Silencing METTL3 inhibits the proliferation and invasion of osteosarcoma by regulating ATAD2

Biomedicine & Pharmacotherapy 125 (2020) 109964

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Silencing METTL3 inhibits the proliferation and invasion of osteosarcoma by regulating ATAD2

T

Lei Zhoua,b,1, Changsheng Yanga,b,1, Ning Zhangc,1, Xin Zhanga,b, Tingbao Zhaoa,b, Jinming Yub,d,* a

Department of Orthopedic Oncology Surgery, Shandong Cancer Hospital and Institute Affiliated to Shandong University, Jinan 250117, China Shandong Cancer Hospital and Institute Affiliated to Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan 250117, China c Department of Orthopedics, Jinan City People's Hospital, Jinan 271100, China d Department of Radiation Oncology, Shandong Cancer Hospital and Institute Affiliated to Shandong University, Jinan 250117, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: METTL3 Osteosarcoma m6A methylation Proliferation Invasion ATAD2

Background: Osteosarcoma is the most common primary malignant bone tumor in children and young adults. RNA N6-methyladenosine (m6A) is the most abundant internal modification in mammalian mRNA, which is involved in tumorigenesis and tumor progression. It has been reported that methyltransferase-like 3 (METTL3), the first reported m6A “writer”, plays critical roles in cancer progression. However, its role and molecular mechanism in osteosarcoma is poor studied. In this study, we aimed to investigate the functional role and underlying mechanism of METTL3 in the progression of osteosarcoma. Methods: We detected the mRNA expression of METTL3 in osteosarcoma cell lines, and immunofluorescence assay was performed to observe the location of METTL3. Cell lines with METTL3 gene overexpression or knockdown were established by pcDNA3.1-METTL3 or siRNA interferences in order to determine the function of METTL3 in osteosarcoma in vitro. Transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of METTL3 in osteosarcoma. Results: We found that METTL3 localized in cytoplasm and nucleus of osteosarcoma cells. Silencing METTL3 in SAOS-2 and MG63 cells significantly inhibited the m6A methylation level, proliferation, migration, and invasion abilities, as well as promoted cell apoptosis. However, up-regulation of METTL3 had no significant effect on the biological behaviors of U2OS cells. Further mechanism analysis suggested that METTL3 knockdown inhibited the expression of ATPase family AAA domain containing 2 (ATAD2). Moreover, ATAD2 knockdown inhibited the proliferation and invasion of SAOS-2 and MG63 cells, while its overexpression showed a significant increase in cell proliferation and invasion. Furthermore, METTL3 knockdown abrogated the promoting effects of ATAD2 overexpression on osteosarcoma cells proliferation and invasion. Conclusion: Overall, our study revealed that METTL3 functions as an oncogene in the growth and invasion of osteosarcoma by regulating ATAD2, suggesting a potential therapeutic target for osteosarcoma treatment.

1. Introduction Osteosarcoma is the most common primary malignant bone tumor in children and young adults, accounting for 2.4 % of malignant tumors in children [1–3]. Osteosarcoma is characterized by high malignancy and rapidly damage the surrounding tissues for metastasis. It has been reported that about 10–20 % of patients with osteosarcoma develop metastatic diseases at the time of diagnosis [4]. Early metastasis is one of the main factors leading to poor prognosis in patients with

osteosarcoma. Although improvement has been developed in current treatment strategies of osteosarcoma, including surgical excision combined with systemic chemotherapy or radiotherapy, the survival rate of osteosarcoma patients remains poor [5]. For patients with localized osteosarcoma, the 5-year event-free survival (EFS) is about 70 %, while the 5-year survival rate of patients with metastatic disease ranges from 15 % to 30 % [3,5]. What’s more, approximately 80 % of osteosarcoma patients with surgical treatment will experience relapse, which seriously affects the prognosis of patients [6,7]. Thus, it is essential to

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Corresponding author at: Department of Radiation Oncology, Shandong Cancer Hospital and Institute affiliated to Shandong University, No.440 Jiyan Road, Jinan 250117, China. E-mail address: [email protected] (J. Yu). 1 These authors contributed equally. https://doi.org/10.1016/j.biopha.2020.109964 Received 15 October 2019; Received in revised form 15 January 2020; Accepted 24 January 2020 0753-3322/ © 2020 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

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further investigate the molecular carcinogenesis of osteosarcoma and identify novel therapeutic targets for osteosarcoma treatment. With the in-depth study of the human cancer genome, epigenetic modifications, such as DNA methylation, chromatin remodeling or N6methyladenosine (m6A), have been found to be involved in tumorigenesis and cancer development, which has become an emerging research hotspot in cancer biology [8–11]. It is generally known that m6A, the most abundant internal modification in mammalian mRNA, is a dynamic reversible process mainly catalyzed by methyltransferaselike 3 and 14 (METTL3 and METTL14) and reversed by ALKBH5 and the FTO [12–15]. It has been revealed that METTL3, the first reported m6A “writer”, is implicated in various stages of the RNA life cycle, including pre-mRNA splicing, 3'-end processing, translational regulation, as well as miRNA processing [16–21]. Recently, emerging evidence has showed that METTL3 plays critical roles in cancer progression, including colorectal cancer [22,23], bladder cancer [24], and ovarian cancer [25]. Peng W et al. show that METTL3 promotes the migration and invasion of colorectal cancer by methylation of pri-miR-1246 to regulate SPRED2 [22]. Li T et al. report that METTL3 also exerts an oncogenic role in colorectal carcinoma by an m6A-IGF2BP2-dependent mechanism [23]. However, whether METTL3 affects the growth and metastasis of osteosarcoma is poor studied. In the present study, we elucidated the role of METTL3 in the progression of osteosarcoma and investigated the underlying mechanism. Our data demonstrated that METTL3 exerts an oncogenic role in promoting osteosarcoma cell proliferation and invasion. Further, we identified ATPase family AAA domain-containing protein 2 (ATAD2) as the downstream target of METTL3, which also functions as an oncogene in osteosarcoma. Overall, our data suggest that METTL3 facilitates the growth and metastasis of osteosarcoma by regulating ATAD2, indicating that METTL3 is a potential therapeutic target in osteosarcoma.

Table 1 Primer sequences. Gene

Primer sequences

human-β-actin

Forward, 5′-CCCGAGCCGTGTTTCCT-3' Reverse, 5′-GTCCCAGTTGGTGACGATGC-3' Forward, 5′-ACCCTGACAGATGATGAGATGC-3' Reverse, 5′-CGTTCATACCCCCAGAGGTTTAG-3' Forward, 5′-GCAGGACTGGATTTCACTTAGA-3' Reverse, 5′-GGCCCACTGCTATACTGATAAA-3' Forward, 5′-CCCAACCCTGCTGTGAATAA-3' Reverse, 5′-GGCGGCTCACCATATGTAAA-3' Forward, 5′-CAGGAAGGCGGATGAAGATAG-3' Reverse, 5′-CTGGATACCATGTGTGTTCTGA-3' Forward, 5′-AGTATGCCCAGCTCCTAGT-3' Reverse, 5′-CGGTTCATGCACAGGTAGAA-3' Forward, 5′-CAGTTTCTGCTCTCAGGAAGAT-3' Reverse, 5′-GGGATGATGTGTGGCTTCA-3' Forward, 5′-TTCTCCTGCAAGACCAAGATAC-3' Reverse, 5′-GTCGAGTCACTACTGTGGATTG-3' Forward, 5′-GGATACCTTGGCGCTAGTATTT-3' Reverse, 5′-CACAGCTGTACTCCTGTTCTG-3'

METTL3 FGF9 E2F8 TP63 FGF18 PYCR1 ATAD2 PCNA

2.3. Western blot analysis Following transfection for 48 h, cells were lysed in RIPA buffer supplemented with protease inhibitors (CWBIO) to exact protein. The protein was quantified by a BCA Protein Assay kit (CWBIO) and separated by 10 % SDS-PAGE. Then, protein was transferred onto PVDF membranes (Millipore, Danvers, MA) which was blocked with skimmed milk for 1 h afterwards. After the incubation with primary antibodies (1:1000; Proteintech, USA) overnight at 4 °C, the membranes were then incubated with HRP-conjugated secondary antibodies (1:5000; Proteintech) at room temperature for 1 h. Protein bands were visualized by an ECL kit (Biovision, USA) and quantified using Quantity One (BioRed, USA).

2. Materials and methods 2.1. Cell culture and transfection

2.4. Immunofluorescence assay

The human osteosarcoma cell lines HOS, SAOS-2, U2OS, and MG63 were obtained from KeyGEN BioTech (Nanjing, China). Cells were maintained in DMEM medium (HyClone, Logan, UT, USA) supplemented with 10 % FBS (Gibco, Thermo Fisher Scientific, USA) and penicillin (100 U/mL; Sigma) and streptomycin (0.1 mg/mL; Sigma) at 37 °C with 5 % CO2. The siRNA-METTL3 (termed as METTL3-KD), the corresponding negative control (termed as KDeNC), pcDNA3.1-METTL3 (termed as METTL3-OE), the corresponding negative control (pcDNA3.1; termed as OEeNC), siRNA-ATAD2 (termed as ATAD2-KD), and pcDNA3.1ATAD2 (termed as ATAD2-OE) were synthetized by KeyGEN BioTech. Osteosarcoma cells were transfected with these siRNAs or vectors using the Lipofectamine 2000 (Invitrogen, USA) according to the instructions. The target sequences of the siRNAs used in this study were as follows: siRNA-METTL3, 5′- GCAAGAATTCTGTGACTAT-3'; siRNA-ATAD2, 5′-ATGATAAAACATCACTTATTCAGAA-3'. Cells without any treatment were used as control group.

Cultured cells were rinsed in PBS for 3 times and fixed with 4 % paraformaldehyde for 15 min. After washing in PBS, cells were permeabilized with PBS containing 0.5 % Triton X-100 at room temperature for 20 min. After three times of PBS immersion and drying, the cells were blocked for 30 min in normal goat serum at room temperature. Then, cells were incubated with diluted primary antibody overnight at 4 °C. After washing 3 times with PBST, the cells were incubated with the diluted fluorescent antibodies at 20−37 °C for 1 h. The cells were then incubated with DAPI for 5 min in the dark, washed with PBS and mounted with a blocking solution containing anti-fluorescent quencher. The images were observed under a fluorescence microscope. 2.5. M6A RNA methylation level quantification After 48 h of transfection, total RNA was collected and the methylation level of each group was measured using the m6A RNA Methylation Quantification Kit (ab185912; Abcam, USA) according to the instructions.

2.2. RT-PCR assay Total RNA was isolated from osteosarcoma cells transfected for 24 h by using TRIzol reagent and reversely transcribed into cDNA by HiFiScript cDNA Synthesis Kit (CWBIO, Beijing, China) according to the manufacturer’s protocol. The real-time PCR analysis was performed using UltraSYBR Mixture Kit (CWBIO) in ABI 7500 Fast system (Applied Biosystems, CA, USA). β-actin was used as an internal control. The primers used in this study was shown in Table 1. Relative gene expression was analyzed by the 2−ΔΔCt method.

2.6. CCK8 assay A CCK8 system (Solarbio, Beijing, China) was performed to measure cell proliferation. About 1 × 103 cells/well transfected cells were seeded into a 96-well plate and incubated at 37 °C for 0, 24, 48, and 72 h, respectively. Before detection, 10 μl of CCK8 reagent was added to each well and the cells were incubated at 37 °C for 1.5 h. Then, the OD450 nm value was measured by a microplate reader. 2

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Fig. 1. Expression of METTL3 in osteosarcoma cells. A. RT-PCR analysis of relation expression of METTL3 mRNA in osteosarcoma cell lines. ΔP < 0.05, by Student’s t-test vs U2OS; n = 3. B. Western blot analysis of relation expression of METTL3 protein in osteosarcoma cell lines. ΔP < 0.05, by Student’s t-test vs U2OS; n = 3. C. Immunofluorescence analysis of METTL3 (green) and DAPI (blue, cell nuclei) in HOS, SAOS-2 and MG63 cells. D. RT-PCR analysis of relation expression of METTL3 mRNA in SAOS-2 and MG-63 cells transfected with siRNA-METTL3 or siRNA-control, and U2OS cells transfected with METTL3-overexpression or control vectors. *P < 0.05, **P < 0.01, by Student’s t-test vs negative control; n = 3. E. Western blot analysis of relation expression of METTL3 protein in METTL3-knockdown or -overexpression osteosarcoma cells. F. M6A RNA methylation level quantification analysis of m6A RNA methylation level in METTL3-knockdown or -overexpression osteosarcoma cells. *P < 0.05, by Student’s t-test vs negative control; n = 3. Control, cells without any treatment; METTL3-KD, cells transfected with siRNA-METTL3, METTL3-knockdown; KD-NC, cells transfected with siRNA-control, knockdown-negative control; METTL3-OE, cells transfected with pcDNA3.1-METTL3, METTL3-overexpression; OE-NC, cells transfected with pcDNA3.1, METTL3-negative control.

2.7. Colony formation assay

the migration assay was 5000.

Following transfection for 24 h, cells were seeded into 60 mm plates at 500 cells/plate and cultured in DMEM medium supplemented with 10 % FBS for 14–20 days. After fixed with methanol, the cells were stained with 0.1 % crystal violet 30 min and then the colonies were imaged and counted. When colonies were visible to the naked eye, cells were fixed with 4 % paraformaldehyde for 30 min and stained with 0.1 % crystal violet for 30 min. Colonies were counted and photographed under inverted microscope (Olympus, Japan).

2.9. Flow cytometry assay Cells transfected for 24 h were incubated in serum-free medium for 24 h. Cells were digested with trypsin digestion without EDTA and resuspended in 1X binding buffer to adjust cell density to 1−5 × 106/ ml. One hundred microliter of cell suspension was incubated with 5 μl of Annexin V/FITC (4A Biotech Co., Ltd, Beijing, China) at room temperature for 5 min in the dark, then incubated with 10 μl of PI. The percentage of apoptosis was analyzed by a flow cytometry (BD FACSCanto™ II; BD Biosciences) and calculated using the BD FACSDiva™ software (BD Biosciences).

2.8. Transwell assay Transwell chambers coated with Matrigel gel (BD Biosciences, USA) were used to assess cell invasion, while chambers without Matrigel gel were used for migration assay. The cells after transfection for 24 h were cultured in serum-free medium to prepare a cell suspension, 100 μl of cell suspension (1 × 105 cells) was added to the upper chamber, and 500 μl of DMEM medium containing 10 % FBS was added to the lower chamber. Following incubation for 24 h, the invaded or migrated cells were fixed with 4 % paraformaldehyde for 30 min and stained with 0.1 % crystal violet for 20 min. The number of invaded and migrated cells were counted under a microscope. In addition, the number of cells in

2.10. RNA-seq analysis After being transfected with siRNA-METTL3 or siRNA negative control for 24 h, MG63 cells were collected and RNA-seq analysis was performed by BGI Genomic Technology Co Ltd (Shenzhen, China). 2.11. Statistical analysis Data were expressed as the means ± SD from triple independent 3

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Fig. 2. METTL3 regulates the proliferation of osteosarcoma cells. Cells were transfected with siRNA-METTL3 or METTL3-overexpression vector and corresponding control for 24 h. A, B and C. CCK8 assay of the viability of SAOS-2 (A), MG63 (B) and U2OS (C) cells. *P < 0.05, by ANOVA vs negative control; n = 3. D. Colony formation assay was performed to detect effect of METTL3 on the proliferation of SAOS-2, MG63 and U2OS cells. E. Quantitative analysis of colony formation assay results. *P < 0.05, by Student’s t-test vs negative control; n = 3.

(Fig. 1F).

experiments. The GraphPad Prism7 software was performed for statistical analysis. Student t-test with Shapiro-Wilk test was used for comparisons between two groups, and ANOVA followed by the LSD post hoc test was used for comparisons between 3 or more groups. P < 0.05 was considered statistically significant.

3.2. Silencing METTL3 inhibits the proliferation of osteosarcoma cells CCK8 assay was performed to assess the effect of METTL3 on the growth ability of osteosarcoma cells. Interestingly, a significant decrease in cell viability was observed in METTL3-inhibited SAOS-2 cells (Fig. 2A). Similarly, METTL3 knockdown also inhibited the viability of MG63 cells (Fig. 2B). However, up-regulation of METTL3 had no significant effect on the viability of U2OS cells (Fig. 2C). Consistent with CCK8 assay, SAOS-2 and MG63 cells colony-forming abilities were also suppressed by silencing METTL3 compared to the corresponding negative control group, but the colony-forming ability of U2OS cells was still not affected by METTL3 overexpression (Fig. 2D and E).

3. Results 3.1. Expression of METTL3 in osteosarcoma cells To evaluate the expression profile of METTL3 in osteosarcoma, we examined the expression of METTL3 in four osteosarcoma cell lines, HOS, SAOS-2, U2OS and MG63. As indicated by RT-PCR and western blot analysis, we observed that among these four cell lines, METTL3 expression was the lowest in U2OS cells at both mRNA and protein levels, while higher in SAOS-2 and MG63 cells (Fig. 1A and B). Thus, U2OS, SAOS-2 and MG63 cells were used for the next experiment. As shown in Fig. 1C, immunofluorescence assays showed that METTL3 localized in cytoplasm and nucleus of osteosarcoma cells. To investigate the functional role of METTL3 in osteosarcoma cells, the expression of METTL3 was knocked down by siRNA-METTL3 interference at mRNA and protein level in SAOS-2 and MG63 cells, while its expression was obviously up-regulated by transfection with pcDNA3.1-METTL3 in U2OS cells (Fig. 1D and E). Consistently, upon METTL3 knockdown, the m6A RNA methylation level showed a significant decrease in both SAOS-2 and MG63 cells, whereas up-regulation of METTL3 increased the m6A RNA methylation level in U2OS cells

3.3. Silencing METTL3 suppresses the migration and invasion of osteosarcoma cells As generally known, metastasis is a key factor in cancer-related death. Therefore, a transwell assay was used to determine the effect of METTL3 on cell migration and invasion in osteosarcoma cells. As shown in Fig. 3A and C, METTL3 knockdown resulted in a significant depression in the migration ability of SAOS-2 and MG63 cells compared to the corresponding negative control group. Up-regulation of METTL3 still had no effect on the migration ability of U2OS cells (Fig. 3B and C). Similarly, the invasion abilities of SAOS-2 and MG63 cells were also suppressed after METTL3 inhibition (Fig. 3D and F), but the invasion 4

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Fig. 3. METTL3 regulates the migration and invasion of osteosarcoma cells. Cells were transfected with siRNA-METTL3 or METTL3-overexpression vector and corresponding control for 24 h. A. Transwell assay was performed to assess the migration abilities of SAOS-2 and MG63 cells. B. Transwell assay was performed to assess the migration ability of U2OS cells. C. Quantitative analysis of Transwell migration assay results. *P < 0.05, by Student’s t-test vs negative control; n = 3. D, E. Transwell assay was performed to assess the invasion abilities of SAOS-2 and MG63 cells (D) and U2OS cells (E). F. Quantitative analysis of Transwell invasion assay results. *P < 0.05, by Student’s t-test vs negative control; n = 3.

3.5. METTL3 targets ATAD2 and regulates its expression

ability of U2OS cells was not impaired (Fig. 3E and F).

To investigate the molecular mechanism of METTL3, we performed RNA-seq in MG63 and METTL3-silenced MG63 cells. The results showed that there were 578 genes were up-regulated in METTL3-silenced MG63 cells, and 481 genes was down-regulated (Fig. 5A). Hierarchical clustering analysis of differentially expressed genes was shown in Fig. 5B. Different gene expression patterns were observed between METTL3-silenced cells and NC cells. Go annotations of the differential expression genes was shown in supplementary Fig. 1A. Classification and statistics of KEGG biological pathways of the differentially expressed genes was shown in supplementary Fig. 1B. The altered genes by METTL3 knockdown could also have an impact on several vital pathways, such as TNF signaling pathway, PI3K/Akt signaling pathway, MAPK signaling pathway, cell cycle-related pathway, and DNA replication-related pathway (supplementary Fig. 1C). To further investigate the underlying mechanism of METTL3, we first selected 7 predicted downstream genes of METTL3, FGF9, E2F8, TP63, FGF18, PYCR1, ATAD2, and PCNA (supplementary Fig. 1D). RTPCR results demonstrated that except for PCNA, the expression of the other six candidate genes were down-regulated with the inhibition of METTL3 in MG63 cells, especially ATAD2 (Fig. 5C). Western blot analysis further revealed that METTL3 knockdown significantly

3.4. METTL3 knockdown promotes cell apoptosis in osteosarcoma cells As indicated by flow cytometry assay, we found that there was a significant increase in the percentage of apoptotic cells of METTL3-silenced cells compared with the negative control cells (Fig. 4A and C). But the apoptosis of U2OS cells was not impaired by METTL3 overexpression (Fig. 4B and C). Western blot analysis was used to detect the expression of apoptosisrelated proteins to investigate the mechanism underlying the induced apoptosis by METTL3 knockdown. Our data showed that the expression of anti-apoptotic protein Bcl-2 was significantly down-regulated by METTL3 knockdown in both SAOS-2 and MG63 cells, while the expression of pro-apoptotic proteins Bax and cleaved Caspase 3 was obviously up-regulated (Fig. 4 D). Obviously, the up-regulation of METTL3 had no significant effect on the expression of these proteins in U2OS cells (Fig. 4D). Therefore, silencing METTL3 may promote apoptosis in osteosarcoma cells by regulating the Bcl-2/Bax axis and Caspase cascade.

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Fig. 4. METTL3 regulates the apoptosis of osteosarcoma cells. Cells were transfected with siRNA-METTL3 or METTL3-overexpression vector and corresponding control. A, B. Following transfection of 24 h, flow cytometry assay was performed to determine METTL3-knockdown or -overexpression on cell apoptosis in SAOS-2 and MG63 cells (A) and U2OS cells (B). C. Quantitative analysis of flow cytometry assay results. *P < 0.05, by Student’s t-test vs negative control; n = 3. D. After transfection of 48 h, western blot analysis of relative expression of apoptosis-related proteins (Bcl-2, Bax and cleaved Caspase 3).

METTL3 has been well studied [22,23,27]. Herein, our results revealed a significant oncogenic role of METTL3 in the growth and invasion of osteosarcoma. Mao W et al. previously report that the m6A methylation level and METTL3 expression are both up-regulated in osteosarcoma tissues and cell lines [28]. METTL3 silence could decrease cell proliferation and migration in HOS and SAOS-2 cells [28]. In our study, we found that METTL3 localized in cytoplasm and nucleus of osteosarcoma cells. We verified the expression of METTL3 in four osteosarcoma cell lines, and METTL3 was knocked down in MG63 and SAOS-2 cells due to the higher METTL3 expression level, while we overexpressed its expression in U2OS cells as its lower expression. Our data showed that silencing of METTL3 could down-regulate the m6A methylation in osteosarcoma cells and inhibit the proliferation, migration and invasion abilities of osteosarcoma cells. These results were consistent with the previous report [28], suggesting that METTL3 functions an oncogenic role in osteosarcoma progression. More importantly, we found that silencing of METTL3 promoted cell apoptosis in osteosarcoma cells by regulating the Bcl-2/Bax axis and Caspase 3 activation. It is well known that evading programmed death is a main feature of tumor cells, promoting tumor cell apoptosis is considered to be an effective strategy to inhibit tumor growth [29]. Therefore, METTL3 might be a potential therapeutic target for osteosarcoma treatment. However, in vivo experiments are needed to further confirm the oncogenic role of METTL3 in the growth and metastasis of osteosarcoma. According to the study of the mechanism of METTL3, METTL3 participates in tumor progression through m6A-dependent or m6A-independent manner. For example, Li T et al. report a network of “writer”-METTL3/“reader”-IGF2BP2/ “target”-SOX2, knockdown of

inhibited the protein expression of ATAD2 in both SAOS-2 and MG63 cells (Fig. 5D). Therefore, ATAD2 might be a downstream target of METTL3 in osteosarcoma cells. 3.6. METTL3 regulates the proliferation and invasion of osteosarcoma cells by regulating ATAD2 The loss- and gain- of-function assays were performed to investigate the biological functional role of ATAD2 in osteosarcoma cells. As shown in Fig. 6A and B, knockdown of ATAD2 significantly inhibited the proliferation of both SAOS-2 and MG63 cells. On the contrary, overexpression of ATAD2 promoted the proliferation of SAOS-2 and MG63 cells (Fig. 6A and B). Transwell assay revealed that silencing of ATAD2 decreased the invasion abilities of both SAOS-2 and MG63 cells, whereas up-regulation of ATAD2 resulted in an increase in the invasion abilities of both SAOS-2 and MG63 cells (Fig. 6C and D). These results suggest an oncogenic role of ATAD2 in osteosarcoma cells. Furthermore, METTL3 knockdown could reverse the promotion of ATAD2 overexpression on osteosarcoma cells proliferation and invasion (Fig. 6A-D). Taken together, ATAD2 is a downstream target of METTL3, METTL3 regulates the proliferation and invasion of osteosarcoma cells by regulating ATAD2. 4. Discussion As mentioned above, METTL3, a main component of m6A methyltransferase complex, is believed to be involved in cancer progression [22–26]. Especially in colon cancer, the tumorigenic function of 6

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Fig. 5. METTL3 targets ATAD2 and regulates its expression. RNA-seq analysis in METTL3-silenced and control MG63 cells. A. Differentially expressed genes in METTL3-silenced cells. B. Hierarchical clustering analysis of differentially expressed genes. C. RT-PCR analysis of relative expression of potential downstream target genes of METTL3. *P < 0.05, by Student’s t-test vs negative control; n = 3. D. Western blot analysis of expression of ATAD2 in control and METTL3-knockdown SAOA-2 and MG63 cells. *P < 0.05 vs NC group.

Fig. 6. METTL3 regulates the proliferation and invasion of osteosarcoma cells by regulating ATAD2. SAOA-2 and MG63 cells were transfected with siRNA-ATAD2, pcDNA3.1-ATAD2, siRNA-METTL3+ pcDNA3.1-ATAD2, respectively. A, B. CCK8 assay was performed to assess cell proliferation in SAOA-2 (A) and MG63 (B) cells. *P < 0.05, by ANOVA vs negative control; n = 3. C. Transwell assay was performed to assess cell invasion in SAOA-2 and MG63 cells. D. Quantitative analysis of Transwell results. *P < 0.05, by Student’s t-test vs negative control; n = 3.

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and approved the final manuscript.

METTL3 inhibits tumorigenesis and metastasis in colorectal carcinoma by regulating SOX2 expression via an m6A-dependent manner [23]. Cheng M et al. reveal that METTL3 plays an oncogenic role in the bladder cancer through regulating the AFF4/NF-κB/MYC signaling network by an m6A-dependent manner [30]. Additionally, METTL3 also implicates miRNAs processes to promote the maturation of miRNAs by m6A dependent manner. METTL3 accelerates the maturation of primiR221/222 in bladder cancer in an m6A dependent manner, thus promoting tumor proliferation [24]. In colorectal cancer, METTL3 is also found to promotes the maturation of pri-miR1246 to regulate SPRED2 expression, thus hampering tumor progression [22]. However, Lin S et al. demonstrate that METTL3 could bind to gene promoter to up-regulate the translation of m6A-containing mRNAs, but not correlated to its methyltransferase activity and m6A modification [31]. A recent study shows that METTL3 is involved in osteosarcoma cell proliferation and migration by modulating the m6A level of LEF1 [28]. In the present study, by RNA-seq analysis, we found that silencing METTL3 can affect a number of genes expression, METTL3 silencing upregulated 578 genes and down-regulated 481 genes in MG63 cells. Moreover, several vital pathways were also impacted by METTL3 silencing, including TNF, PI3K/Akt and MAPK signaling pathways. These results indicate that METTL3 silencing might induce a complex expression pattern in transcriptional level, and some genes might function as downstream effectors of METTL3. Thus, further experiments confirmed that ATAD2 was a downstream effector of METTL3, which was positively regulated by METTL3. Whether other genes participate in the oncogenic role of METTL3 as downstream effectors will be studied in our future research. It has been reported that ATAD2, a member of ATPase family, is an epigenetic regulator and is associated with various cellular processes [32–34]. ATAD2 could activate transcription factors, such as E2F family member, to regulate genes expression or chromatin modification [35–37]. Increasing studies have demonstrated that ATAD2 is aberrantly expressed in some types of cancer and involved in tumor progression, for instance, papillary thyroid cancer [38], colorectal cancer [39], pancreatic cancer [40], and lung cancer [41]. However, the functional role of ATAD2 in osteosarcoma remains unclear. In this study, our data first demonstrated that down-regulation of ATAD2 inhibited proliferation and invasion of osteosarcoma cells, while its overexpression showed a promoting effect, suggesting that ATAD2 exerts an oncogenic role in the progression of osteosarcoma. Moreover, METTL3 silence could reverse the promotion of ATAD2 overexpression on osteosarcoma cells proliferation and invasion. Collectively, METTL3 could target and regulate the ATAD2 expression, thus promoting the growth and metastasis of osteosarcoma. However, whether METTL3 regulates ATAD2 expression through m6A modification or direct binding effects remains unclear, which is also the focus of our future study. In conclusion, our results demonstrated that METTL3 acts as an oncogene in the progression of osteosarcoma, knockdown of METTL3 could inhibit cell growth and metastasis abilities of osteosarcoma cells. Moreover, our study firstly identified the oncogenic role of ATAD2 in osteosarcoma, and ATAD2 is a downstream target of METTL3, which is involved in the effects of METTL3. These findings indicate that METTL3 may be a potential therapeutic target for osteosarcoma treatment.

Funding Not applicable. Declaration of Competing Interest The authors confirm that there are no any conflicts of interest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2020.109964. References [1] R.A. Durfee, M. Mohammed, H.H. Luu, Review of osteosarcoma and current management, Rheumatol. Ther. 3 (2) (2016) 221–243. [2] K.R. Duchman, G. Yubo, B.J. Miller, Prognostic factors for survival in patients with Ewing’s sarcoma using the surveillance, epidemiology, and end results (SEER) program database, Cancer Epidemiol. 39 (4) (2015) 593–599. [3] L.A. Duong, L.C. Richardson, Descriptive epidemiology of malignant primary osteosarcoma using population-based registries, United States, 1999-2008, J. Registry Manag. 40 (2) (2013) 59. [4] L. Kager, A. Zoubek, U. Pötschger, U. Kastner, S. Flege, B. Kempfbielack, D. Branscheid, R. Kotz, M. Salzerkuntschik, W. Winkelmann, Primary metastatic osteosarcoma: presentation and outcome of patients treated on neoadjuvant Cooperative Osteosarcoma Study Group protocols, J. Clin. Oncol. 21 (10) (2003) 2011–2018. [5] D.J. Harrison, D.S. Geller, J.D. Gill, V.O. Lewis, R. Gorlick, Current and future therapeutic approaches for osteosarcoma, Expert Rev. Anticancer Ther. 18 (1) (2017) 39. [6] M. Neyssa, G. Mark, T. Lisa, G. Richard, Biology and therapeutic advances for pediatric osteosarcoma, Oncologist 9 (4) (2004) 422. [7] A. Briccoli, M. Rocca, M. Salone, G.A. Guzzardella, A. Balladelli, G. Bacci, High grade osteosarcoma of the extremities metastatic to the lung: long-term results in 323 patients treated combining surgery and chemotherapy, 1985–2005, Surg. Oncol. 19 (4) (2010) 193–199. [8] X. Hao, H. Luo, M. Krawczyk, W. Wei, W. Wang, J. Wang, K. Flagg, J. Hou, H. Zhang, S. Yi, DNA methylation markers for diagnosis and prognosis of common cancers, Proc Natl Acad Sci U S A 114 (28) (2017) 7414–7419. [9] R.H. Xu, W. Wei, M. Krawczyk, W. Wang, H. Luo, K. Flagg, S. Yi, W. Shi, Q. Quan, K. Li, Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma, Nat. Mater. 16 (11) (2017) 1155. [10] W.A. Flavahan, E. Gaskell, B.E. Bernstein, Epigenetic plasticity and the hallmarks of cancer, Science 357 (6348) (2017) eaal2380. [11] X. Deng, R. Su, X. Feng, M. Wei, J. Chen, Role of N6-methyladenosine modification in cancer, Curr. Opin. Genet. Dev. 48 (2018) 1–7. [12] X.L. Ping, B.F. Sun, L. Wang, W. Xiao, X. Yang, W.J. Wang, S. Adhikari, Y. Shi, Y. Lv, Y.S. Chen, Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase, Cell Res. 24 (2) (2014) 177–189. [13] S. Schwartz, M. Mumbach, M. Jovanovic, T. Wang, K. Maciag, G.G. Bushkin, P. Mertins, D. Ter-Ovanesyan, N. Habib, D. Cacchiarelli, Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5′ sites, Cell Rep. 8 (1) (2014) 284–296. [14] G. Jia, Y. Fu, X. Zhao, Q. Dai, G. Zheng, Y. Yang, C. Yi, T. Lindahl, T. Pan, Y.G. Yang, N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO, Nat. Chem. Biol. 7 (12) (2011) 885–887. [15] G. Zheng, J.A. Dahl, Y. Niu, P. Fedorcsak, C.M. Huang, C.J. Li, C.B. Vågbø, S. Yue, W.L. Wang, S.H. Song, ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility, RNA Biol. 49 (1) (2013) 18–29. [16] I.U. Haussmann, Z. Bodi, E. Sanchez-Moran, N.P. Mongan, N. Archer, R.G. Fray, M. Soller, m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination, Nature 540 (2016) 301. [17] L. Nian, D. Qing, Z. Guanqun, H. Chuan, P. Marc, P. Tao, N(6)-methyladenosinedependent RNA structural switches regulate RNA-protein interactions, Nature 518 (7540) (2015) 560–564. [18] K. Shengdong, E.A. Alemu, M. Claudia, G. Emily Conn, J.J. Fak, M. Aldo, H. Bhagwattie, Z.S. Ilana, M.J. Moore, C.Y. Park, A majority of m6A residues are in the last exons, allowing the potential for 3’ UTR regulation, Genes Dev. 29 (19) (2015) 2037–2053. [19] B. Molinie, J. Wang, K.S. Lim, R. Hillebrand, Z. Lu, N.V. Wittenberghe, B.D. Howard, K. Daneshvar, A.C. Mullen, P. Dedon, m6A-LAIC-seq reveals the census and complexity of the m6A epitranscriptome, Nat. Methods 13 (8) (2016) 692–698. [20] X. Wang, B.S. Zhao, I. Roundtree, Z. Lu, D. Han, H. Ma, X. Weng, K. Chen, H. Shi, C. He, N 6 -methyladenosine Modulates Messenger RNA Translation Efficiency, Cell 161 (6) (2015) 1388–1399. [21] C.R. Alarcón, L. Hyeseung, G. Hani, H. Nils, S.F. Tavazoie, N6-methyladenosine

Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Authors' contributions This study was designed and conceived by LZ, CY and NZ. The experimental procedures and data analysis were carried out by all authors. The manuscript was prepared by LZ, CY and NZ. All authors read 8

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