Effect of lncRNA XIST on acute myeloid leukemia cells via miR-142-5p-PFKP axis

ABSTRACT

Acute myeloid leukemia (AML) is the common blood cancer in hematopoietic system-related diseases and has a poor prognosis. Studies have shown that long non-coding RNAs (lncRNAs) are closely related to the pathogenesis of a variety of diseases, including AML. However, the specific molecular mechanism remains unclear. Hence, the objective of this study was to investigate the effect and mechanism of lncRNA X inactive specific transcript (lncRNA XIST) on AML. To achieve our objective, some tests were performed. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to detect the expression of lncRNA XIST, miR-142-5p and the platelet isoform of phosphofructokinase (PFKP). The targeting relationship between miR-142-5p and lncRNA XIST and PFKP was verified by Pearson correlation analysis, dual-luciferase reporter assay, and pull-down assay. Functional experiments were used to analyze the effect and mechanism of action of knocking down lncRNA XIST on THP-1 and U937 cells. Compared with bone marrow cells, lncRNA XIST and PFKP expression levels were up-regulated and miR-142-5p expression levels were down-regulated in AML. Further analysis revealed that lncRNA XIST targeted and bound to miR-142-5p, and PFKP was a target gene of miR-142-5p. Knockdown of lncRNA XIST significantly promoted miR-142-5p expression to down-regulate PFKP in THP-1 and U937 cells, while the cell proliferation, cell viability, and cell cycle arrest were inhibited and apoptosis was increased. Knockdown of miR-142-5p reversed the functional impact of lncRNA XIST knockdown on AML cells. In conclusion, down-regulation of lncRNA XIST can affect the progression of AML by regulating miR-142-5p.

KEYWORDS:

• Acute myeloid leukemia (AML)
• lncRNA X inactive specific transcript (lncRNA XIST)
• miR-142-5p
• the platelet isoform of phosphofructokinase (PFKP)
• proliferation
• apoptosis
• blood cancer
• cell viability

1. Introduction

Acute myeloid leukemia (AML) belongs to a malignant clonal disorder of hematopoietic stem and progenitor cells of bone marrow [1, 2]. Generally speaking, AML is characterized by abnormal proliferation of hematopoietic progenitor cells in the bone marrow, peripheral blood, or other tissues [3, 4]. Currently, standard therapies for AML are chemotherapy and stem cell transplantation [5, 6]. Although most AML patients achieve complete remission after receiving chemotherapy, the overall 5-year survival rate remains very poor [7]. Specifically, the five-year overall survival rate for AML patients under 60 years of age is less than 50%, and for older patients, less than 20% [8]. In addition, with the rapid development of medical, science and technology, great progress has been made in drug therapy and chemotherapy [9]. Nevertheless, resistance to chemotherapy in AML patients is the main cause of treatment failure and poor prognosis [10]. Therefore, there is an urgent need to improve efficacy by further studying the mechanisms of AML and developing new therapies.

Long non-coding RNAs (lncRNAs) are non-coding RNAs greater than 200 nucleotides in length [11]. It has been shown that lncRNAs participate in both the normal physiological process of cells and the pathological process of tumor development [12]. In recent years, more and more functional lncRNAs have been gradually mined and have become the focus of current research [13]. Actually, lncRNAs are differentially expressed in human cancers, significantly affect cancer development and progression [14]. Besides, lncRNAs involved in the development of AML mainly include the lncRNA HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) [15], lncRNA plasmacytoma variant translocation gene 1 (PVT1) [16] and lncRNA Antisense noncoding RNA in the INK4 Locus (ANRIL) [17]. Therefore, lncRNAs may be potential biomarkers for targeted therapy in AML [18]. Studies have pointed out that lncRNA XIST is an lncRNA of about 17 kb [19] and plays an important role in a variety of tumors. Therefore, the value of lncRNA XIST as a tumor biomarker can be assessed by studying its expression level, prognostic value and mechanism of action in tumors. It’s reported that lncRNA XIST can promote the growth and metastasis of bladder cancer cells by regulating miR-139-5p-mediated Wnt/β-catenin signaling pathway [20]. The lncRNA XIST also interacts with miR-124 to regulate bladder cancer progression [21]. Also, the lncRNA XIST regulates the expression of cyclin-dependent kinase 6 (CDK6) through sponging miR-494, thereby promoting esophageal cancer [22]. Additionally, silencing lncRNA XIST can inhibit drug resistance in AML by up-regulating the expression of microRNA-29a and down-regulating myelocytomatosis oncogene (MYC) [23]. However, the role of lncRNA XIST in AML tumorigenesis is not fully defined. In addition, the suppression role of miR-142-5p in a variety of tumors has been demonstrated by some reports [24, 25]. For instance, Liu et al. claimed that miR-142-5p was significantly down-regulated in AML by bioinformatics analysis of microRNA expression datasets from adult and pediatric AML patients [26]. However, the mechanism of miR-142-5p in AML has not yet been reported. In this study, miR-142-5p was predicted to be a target gene of lncRNA XIST by bioinformatics. Briefly speaking, we hypothesized that lncRNA XIST could be involved in the progression of AML by regulating the expression of miR-142-5p. By virtue of this study, new targets and directions are expected to be provided for the treatment of human AML.

2. Materials and methods

2.1. Cell culture

Human normal bone marrow cells (HS-5) and AML cells (THP-1, HL-60, TF-1, NB4 and U937) were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). THP-1, HL-60, TF-1, NB4 and U937 were cultured in RPMI-1640 culture medium (11875119, Thermo Fisher Scientific, Waltham, USA), and HS-5 was cultured in formulated Dulbecco's Modified Eagle Medium (DMEM) culture medium (11965092, Thermo Fisher Scientific, Waltham, USA). Both mediums were supplemented with 10% fetal bovine serum (FBS, A31608-02, Gibco, USA) and 1% penicillin streptomycin (15140148, Gibco, USA), and the cells were cultured in an incubator with 5% CO₂ at 37 °C. When cultured cells reached the log phase, they were passaged by centrifugation for seed preservation or experimental studies.

2.2. Cell transfection

THP-1 and U937 cells in the logarithmic growth phase were collected, diluted to 2× l0⁶ cells/ml, then seeded in a 6-well plate. Transfection was performed when the cell growth confluence was up to 80–90%. LncRNA XIST shRNA (sh-XIST) and its control (sh-NC) were designed and synthesized by Guangzhou RiboBio Co., Ltd. MiR-142-5p mimics, miR-142-5p inhibitor and their negative controls (mimics NC and inhibitor NC) were purchased from GenePhama (China). The interference vectors, mimics or inhibitors were transfected into the cells by Lipofectamine ™ 2000 Transfection Kit (11668019, Invitrogen, USA). After 6 hours of incubation, a new culture medium was used for another 48-hour culture, followed by the cell collection.

2.3. Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from the serum or cells by a Total RNA kit (AM1924, ThermoFisher Scientific, America) then stored at −80 °C. Next, the cDNA was synthesized according to the instructions of reverse-transcription PCR kit (RR047A, Takara, Japan), subsequent to the determination of the concentration and purity. Later, referring to the instructions of One Step PremieScript™ RT–PCR kit (RR064A, Takara, Japan), the synthesized cDNA was subject to qRT-PCR. The reaction program was shown as follows: 95 °C for 1 min; 95 °C for 40 s, 58 °C for 40 s, 72 °C for 45 s, for 35 cycles; then 72 °C for 10 min. Data analysis was performed by 2^−ΔΔCt. The primer sequences were displayed in Table 1.

Table 1. Primer sequences of qRT-PCR.

Gene	Primer sequences
lncRNA XIST	F 5′-TTTCATCGCCCATCGGTG-3′
	R 5′-CTGCCTGACCTGCTATCATC-3′
PFKP	F 5’-GGAGTGTGAGTGTGTCCTGG-3’
	R 5’-ACGCTATGCAGTAGCCTTCC-3’
miR-142-5p	F 5′-AACTCCAGCTGGTCCTTAG-3′
	R 5′-TCTTGAACCCTCATCCTGT-3′
GAPDH	F 5′-AATCCCATCACCATCTTC-3′
	R 5′-AGGCTGTTGTCATACTTC-3′
U6	F 5′-CTCGCTTCGGCAGCACA-3′
	R 5′-AACGCTTCACGAATTTGCGT-3′

2.4. Cell-Counting-Kit-8 (CCK-8)

After transfection, THP-1 and U937 cells were collected, and the cell concentration was adjusted to 1 × 10⁴/ml. Subsequently, the cells were seeded in a 96-well micro-plate. After the cells were cultured to 24, 48 and 72 h, respectively, 10 μl CCK-8 solution (CA1210, Solarbio, China) was added to each well. Finally, the absorbance value at 450 nm was measured by a microplate reader (Thermo Fisher Scientific, Waltham, USA).

2.5. Colony forming cell assay

The transfected THP-1 cells and U937 cells were continuously diluted and quantified with 10 times sterile physiological saline, and then inoculated onto Middlebrook 7H10 agar (medium 173, ATCC) plates supplemented with 10% OADC. After 3 weeks of culture, the cells were fixed with 4% paraformaldehyde for 15 min. When macroscopically visible colonies were formed, 0.1% crystal violet was added for 10 min of staining. Finally, under an inverted microscope (Nikon, Japan), the colonies were photographed, the number of colonies > 50 was counted and the colony-forming efficiency was calculated.

2.6. Apoptosis

Annexin V-allophycocyanin (APC) apoptosis detection kit (559763, BD Pharmingen, San Jose, USA) was applied to detect apoptosis. To be specific, cells from each group were collected 48 hours after transfection, and the cell concentration was adjusted to 1 × 10⁶/ml. Next, the cells were stained with 5 µl of Annexin V-FITC and 10 µl of PI then placed in the dark at ambient temperature (20–25 °C) for 15 min. After washing twice with PBS, the treated cells were collected and analyzed with FACScan flow (Becton Dickinson, San Diego, CA, USA) within 1 hour.

2.7. Dual luciferase reporter assay

Dual luciferase reporter plasmid was combined with lncRNA XIST-wild type (lncRNA XIST-WT)/the platelet isoform of phosphofructokinase (PFKP)-wild type (PFKP-WT) or 3’-UTR-mutant fragments (lncRNA XIST-MUT, PFKP-MUT). Lipofectamine ™ 2000 Transfection Kit (11668019, Invitrogen, USA) was applied to co-transfect the constructed lncRNA XIST-WT, lncRNA XIST-MUT, PFKP-WT and PFKP-MUT dual luciferase reporter vectors into 293 T cells with miR-142-5p mimics, respectively. After 48-hour culture, the cells were collected and lysed at ambient temperature for 20 min. Later, a luciferase substrate was added and the luciferase activity was measured by a luminescence instrument. Finally, relative firefly luciferase activity was calculated by using the activity of renilla luciferase as an internal control.

2.8. Pull-down assay

Biotin-labeled lncRNA XIST (Bio-XIST) and lncRNA XIST-wild type (Mut-Bio-XIST) RNA fragments were synthesized. Then the fragments were transfected into the cells with Lipofectamine ™ 2000 Transfection Kit (11668019, Invitrogen, USA). After 48-hour transfection, the cells were collected and then dissolved with Dnase I (Rnase free, EN0525, Invitrogen, USA) and complete protease inhibitors. Subsequently, the cell lysate was incubated with Rnase-free BSA and tRNA-coated streptavidin agarose beads (88817, Invitrogen, USA), and the ice bath was conducted for 2 h. Combined with RNA, Trizol was utilized for the purification. Next, qRT-PCR was used to measure the level of miR-142-5p pulled down by Bio-XIST and Mut-Bio-XIST.

2.9. Western blot

The cells were lysed with RIPA lysate for 20 min. After an ice bath and ultrasonic breaking, the cells were centrifuged at 12000rpm at 4 °C for 15 min. Then, the total protein was collected, and the protein concentration was measured. Next, the proteins were separated by 12% of SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk at ambient temperature. After 1 h, the primary antibodies were added, and the membranes were incubated at 4 °C overnight. Subsequently, the membranes were washed twice and diluted with enzyme-labeled secondary antibodies were added for another 1-hour incubation at ambient temperature. Later, bands were detected with an ECL chemiluminescence substrate kit (P0018FM, Beyotime, China) and semiquantitative analysis was performed using ImageJ software (National Institutes of Health, USA).

2.10. Statistical analysis

The result was expressed as mean ± standard deviation (SD). Statistical analysis of the experimental data was performed by SPSS 22.0. All data conformed to a normal distribution and were analyzed using a T-test when the experiment had only two groups. A one-way analysis of variance followed by Tukey’s post-hoc test was used for multiple comparisons. P < 0.05 was considered statistically significant.

3. Results

3.1. High expression of lncRNA XIST in acute myeloid leukemia cells

Firstly, lncRNA XIST expression in AML cell lines (THP-1, HL-60, TF-1, NB4 and U937) was increased obviously compared with that in human normal bone marrow cells (HS-5). Furthermore, lncRNA XIST expression was the highest in THP-1 cells and reached the lowest in U937 cells (Figure 1A). Therefore, THP-1 cells and U937 cells were used in the subsequent experiments. The above results suggested that lncRNA XIST might play an important role in AML.

Figure 1. Expression of lncRNA XIST in acute myeloid leukemia cell lines. A, QRT-PCR to determine lncRNA XIST expression in cell lines (THP-1, HL-60, TF-1, NB4 and U937) of acute myeloid leukemia and human normal bone marrow cells (HS-5) (n = 3). *P < 0.05, **P < 0.01 vs. HS-5 group. Values were expressed mean ± standard deviation.

3.2. Knockdown of lncRNA XIST can inhibit proliferation and promote apoptosis of acute myeloid leukemia cells

To further examine the effect of lncRNA XIST expression on the functions of AML cells, cell proliferation and apoptosis were detected after knockdown of lncRNA XIST. The results showed that lncRNA XIST expression was notably decreased in THP-1 and U937 cells in the sh-XIST#1 and sh-XIST#2 groups compared with that in the sh-NC group, and lncRNA XIST exhibited a better decreasing effect in the sh-XIST#1 group (Figure 2A). In addition, compared with the sh-NC group, the proliferation rate and cell viability of THP-1 and U937 cells were significantly reduced while the apoptosis rate was significantly increased after knockdown of lncRNA XIST (Figure 2B–D). Moreover, B-cell lymphoma 2 (Bcl-2) expression was obviously decreased while the expression of apoptosis-related proteins cleaved cysteinyl aspartate specific proteinase (caspase) 3 and cleaved caspase 9 was significantly increased in the cells of the sh-XIST#1 group (Figure 2E). The results of cell cycle assay also showed a significant increase in the proportion of cells in the G0/G1 phase after knockdown of lncRNA XIST and a notable decline in the proportion of S phase cells in UP937 cells (Figure 2F). In a word, interference with lncRNA XIST expression inhibited AML cell proliferation, cloning and cycle, and promoted apoptosis.

Figure 2. Effects of lncRNA XIST knockdown on proliferation, apoptosis and cell cycle of acute myeloid leukemia cells. After THP-1 and U937 cells were transfected with sh-lncRNA XIST and sh-NC, respectively, A, Expression of lncRNA XIST in THP-1 and U937 cells detected by qRT-PCR ; B, The proliferation rate of THP-1 and U937 cells measured by MTT assay; C, Colony forming cell assay to observe the proliferation of THP-1 and U937 cells; D, Apoptosis of THP-1 and U937 cells determined by flow cytometry; E, Protein expression of cleaved Caspase3, cleaved Caspase9 and Bcl-2 in THP-1 and U937 cells detected by western blot; F, Cell cycles of THP-1 and U937 tested by flow cytometry. *P < 0.05, **P < 0.01 vs. sh-NC group.

3.3. LncRNA XIST can target miR-142-5p expression in acute myeloid leukemia cells

The ENCORI database (http://starbase.sysu.edu.cn/) was utilized to expect the target gene of lncRNA XIST. The results indicated that there was a targeting relationship between lncRNA XIST and miR-142-5p (Figure 3A). Besides, according to the outcome of dual luciferase reporter assay, over-expression of miR-142-5p inhibited the luciferase activity of lncRNA XIST-WT. However, after lncRNA XIST mutation, luciferase activity was not obviously affected (Figure 3B). Also, the pull-down assay showed that lncRNA XIST mainly precipitated miR-142-5p in THP-1 and U937 cells (Figure 3C), further demonstrating that there was a targeting correlation between lncRNA XIST and miR-142-5p. Further, the qRT-PCT results showed a significant decrease in miR-142-5p expression in AML cell lines compared to HS-5 cells, with miR-142-5p being the lowest expressed in THP-1 cells and the highest expressed in U937 cells (Figure 3D). Additionally, miR-142-5p expression was observed to be significantly elevated after knockdown of lncRNA XIST in THP-1 and U937 cells (Figure 3E). In a nutshell, LncRNA XIST targeted and bound miR-142-5p in AML.

Figure 3. Targeting relationship between lncRNA XIST and miR-142-5p. A, ENCORI database to predict the target gene of lncRNA XIST; B, Dual luciferase reporter assay to determine the targeting relationship between lncRNA XIST and miR-142-5p; C, Pull-down assay to validate the binding of lncRNA XIST and miR-142-5p in THP-1 and U937 cells; **P < 0.01 vs. MUT-Bio-XIST group; D, QRT-PCR to measure miR-142-5p in cell lines (THP-1, HL-60, TF-1, NB4 and U937) of acute myeloid leukemia and human normal bone marrow cells (HS-5) (n = 3), **P < 0.01 vs. HS-5 group; E, MiR-142-5p expression in THP-1 and U937 cells in each group detected by qRT-PCR, **P < 0.01 vs. Si-NC group. Values were expressed as mean ± standard deviation.

3.4. Down-regulation of lncRNA XIST inhibits the proliferation and induces apoptosis of acute myeloid leukemia cells by regulating miR-142-5p

To further validate the role of miR-142-5p in the effect of lncRNA XIST on AML cells, the miR-142-5p expression was inhibited and observed. On the basis of the observation results, compared with the sh-XIST1 + NC inhibitor group, inhibition of miR-142-5p in THP-1 and U937 cells not only reversed the promoting effects of knockdown of lncRNA XIST on cell proliferation, viability reduction, apoptosis, but also slowed cell cycle arrest (Figure 4A–D). Besides, down-regulation of miR-142-5p inhibited the down-regulation of Bcl-2 protein and the up-regulation of protein level of cleaved Caspase 3 and cleaved Caspase 9 mediated by knockdown of lncRNA XIST (Figure 4E). Briefly speaking, down-regulation of lncRNA XIST inhibited the proliferation and induced apoptosis of AML cells by regulating miR-142-5p.

Figure 4. The role of miR-142-5p in the effect of lncRNA XIST on the proliferation and apoptosis of acute myeloid leukemia cells. A, MTT assay to detect the proliferation rate of THP-1 and U937 cells in each group; B, Colony forming cell assay to observe the cell viability of THP-1 and U937 cells in each group; C-D, Flow cytometry to determine apoptosis (C) and cell cycles (D) of THP-1 and U937 cells; E, Western blot to measure the protein expression level of cleaved Caspase 3, cleaved Caspase 9 and Bcl-2 in THP-1 and U937 cells. *P < 0.05, **P < 0.01 vs. NC inhibitor group; #P < 0.05, ##P < 0.01 vs. sh-XIST#1 + NC inhibitor group. Values were expressed as mean ± standard deviation.

3.5. LncRNA XIST can affect the occurrence and development of acute myeloid leukemia through regulating the miR-142-5p/PFKP axis

According to the prediction of Targetscan database (http://targetcscan.org/vert-72/), there was a targeting relationship between miR-142-5p and PFKP. The dual luciferase reporter assay outcomes revealed that co-transfection of miR-142-5p mimics significantly inhibited the luciferase activity of PFKP-WT vector, but didn’t affect that of PFKP-MUT vector, indicating a targeting relationship between PFKP and miR-142-5p (Figure 5A). Further, the results of qRT-PCR results showed that PFKP expression was significantly increased in AML cell lines compared to HS-5 cells, where PFKP expression was highest in THP-1 cells and lowest in U937 cells (Figure 5B). Additionally, both mRNA and protein levels of PFKP were notably down-regulated after over-expression of miR-142-5p while up-regulated after inhibition of miR-142-5p in THP-1 and U937 cells (Figure 5C,D). Correlation analysis also showed a negative correlation between miR-142-5p and PFKP expression (Figure 5E). Moreover, knockdown of lncRNA XIST also revealed that the mRNA and protein expression levels of PFKP were significantly reduced in AML cells (Figure 5F,G). Next, the correlations of PFKP, miR-142-5p and lncRNA XIST were further determined by western blot. The determination outcomes showed that inhibition of miR-142-5p reversed the inhibitory effect of knockdown of lncRNA XIST on PFKP expression (Figure 5H), indicating that lncRNA XIST could regulate the miR-142-5p/PFKP axis.

Figure 5. The targeting relationship between miR-142-5p and PFKP in acute myeloid leukemia cells. A, Dual luciferase reporter assay to verify the targeting relationship between miR-142-5p and PFKP, **P < 0.01 vs. NC mimics group; B, qRT-PCR to detect the expression of PFKP in in cell lines (THP-1, HL-60, TF-1, NB4 and U937) of acute myeloid leukemia and human normal bone marrow cells (HS-5) (n = 3), **P < 0.01 vs. HS-5 group; C, qRT-PCR to measure the expression of PFKP mRNA after overexpression or inhibition of miR-142-5p in THP-1 and U937 cells, **P < 0.01 vs. NC mimics group, ##P < 0.01 vs. NC inhibitor group; D, Western blot to determine the protein expression of PFKP after overexpression or inhibition of miR-142-5p in THP-1 and U937 cells; E, Correlation analysis to test the association between miR-142-5p and PFKP (n = 30); F, qRT-PCR to measure the expression of PFKP mRNA after inhibition of lncRNA XIST in THP-1 and U937 cells; G, Western blot to evaluate the expression of PFKP protein after inhibition of lncRNA XIST in THP-1 and U937 cells, **P < 0.01 vs. si-NC group; H, Western blot to detect the PFKP expression in THP-1 and U937 cells in each group, **P < 0.01 vs. NC mimics group, ##P < 0.01 vs. si-XIST#1 + NC inhibitor group. Values were expressed as mean ± standard deviation.

4. Discussion

AML is a malignant hematopoietic disease that seriously threatens public health and life safety [26–28]. Despite the gradually improvement of the therapeutic effect with the development of medical technology, AML still has a poor clinical prognosis. At present, chemotherapy, with some side effects on the human body, is the main therapeutic method for AML [29, 30]. Therefore, it is essential to find effective targets and strategies for early diagnosis and treatment of AML. In recent years, the effect of lncRNAs on AML has attracted the high attention of researchers. For example, some scholars have reported the important role of lncRNAs in regulating gene expression [31]. LncRNA XIST, a transcription product of chromosome Xq13.2 that is located in the X-chromosome inactivation center region, can affect the activation of X-chromosome-associated genes [32]. Some studies have discovered that lncRNA XIST is closely related to the occurrence and development of AML and may serve as a prognostic marker and therapeutic target in AML patients [33]. In this study, lncRNA XIST was up-regulated in AML cell lines; Knockdown of lncRNA XIST significantly inhibited the proliferation and viability while promoting apoptosis in AML cells. The results of this study were consistent with previously reported findings, confirming high lncRNA XIST expression in tumor tissues.

MicroRNAs (miRNAs) belong to endogenous non-coding RNAs of 18–25 nucleotides in length. Generally, miRNAs control a series of biological processes such as gene expression, cell differentiation, proliferation, and apoptosis by specifically binding to mRNA 3'UTR [34]. Some reports have stated that miRNAs play an important role in leukemogenesis and hematopoietic stem cell function [35]. Recent studies have claimed that miR-142 is the most commonly mutated miRNA gene, with mutations occurring mainly in blood cancers. In AML, point mutations in the miR-142-3p seed sequence result in a loss of miR-142-3p function and a decreased in miR-142-5p expression, thereby enhancing the pathogenic activity and myeloid potential of multipotent hematopoietic progenitor cells; besides, the mutations also reduce lymphoid potential, cause sustained expression of myeloid progenitor cells/myeloblasts, and ultimately transformation to leukemia [35, 36]. Shortly speaking, miR-142-5p is closely associated with AML pathogenesis and is a potential therapeutic target for AML. In this study, miR-142-5p expression levels were significantly reduced in AML cell lines, and further analysis showed that lncRNA XIST was negatively correlated with miR-142-5p expression. Also, dual luciferase reporter assay and pull-down assay demonstrated a competitive targeting relationship between lncRNA XIST and miR-142-5p. In addition, inhibition of miR-142-5p reversed the effects of lncRNA XIST knockdown on AML cell proliferation, cell viability, apoptosis, and cell cycle. In other words, lncRNA XIST may promote the progression of AML by inhibiting miR-142-5p expression.

PFKP is a rate-limiting enzyme during glycolysis. The level of gene transcription and translation of PFKP can cause changes in glucose metabolism [37]. At present, up-regulated PFKP expression has been revealed in tumor studies such as nerve cells [38], human cervix [39], liver cancer [40], renal cancer [41] and breast cancer [42], while few studies report PFKP expression in leukemia. In this study, PFKP expression was up-regulated in AML cell lines; moreover, PFKP expression was positively correlated with lncRNA XIST expression and negatively correlated with miR-142-5p expression. Additionally, according to the results of dual luciferase reporter assay, PFKP was a target gene of miR-142-5p. Furthermore, inhibition of miR-142-5p reversed the suppression of lncRNA XIST knockdown on PFKP. All the above results indicate that lncRNA XIST regulated the biological process of AML through the miR-142-5p/PFKP axis. However, the specific mechanism by which lncRNA XIST regulates the miR-142-5p/PFKP axis still requires further experimental investigation.

However, this study can preliminarily show that lncRNA XIST affects the progression of AML by regulating the miR-142-5p/PFKP axis. But there are still many deficiencies in our research. First, we did not collect the age, sex, and average age of the AML patients and healthy individuals. Then, we did not detect the expression of lncRNA XIST in AML patients of different age groups. The expression of lncRNA XIST may vary with the age of AML patients in the study. In addition, our study lacks a correlation between the expression of lncRNA XIST and the clinicopathological information. Finally, we did not explore the regulatory mechanism and influence of lncRNA XIST in AML in vivo.

5. Conclusion

In summary, lncRNA XIST is up-regulated in AML. Inhibition of lncRNA XIST inhibits the proliferation but promotes apoptosis in AML cells. Specifically, lncRNA XIST may affect the occurrence and development of AML by competitive binding to miR-142-5p and targeted regulation of PFKP. All in all, lncRNA XIST may be a potential target for early diagnosis and targeted therapy of AML.

Author contributions

Zhaozhi Jiang and Jiao Li conceived the study. Tingting Liu extracted all dada. Lusheng Guo co-wrote the paper. Youhong Wang undertook the statistical analyses. All authors read and approved the final manuscript.

Competing interests

The authors have no competing interests to declare.

Data availability statement

All data generated or used during the study are available from the corresponding author by request.

Additional information

Funding

This work was supported by Department of Education of Jilin Province (grant numbers JJKH20170417KJ).