lncRNA TP73-AS1 Regulates miR-21/PTEN Axis to Affect Cell Proliferation in Acute Myeloid Leukemia

Participants and bone marrow

This study passed the review of the Ethics Committee of the First Affiliated Hospital of Kunming Medical University (no. FAH20160423896532KMU).

Bone marrow (BM) samples were obtained from 50 AML patients (30 males and 20 females, 51–75 years old, mean age 63.6 ± 5.1 years) and 50 healthy controls (30 males and 20 females, 51–75 years old, mean age 63.8 ± 5.3 years) who were admitted at the aforementioned hospital between May 2016 and May 2019. AML with maturation (M2) is a subtype of AML. In this study, 40 patients were diagnosed as M2. All AML patients are newly diagnosed cases and no other severe clinical disorders were diagnosed. No therapies were initiated before the collection of BM. BM mononuclear cells (BMMNCs) were separated from BM using the Lymphocyte Separation Medium (TBD Sciences). All samples of BMMNCs were stored in liquid nitrogen before use.

AML cell line and transient transfections

The AML human Kasumi-6 cell line (M2) from ATCC was used in this study. Cells were cultivated in Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% fetal bovine serum and 100 U/mL penicillin/streptomycin at 37°C with 5% CO₂. Cells used for transfections were harvested at 75%–85% confluence.

Expression vectors of TP73-AS1 and PTEN were constructed by RiboBio (Guangzhou, China) using pcDNA3.1 vector as the backbone. Negative control (NC) miRNA and miR-21 mimic were also synthesized by RiboBio. Lipofectamine 2000 (Invitrogen) was used to transfect siRNA (50 nM) or vector (10 nM) into 10⁶ cells. Transfection efficiency was measured by quantitative polymerase chain reaction (qPCR) at 24 h post-transfection. Untransfected cells were used as control (C) cells. Cells transfected with NC miRNA or empty pcDNA3.1 vector were NC cells. Cells were collected at 24 h post-transfection to perform the following experiments.

Luciferase activity assay

pGL3 Luciferase Reporter Vector (Basic; Promega Corporation) was used as the backbone to construct TP73-AS1 luciferase vector. Transfections were performed to cotransfect TP73-AS1 vector + NC miRNA or TP73-AS1 vector + miR-21 mimic into 10⁶ cells. Cells were harvested at 48 h post-transfection. Luciferase activity assay was performed using the Dual-Luciferase Reporter Assay System (Promega Corporation). Renilla luciferase activity was normalized to Firefly luciferase expression.

RNA extractions and RT-qPCR

Total RNAs were extracted from tissue samples and cultivated cells using the GenElute™ Total RNA Purification Kit (Sigma-Aldrich). RNA samples were digested by DNA eraser (Takara) to remove genomic DNAs. Reverse transfections were performed using the PrimeScript^® RT reagent kit (TaKaRa). qPCRs were prepared using the QuantiFast SYBR Green PCR Kit (QIAGEN) to measure the expression levels of TP73-AS1 and PTEN. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as endogenous control. High Pure miRNA Isolation Kit (Sigma-Aldrich) was used to extract miRNAs. The expression levels of mature miR-21 were measured using the All-in-One™ miRNA qRT-PCR Reagent Kit (GeneCopoeia). Fold changes of gene expression were calculated using 2^−ΔΔCT methods. All PCRs were repeated three times.

Western blot

Total proteins were extracted from cells harvested at 24 h post-transfection using RIPA buffer (Cell Signaling Technology). Protein concentrations were measured using the BCA Protein Assay Kit (Cell Signaling Technology). After protein denaturation at 95°C for 10 min, 12% sodium dodecyl sulfate/polyacrylamide gel was used to separate proteins, followed by gel transfer to polyvinylidene fluoride membranes. After blocking in phosphate-buffered saline containing 5% fat-free milk at room temperature for 2 h, membranes were incubated with rabbit primary antibodies of GAPDH (ab9845; Abcam) and PTEN (ab31392; Abcam) at 4°C for 18 h, followed by incubation with the HRP goat anti-rabbit (IgG) secondary antibody (ab6721; Abcam) at room temperature for 2 h.

Signals were produced using enhanced chemiluminescence (Santa Cruz Biotechnology), and data were processed using the ImageJ v1.46 software.

Cell proliferation assay

Cell proliferation was assessed at 24 h post-transfection using the Cell Counting Kit-8 kit (Dojindo). Briefly, each well of a 96-well plate was added with 0.1 mL cell suspension containing 10⁴ cells. Cells were cultivated under aforementioned conditions and 0.01 mL WST-8 solution was added into each well at 4 h before cell collection. Cells were collected every 24 h for a total of 96 h. Absorbance was measured at 450 nm using a spectrophotometer.

Statistical analysis

Data of three biological replicates are expressed as mean ± standard deviation and were analyzed by the SPSS 18.0 software (SPSS, Inc.). Unpaired t-test was used to compare differences between two groups. Differences among multiple groups were compared by analysis of variance (ANOVA) (one-way) and Tukey test. p < 0.05 was statistically significant.

TP73-AS1 was downregulated in AML

The TCGA data set was the first to explore the differential expression of TP73-AS1 in AML. It was observed that TP73-AS1 was significantly downregulated in AML in comparison with that in nontumor tissues (12.01 vs. 24.51). Downregulation of TP73-AS1 in AML was further confirmed by detecting the expression levels of TP73-AS1 in BMMNCs from both AML patients (n = 50) and healthy controls (n = 50) (Fig. 1). Unpaired t-test showed that the expression levels of TP73-AS1 were significantly lower in AML patients than that in the controls (p < 0.001).

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FIG. 1.

TP73-AS1 was downregulated in AML. The downregulation of TP73-AS1 in AML was analyzed by measuring the expression levels of TP73-AS1 in bone marrow mononuclear cells from both AML (n = 50) and controls (n = 50). Unpaired t test was used for data comparison. Polymerase chain reactions were repeated three times and data were expressed by mean values.***p < 0.001. AML, acute myeloid leukemia.

TP73-AS1 can directly interact with miR-21

The interactions between TP73-AS1 and human miRNAs in the miRBase database were predicted using the online RNA-RNA interaction prediction program IntaRNA. Among human miRNAs, TP73-AS1 was predicted to form strong base-pairing with miR-21 (Fig. 2A). Dual-luciferase reporter assay showed that compared with Kasumi-6 cells transfected with TP73-AS1 vector and miRNA NC (NC group), cells transfected with TP73-AS1 vector and miR-21 mimic had a significantly lower relative luciferase activity (Fig. 2B, p < 0.05).

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FIG. 2.

TP73-AS1 can directly interact with miR-21. The interactions between TP73-AS1 and human miRNAs from miRBase were analyzed using an online RNA-RNA interaction prediction program named IntaRNA. The potential base-paring formed by TP73-AS1 and miR-21 was presented (A). Dual luciferase reporter assay was performed by cotransfecting TP73-AS1 vector with miRNA NC (NC group) or miR-21 mimic (miR-21 group) into Kasumi-6 cells. Experiments were repeated three times and data were expressed as mean values (B). *p < 0.05. NC, negative control.

TP73-AS1 upregulates PTEN, a target of miR-21

Kasumi-6 cells were transfected with TP73-AS1 expression vector or miR-21 mimic to further assess the interaction between TP73-AS1 and miR-21. Overexpression of TP73-AS1 and miR-21 was confirmed by qPCR at 24 h post-transfection (Fig. 3A, p < 0.05). The effects of overexpressing TP73-AS1 and miR-21 on the expression of each other were also evaluated by qPCR at 24 h post-transfection. Compared with the NC and C groups, overexpression of TP73-AS1 and miR-21 did not affect the expression of each other (Fig. 3B, p < 0.05). Western blot and qPCR were performed to evaluate the effects of overexpressing TP73-AS1 on the expression of PTEN at both protein and mRNA levels, respectively. Compared with the C and NC groups, overexpression of TP73-AS1 led to upregulated PTEN at both protein (Fig. 3B) and mRNA (Fig. 3C) levels (p < 0.05).

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FIG. 3.

TP73-AS1 upregulates PTEN, a target of miR-21. Kasumi-6 cells were transfected with TP73-AS1 expression vector or miR-21 mimic to further analyze the interaction between TP73-AS1 and miR-21. Overexpression of TP73-AS1 and miR-21 was confirmed by qPCR at 24 h post-transfection (A). The effects of TP73-AS1 and miR-21 overexpression on the expression of each other were also analyzed by qPCR at 24 h post-transfection (B). Western blot and qPCR were performed to analyze the effects of TP73-AS1 overexpression on the expression of PTEN at both mRNA (C) and protein (D) levels. Experiments were repeated three times and data were expressed as mean values. *p < 0.05. PTEN, phosphatase and tensin homologue; qPCR, quantitative polymerase chain reaction.

TP73-AS1 regulated miR-21/PTEN axis to suppress the proliferation of Kasumi-6 cells

The effects of overexpressing TP73-AS1, miR21, and PTEN on the proliferation of Kasumi-6 cells were assessed by CCK-8 assay. Compared with the C group, overexpression of TP73-AS1 and PTEN led to decreased proliferation rates of AML cells. Overexpression of miR-21 played an opposite role and reduced the effects of overexpressing TP73-AS1 and PTEN (Fig. 4, p < 0.05).

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FIG. 4.

TP73-AS1 regulated miR-21/PTEN axis to suppress the proliferation of Kasumi-6 cells. The effects of TP73-AS1, miR21, and PTEN overexpression on the proliferation of Kasumi-6 cells were analyzed by CCK-8 assay. Experiments were repeated three times and data were expressed as mean values. *p < 0.05. PTEN, phosphatase and tensin homologue.