Bone marrow containing malignant cells was obtained from 32 patients with pediatric T-ALL and 32 age- and sex-matched healthy volunteers. Patients were admitted to The First Clinical Hospital Affiliated to Harbin Medical University (Heilongjiang, China) between June 2014 and July 2018. The inclusion criteria for recruitment were: i) Patients were diagnosed with ALL for the first time; and ii) patients had otherwise normal function of the major organs. The exclusion criteria were: i) Patients had received treatment for ALL in the past 3 months; ii) other clinical disorders were observed; and iii) patients failed to cooperate with the researchers. The ALL group included 19 males and 13 females (age range, 7–14 years; mean age, 10.8±1.9 years). In the control group, there were 18 males and 14 females (age range, 7–14 years; mean age, 10.6±1.7 years). The present study was approved by the Ethics Committee of The First Clinical Hospital Affiliated to Harbin Medical University. All participants' guardians signed informed consent.

Cells of Loucy (cat. no. CRL-2629) were obtained from the American Type Culture Collection. Cells were cultured in RPMI-1640 medium (cat. no. 30–2001; American Type Culture Collection) supplemented with 10% FBS (Sigma-Aldrich; Merck KGaA) and Penicillin-Streptomycin (100 U/ml; Sigma-Aldrich; Merck KGaA) at 37°C and 5% CO₂.

The GenElute™ Total RNA Purification kit (Sigma-Aldrich; Merck KGaA) was used for RNA extraction from bone marrow samples and in vitro cultured cells, followed by preparation of cDNA samples using AMV Reverse Transcriptase (VWR International) with the following thermocycling conditions: 25°C for 5 min, 53°C for 20 min and 75°C for 10 min. All PCR mixtures were prepared using the SuperScript III Platinum One-Step qRT-PCR kit (Thermo Fisher Scientific, Inc.). The ABI 7500 system was used to perform all PCR reactions with the following conditions: 1 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 40 sec at 57.5°C. C_t values were normalized using the 2^−ΔΔCq method (12). Primer sequences were as follows: lncRNA AWPPH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′; ROCK2 forward, 5′-GTGTCGGCTCCTCTGATCTC-3′ and reverse, 5′-GGCATGTCTGGATGACCTCT-3′; and GAPDH (reference gene) forward, 5′-CTGGATGGTCGCTGCTTTTTA-3′ and reverse, 5′-AGGGGGATGAGTCGTGATTT-3′.

pcDNA3.1 vectors expressing ROCK2 and lncRNA AWPPH were designed and prepared by Sangon Biotech Co., Ltd., along with empty vectors. Cells were cultivated overnight to reach 70–80% confluence before transfection. Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used to perform cell transfection using 15 nM vectors. Transfection with empty vectors was used as the negative control (NC). Treatment with the Lipofectamine^® 2000 reagent alone was also used as another control (C). Cells were cultivated in fresh medium for 48 h prior to the subsequent assays.

Cell proliferation was measured using the CCK-8 assay (Sigma-Aldrich; Merck KGaA) at 24 h after transfection, according to the manufacturer's instructions. Briefly, 3×10³ cells/well were added to a 96-well plate, followed by the addition of 10 µl CCK-8 solution every 24 h up to 96 h. Following incubation at 37°C for 4 h, a microplate reader (Bio-Rad Laboratories, Inc.) was used to measure the absorbance at 450 nm.

Cell apoptosis was analyzed using a cell apoptosis assay 24 h after transfection. Cells were centrifuged at room temperature at 1,500 × g for 10 min to remove the supernatant. Cells were washed with PBS and counted. Subsequently, 1×10⁶ cells were stained with 500 µl binding buffer, 5 µl FITC-labeled Annexin V and 5 µl propidium iodide solution. After incubation in the dark for 10 min, apoptotic cells were detected using the FACSCalibur flow cytometry system (BD Biosciences). Data was analyzed using Flowing Software version 2.5 (Turku Bioscience).

Total protein was extracted from in vitro cultivated Loucy cells at 48 h after transfection using a Total Protein Extraction kit (cat. no. NBP2-37853; Novus Biologicals, LLC). BCA assay (Invitrogen; Thermo Fisher Scientific, Inc.) was performed to measure protein concentration. Following protein denaturation (5 min in boiling water), electrophoresis was performed (30 µg per lane) using 10% SDS-PAGE, and PVDF membranes were used for gel transfer. Following incubation with 5% skimmed milk for 2 h at 22°C, membranes were incubated with ROCK2 (dilution, 1:2,000; cat. no. ab71598; Abcam) and GAPDH (dilution, 1:1,000; cat. no. ab9485; Abcam) rabbit anti-human primary antibodies for 18 h at 4°C, followed by incubation with immunoglobulin G-horseradish peroxidase goat anti-rabbit secondary antibody (dilution, 1:1,000; cat. no. MBS435036; MyBioSource, Inc.) for 2 h at room temperature. Subsequently, membranes were incubated with ECL reagent (Sigma-Aldrich; Merck KGaA) to detect signals. Data were analyzed using ImageJ v1.46 (National Institutes of Health).

GraphPad Prism 6 software (GraphPad Software, Inc.) was used for data analysis. Data are presented as the mean ± standard deviation of three biological replicates. Differences between two groups were determined using an unpaired Student's t-test. Differences among three or more groups were analyzed using one-way ANOVA with Tukey's post hoc test. Correlations between lncRNA AWPPH and ROCK2 expression were determined using Pearson's correlation coefficient. Diagnostic values of lncRNA AWPPH and ROCK2 for T-ALL were determined using receiver operating characteristic (ROC) curve analysis. P<0.05 was considered to indicate a statistically significant difference.

lncRNA AWPPH and ROCK2 mRNA expression in bone marrow was detected by RT-qPCR. Compared with the control group, lncRNA AWPPH (Fig. 1A) and ROCK2 mRNA (Fig. 1B) expression was significantly upregulated in patients with T-ALL. In addition, western blotting results revealed that ROCK2 protein levels were higher in patients (n=4) than in controls (n=2) (P<0.05; Fig. 1C). It is worth noting that due to a lack of quality protein samples, the number of assayed patients was limited.

The diagnostic values of lncRNA AWPPH and ROCK2 for T-ALL were analyzed by ROC curve analysis. In the present analysis, true positive cases were patients with T-ALL, while true negative cases were healthy controls. For lncRNA AWPPH, the area under the curve (AUC) was 0.8954 (95% CI, 0.8168–0.9740; standard error, 0.04008; Fig. 2A). For ROCK2 mRNA, the AUC was 0.9194 (95% CI, 0.8537–0.9851; standard error, 0.03351; Fig. 2B).

The correlation between lncRNA AWPPH and ROCK2 expression was analyzed using Pearson's correlation coefficient. As shown in Fig. 3, the expression levels of lncRNA AWPPH and ROCK2 were positively correlated in patients with T-ALL (Fig. 3A) and in healthy controls (Fig. 3B).

Expression levels of lncRNA AWPPH and ROCK2 were detected 24 h after transfection. As shown in Fig. 4A, overexpression of lncRNA AWPPH and ROCK2 was achieved in the human T-ALL Loucy cell line (P<0.05; Fig. 4A). Compared with the C and NC groups, overexpression of ROCK2 significantly upregulated lncRNA AWPPH expression (P<0.05; Fig. 4B). In addition, overexpression of lncRNA AWPPH upregulated ROCK2 expression at both the mRNA and protein levels (P<0.05; Fig. 4C).

Cell proliferation and apoptosis were analyzed by CCK-8 and cell apoptosis assays, respectively, at 24 h after transfection. Compared with the C and NC groups, overexpression of either lncRNA AWPPH or ROCK2 significantly promoted proliferation (Fig. 5A) and inhibited apoptosis (Fig. 5B) of Loucy cells (P<0.05).