Circular RNA circPRKCI contributes to malignant progression of T-cell acute lymphoblastic leukemia by modulating miR-20a-5p/SOX4 axis

The expression levels of circPRKCI are positive correlated with SOX4 and are negative correlated with miR-20a-5p in T-ALL samples

To determine the correlation of circPRKCI, miR-20a-5p, and SOX4 in the development of T-ALL, we evaluated the expression levels of circPRKCI, miR-20a-5p, and SOX4 in clinical T-ALL samples. Significantly, the expression of circPRKCI was enhanced while the expression of miR-20a-5p was reduced in the clinical T-ALL samples (Figure 1A, 1B). In addition, the expression of SOX4 was also induced in the clinical T-ALL patients relative to the healthy controls (Figure 1C). Moreover, the expression of miR-20a-5p was negatively associated with circPRKCI and SOX4 in the T-ALL patients (Figure 1D, 1E). Importantly, the expression of circPRKCI was positive correlated with SOX4 in the T-ALL patients (Figure 1F).

CircPRKCI knockdown suppresses T-ALL cell survival in vitro

We then explored the function of circPRKCI in the mediation of T-ALL cell survival in vitro. To this end, the T-ALL MOLT-4 and JURKAT cell lines were treated with circPRKCI shRNA and the treatment efficacy of circPRKCI shRNA was validated in the cells (Figure 2A). The silencing of circPRKCI using shRNA suppressed the viability of MOLT-4 and JURKAT cells (Figure 2B). Conversely, the knockdown of circPRKCI promoted the apoptosis of MOLT-4 and JURKAT cells (Figure 2C, 2D). Consistently, the levels of cleaved PARP and cleaved caspase3 were induced by the depletion of circPRKCI in MOLT-4 and JURKAT cells (Figure 2E), indicating that CircPRKCI knockdown suppresses T-ALL cell survival in vitro.

CircPRKCI serves as a ceRNA of miR-20a-5p in T-ALL cells

Next, we verified the relationship of circPRKCI and miR-20a-5p. We identified the predicted binding site between circPRKCI and miR-20a-5p in the bioinformatics analysis (Figure 3A). The treatment of miR-20a-5p mimic remarkably increased miR-20a-5p expression in MOLT-4 and JURKAT cells (Figure 3B). The luciferase activity of circPRKCI was significantly decreased in MOLT-4 and JURKAT cells after the treatment of miR-20a-5p mimic (Figure 3C, 3D).

Meanwhile, the silencing of circPRKCI using shRNA promoted the expression of miR-20a-5p in MOLT-4 and JURKAT cells (Figure 3E, 3F), implying that circPRKCI serves as a ceRNA of miR-20a-5p in T-ALL cells.

CircMRPS35 regulates PD-L1 expression and cell growth in TNBC

MiR-20a-5p represses T-ALL cell survival in vitro

Then, we validated the function of miR-20a-5p in the modulation of T-ALL cell survival in vitro. For this purpose, the T-ALL MOLT-4 and JURKAT cell lines were treated with miR-20a-5p mimic and the treatment efficacy of miR-20a-5p mimic was confirmed in the cells (Figure 4A). The treatment of miR-20a-5p mimic inhibited the viability of MOLT-4 and JURKAT cells (Figure 4B). Reversely, miR-20a-5p mimic enhanced the apoptosis of MOLT-4 and JURKAT cells (Figure 4C, 4D). Similarly, the levels of cleaved PARP and cleaved caspase3 were increased by miR-20a-5p mimic in MOLT-4 and JURKAT cells (Figure 4E), indicating that miR-20a-5p represses T-ALL cell survival in vitro.

MiR-20a-5p can target SOX4 in T-ALL cells

We then investigated the interaction of miR-20a-5p and SOX4. We found the predicted binding site between SOX4 and miR-20a-5p in the bioinformatics analysis (Figure 5A). The luciferase activity of SOX4 was significantly suppressed in MOLT-4 and JURKAT cells after the treatment of miR-20a-5p mimic (Figure 5B, 5C). Both mRNA and protein levels of SOX4 were remarkably repressed by the treatment of miR-20a-5p mimic in MOLT-4 and JURKAT cells (Figure 5D–5F). Crucially, the expression of SOX4 was decreased in MOLT-4 and JURKAT cells after the depletion of circPRKCI using shRNA, while the inhibitor of miR-20a-5p could rescue the decrease in the cells (Figure 5G).

SOX4 knockdown suppresses T-ALL cell survival in vitro

Next, we further validated the function of SOX4 in T-ALL cell survival in vitro. The depletion efficacy of SOX4 shRNA was verified in MOLT-4 and JURKAT cells (Figure 6A). The knockdown of SOX4 using shRNA decreased the viability of MOLT-4 and JURKAT cells (Figure 6B). Meanwhile, the silencing of SOX4 induced the apoptosis of MOLT-4 and JURKAT cells (Figure 6C, 6D). In addition, the levels of cleaved PARP and cleaved caspase3 were promoted by the depletion of SOX4 in MOLT-4 and JURKAT cells (Figure 6E), indicating that SOX4 knockdown suppresses T-ALL cell survival in vitro.

CircPRKCI/ miR-20a-5p/SOX4 axis regulates T-ALL cell survival in vitro

Then, we tried to assess the correlation of circPRKCI, miR-20a-5p, and SOX4 in the modulation of T-ALL cell survival in vitro. As expected, the MOLT-4 and JURKAT cell viability was reduced by circPRKCI silencing, while the overexpression of SOX4 or suppression of miR-20a-5p rescued the reduced viability in MOLT-4 and JURKAT cells (Figure 7A, 7B). Besides, the knockdown of circPRKCI enhanced MOLT-4 and JURKAT cell apoptosis, and SOX4 re-expression or miR-20a-5p inhibition could reverse this enhancement (Figure 7C, 7D).

Clinical specimen

The bone marrow tissues were collected from patients newly diagnosed as T-ALL (n=27) who hospitalized in our hospital from February 2017 to July 2020, and healthy donors as control (n=27). All participants have signed the informed consent. The experiment was approved by the Clinical Ethics Committee of Shaanxi Provincial People’s Hospital. Patients were selected by following criteria: patients with arrhythmia, heart dysfunction, hepatic or renal dysfunction, lung dysfunction and other tumor history were excluded. Flowcytometry analysis identified positive staining of T cell biomarkers (CD2, CD3, CD4, CD8) and stem cell biomarker (CD34 and CD38), and negative staining of myeloid biomarkers such as CD13, CD15, CD33 and CD117 were diagnosed as T-ALL patients. The T-ALL patients included 9 females and 18 males, averagely aged 32-years old. The healthy donors included 15 females and 12 males, averagely aged 30-years old.

Cell culture and transfection

The human T-ALL cell lines MOLT-4 and JURKAT were purchased from American Type Cell Collection (ATCC, USA), and cultured in RPMI-1640 medium which contains 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin mixture (Thermo Fisher, USA). Cells were maintained in humidified 5% CO₂ atmosphere at 37° C. For cell transfection, the short hairpin RNA (shRNA) targeting CircPRKCI (shCircPRKCI), shRNA targeting SOX4 (shSOX4), shRNA negative control (shNC), SOX4 overexpressing plasmid (pcDNA-SOX4) and control plasmid (pcDNA-NC), miR-20a-5p mimics and inhibitors were purchased from GenePharma (China). The transfection reagent Lipofectamine 2000 was obtained from Invitrogen (USA) and used for cell transfection according to manufacturer’s instruction. The dose of oligonucleotides used for transfection was 50 nM.

CCK-8 assay

MOLT-4 and JURKAT cells were placed in a 96-well plate (3000/well) after transfection with indicated oligonucleotides. After incubation for 0, 24, 48, and 72 hours, Cell Counting Kit-8 (CCK-8, Thermo Fisher) reagent was added into each well, and cultured for 1 hour. Subsequently, optical density of 450 nm was detected by a microplate detector (BD Biosciences, USA).

Apoptosis

Cell apoptosis was determined by a FITC-Annexin V/PI Apoptosis detection kit (Thermo Fisher) following manufacturer’s protocol. In brief, MOLT-4 and JURKAT cells were digested and suspended in binding buffer after cell transfection. Then FITC-Annexin V (5 μL/well) and PI were added into each well, and hatched at room temperature for 30 minutes. The apoptotic cell portion was detected by a flow cytometer (BD Biosciences).

RNA quantification

Total RNA was extracted from patient samples, MOLT-4 and JURKAT cells via TRIzol reagent (Invitrogen) in accordance with manufacturer’s instruction. The complementary DNA was synthesized from total RNA (1 μg) in a total volume of 20 μl by using a High-Capacity cDNA reverse transcription kits (Thermo Fisher). The reaction mixture was incubated at 25° C for 5 min for priming, then at 42° C for 30 min for reverse transcription, and finally at 85° C for 5 min for reverse transcriptase inactivation. The complementary DNA (cDNA) was stored at −20° C until further use. Then the samples were subjected to quantification by SYBR Green Master Mix (Thermo Fisher). 400nM specific sense primer, 400 nM specific antisense primer, RNase/DNase-free water, and cDNA template (previously isolated and reverse-transcribed RNA of 1 ng/well) up to final volume of 10 μl. Amplifications were performed starting with a 30 sec enzyme activation at 95° C, followed by 40 cycles of denaturation at 95° C for 5 sec, and then annealing/extension at 60° C for 30 sec. At the end of each run a melting curve analysis was performed from 65–95° C. All samples and negative controls were amplified in triplicate, and the obtained mean value was then used for further analysis. Cycle of quantification (Cq) values of >35 was excluded from further mathematical calculations. Relative gene expression was normalized to GAPDH or U6 and calculated by 2^−ΔΔCt method. The experiments were repeated at least for 3 times. Primer sequences were as following:

circPRKCI, F, 5’-ATTCAGGGACACCCGTTCTT-3’, R, 5’-CTCTTCAGAACACTTGCAGCTT-3’;

miR-20a-5p, F, 5’-UAAAGUGCUUAUAGUGCAGGUAG-3′, R, 5′-CUACCUGCACUAUAAGCACUUUA-3′;

SOX4, F, 5’-AGCGACAAGATCCCTTTCATTC-3’, R, 5’-CGTTGCCGGACTTCACCTT-3’;

U6, F, 5′-CTCGCTTCGGCAGCACATATACT-3′, R, 5′-ACGCTTCACGAATTTGCGTGTC-3′;

GAPDH, F, 5′-CGGAGTCAACGGATTTGGTCGTAT -3′, R, 5′-AGCCTTCTCCATGGTGGT GAAGAC -3′.

Western blotting

Cleaved PARP, cleaved Caspase3, β-actin, SOX4, Total proteins were extracted from patient samples, MOLT-4 and JURKAT cells via RIPA lysis buffer (Invitrogen), and quantified by using a BCA kit (Beyotime, China). The proteins were separated by sodium dodecylsul-fate polyacrylamide gel electrophoresis (SDS-PAGE) and shifted onto a PVDF membrane (Millipore, USA). The membranes were then blocked in 5% defatted milk, followed by incubation with the primary antibodies against Cleaved PARP (1:1000, Proteintech, China), cleaved Caspase3 (1:1000, Proteintech), β-actin (1:1000, Proteintech) and SOX4 (1:1000, Proteintech) at 4° C overnight. Next day, the membranes were hatched in the secondary anti-rabbit antibody. The chemiluminescence was developed with ECL kit (Thermo Fisher).

Dual luciferase reporter assay

Binding sequences of miR-20a-5p to circPRKCI and SOX4 3’UTR were predicted via bioinformatics analysis. wild type reporter gene plasmids (WT) and mutated reporter gene plasmids (MUT) of circPRKCI and SOX4 were constructed by inserting corresponding sequences into pmirGLO reporter plasmid (Promega, USA). MOLT-4 and JURKAT cells were seeded in 12-well plates and transfected with MUT or WT and miR-20a-5p mimics or NC. The luciferase activity was detected by a Dual-Luciferase Reporter Assay System (Promega, USA).

Statistics

Data in this work were presented as mean ± SD of three replicates. SPSS software (Version 22.0) was adopted for comparison between two or more groups via Student’s t-test or one-way ANOVA method. P < 0.05 was taken as statistically significant.