Linoleic acid derivatives target miR‐361‐3p/BTG2 to confer anticancer effects in acute myeloid leukemia

Acute myeloid leukemia (AML) is a deadly hematologic malignancy. In this study, miR‐361‐3p and BTG2 gene expression in AML blood and healthy specimens were analyzed using quantitative real‐time reverse transcription polymerase chain reaction. A significant negative correlation between miR‐361‐3p and BTG2 was observed. The cell viability and apoptosis were measured by CCK‐8 assay, EdU incorporation assay and flow cytometry. A dual‐luciferase reporter gene assay was performed to confirm the binding sequence between miR‐361‐3p and BTG2 messenger RNA 3ʹ‐untranslated region. 9s‐Hydroxyoctadecadienoic acid (9s‐HODE), a major active derivative of linoleic acid, reduced the viability and induced cell apoptosis of HL‐60 cells. Furthermore, the miR‐361‐3p mimics and siBTG2 reversed the above effects of 9s‐HODE. 9s‐HODE exerted an anti‐AML effect through, at least partly, regulating the miR‐361‐3p/BTG2 axis.


| INTRODUCTION
Acute myeloid leukemia (AML) is characterized by uncontrolled proliferation of clonal neoplastic cells and excessive accumulation of immature myeloid blasts in the bone marrow. [1]The incidence rate for AML is approximately 2.8/100,000 per year worldwide. [2]Current therapies for AML include chemotherapy and radiotherapy (monotherapy or in combination) as well as hematopoietic stem cell transplantation. [3]However, the adverse effects associated with current therapies require the urgent development of alternative strategies. [4]croRNAs (miRNAs) are noncoding small RNAs, which are generally 18-25 nucleotides in length. [5]miRNAs regulate the expression of targeted genes by degradation of mRNAs or inhibition of translation. [6]9][10][11] miRNAs can function as either oncomiRs or tumor suppressors in AML. [12]Several studies involving the use of miR-based therapeutics have shown encouraging results in preclinical in vitro and animal models, suggesting manipulating miRNA may improve AML management. [12]noleic acid (LA) is an essential polyunsaturated fatty acids for growth and development. [13]LA and its oxidized derivatives exert a variety of biological effects, such as antimicrobial or antioxidative effects. [14]9-and 13-hydroxyl-octadecadienoic acids (HODEs) are two major metabolic derivatives of LA and are often present in two enantiomeric forms, 9/13-S-HODE and 9/13-R-HODE, respectively. [15]ese two HODEs were associated with metabolic disorders and cancer. [16,17]For instance, HODEs suppress cell proliferation and induces apoptosis in colorectal cancer cells by targeting peroxisome proliferator-activated receptor gamma. [18]Moreover, in our previous study, we found HODEs inhibited the proliferation and migration of AML cells (HL-60). [19]However, the underlying mechanism of this effect in AML remains to be fully established.
In the present study, we investigated the potential role of miRNAs in the treatment of AML.We show that miR-361-3p is a biomarker and therapeutical target of AML.Herein, through an in vitro model, we prove that 9s-HODE can inhibit AML cell proliferation and induce cell apoptosis via regulating miR-361-3p/BTG2 axis.

| Patients and clinical specimens
This study was conducted in accordance with the Declaration of Helsinki, and approved by the Southern Medical University-nanfang Hospital Ethics Board (NFEC-2022-014).All participants had written informed consent.All methods were performed in accordance with the relevant guidelines and regulations.Blood samples (3-5 mL) from 34 AML patients (18-65 years) and five healthy (24-40 years) donors were collected.

| Cell cultures
Human acute myeloid leukemia cell line (HL-60) and HEK293 cell line were obtained from the Chinese Academy of Sciences Cell Bank.Cells were cultured in Dulbecco's modified Eagle's medium media containing 10% fetal bovine serum and antibiotics (100 units/mL penicillin G and 100 mg/mL streptomycin) at 5% CO 2 and 37°C.Cells were then subcultured and treated with 9s-HODE (≥98.0%by HPLC; Larodan Fine Chemicals AB) dissolved in ethanol.

| miRNA expression profiling
HL-60 cells with or without 9s-HODE treatment were lysed in Trizol (Invitrogen) for total RNA extraction.DNA was removed by using RNase-free DNase I (Life Technologies).The RNA yield was measured using UV absorbance spectroscopy (Kaiao), and the RNA quality was checked by gel electrophoresis.miRNA expression profiling was done using the Agilent miRNA expression platform (Agilent).

| RNA isolation and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from HL-60 cells or blood samples using TRIzol reagent (Servicebio) and the concentration was determined.The RNA was reverse-transcribed into complementary DNA and measured by qPCR using an ABI PRISM 7500 Sequence Detection System with miRNA Expression Assay primer and probe sets (Applied Biosystems).miRNA expression levels were normalized using the RNU6B small nuclear RNA (U6) as an endogenous control using the 2 C t ∆∆ method.The expression of the corresponding miRNA target genes was quantitated using DBI Bestar ® SybrGreen qPCR MasterMix with β-Actin and glyceraldehyde 3phosphate dehydrogenase (GAPDH) as internal controls.The primers used for qRT-PCR analysis were as follows (Table 1).All procedures were run in triplicates in at least three independent experiments.

T A B L E 1
The primers used for qRT-PCR analysis (human).
Normalized luciferase activity was reported as Renilla Luciferase activity.

| Cell viability assay
HL-60 cells were transfected with pcDNA-BTG2, the mimics or inhibitor of miR-361-3p for 12 h, and then 25 μM of 9s-HODE was added for additional 24-72 h.Cell viability was assayed by using Cell Counting Kit-8 (Dojindo), according to the manufacturer's instructions.All experiments were performed in triplicate and three independent experiments were performed.

| Flow cytometric analysis
For flow cytometry analysis, HL-60 cells in each group were collected after washing twice with cold phosphate-buffered saline, and resuspended in binding buffer, the final concentration was about

| Western blot analysis
HL-60 cells were split with lysis buffer.The protein was separated in parallel lanes of 10% SDS-gels and transferred to a polyvinylidene difluoride membrane (IPVH00010, Millipore).After blocking in 5% nonfat dry milk in TBST (0.1% tween-20 in TBS) for 1 h, the membrane was incubated overnight with 1: 1000 diluted anti-BTG2 (Abcam), followed by incubation in 1: 1000 HRP-conjugated anti-rabbit secondary antibody (BOSTER) for 1 h.GAPDH primary antibody (Abcam) was used as a standard control for protein expression levels.The proteins were analyzed using ImageJ gel analysis software.

| Statistical analysis
The data are presented as the mean ± standard deviation (SD) of at least three independent experiments.Two-tailed student's t test was performed with GraphPad Prism (version 5.0; GraphPad Software) and a p < 0.05 was considered a statistically significant difference.

| miR-361-3p is oncogenic in AML
[22] To investigate its role in AML, we first examined its expression in AML patients.Compared with its expression in the blood of healthy donors (n = 5), the expression of miR-361-3p was massively upregulated (>200-fold) in the blood of AML patients (n = 34).
These results indicated a probable oncogenic role of miR-361-3p in AML (Figure 1A).Then we examined its expression in an AML cell line, HL-60.Compared with its expression in blood from healthy humans, miR-361-3p expression was more than eightfold higher in HL-60 (Figure 1B).So we used HL-60 cell as an in vitro model.
We next manipulated miR-361-3p endogenous expression by specific microRNA mimic and inhibitor (Figure 1C) to evaluate its effect on tumor-associated properties of HL-60, including proliferation and apoptosis.Cell counting kit-8 (CCK-8) assay and EdU assay were employed to measure the proliferation of HL-60.In the case of proliferation, inhibition of miR-361-3p in HL-60 cells by a specific inhibitor decreased cell proliferation, whereas overexpression of miR-361-3p by a specific mimic increased cell proliferation (Figure 1D,E).In comparison, inhibition of miR-361-3p increased apoptosis in HL-60 cells as measured by Annexin V/PI staining and flow cytometry assay.In contrast, overexpression of miR-361-3p inhibited apoptosis (Figure 1F).
These data together suggest that miR-361-3p is oncogenic in AML.

| miR-361-3p directly targets BTG2
miRNAs function by negatively regulating their target genes.To further understand the anticancer effect of miR-361-3p in AML, we explored the direct targets of miR-361-3p.We first approached public databases, including miRBase (http://www.mirbase.org/)and Starbase (http://starbase.sysu.edu.cn/index.php)for potential target genes of miR-361-3p.Three genes, BTG2, cytoplasmic polyadenylation element binding protein (CPEB) and myelin transcription factor 1 (MYT1), were picked up for further investigation due to their prediction with high confidence as well as their function as tumor suppressors. [23]All of these three genes showed high minimum free energy (MFE) scores generated after structure prediction (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) as shown in Figure 2A, indicating that the binding affinity between miR-361-3p and target genes are strong. [24]Among them, BTG2 has the highest MFE and affinity with miR-361-3p, suggesting BTG2 is more likely to be directly regulated by miR-361-3p than the other two putative targets.
We then investigated whether miR-361-3p can directly bind to BTG2, CPEB, and MYT1 3′-UTR, which is the gold standard to validate direct regulation.Luciferase reporter plasmids containing the wild-type or mutant 3′-UTR for either BTG2, CPEB, or MYT1 were constructed (Figure 2B and Supporting Information: Figure S1).Cotransfection of miR-361-3p mimic and luciferase reporter plasmids containing the wild-type BTG2 3′-UTR into HEK293 cells significantly repressed the relative luciferase activity compared to the negative control mimic.Such inhibition was completely abrogated when BTG2 3′-UTR was mutated, suggesting miR-361-3p can directly bind to BTG2 3′-UTR (Figure 2C).Similar trends were observed for CPEB2 and MYT1 (Supporting Information: Figure S1).
However, the regulation was not significant (Supporting Information: Figure S1).Consistently, overexpression of miR-361-3p decreased BTG2 expression at both mRNA and protein levels, whereas inhibition of miR-361-3p caused an upregulation of BTG2 at both levels (Figure 2D,E).
We further explored the clinical relevance of miR-361-3p/ BTG2 axis in AML patients.Opposite to the upregulated expression of miR-361-3p in the blood of AML patients (Figure 1B), mRNA expression level of BTG2 was markedly lower in the AML group than its expression in the blood of healthy donors, suggesting an inverse correlation between miR-361-3p and BTG2 in AML patients (Figure 2F).Taking together, these results confirm that BTG2 is a direct target of miR-361-3p in AML.
3.4 | miR-361-3p/BTG2 axis contributes to the anti-AML effect of 9s-HODE We finally evaluated the effect of 9s-HODE on AML with a focus on the effect on cell proliferation and apoptosis.9s-HODE treatment significantly reduced cell growth and apoptosis of HL-60 cells compared with control (Figure 4A-C).Overexpression of miR-361-3p by a specific mimic eliminated the inhibitory effects of 9s-HODE on cell proliferation (Figure 4D,E).Consistent with the results shown in Figure 3E and 3F that miR361-3p and BTG-2 were involved in 9s-HODE-mediated apoptotic signaling pathways, increasing miR-361-3p or inhibitor of BTG2 elimiated 9s-HODE -mediated apoptotic phenotype (Figure 4F).These results strongly support that 9s-HODE elicits anti-AML function through, at least partly, targeting miR-361-3p/BTG2 axis.

| DISCUSSION
AML is one of the most common types of leukemia.Despite the recent progress in developing AML-targeted drugs, the development of effective therapy towards AML remains a challenge.7][28] However, the unknown mechanisms underlying these effects hinder their clinical translation.[31] In the present study, we showed miR-361-3p was highly upregulated in AML patients.9s-HODE could rescue miR-361-3p expression and exert an antitumor effect in AML cells.These effects can be reversed by overexpressing miR-361-3p or inhibiting its direct target gene BTG2, suggesting the effect is miR-361-3p/BTG2 mediated.
These results are consistent with the reports that miR-361-3p acts as an oncogene and can be used as a potential target for the treatment of cancers. [32]miR-361-3p has been first demonstrated as a novel oncogene. [23]Additional works continue to elucidate that miR-361-3p regulates the proliferation, migration, and invasion of human liver cancer, [33] prostate cancer cells, [21] and oral squamous cell carcinoma (OSCC). [20]For instance, miR-361-3p induced cell proliferation and promoted EMT through negative regulation of dual specificity phosphatase-2 (DUSP2), thereby activating ERK signaling in pancreatic cancer cells. [21]This study also provides clinical evidence that a high level of miR-361-3p is associated with an advanced stage of tumor and shorter overall survival.In addition, miR-361-5p was reported to be significantly increased in plasma of newly diagnosed AML patients at diagnosis compared to healthy controls and decreased after chemotherapy. [34]In our study, similar results were found that the miR-361-3p was higher in AML patients compared to healthy individuals.These findings indicate that targeting miR-361-3p may be a useful therapeutic approach for patients with AML.We examined the regulatory role of 9S-HODE on miR-361-3p, and found that 9S-HODE significantly decreased miR-361-3p expression in HL-60.Bioinformation screening showed that miR-361-3p interacted with BTG2.Dual-luciferase experiments confirmed that miR-361-3p targeted with BTG2.Namely, miR-361-3p negatively regulated BTG2, and silencing BTG2 promoted AML cell proliferation and inhibited cell apoptosis.BTG2 is the first gene identified in the BTG/TOB gene family, which belongs to the antiproliferative gene families. [35]The loss of BTG2 in many human cancers is associated with a prognosis of more aggressive cancer development. [36]Moreover, BTG2 regulates cell growth, death, migration, apoptosis, and radio sensitization through several important signaling pathways. [37]Here we found that miR-361-3p/BTG2 played a crucial role in AML tumorigenesis.Importantly, 9S-HODE, as a strong inhibitory drug, downregulated miR-361-3p expression while upregulated BTG2.Moreover, the present study verified that the effect of 9s-HODE in HL-60 was eliminated through the overexpression of miR-361-3p and silencing BTG2.This supports our hypothesis that 9s-HODE affects the proliferation and apoptosis in HL-60 by miR-361-3p/BTG2 axis.
In summary, the present study provided evidence to support the fact that 9S-HODE could inhibit proliferation and induce apoptosis in HL-60 via the miR-361-3p/BTG2 axis.Both miR-361-3p and BTG2 may be potential therapeutic targets for future AML treatment.

1 × 10 6
cells/mL.Then the cell suspension was mixed with Annexin V-fluorescein isothiocyante (FITC)/propidium iodide (PI) staining solution using the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences), and incubated for 15 min at room temperature in the dark.Then, the cells were evaluated by flow cytometry (BD Biosciences).The percentage of apoptosis cells was analyzed using Cell Quest v.3.1 Software (Becton Dickinson).
is targeted by miR-361-3p.(A) Predicted dot-bracket structures of miRNA-mRNA complexes and their MFE scores by the RNAfold web server.(B) Binding sites of miR-361-3p and the mutated site in the BTG2 3′-UTR.(C) HEK293 cells were transfected with luciferase reporter plasmid containing wild-type (WT) or mutant BTG2 3′-UTR together with miR-361-3p mimic or mimic NC, and luciferase activity was measured 48 h later.n = 3 independent experiments.(D) Inhibition of miR-361-3p by inhibitor increases BTG protein.A representative Western blot analysis of BTG2 normalized to GDPDH protein at 48 h.All bands were cropped and full-length blots are shown in Supporting Information: Figure3.(E) Inhibition of miR-361-3p by inhibitor increases BTG mRNA expression.In contrast, overexpression of miR-361-3p by mimic inhibits BTG2 expression at both protein (D) and mRNA levels (E).(F) BTG2 mRNA expression level is lower in the blood from AML patients (n = 34) compared with the expression in the blood from health donors (n = 5).* * p < 0.01, * p < 0.05, ns > 0.05.3′-UTR, untranslated region; 9s-HODE, 9-hydroxyl-octadecadienoic acids.

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I G U R E 4 (See caption on next page).LIU ET AL. | 7 of 10