each group. The wells were firstly coated with 50 μL serum-free-medium diluted Matrigel (1:5, Sigma-Aldrich Chemical Company, St Louis, MO, USA). Followed by 4-5 h of air drying at 37°C, the apical chamber was added with serum-free RPMI 1640 medium to produce 200 μL cell suspension with the cell density adjusted to 1 × 10 5 cells/mL, while the basolateral chamber was added with 500 μL RPMI 1640 complete medium containing 10% FBS. Twenty-four hours later, the transwells were taken out, washed twice with PBS, and fixed with 5% glutaraldehyde for 30 min, which was followed by gentian violet staining for 5 min. Then the number of stained cells was calculated under an inverted microscope (Olympus Optical Co., Ltd., Tokyo, Japan). The number of invaded cells was observed under a fluorescence microscope with five views randomly selected. Three duplicated wells were set for each group, and the experiments were performed three times with the average value calculated.

Statistical analysis
The Statistical Package for the Social Sciences (SPSS) 21.0 (IBM Co. Armonk, NY, USA) was applied for data analysis. Measurement data were expressed as mean ± standard deviation. Differences between every group pair were analyzed using the t-test, while among multiple groups were compared using one-way analysis of variance (ANOVA) and Bonferroni correction was applied for pair comparisons after ANOVA. p < 0.05 (two-tailed test) was considered to show a statistically significant difference.

The relative expression of miR-145-3p is lower in Jurkat cells opposite to NF-κB-expression in comparison to MNCs
RT-qPCR was applied to measure the miR-145-3p and NF-κB-p65 expression in MNCs and Jurkat cells, which suggested that miR-145-3p expression was relatively lower while NF-κB-p65 expression was relatively higher in Jurkat cells than in MNCs (Figs. 1(A)-1(B)) (all p < 0.05). Correspondingly, the Western blot analysis results presented the same trends (Fig.

Western blot analysis
Total proteins of cells were extracted, and then the protein concentration was detected using a bicinchoninic acid (BCA) kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Extracted proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore Co., Billerica, MA, USA) with the voltage from 80 V (35 min) to 120 V (45 min). Followed by 5% bovine serum albumin (BSA) sealing for 1 h at room temperature and PBST (phosphate buffer saline (PBS) + 0.1% Tween 20) washing, the membranes were incubated with the following primary antibodies at 4°C overnight: NF-κB-p65 rabbit monoclonal antibody (1: 5000, ab51248, Abcam Inc., Cambridge, MA, USA), Bcl-2 (1:1000, 4223, Cell Signaling Technology (CST), Beverly, MA, USA) and Bax rabbit polyclonal antibody (1:1000, 2774, Abcam). Next, the membranes were washed with PBST three times, 10 min each. Thereafter, the membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit and goat antimouse secondary antibodies (1:10000, Jackson, USA) at room temperature for 1 h, after which they were washed with PBST buffer three times, 10 min each. Then the membranes were soaked into an enhanced chemiluminescence (ECL) system (Pierce, Waltham, MA, USA) at room temperature for 1 min. Then the liquid was discarded, and the membranes were covered with plastic wraps, exposed, visualized, and fixed in the dark, after which the bands were analyzed using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). One-way analysis of variance (ANOVA) was applied for data analysis, and each experiment was repeated three times.

Flow cytometry
Cell apoptosis was detected using flow cytometry. In brief, 48 h after transfection, cells were detached with trypsin, and then the cell suspension was collected, centrifuged, washed twice with PBS and centrifuged again. Then the cells were collected for further experiments. Cell apoptosis was detected as follows: cells were resuspended with PBS, and the resuspension (about 1 × 10 4 cells) was centrifuged with the supernatant discarded. After that, the cells were gently resuspended with 195 μL binding buffer, and then added with 5 μL annexin V-fluorescein isothiocyanate (FITC) and 10 μL propidium iodide (PI). The mixture was then allowed to stand at room temperature in the dark for 20 min, ice bathed, and then detected on a flow cytometer (C1063, Beyotime 1(C)) (p < 0.05).

Up-regulated miR-145-3p inhibits Jurkat cell proliferation and growth
The MTT assay results suggested that over-expression of miR-145-3p significantly reduced the Jurkat cell proliferation 96 h after transfection, while over-expression of NF-κB-p65 notably elevated cell proliferation. Co-transfection with miR-145-3p mimic and oe-NF-κB-p65 led to significantly enhanced cell proliferation compared to miR-145-3p mimic transfection alone, and correspondingly, this co-transfection led to an obvious, reduced cell proliferation compared to oe-NF-κB-p65 transfection alone (all p < 0.05) (Fig. 3). These results suggested that up-regulated NF-κB-p65 could promote Jurkat cell proliferation, while overexpression of miR-145-3p could suppress this promotion.

Up-regulated miR-145-3p induces cell cycle arrest and apoptosis of Jurkat cells
Cell apoptosis and cell cycle were measured using flow cytometry. The apoptosis detection suggested that cells in the control group and mimic group presented no obvious difference. While up-regulated miR-145-3p markedly enhanced cell apoptosis, but over-expressed NF-κB-p65 presented an opposite trend (all p < 0.05). Meanwhile, the co-transfection of miR-145-3p mimic and oe-NF-κB-p65 led to reduced apoptosis compared to miR-145-3p transfection alone, while it led to enhanced cell apoptosis compared to oe-NF-κB-p65 transfection alone (all p < 0.05) (Fig.  4(A)). Likewise, cells in the control group and mimic group presented no obvious difference in terms of the cell cycle. But the proportion of Jurkat cells in the G 0 /G 1 phase in those with over-expression of miR-145-3p was notably increased compared to the control, while cells with over-expression of NF-κB-p65 presented an opposite trend. Moreover, the co-transfection of miR-145-3p mimic and oe-NF-κB-p65 led to reduced G 0 /G 1 proportion of cells compared to miR-145-3p mimic transfection alone while increased the G 0 / G 1 proportion in cells transfected with oe-NF-κB-p65 alone (all p < 0.05) (Fig. 4(B)). These results identified that overexpressed miR-145-3p or down-regulated NF-κB-p65 could induce cell cycle arrest and apoptosis of Jurkat cells.

Discussion
Leukemia is a result of aberrant regulation of myeloid or lymphoid commitment during hematopoiesis (Lv et al., 2012). While differential miRNA expression found during hematopoiesis suggests that abnormal expression of miRNAs may be correlated with leukemogenesis (Yendamuri and Calin GA, 2009;Garzon and Croce, 2008), this study was carried out to investigate the role of miR-145-3p in T-ALL development and found that over-expression of miR-145-3p could inhibit the growth and malignant behaviors of T-ALL cells.
The initial finding of the current study was that miR-145-3p expression was relatively higher, while NF-κB-p65 was relatively lower in T-ALL Jurkat cells than in human MNCs. It has been suggested that miR-145 expression was significantly inhibited in ALL cells (Batliner et al., 2012). Moreover, we found that NF-κB-p65 is a target gene of miRNA-145-3p according to the evidence of online software analysis and dual-luciferase gene reporter assay, which was in coincidence with a previous study (Wen et al., 2014). Meanwhile, a recent study mentioned that human T-cell leukemia virus proteins may regulate NF-κB activation (Fochi et al., 2019). Also, NF-κB-p65 has been revealed to be upregulated in nucleated peripheral blood cells from leukemia patients (Zhang et al., 2015).
Following the results above, the focus of the experiment shifted to figuring out the roles of miR-145-3p and NF-κB-p65 in Jurkat cell growth. Our study identified that overexpression of miR-145-3p inhibited Jurkat cell proliferation and invasion and resistance to apoptosis. A former study identified that miR-145 specifically could suppress tumor cell growth, and its down-regulation could lead to worse prognosis and a lower overall survival rate for T-ALL patients (Xia et al., 2014). Meanwhile, miR-145 has also been demonstrated to act as a tumor suppressor in other cancers. For instance, miR-145 could inhibit drug-resistant prostate cancer pathogenesis . Similarly, miR-145 has been demonstrated to suppress bladder cancer growth via specific signaling pathways (Yoshino H et al., 2013). miR-145-3p is the passenger strand of miR-145 and is down-regulated in several types of cancer types as well as the guide strand miR-145-5p (Misono et al., 2019). Similarly, down-regulation of miR-145-3p has been observed in tissues and cells of several types of metastatic cancers such as breast, prostate, and lung cancers (Kumoğlu et al., 2019). On the other hand, NF-κB-p65 activation could rescue the leukemia cells from apoptosis (Nugues et al., 2014). Inhibition of NF-κB has also been suggested to markedly enhance apoptosis of leukemia cells (Dai et al., 2005). Besides, overexpression of miR-145-3p and the corresponding NF-κB-p65 significantly enhanced Bax expression while reduced Bcl-2 expression in Jurkat cells. miR-145 has been shown to up-regulate the Bax/Bcl2 ratio in non-small cell lung cancer cells (Pan et al., 2018). Bax are well-recognized apoptosis-related factors whose high expression could lead to apoptosis (Szobi et al., 2014;Fan et al., 2016). Bcl-2 is an anti-apoptotic protein that modulates the permeability of the mitochondrial membrane, while Bax could damage the outer mitochondrial membrane, thus promoting cell apoptosis (Lv et al., 2018). Moreover, our study found that the up-regulation of miR-145-3p led to decreased MMP-9 expression but elevated E-cadherin expression. MMP-9, a member of the MMP family, is wellknown for the promoting effect on cell invasion (Ogasawara Nobutaka et al., 2018). E-cadherin is a junction protein between cells and always depleted during the epithelial-tomesenchymal transition and further cell invasion (Zhao et al., 2014). Hence, it can be concluded that overexpression of miR-145-3p is capable of inhibiting proliferation and invasion and promoting apoptosis of T-ALL Jurkat cells.
As a minor strand of miR-145, the roles of miR-145-3p in diseases are less studied, and the roles of miR-145-3p in T-ALL have never been elucidated before. Here, our study identified that miR-145-3p may suppress the proliferation, invasion, and resistance to apoptosis growth of T-ALL Jurkat cells, possibly through inactivating the NF-κB signaling pathway. We hope these findings could provide new insights into the pathogenesis of T-ALL and the development of novel therapeutic options for T-ALL treatment.

acknowledgement
Not applicable.

Conflicts of Interest
We declare no conflicts of interest.