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Introduction

T cell acute lymphoblastic leukemia (T-ALL) is a malignant hematological disease involving the infinite expansion of defective naive T cells (1,2). Children diagnosed with T-ALL account for ~15% of all cases of ALL (3). Currently, chemoradiotherapy and hematopoietic stem cell transplantation (HSCT) are the main therapeutic regimens used for ALL (1,4). With regards to the use of chemoradiotherapy, drug resistance and the tolerance of patients to the drugs used in the later stages of treatment are the main drawbacks for its use (5). On the other hand, HSCT, is associated with high costs and severe complications, such as graft-versus-host disease, which greatly limits its use and effectiveness (6-8). Even in cases in which initial treatment may seem effective, relapses can often occur unexpectedly (9). As such, effective treatment regimens for T-ALL are urgently required, particularly for children who are diagnosed with early T cell progenitor ALL (10). Currently, the development of novel strategies which can overcome the current obstacles is a major challenge to effectively treat the disease (11).

MicroRNAs (miRNAs/miRs), as non-coding RNAs, play an important role in regulating mRNA expression (12,13). Researchers have found that miRNAs are involved in various cellular processes, such as cell cycle progression and determining cell fate, through affecting proliferation, differentiation, metabolism and apoptosis (14). Consequently, miRNAs are regarded as targets for cancer therapeutic intervention, rendering them promising candidates (15,16). The focus of research on miRNAs is increasing. Previous studies have demonstrated that miR-325 can potently inhibit cell growth, such as that of hepatocellular carcinoma and non-small cell lung cancer (NSCLC), by targeting high mobility group box1 and aquaporin 5 (17-19).

Bcl-2-associated athanogene (BAG)2 is an anti-apoptotic oncogene, which plays a pivotal role in various diseases, such as numerous types of cancer, Alzheimer's disease, Parkinson's disease and spinocerebellar ataxia type-3(20). Previous studies have demonstrated that BAG2 is highly expressed in a number of tumor types, such as multiple myeloma, colorectal cancer, ovarian cancer and lung cancer (21-24). However, the roles of miR-325 and BAG2 in T-ALL, as well as the interaction between the two, remain to be determined. As such, the present study aimed to investigate the roles of miR-325 and BAG2 in a T-ALL cell line (Jurkat cells) and to further explore the underlying mechanisms of action.

Materials and methods

Cell lines and clinical samples

In the present study, human T-ALL cell lines, including TALL-1, KOPTK1, Jurkat, CCRF-CEM and Molt16, were purchased from the American Type Culture Collection (ATCC). The cells mentioned above were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, (NanJing SunShine Biotechnology Co., Ltd.) and 100 µg/ml streptomycin (Sunshine Biotechnology, Nanjing, China). The cells were maintained in a humidified atmosphere with 5% CO2 at 37˚C.

Clinical samples were obtained from Zibo Central Hospital (Zibo, China) from February 2018 to April 2019. Blood samples were obtained from 20 pediatric patients (age range, 3.6-14 years; 10 males, 10 females) who were diagnosed with T-ALL and 20 healthy donors (age range, 3-15.2 years; 10 males, 10 females). All fresh blood samples were immediately separated into several portions, snap-frozen in liquid nitrogen and stored at -80˚C prior to protein and RNA extraction. The present study was approved by the Ethical Review Committee of Zibo Central Hospital. All participants and their legal guardians agreed to the use of their samples in the present study and written informed consents were obtained from all of the legal guardians of all participants.

Transient transfection

0.2 µM BAG2-specific small interfering RNA (BAG2-siRNA; 5'-GGGAAGAACUCUCACCGUUTT-3'; Santa Cruz Biotechnology, Inc.), 0.2 µM control-siRNA (5'-UUCUCCGAACGUGUCACGUTT-3'; Santa Cruz Biotechnology, Inc.), 1 µg control-plasmid (cat. no. sc-437275; Santa Cruz Biotechnology, Inc.), 1 µg BAG2-plasmid (cat. no. sc-404540-ACT; Santa Cruz Biotechnology, Inc.), 100 nM mimic control (Shanghai GenePharma Co., Ltd.) and 100 nM miR-325 mimic (Shanghai GenePharma Co., Ltd.) were transfected into the Jurkat cell line, separately, using Lipofectamine® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol at 37˚C. Immediately following 48 h of transfection, the cells were collected for protein and RNA extraction.

Bioinformatics analysis

Bioinformatics analysis software TargetScan version 7.2 (http://www.targetscan.org/vert_72/) was used to predict the potential targets of miR-325.

Luciferase reporter assay

The pGL-3 plasmid vector (Promega Corporation) was employed in the luciferase reporter assay, which contained the wild-type (BAG2-WT) and mutant-type BAG2-mutant, containing mutations in the binding region of miR-325 with the BAG2 gene. The BAG2 gene was designed to contain the predicted miR-325 binding site. For the luciferase assay, 293T cells were cultured in a 24-well plate at a density of 5x104 cells/well overnight at 37˚C prior to transfection. The cells were co-cultured with the pGL-3 plasmid vectors and miR-325 mimic using a Lipofectamine® 3000 reagent kit at 37˚C for 48 h. After 48 h of transfection, the luciferase activity was measured using the Dual-luciferase Reporter Assay system (Promega Corporation) according to the manufacturer's protocol, and the results were normalized to Renilla luciferase activity.

RNA extraction and reverse transcription-quantitative PCR (RT-qPCR)

To further confirm the expression levels of miR-325 and BAG2, RT-qPCR was performed. Total RNA was extracted from the cell lines using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and RNA was extracted from clinical samples using the miRNeasy Mini kit (Qiagen, Inc.), according to the manufacturer's instructions. A NanoDrop-1000 spectrophotometer (Thermo Fisher Scientific, Inc.) was applied to quantify the extracted RNA. RNA was reverse transcribed into cDNA using Superscript III Reverse Transcriptase (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The reaction conditions for RT were as follows: 70˚C for 5 min, 37˚C for 5 min and 42˚C for 60 min. The quantitative expression of each gene was determined using SYBR-Green I (Thermo Fisher Scientific, Inc.). Thermocycling conditions used for the qPCR were as follows: Initial denaturation at 95˚C for 5 min; followed by 38 cycles of 15 sec at 95˚C, 1 min at 60˚C and 30 sec at 72˚C; and a final extension for 10 min at 72˚C. The primers were synthesized and purified by Sangon Biotech (Shanghai) Co., Ltd. Primer sequences were listed as following: miR-325 forward, 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACACUUAC-3' and reverse, 5'-ACACTCCAGCTGGGCCUAGUAGGUGUCCAGU-3'; BAG2 forward, 5'-CTTTGAGAGAAGCAGCAACTG-3' and reverse, 5'-TGACACTTCAACGGTGAGAG-3'; U6 forward, 5'-ATACAGAGAAAGTTAGCACGG-3' and reverse, 5'-GGAATGCTTCAAAGAGTTGTG-3'; GAPDH forward, 5'-TTTGGTATCGTGGAAGGACTC-3' and reverse, 5'-GTAGAGGCAGGGATGATGTTCT-3'. GAPDH (for mRNA) and U6 (for miRNA) were used as the internal controls. Gene expression was calculated using the 2-ΔΔCq method (25).

Western blot analysis

Following transfection, the Jurkat cells were harvested and lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) containing protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). The clinical samples were lysed using a Total Protein Extraction kit (Beijing Solarbio Science & Technology Co., Ltd.). The protein concentration was then determined using a BCA kit (Beyotime Institute of Biotechnology). Individual samples (15 µg/lane) were separated by 12% SDS-PAGE and transferred electrophoretically onto PVDF membranes (EMD Millipore). The membranes were blocked using 5% non-fat milk contained in TBST (0.1% Tween 20) for 1 h at room temperature, followed by incubation with primary antibodies against BAG2 (cat. no. ab79406; Abcam; working dilution, 1:1,000), Bcl-2 (cat. no. ab182858; Abcam; working dilution, 1:1,000), Bax (cat. no. ab32503; Abcam; working dilution, 1:1,000) and GAPDH (cat. no. ab9485; Abcam; working dilution, 1:1,000) overnight at 4˚C. The following day, the membranes were incubated with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (cat. no. 7074; Cell Signaling Technology, Inc.; working dilution, 1:2,000) at room temperature for 1 h. To observe the protein bands, an ECL kit (Amersham Pharmacia Biotech) was used for staining and the membranes were then photographed immediately. Band densities were quantified using Gel-Pro Analyzer densitometry software (version 6.3; Media Cybernetics, Inc.).

In vitro proliferation assay

To assess the effects of BAG2-siRNA and miR-325 on proliferation in vitro, a standard MTT assay was performed. Briefly, the Jurkat cells were seeded in 96-well plates at a density of 4,000 cells/well in complete medium, as described above, and cultured overnight. The following day, the cells were transfected with control-siRNA, BAG2-specific siRNA, control-plasmid, mimic control, miR-325 mimic, miR-325 mimic + control-plasmid, or miR-325 mimic + BAG2-plasmid in fresh medium at 37˚C for 48 h following the same conditions as aforementioned. The Jurkat cells were separately centrifuged and examined at 0, 12, 24 and 48 h immediately following the end of the transfection. A total of 150 µl DMSO (Sigma-Aldrich; Thermo Fisher Scientific, Inc.) was added to dissolve the purple formazan. Untreated cells were used as controls and regarded to have 100% viability. The data were measured using a BioTek microplate reader (BioTek Instruments, Inc.) at the absorbance of 570 nm.

Flow cytometry (FCM) for apoptosis

To verify the effects on apoptosis in vitro, Jurkat cells were harvested following transfection at the concentration of 1x106 cells/tube. The cells were then washed, pelleted and stained with Annexin V-FITC (Beyotime Institute of Biotechnology) and PI on ice in the dark for 15 min. The samples were then analyzed using a flow cytometer (FACSCalibur; BD Biosciences) and quadrants 2 and 3 (Q2 + Q3) were used for calculating the extent of apoptosis. FlowJo software (version 7.6.1; FlowJo LLC) was applied to analyze the data.

Statistical analysis

Unless otherwise stated, the data in the present study are expressed as the means ± SD, with n=3 or more replicates. GraphPad Prism 6.0 (GraphPad Software) was used for statistical analysis. The statistical significance of differences between groups was determined using unpaired Student's t-tests or one-way ANOVAs followed by Tukey's post hoc tests. P<0.05 was considered to indicate a statistically significant difference.

Results

Expression of miR-325 in clinical samples and human T-ALL cell lines

Previous studies have demonstrated that miR-325 can suppress cancer cell proliferation and accelerate apoptosis (17-19); however, to the best of our knowledge, there are no previous studies investigating the role of miR-325 in T-AL. In the present study, miR-325 expression levels in clinical samples from healthy donors and pediatric patients diagnosed with T-ALL, as well as in T-ALL cell lines (Jurkat, CCRF-CEM, TALL-1 and KOPTK1) were detected using RT-qPCR. The results revealed that, compared with the healthy samples, the levels of miR-325 in the patient samples were significantly lower (Fig. 1A). A similar trend was observed in the cell lines, with the Jurkat cells exhibiting the lowest expression levels of miR-325 (Fig. 1B). For this reason, Jurkat cells were selected for use in further in vitro experiments.

Figure 1 Expression of miR-325 in clinical samples and T-ALL cell lines. Expression of miR-325 in blood samples of patients and healthy donors was detected using RT-qPCR (n=20). (B) Expression of miR-325 in T-ALL cell lines was detected using RT-qPCR. **P<0.01 vs. healthy controls; ##P<0.01 vs. T cells. miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; T-ALL, T cell acute lymphoblastic leukemia.

Verification of the interaction between miR-325 and BAG2

To date, the experimental approach available for the verification of miRNAs and their target gene include the dual-luciferase report assay, which can detect the interaction between miRNAs and their corresponding target genes (26). In the present study, TargetScan, which is a popular miRNA target predication algorithm, was used to identify the target of miR-325. The results revealed that miR-325 shared a binding site with BAG2 (Fig. 2A). In addition, a dual-luciferase report assay was performed and the results further verified the interaction between miR-325 and BAG2 (Fig. 2B). In view of this hypothesis, it was confirmed that BAG2 was a direct target gene of miR-325.

Figure 2 miR-325 is directly targeted by the BAG2 gene. (A) Prediction of the binding site between miR-325 and BAG2 using TargetScan software. (B) Interaction between miR-325 and BAG2 assessed by a luciferase report gene assay. **P<0.01 vs. mimic control. BAG2, Bcl-2-associated athanogene; miR, microRNA; MUT, mutant; UTR, untranslated region; WT, wild-type.

Expression of BAG2 in clinical samples and human T-ALL cell lines

As it was found that BAG2 was a target of miR-325, as mentioned above, it was hypothesized that the expression levels of BAG2 in patient samples and T-ALL cell lines would exhibit an opposite trend to that of miR-325. To verify this hypothesis, the mRNA expression levels of BAG2 in patient samples and T-ALL cell lines was detected using RT-qPCR. The results revealed that the expression of BAG2 in patients diagnosed with T-ALL was significantly higher compared with the healthy donors (Fig. 3A). It was also found the trend for the expression levels of miR-325 in T-ALL cell lines was in accordance with that in patient samples, with significantly higher levels in all T-ALL cell lines (Fig. 3B). Moreover, the BAG2 expression levels were highest in the Jurkat cells compared with all other T-ALL cell lines (Fig. 3B).

Figure 3 Expression of BAG2 in clinical samples and T-ALL cell lines. (A) Expression of BAG2 mRNA in the blood samples of patients and healthy donors was detected using RT-qPCR (n=20). (B) Expression of BAG2 mRNA in T-ALL cell lines was detected using RT-qPCR. **P<0.01 vs. healthy controls; ##P<0.01 vs. T cells. BAG2, Bcl-2-associated athanogene; RT-qPCR, reverse transcription-quantitative PCR; T-ALL, T cell acute lymphoblastic leukemia.

BAG2 knockdown inhibits the proliferation and promotes the apoptosis of Jurkat cells

To explore the role of BAG2 in cell proliferation and apoptosis, siRNA technology was applied in the present study. BAG2-specific siRNA and control-siRNA were generated and co-cultured with Jurkat cells for 48 h, respectively. The RT-qPCR and western blotting data revealed that the expression of BAG2 in Jurkat cells was notably decreased following transfection with BAG2-siRNA, compared with the control-siRNA transfected group, demonstrating a successful transfection (Fig. 4A and B). Subsequently, the biological behaviors, namely the proliferation and apoptosis of Jurkat cells, following BAG2 knockdown were assessed. Firstly, an MTT assay was performed at 0, 24, 48 and 72 h. The analysis revealed that the knockdown of BAG2 markedly inhibited the proliferation of Jurkat cells (Fig. 4C). Secondly, using Annexin V-FITC/PI double staining, the cells were analyzed using FCM. It was found that the percentage of apoptotic cells was markedly increased in the group transfected with BAG2-siRNA (Fig. 4D and E). Finally, the expression levels of the apoptosis-related proteins, Bcl-2 and Bax, were examined using western blot analysis. The data demonstrated that the protein expression levels of Bcl-2 were decreased and the Bax protein expression levels were increased (Fig. 4F), with the ratio of Bcl-2/Bax being significantly decreased (Fig. 4G) compared with the control-siRNA group. Taken together, these data showed that BAG2 is a key target which can directly influence the cell proliferation and apoptosis of Jurkat cells.

Figure 4 Biological functions of BAG2 in Jurkat cells. Transfection efficiency of BAG2-siRNA, detected using (A) reverse transcription-quantitative PCR and (B) western blotting. Effect of BAG2 downregulation on (C) cell proliferation was determined using MTT assays. (D) Apoptosis was determined and (E) quantified using flow cytometry. (F) The protein expression levels of Bcl-2 and Bax proteins were determined using and western blot analysis. (G) The ratio of Bcl-2/Bax was calculated. **P<0.01 vs. control-siRNA. BAG2, Bcl-2-associated athanogene; OD, optical density; siRNA, small interfering RNA.

Overexpression of miR-325 can partially inhibit cell proliferation and induce apoptosis by downregulating the expression of BAG2

Based on the previous results, it was hypothesized that miR-325 might protect Jurkat cells from proliferation and accelerate apoptosis in a BAG2-dependent manner. Then, to determine whether the overexpression of miR-325 can protect Jurkat cells from proliferation and accelerate apoptosis in a BAG2-dependent manner, Jurkat cells were first transfected with various plasmids, miRNA mimics or a combination of these for 48 h. RT-qPCR and western blot analyses were carried out to identify the transfection efficiency. It was found that, compared with the control group, the expression levels of BAG2 and miR-325 were significantly increased following transfection with BAG2-plasmid and miR-325 mimic alone (Fig. 5A-C), demonstrating the successful transfections. It was also found that the expression levels of BAG2 were notably decreased in the group transfected with miR-325 mimic (Fig. 5D and E), as hypothesized.

Figure 5 miR-325 negatively regulates BAG2 expression in Jurkat cells. The expression level of (A) BAG2 mRNA and (B) BAG2 protein in Jurkat cells transfected with BAG2-plasmid was determined using RT-qPCR and western blot analyses, respectively. (C) miR-325 mimic increased the expression levels of miR-325 in Jurkat cells transfected with miR-325 mimic, as determined using RT-qPCR. The (D) mRNA and (E) protein expression levels of BAG2 in Jurkat cells transfected with mimic control, miR-325 mimic, miR-325 mimic + control-plasmid or miR-325 mimic + BAG2-plasmid was determined using RT-qPCR and western blot analyses, respectively. **P<0.01 vs. control-plasmid; ##P<0.01 vs. mimic control; &&P<0.01 vs. miR-325 mimic + control-plasmid. BAG2, Bcl-2-associated athanogene; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR.

The analysis revealed that the overexpression of miR-325 reduced the expression levels of BAG2 in Jurkat cells and the downregulatory effect was reversed by the introduction of the BAG2-plasmid (Fig. 5D and E). To better understand the biological effects of miR-325 mimic on cell proliferation and apoptosis following knocking down of the expression levels of BAG2, an MTT assay, western blot analysis and FCM were performed. In the group transfected with the miR-325 mimic alone, the cell proliferation of the Jurkat cells was inhibited (Fig. 6A) and the percentage of apoptotic cells was significantly increased (Fig. 6B and C). In addition, the effect on the protein expression levels of Bcl-2 and Bax, was investigated. A decreased Bcl-2 expression and an increased Bax expression were observed (Fig. 6D and E). Moreover, the data also demonstrated that the overexpression of BAG2 reversed the biological behaviors caused by miR-325 mimic transfection. These results demonstrated that expression of BAG2 was necessary for the induction of apoptosis of Jurkat cells by miR-325.

Figure 6 Biological functions of miR-325 in Jurkat cells. Jurkat cells were transfected with mimic control, miR-325 mimic, miR-325 mimic + control-plasmid or miR-325 mimic + BAG2-plasmid for 48 h. Subsequently, (A) cell proliferation, (B and C) cell apoptosis and (D) the protein expression levels of Bcl-2 and Bax were determined using an MTT assay, flow cytometry and western blot analysis, respectively. (E) The ratio of Bcl-2 to Bax was calculated and presented. **P<0.01 vs. mimic control; ##P<0.01 vs. miR-325 mimic + control-plasmid. BAG2, Bcl-2-associated athanogene; miR, microRNA; OD, optical density.

Discussion

It has previously been demonstrated that BAG2 plays a crucial role in the progression of tumor cell growth (27). BAG2, as an anti-apoptotic gene, can promote cell proliferation, inhibit cell apoptosis and arrest the cell cycle, and a raised BAG2 expression is found in a number of tumor types, such as thyroid cancer (28) and breast cancer (22). Ge et al (29) found that upregulation of BAG2 might be associated with underlying TNF-related apoptosis-inducing ligand (TRAIL)-resistance mechanisms in NSCLC. However, its biological function in T-ALL is not yet well understood.

In the present study, preliminary analysis with RT-qPCR and western blot analysis suggested that the mRNA and protein expression levels of BAG2 were higher in the blood samples of patients diagnosed with T-ALL and in T-ALL cell lines compared with those in healthy donor samples. To discover the role of BAG2 on proliferation and apoptosis, BAG2 expression was silenced in Jurkat cells using siRNA technology. The data displayed a significant decrease in cell expansion and an increased portion of apoptotic cells in the group transfected with BAG2-siRNA. This suggested that BAG2 indeed affects cell growth and survival.

miRNAs, as non-coding small RNAs, are regulators of gene expression at the mRNA level (28,30). An increasing number of studies have focused on oncogenic miRNAs, which determine the tumor cell fate and oncogenic miRNAs have been shown to be promising targets for therapy (31,32). T-ALL is a refractory and relapsing malignant cancer which has been the focus of numerous clinicians and studies in recent years (33,34). In recent years, the research of miRNA in T-ALL has attracted more and more attention (1). In 2004, Chen et al (35) identified three miRNAs that are specifically expressed in hematopoietic cells, which were dynamically regulated in the period of early hematopoiesis and lineage commitment. miR-142-3p (36), miR-155(37), miR-146a (38) and miR-150(39) have been reported to play roles in T-ALL. The present study found that miR-325 shared a binding site with BAG2 using TargetScan and the result was in accordance with the results of the dual-luciferase reporter assay. As previously reported, miRNAs are small RNAs which play a significant role in regulating gene expression by interfering with mRNA translation or promoting mRNA degradation (40). The results described above demonstrated the following 3 points: i) Compared with healthy donor samples, the expression of miR-325 was markedly lower and the level of BAG2 was markedly higher in patients with T-ALL and in T-ALL cell lines; ii) BAG2 knockdown can influence cell proliferation and apoptosis; and iii) miR-325 shares a binding site with BAG2. Taken together, it was hypothesized that BAG2 may be targeted by miR-325 in T-ALL. To verify this hypothesis, Jurkat cells were transfected with miR-325 mimics in vitro and cell proliferation was significantly inhibited compared with the control group. This was consistent with previous studies (17-19), demonstrating that miR-325 has an inhibitory effect on the proliferation of cancer cells. Additionally, the biological effects of miR-325 on the proliferation and apoptosis of Jurkat cells were reversed by the introduction of BAG2.

The BAG family was first identified as a group of proteins that prevent cell death through their interaction with Bcl-2(20). The Bcl-2 family genes (Bcl-2 and Bax) play critical roles in the apoptosis of T-ALL cells (41,42). In the present study, the results also demonstrated that the protein expression levels of Bcl-2 were decreased and the levels of Bax were increased simultaneously following the downregulation of the levels of BAG2. This indicated that Bcl-2/Bax may be the downstream genes of BAG2 regulated by miR-325. The antitumor biological effects may be dependent on the miR-325/BAG2/Bcl-2/Bax pathway; however, this requires verification in future studies.

In conclusion, the findings of the present study demonstrated that the miR-325/BAG2 axis may be a promising therapeutic target for the treatment of T-ALL. miR-325 inhibited the proliferation and promoted the apoptosis of T-ALL cells in a BAG2-dependent manner.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

All datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

FYW contributed to study design, data collection, statistical analysis, data interpretation and manuscript preparation. FLW and SZ contributed to data collection and statistical analysis. XX contributed to data collection, statistical analysis and manuscript preparation. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethical Review Committee of Zibo Central Hospital. All participants and their legal guardians agreed to the use of their samples in the present study and written informed consents were obtained from all of the legal guardians of all participants.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References