Tumor suppressor miR-520a inhibits cell growth by negatively regulating PI3K/AKT signaling pathway in acute myeloid leukemia

Background. Short regulatory RNAs, called microRNAs (miRNAs), have been found to possess regulatory functions in cancer and, as such, have recently been evaluated for their therapeutic role against various human malignancies. Objectives. The present work aimed to investigate whether miR-520a can play a therapeutic role in the treat-ment of human acute myeloid leukemia. Materials and methods. Human myeloid leukemia cell lines (Kasumi-1, Kasumi-3, Kasumi-6, BDCM, and K562) and a normal myeloid cell line (NCI-H5N6) were used for the study. Cell lines were subjected to real-time quantitative polymerase chain reaction (RT-qPCR), evaluation of cell viability and proliferation by MTT assay and colony formation assays. Dual acridine orange (AO)/ethidium bromide (EB) staining was applied for transfected K562 cells with miR-negative control (NC) or miR-520a mimics, and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dual staining and flow cytometry were performed to analyze cancer cell apoptosis followed by western blot. Results. Cancerous cell lines exhibited lower gene expression of miR-520a, and its overexpression significantly reduced (p < 0.05) the proliferation and viability of cancer cells. Cancer cells demonstrated the induction of Bax/Bcl-2-mediated apoptosis following miR-520a overexpression. The miR-520a was shown to target the PI3K/AKT signaling pathway in human acute myeloid leukemia to exercise its regulatory role in cancer. Conclusions. The study showed that miR-520a actively regulated cell proliferation in acute myeloid leukemia and illustrated the mechanism by which it exerts its regulatory role, emphasizing the possibility of targeting miR-520a as an efficient therapeutic strategy against human acute myeloid leukemia.


Background
There are many types of leukemia, but acute leukemia is one of the most common hematologic malignancies.It has been shown that acute leukemia occurs due to the abnormal proliferation of hematopoietic stem cells in the bone marrow and other hematopoietic tissues.Moreover, malignant cells have also been shown to accumulate in these tissues. 1 Acute myeloid leukemia results in impaired blood cell production and bone marrow failure. 2 If left untreated, patients with this disorder can die in a few weeks due to increased susceptibility to blood infections or uncontrolled blood loss caused by excessive bleeding. 3Acute leukemia is generally classified as either acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML), according to its distinctive morphology, prognosis and preferred treatment protocols to differentiate them. 4Among hematopoietic disorders, AML is characterized by the presence of numerous cytogenetic and molecular abnormalities, 5,6 and there are high morbidity and mortality rates associated with AML when compared to other cancers.The main therapeutic strategies used in the treatment of AML are chemotherapy and allogeneic stem cell transplantation. 7However, in general, the 5-year survival rate of patients with AML is still unsatisfactory. 8Therefore, the etiology of the disease needs to be investigated, and new therapeutic strategies must be formulated.
Several studies have shown that microRNA (miR) has an important role to play in regulating the expression of genes by the regulation of post-translational expression.Furthermore, miRs may play a role in the progression of a significant number of diseases. 9According to recent studies, miRNAs are involved in the regulation of leukemia progression, including both AML and chronic myeloid leukemia (CML). 10The miR-520a controls the growth and progression of many human cancers. 11A recent study has demonstrated that miR-520a suppresses the progression of non-small cell lung cancer by targeting the RRM2/Wnt pathway. 12Based on investigations carried out in HCT116 and SW480 cells, silencing of ATAD2 modulates vascular endothelial growth factor A (VEGFA) and miR-520a in colorectal cancer. 13Moreover, a study found that miR-520a regulates endoplasmic reticulum (ER) stress, proliferation, and the AKT1/NF-κB or PERK/eIF2 signaling pathways in Raji cells. 14Another study showed that piperine significantly decreased analgesia in the rat model without compression of the lumbar disc herniation by specifically and directly targeting P65 with miR-520a to treat sciatica.
However, the regulatory function of miR-520a in AML is scarce, which prompted us to evaluate the potential underlying molecular mechanisms.Furthermore, in this study, we present the therapeutic benefits of miR-520a in activating the PI3K/AKT signaling pathway for AML and suggest that targeting miR-520a could be an effective anticancer strategy against this disease.

Objectives
We aimed to investigate whether miR-520a can play a therapeutic role in the treatment of human AML by activating the PI3K/AKT signaling pathway.

Tissue samples
Samples of blood from both AML patients (24 samples) and healthy controls (22 samples) were collected from Renmin Hospital (Hubei University of Medicine, Shiyan, China) between August 2019 and September 2020.The healthy control samples were collected from normal blood donors.There was no chemotherapy or radiation therapy administered to these patients prior to sample collection, and there was no evidence of infections or multiple cancers, indicating that there was no history of multiple cancers among these patients.Patient and healthy control demographics are outlined in Table 1.All patients signed an informed consent form before the procedure.The study was approved by the Ethics Committee of Renmin Hospital (protocol No. FPH-34/2341/22), and the experimental procedure was carried out in accordance with the principles of the Declaration of Helsinki.

Cell lines
Human myeloid leukemia cell lines (Kasumi-1, Kasumi-3, Kasumi-6, BDCM, and K562) and a normal myeloid cell line (NCI-H5N6) were obtained as donations from the Department of Hematology of the Third Hospital of Shanxi Medical University (Taiyuan, China).Cell lines were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, USA), containing 10% fetal bovine serum (FBS) in 5% CO 2 at 37°C.Plasma cells were isolated and cultured from peripheral blood smears according to previously published methods. 15A colorimetric detection method (InvivoGen, San Diego, USA) was used to identify mycoplasma contamination in cell cultures, and the used cell cultures were free of mycoplasma contamination.

Cell transfection
The cells were seeded in 6-well plates at a density of 3×10 5 cells per well.According to our previous report, 5 µL of miRNA (miR-negative control (NC) or miR-520a mimics, 50 nM; Thermo Fisher Scientific, Waltham, USA) were transfected into K562 cells during the logarithmic growth phase using HiPerFect Transfection Reagent (5 µL in 300 µL of Dulbecco's modified Eagle medium (DMEM); QIAGEN, Germantown, USA) without serum, and incubated at 37°C for 20 min following the manufacturer's instructions.The cells were cultured in a CO 2 incubator for 24 h at 37°C to determine whether the transgene was expressed in cells.
In this study, a control plasmid containing empty sequences was used as a control.All plasmids were obtained from Invitrogen.

Real-time PCR
We used an Agilent High Sensitivity RNA Screen Tape (Agilent Technologies, Santa Clara, USA) as a tool to determine the quality of the RNA.The RNA was subjected to reverse transcription as soon as it was isolated from cells. 16otal RNA was extracted using TRIzol reagent to conduct RNA analysis in clinical samples and cell lines.This was done by following the manufacturer's instructions.To quantify the level of miR-520a transcript, DNase I was used to remove any DNA contamination, and approx. 1 µg of purified RNA was used to produce complementary DNA (cDNA) using an iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, USA).Real-time polymerase chain reaction (PCR) was then performed using the SYBR Green Master mix (Thermo Fisher Scientific, Waltham, USA).Three replicates were used for each real-time reaction, and the relative expression levels were quantified using the 2 −ΔΔCt method.Human β-actin was used as an internal control for miR-520a expression.Primers (Table 2) were synthesized by Suzhou Ruibo Biotechnology Co., Ltd.(Guangzhou, China).

MTT proliferation assay
The proliferation of K562 cancer cells, transfected with miR-NC or miR-520a mimics using Lipofectamine 2000 (Thermo Fisher Scientific), was estimated by the MTT assay.In brief, transfected K562 cancer cells (2×10 5 per well) were placed in a 96-well plate, then cultured in RPMI-1640 medium for 0, 12, 24, 48, or 96 h at 37°C.Each well had 10 μL of MTT reagent (dissolved in phosphate-buffered saline (PBS) (5 mg/mL)) added, followed by prolonged incubation for 4 h at 37°C.The culture medium was removed and dimethyl sulfoxide (DMSO) (150 µL) was added to each well.Then, the samples were processed for absorbance measurement at 570 nm to examine cell proliferation rates. 16

Acridine orange/ethidium bromide staining
The K562 cells (2×10 5 cells) with miR-NC or miR-520a mimics to stimulate miRNA expression were seeded in the 12-well plate.The cells were harvested after 24 h of culture at 37°C and washed twice with PBS.Then, they were fixed with methanol, and dual staining with acridine orange/ethidium bromide (AO/EB) (Cat No. E607308; Sangon Biotech Co., Ltd., Shanghai, China) was applied.A fluorescent microscope (Olympus Corp., Tokyo, Japan) was used to analyze their nuclear morphology. 16

Annexin V-FITC/PI dual staining and flow cytometry
Flow cytometry was performed to assess cell apoptosis.Briefly, miR-NC or miR-520a mimics were transfected and 2×10 6 cells per well were cultured for 48 h in a 12-well plate.The K562 cells were incubated at 37°C for 2 h with 10 µL of fluorescein isothiocyanate (FITC) and 5 µL of propidium iodide (PI) (Beyotime, Nanjing, China).After centrifugation and washing with PBS, the cells were harvested and fixed in ethanol.Then, the apoptosis was detected by flow cytometry (BD Biosciences, San Diego, USA), following the manufacturer's instructions.Apoptotic subpopulations were differentiated as follows: early apoptosis (Annexin V+/PI−), late apoptosis (Annexin V+/PI+), or necrotic/dead (Annexin V−/PI+).A total of 10,000 cells were analyzed per replicate. 16

Statistical analyses
GraphPad Prism (v.9.1; GraphPad Software, Boston, USA) was used to analyze the data, and a minimum of 3 or 5 independent replications were carried out for each experiment (n = 3-5).The Mann-Whitney U test was performed with respect to age and sex distribution and peripheral blast (PB) count.The Shapiro-Wilk normality test and Kolmogorov-Smirnov test were performed to determine whether the data conformed to a normal distribution.We found that the data were non-normally distributed.Therefore, data are presented as a median with an interquartile range (Q1-Q3).The Mann-Whitney U test was employed to compare 2 groups, and the Kruskal-Wallis test, followed by Dunn's multiple comparison test were used for comparing multiple groups.A value of p < 0.05 was considered statistically significant.

The expression of miR-520a is negatively regulated in AML
Real-time PCR expression analysis was used to investigate the relative expression of miR-520a in tissue samples.We found that miR-520a displayed significantly reduced expression in cancerous tissues compared to normal bone marrow tissue specimens (Fig. 1A, Table 3; p < 0.0001; Mann-Whitney test, U = 75.5).Similarly, cancer cell lines showed a substantially lower expression of miR-520a transcripts compared to the normal myeloid cell line (Fig. 1B, Table 4; p = 0.0004; Kruskal-Wallis test followed by Dunn's multiple comparison tests; χ 2 (5) = 22.87).The K562 cells possessed the lowest expression values (p = 0.0002) among all cancer cell lines and were therefore selected for further characterization.Therefore, the results showed that miR-520a was negatively regulated in AML and may play a prognostic role in this malignancy.

Overexpression of miR-520a decreases proliferation and viability
To examine the function of miR-520a in AML, miR-520a was overexpressed in K562 cells, and its overexpression was confirmed by real-time PCR (Fig. 2A, Table 5; p = 0.0006; Mann-Whitney test, U = 0).An MTT assay was used to investigate the proliferative impact of miR-520a impact on AML.The miR-520a-overexpressing K562 leukemia cells were found to exhibit significantly lower proliferation rates (Fig. 2B, Table 6; p = 0.031; Mann-Whitney test,   U = 2).The viability of K562 cancer cells was negatively affected by miR-520a.Cancer cells transfected with miR-520a mimics showed markedly lower colony formation potential compared to normal cells transfected with miR-NC (Fig. 2C, Table 5; p = 0.002; Mann-Whitney test, U = 0).These findings demonstrate that miR-520a negatively regulates cancer cell proliferation in AML, indicating its anticancer therapeutic potential (Fig. 2D).

Overexpression of miR-520a induces apoptosis in leukemia cells
Evaluation of the nuclear morphology of K562 leukemia cells using the AO/EB staining method showed that cancer cells exhibit a loss of nuclear viability when transfected with miR-520a mimics (Fig. 3A,B, Table 7; p = 0.002; Mann-Whitney test, U = 0).Consistent with these findings, Annexin V-FITC/PI dual staining revealed that K562 cells transfected with miR-520a mimics demonstrated apoptotic cell death (Fig. 3C,D, Table 7; p = 0.002; Mann-Whitney test, U = 0).Moreover, the expression of Bax was found to increase, while Bcl-2 protein expression decreased due to the overexpression of miR-520a, which acts as a positive signal for the induction of apoptosis (Fig. 4A, Table 8; p = 0.002; Mann-Whitney test, U = 0).Together,   p-value 0.031* *p = 0.0317 compared to miR-NC; Q1 -1 st quartile; Q3 -3 rd quartile; 95% CI -95% confidence interval.The Mann-Whitney test was used to compare the 2 groups (n = 3-5 for both groups).
the findings support our hypothesis that miR-520a positively regulates cancer cell apoptosis in AML, further confirming its anticancer therapeutic potential against this disorder.

miR-520a targets the PI/AKT signaling pathway in AML
To find a possible mechanism for the regulatory role of miR-520a in AML, we performed western blotting for both AKT and PI3K proteins (non-phosphorylated and phosphorylated).The overexpression of miR-520a did not significantly affect non-phosphorylated PI3K and AKT protein levels.However, a substantial decrease in the expression levels of the phosphorylated AKT and PI3K (p-AKT and p-PI3K) was observed due to the overexpression of miR-520a (Fig. 4A-D, Table 8; p = 0.002; Mann-Whitney test, U = 0).This indicates that miR-520a targets and blocks the PI3K/AKT signaling pathway in AML to exert its anti-proliferative role.

Discussion
Recently, miRs have been found to be crucial regulatory molecules in almost all eukaryotic organisms. 17They are seen to influence many developmental and physiological processes in humans, 18 such as proliferation, differentiation, programmed cell death, and apoptosis. 19MicroRNAs exert their regulatory role through post-transcriptional silencing of target protein-coding genes. 20Dysregulation of many miRs has been associated with various cancers, prompting researchers to investigate their regulatory role in malignancy. 21MicroRNAs are also important prognostic markers for human cancer. 22Moreover, the dysregulation of miRs has been reported in AML. 23In a recent report, miR-98 was highly expressed in AML, and the study findings were helpful in determining the prognostic importance of miR biomolecules. 24The miR-520a is a tumor suppressor gene in many human cancers, 25,19 and its downregulation has been associated with human breast cancer. 26In the present investigation, we found a similar dysregulation of miR-520a in AML.Previously, the upregulation of miR-520a was shown to negatively affect cancer cell proliferation, 27 which is supported by our results showing that the overexpression of miR-520a decreases AML cell proliferation.
Furthermore, we found that the reason behind the decrease in leukemia cell proliferation under miR-520a overexpression was the induction of apoptotic cell death in leukemia cells.Similar conclusions were drawn from previous research studies. 11The PI3K/AKT signaling pathway has shown involvement in the regulation of cell proliferation and apoptosis. 28The abnormal activation of this pathway is coupled with increased cell proliferation and survival of cells in many human cancers. 29Furthermore, it is important to highlight that phosphorylation of the PI3K and AKT proteins is responsible for stimulating the PI3K/AKT signaling pathway. 28nterestingly, the overexpression of miR-520a in leukemia cells considerably decreased the levels of phosphorylated AKT and PI3K proteins.This indicates that miR-520a inhibits the phosphorylation of these proteins and blocks the PI3K/AKT signaling cascade, leading to decreased proliferation of leukemia cells and the initiation of apoptosis.A similar mechanism of miR-520a has been reported previously. 25In summary, the current study explored the possibility of using the drug-based targeting of miR-520a for its transcriptional enhancement, as an alternative anticancer approach that could be investigated in the future against human AML.Relative protein expression levels of Bax and Bcl-2 proteins from miR-negative control (NC) or miR-520a mimics-transfected K562 cells.The Mann-Whitney test was used to compare the 2 groups (n = 6 for both groups); C-F.Relative protein expression levels of p-PI3K, PI3K, p-AKT, and AKT proteins from miR-NC or miR-520a mimics-transfected K562 cells (n = 3-5 for both groups); **p = 0.002 compared to miR-NC; Q1 -1 st quartile; Q3 -3 rd quartile.

Limitations
There were 3 limitations to this study.First, a limited number of cell lines were used in the present study to examine the effects of miR-520a.Second, inhibitors of miR-52a are not investigated in the present study.Third, other than PI3K/AKT, other significant signaling mechanisms are not explored in the current study.

Conclusions
The results of the present research established the mechanism by which miR-520a regulates the progression of human AML.The study also revealed the tumor-suppressing role of miR-520a in AML, which can be amplified by activating the PI3K/AKT pathway that specifically targets regulatory molecules to prevent proliferation of human AML efficiently.

Fig. 2 .
Fig. 2. Overexpression of microRNA (miR)-520a reduces leukemia cell proliferation and viability.A. Relative gene expression of miR-520a in miRnegative control (NC) or miR-520a mimics-transfected K562 cells.The data were not normally distributed and were tested by the Shapiro-Wilk test and Kolmogorov-Smirnov test with p-values for miR-NC (p = 0.013; p = 0.021) and miR-520a (p = 0.036; p = 0.047).The Mann-Whitney U test was used to compare the 2 groups.The data are presented as a median with an interquartile range (Q1-Q3).The upper and lower lines of each box indicate the 25 th (Q1) and 75 th (Q3) percentiles.The line inside each box indicates the median; B. MTT assay for proliferation analysis of miR-NC or miR-520a mimics-transfected K562 cells.The data were not normally distributed and were tested by the Shapiro-Wilk test and Kolmogorov-Smirnov test with p-values for miR-NC (p = 0.014; p = 0.032) and miR-520a (p = 0.034; p = 0.027), indicating significant difference at time intervals.The Mann-Whitney test was used to compare the 2 groups.The data are presented as a median with an interquartile range (Q1-Q3); C. Colony forming assay for viability analysis of miR-NC or miR-520a mimics-transfected K562 cells; D. Percentage number of colonies formed by miR-NC or miR-520a mimics-transfected K562 cells.The data were not normally distributed and were tested by the Shapiro-Wilk test and Kolmogorov-Smirnov test with p-values for miR-NC (p = 0.01; p = 0.031) and miR-520a (p = 0.015; p = 0.025).The upper and lower lines of each box indicate the 25 th (Q1) and 75 th (Q3) percentiles.The line inside each box indicates the median.The Mann-Whitney test was used to compare the 2 groups.The data are presented as a median with an interquartile range (Q1-Q3) (n = 3-5) ***p = 0.0006; *p = 0.031; **p = 0.002 compared to miR-NC.

Fig. 3 .
Fig. 3. Overexpression of microRNA (miR)-520a induces apoptosis in leukemia cells.A. Acridine orange/ethidium bromide (AO/EB) staining for the analysis of nuclear morphology of miR-negative control (NC) or miR-520a mimics-transfected K562 cells; B. Quantification of the percentage of apoptotic cells based on nuclear morphology in miR-NC and miR-520a mimics-transfected K562 cells.The data were not normally distributed and were tested by the Shapiro-Wilk test and Kolmogorov-Smirnov test with p-values for miR-NC (p = 0.011; p = 0.021) and miR-520a (p = 0.022; p = 0.036).The Mann-Whitney test was used to compare the 2 groups.The data are presented as a median with an interquartile range (Q1-Q3).The upper and lower lines of each box indicate the 25 th (Q1) and 75 th (Q3) percentiles.The line inside each box indicates the median; C. Flow cytometry-based analysis of apoptosis of miR-NC or miR-520a mimics-transfected K562 cells stained with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dual staining mix; D. Quantification of the percentage rate of apoptosis of miR-NC or miR-520a mimics-transfected K562 cells based on flow cytometry analysis.The data were not normally distributed and were tested by the Shapiro-Wilk test and Kolmogorov-Smirnov test with p-values for miR-NC (p = 0.029; p = 0.028) and miR-520a (p = 0.034; p = 0.017).The Mann-Whitney test was used to compare the 2 groups.The data are presented as a median with an interquartile range (Q1-Q3).The upper and lower lines of each box indicate the 25 th (Q1) and 75 th (Q3) percentiles.The line inside each box indicates the median (n = 3-5) **p = 0.002 compared to miR-NC.

Table 1 .
Demographics and clinical characteristics of patients and healthy blood donors AML -acute myeloid leukemia; Q1 -1 st quartile; Q3 -3 rd quartile.In healthy volunteers, there were usually no blast cells in the blood.

Table 2 .
Primers used in the study

Table 3 .
Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of microRNA (miR)-520a in tissue specimens of acute myeloid leukemia (AML) patients and healthy volunteers

Table 4 .
Real-time quantitative polymerase chain reaction (RT-qPCR) analysis in normal myeloid cells (NCI-H5N6) and various myeloid cancerous cell lines Q3 -3 rd quartile.The Kruskal-Wallis test was performed, followed by Dunn's multiple comparison test to compare the multiple groups.

Table 5 .
Relative gene expression of microRNA (miR)-520a in miR-negative control (NC) or miR-520a mimics-transfected K562 cells and the percentage number of colonies

Table 7 .
Quantification of the percentage of apoptotic cells in microRNA-negative control (miR-NC) and miR-520a mimics-transfected K562 cells based on nuclear morphology and flow cytometry analysis Quantification of the percentage of apoptotic cells based on nuclear morphology in miR-NC and miR-520a mimics-transfected K562 cells.The Mann-Whitney test was used to compare the 2 groups.The data are presented as median with interquartile range (Q1-Q3); B. Quantification of the percentage rate of apoptosis of miR-NC or miR-520a mimics-transfected K562 cells based on flow cytometry analysis.The Mann-Whitney test was used to compare the 2 groups; **p = 0.002 compared to miR-NC; Q1 -1 st quartile; Q3 -3 rd quartile.