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The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1 Wujun Miao a, 1, Jiajia Chen c, d, 1, Lianshun Jia a, *, Jun Ma a, **, Dianwen Song b, c, *** a
Department of Orthopedics, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai, 200003, China Department of Orthopedics, The First People's Hospital, Shanghai Jiao Tong University, 650 Xinsongjiang Road, Shanghai, 201620, China c Department of Orthopedics, Shanghai General Hospital of Nanjing Medical University, 650 Xinsongjiang Road, Shanghai, 201620, China d Department of Spine Surgery, The Second Affiliated Hospital of Nantong University, 6Haier Lane North Road, Nantong, Jiangsu, 226001, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 May 2019 Accepted 22 June 2019 Available online xxx
Osteosarcoma(OS) is the most common and aggressive malignant bone sarcoma,which occurs in rapidly growing bones in children and adolescents. However, the underlying molecular mechanisms of OS development have not been fully illustrated. N6-Methyladenosine (m6A) is the most prevalent internal chemical modification of mRNAs, which is involved in many pathological processes in cancer development. However, its role and regulatory mechanism in OS remain unknown. In this study, we aimed to investigate the roles of m6A and its methyltransferase METTL3 in OS development. The results showed that m6A level for RNA methylation and the expression level of METTL3 were up-regulated in human OS tissues and OS cell lines. Functionally, lentivirus-mediated METTL3 silence in HOS and SAOS-2 cells inhibited the cell proliferation, migration and invasion ability. Further mechanism analysis suggested that METTL3 silence decreased the m6A methylation and total mRNA level of lymphoid enhancerbinding factor 1 (LEF1), followed by inhibited the activity of Wnt/b-catenin signaling pathway. Moreover, LEF1 over-expression abrogates the repressive effects of METTL3 silence on the proliferation, migration and invasion abilities of OS cells. Together, these results revealed that the m6A methyltransferase METTL3 promotes osteosarcoma cell progression by regulating the m6A level of LEF1 and activating Wnt/b-catenin signaling pathway. © 2019 Published by Elsevier Inc.
Keywords: Osteosarcoma METTL3 m6A LEF1
1. Introduction As one of the most common cancers, human osteosarcoma (OS) has a high mortality and heterogeneous presentation [1,2]. Amputation was the most common treatment for osteosarcoma over the past decades [3]. Though the 5-year survival rate of patients with OS has already significantly improved, there are still many patients with poor prognosis [4]. Therefore, a more comprehensive understanding of the underlying molecular
* Corresponding author. Department of Orthopedics, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai, 200003, China. ** Corresponding author. Department of Orthopedics, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai, 200003, China. *** Corresponding author. Department of Orthopedics, The First People's Hospital, Shanghai Jiao Tong University, 650 Xinsongjiang Road, Shanghai, 201620, China. E-mail addresses: [email protected] (L. Jia), [email protected] (J. Ma), [email protected] (D. Song). 1 Wujun Miao and Jiajia Chen contributed equally.
mechanisms is urgently required for the treatment of OS. In eukaryotes, N6-Methyladenosine (m6A) is one of the most common internal chemical modifications of mRNAs [5], which is catalyzed by a methyltransferase complex, including of methyltransferase-like 3 and 14 (METTL3 and METTL14) and their cofactors, such as Wilms tumor 1-associated protein (WTAP), VIRMA (KIAA1429)and RBM15 [6]. On the other hand, this modification is reversed by two mammalian RNA demethylases, alkylation repair homolog protein 5(ALKBH5) [7] and the fat mass and obesity-associated protein(FTO) [8], which maintain the dynamic balance of m6A methylation and demethylation in mammalian cells. By methylating its target mRNAs, METTL3 is involved in a variety of pathological processes, including various cancers [9e12]. However, the roles and underlying molecular basis of METTL3 in the progress of human osteosarcoma remain unknown. Previous studies demonstrated that the Wnt signaling pathway plays critical roles in tissue homeostasis, stem cell biology, organogenesis as well as cancer progression [13,14]. The binding between LRP5/6 transmembrane receptors with secreted Wnt ligands to
https://doi.org/10.1016/j.bbrc.2019.06.128 0006-291X/© 2019 Published by Elsevier Inc.
Please cite this article as: W. Miao et al., The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.128
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Frizzled hold back the degradation of the signaling pool of cytoplasmic b-catenin. Avoiding from degradation, b-catenin gradually accumulates in the nucleus followed by binding to transcription factors, the LEF/TCF (lymphoid enhancer-binding factor and T cell factor) family, to stimulate the downstream gene expression. Moreover, previous studies have shown that LEF1 contributes to metastasis and chemoresistance in several cancers including osteosarcoma [15,16]. However, the underlying mechanism by which LEF1 is regulated, especially m6A modification of mRNA, in human osteosarcoma is still largely unknown. Here, our data indicated that total m6A methylated RNA level and the expression level of METTL3 were elevated in human OS tissues and cell lines. METTL3 silence in HOS and SAOS-2 cells inhibited the cell proliferation, migration and invasion ability. Mechanism analysis further suggested that METTL3 silence decreased the m6A methylation and total mRNA level of lymphoid enhancer-binding factor 1 (LEF1), subsequently inhibited the activity of Wnt/b-catenin signaling pathway. Furthermore, LEF1 overexpression abrogates the repression effect of METTL3 silence on the abilities of proliferation, migration and invasion in OS cells. Taken together, these results reveal that the m6A methyltransferase METTL3 promotes osteosarcoma cell progression through regulating the m6A level of LEF1 and activating Wnt/b-catenin signaling. Our data indicate that METTL3 may be a therapeutic target which is potential to be used for the treatment of human OS. 2. Materials and methods 2.1. Patient and clinical samples with ethical approval A total of 40 pairs of patient specimens were used in this research. These human samples were obtained according to the principles of the Declaration of Helsinki and approved by Changzheng Hospital (Shanghai, China). Written informed consent was obtained from all patients between 2000 and 2014. 2.2. Quantitative real-time PCR (qRT-PCR)
antibody, followed by incubation with diaminobenzene as the chromogen, and hematoxylin as the nuclear counterstain. 2.5. Cell lines and cell culture Human OS cell lines (U2OS, MG-63, Saos-2 and HOS) and normal human osteoblast cells (NHOst) were obtained from the Chinese Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All these cell lines were maintained in DMEM supplemented with 10% fetal bovine serum and 1% PS (penicillin and streptomycin) under standard culture conditions (5% CO2, 37 C). 2.6. Cell proliferation assay Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China) was used to measure the cell proliferation as previously described [12]. 2.7. Migration and invasion assay The cell migration ability was detected by wound healing assay as previously reported [17]. The invasion assay was analyzed using transwell chamber (8-mm pores, BD Falcon, Franklin Lakes, NJ, USA). 2.8. TOP/FOP-Flash reporter assay TOP/FOP-Flash reporter assay was performed as previously reported [19]. Briefly, the OS cells were seeded in 24-well plates followed by transfection with TOP/FOP flash plasmids (Simo Biomedical Technology, Shanghai, China). The luciferase activity was examined by the Dual Luciferase Assay kit (Promega, Madison, WI). 2.9. m6A RNA methylation quantification The m6ARNA Methylation Assay Kit (Abcam, ab185912) was used to evaluate the content of m6A in total RNA as previously reported [12].
Total RNA was lysed using TRIzol reagent and used for the synthesis of the cDNA with One-Step RT-PCR Kit (Thermo Fisher). The real-time PCR was performed using the ABI Vii7 system (Applied Biosystems, USA). GAPDH was used as a housekeeping gene. Primers were synthesized as follows, the human METTL3 forward primer:50 -CAAGCTGCACTTCAGACGAA-30 ,and human METTL3 reverse primer: 50 -GCTTGGCGTGTGGTCTTT-30 ; the human LEF1 forward primer:50 -CTACCCATCCTCACTGTCAGTC-30 ,and human LEF1reverse primer: 50 -GGATGTTCCTGTTTGACCTGAGG-30 ; GAPDH forward: 50 -TGACTTCAACAGCGACACCCA-30 ,and GAPDH reverse: 50 -CACCCTGTTGCTGTAGCCAAA-30 . Relative gene expression was calculated by the comparative CT method (DDCT).
2.10. Me-RIP assay
2.3. Western blot Western blotting was performed with antibodies against METTL3, LEF1 and GAPDH (Abcam, Cambridge, MA, USA)as previously described [17]. Human GAPDH was used as an endogenous control to normalize the protein loading.
The Metastatic Bone Tumor Model was established via injecting HOS cells into the right tibia of each animal as described previously [21]. Two weeks after the injection, the bioluminescence images of bone tumors in the tibia of each animal were observed by an in vivo imaging system (Xenogen IVIS-200, Caliper Life Sciences, Hopkinton).
2.4. Immunohistochemistry
2.13. Statistical analysis
The protocol for the immunohistochemistry was performed as previously described [18]. Briefly, the tissue section was blocked with 5% BSA and then incubated with rabbit anti-human METTL3 antibody at 1:200 dilution at 4 C overnight. Finally, the tissue section was incubated with HRP-labeled goat anti rabbit secondary
Data were expressed as the means ± SEM. The unpaired, 2-tailed t-test was used for comparisons between 2 groups. For multiple comparisons, ANOVA or repeated ANOVA followed by the Bonferroni post hoc test was used with GraphPad Prism® version 6.0 software. A P value < 0.05 was considered statistically significant.
The methylated m6A RNA immunoprecipitation (me-RIP) was performed as described previously [12]. The methylated LEF1RNA was evaluated by qRT-PCR. 2.11. RNA stability assay The mRNA stability of LEF1 is analyzed as previously reported [20]. 2.12. Establishment of the metastatic bone tumor model
Please cite this article as: W. Miao et al., The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.128
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3. Results 3.1. The m6A methylated RNA level and its methyltransferase METTL3 is elevated in in human OS tissues and cell lines To explore the potential role of m6A in human OS, we firstly analyzed the total methylated RNA (m6A) level in human OS tissues. We collected 40pairs of fresh OS tissues and corresponding normal tissues and analyzed the total methylated RNA (m6A) level. As shown in the Fig. 1A, the m6A methylated RNA level of was remarkedly increased in human OS tissues compared with corresponding adjacent normal tissues. Similarly, the m6A methylated RNA level was elevated in the human OS cell lines (U2OS, MG-63, Saos-2 and HOS) compared with the normal osteoblast cells (NHOst) (Fig. 1B). Next, we screened the critical methyltransferase and found that the mRNA expression of METTL3 was consistently elevated in human OS tissues (Fig. 1C) and cell lines (Fig. 1D). The increased expression of METTL3 protein in human OS tissues was further confirmed by western blot (Fig. 1E) and immunohistochemistry (Fig. 1F). These results showed that METTL3 may be a cancer-promoting gene in the development of human OS via m6A modification. 3.2. Silence of METTL3 inhibits the abilities of proliferation, migration and invasion in OS cells To further explore the functional role of METTL3 in human OS, we silenced the expression of METTLL3 with lentivirus-mediated shRNA in human OS cells HOS and SAOS-2. The efficiency of METTL3 silence in HOS and SAOS-2 cells were confirmed by quantitative real-time PCR (Fig. 2A and F) as well as western blot (Fig. 2B and G). Cell proliferation assay was then performed and the data indicated that METTL3 silence obviously inhibited proliferation ability of HOS (Fig. 2C) and SAOS-2 (Fig. 2H) cells. To investigate its role in OS migration and invasion, wound healing and invasion
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assays were conducted. The results suggested that METTL3 silence dramatically repressed the abilities of migration (Fig. 2D and I) and invasion (Fig. 2E and J) in HOS and SAOS-2 cells. These results demonstrated that METTL3 silence inhibits the proliferation, migration and invasion abilities of OS cells in vitro.
3.3. Silence of METTL3 represses the expression and m6A methylated level of LEF1 and inhibits the activity of wnt/b-catenin signaling pathway To explore the underlying mechanisms of METTL3 silencemediated tumor suppression, we analyzed whether LEF1, a downstream component of the Wnt signaling pathway, was involved in oncogenic role of METTL3 in human OS. We found that LEF1 mRNA level was also obviously elevated in human OS tissues (Fig. 3A). Moreover, the level of LEF1 was positively correlated with the level of METTL3 in human OS tissues by linear regression analysis (Fig. 3B). We next examined whether METTL3 could regulate endogenous LEF1 mRNA expression in OS cells. The data showed that METTL3 silence reduced the mRNA (Fig. 3C) and protein(Fig. 3D) levels of LEF1 in HOS and SAOS-2 cells. Furthermore, Wnt reporter activity assay showed that the activity of Wnt/ b-catenin signaling pathway was dramatically decreased in METTL3 silenced HOS and SAOS-2 cells (Fig. 3E). Meanwhile, the methylated level of LEF1mRNAin HOS and SAOS-2 cells was reduced by METTL3 silence. To further analyze the effect of m6A level decrease on the stability of LEF1 mRNA, we conducted RNA stability assays and found that knockdown of METTL3 shortened the half-life of LEF1 mRNA transcript in HOS and SAOS-2 cells. Thus, METTL3 knockdown-induced repression of LEF1 expression is at least in part due to the decreased stability of LEF1 mRNA transcript upon METTL3-mediated m6A methylation of LEF1 mRNA (Fig. 3G). Collectively, these findings suggested that METTL3 silence represses the expression of LEF1 by methylating its mRNA.
Fig. 1. The m6A methylated RNA level ant its methyltransferase METTL3 is elevated in human osteosarcoma. (A)The methylated RNA (m6A) level in 40 paired human OS tissues and adjacent normal tissues. **P < 0.01. (B)The methylated RNA (m6A) level in the human OS cell lines (U2OS, MG-63, Saos-2 and HOS) and the normal osteoblast cells (NHOst). *P < 0.05; **P < 0.01. (C) The mRNA level of METTL3 in 40 paired human OS tissues and adjacent normal tissues were analyzed by qRT-PCR. **P < 0.01.(D) The mRNA level of METTL3 in the human OS cell lines (U2OS, MG-63, Saos-2 and HOS) and the normal osteoblast cells(NHOst) were analyzed by qRT-PCR. *P < 0.05; **P < 0.01. (E) The protein level of METTL3 in 4 paired human OS tissues and adjacent normal tissues were analyzed by western blot. **P < 0.01. (F) The protein level of METTL3 in 4 paired human OS tissues and adjacent normal tissues were analyzed by IHC.
Please cite this article as: W. Miao et al., The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.128
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Fig. 2. Silence of METTL3 inhibits the proliferation, migration and invasion abilities of OS cells in vitro. (A, F) The efficiency of METTL3 silence in HOS and SAOS-2 cells were confirmed by quantitative real-time PCR. *P < 0.05.(B, G) The efficiency of METTL3 silence in HOS and SAOS-2 cells were confirmed by western blot.*P < 0.05. (C, H) The cell viability of HOS and SAOS-2 cells were analyzed by CCK8 assay. (D, I) The migration ability of HOS and SAOS-2 cells were analyzed by wound healing assay. *P < 0.05. (E, J) The invasion ability of HOS and SAOS-2 cells were analyzed by invasion chamber assays. *P < 0.05.
3.4. LEF1over-expression abrogates the repression effect of METTL3 silence on the abilities of proliferation, migration and invasion in OS cells To further explore whether LEF1 is critical to the repressive effects of METTL3 silence on OS cells, we next overexpressed LEF1 in HOS and SAOS-2 cells. The efficiency of LEF1 over-expression in HOS and SAOS-2 cells were confirmed by quantitative real-time PCR (Fig. 4A) and western blot (Fig. 4B). Next, the cell proliferation, migration and invasion were examined. Our data showed that over-expression of LEF1 dramatically abrogated the tumorsuppressive effects of METTL3 silence on cell proliferation (Fig. 4C), migration (Fig. 4D) and invasion (Fig. 4E). We further evaluated these effects in a bone tumor model. HOS cells stably expressing the firefly luciferase gene and the indicated genes were injected into the tibia of nude mice to establish the metastatic bone tumor model. The mice were examined with an in vivo imaging
system two weeks later. The results showed that the animals in the METTL3 silence group have much lower luminescence intensities compare with the control group (Fig. 4F), suggesting decreased progression of the bone tumors' formation. In comparison, the animals injected with LEF1 overexpressed HOS cells showed similar luminescence intensities with control group. However, LEF1 overexpression significantly reversed the repression of METTL3 silence on the bone tumors’ formation (Fig. 4F). 4. Discussion In this study, our results provide insight into the biological role of METTL3 and m6A modification in OS progression for the first time. We found a significantly elevated expression of METTL3 in primary OS tissues and cell lines. In addition, our data showed that the total m6A methylated RNA level and the expression level of METTL3 were elevated in human OS tissues and cell lines.
Please cite this article as: W. Miao et al., The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.128
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Fig. 3. Silence of METTL3 inhibits the expression and m6A methylated level of LEF1 and inhibits the activity of wnt/b-catenin signaling pathway. (A) The mRNA level of LEF1in 40 paired human OS tissues and adjacent normal tissues were analyzed by qRT-PCR. **P < 0.01. (B)Linear regression analysis is used to evaluate the positive correlation between METTL3 expression and LEF1 mRNA expression. (C) The mRNA level of LEF1 in the METTL3 silenced HOS and SAOS-2 cell lines were analyzed by quantitative real-time PCR. *P < 0.05. (D) The protein level of LEF1 in the METTL3 silenced HOS and SAOS-2 cell lines were analyzed by western blot. *P < 0.05. (E) The activity of wnt/b-catenin signaling pathway in the METTL3 silenced HOS and SAOS-2 cell lines were analyzed by TOP/FOP-Flash reporter assay. *P < 0.05. (F) The methylated LEF1 mRNA level in the METTL3 silenced HOS and SAOS-2 cell lines were analyzed by Me-RIP assay. *P < 0.05.(G) The mRNA half-life (T1/2) of LEF1 in HOS and SAOS-2 cells transduced with METTL3 shRNA (Lv-shMETTL3) or control vector (Lv-NC)after transcription inhibition (TI).
As one of the most common chemical modifications of human mRNA, m6A modification is believed to play a crucial role in cancer progression. In this study, we found that the expression of METTL3 was elevated inhuman OS tissues compared with adjacent normal tissues and in OS cell lines compared with normal osteoblast cells. By silencing METTL3, we found that METTL3 silence repressed the cell abilities of proliferation, migration and invasion in vitro and in vivo. For the first time, these findings demonstrated that METTL3 serves as oncogenic role in human OS. Previous studies have shown that there are two distinct mechanisms of METTL3's involvement in tumor progression: m6Adependent as well as m6A-independent.For instance, knockdown of METTL3 in hepatocellular carcinoma (HCC) can suppress m6A methylation-mediated degradation of SOC2 followed by increased expression, thus hampering the progression of HCC [22]. Moreover, METTL3 heightens m6A modification of HBXIP and upregulates its expression, which further promotes the development of breast cancer [23]. However, METTL3 is also found to bind to gene
promoters and promote the expression of oncogenes [24]. Aberrant activation of Wnt/b-catenin pathway is recognized to plays a critical role in the development of OS. In this study, we analyzed the underlying mechanism of METTL3 in human osteosarcoma by focusing on the its effects on Wnt signaling pathway through regulating m6A modification of LEF1. Our results indicate that METTL3 silence decreased the m6A level and mRNA level of lymphoid enhancer-binding factor 1 (LEF1), subsequently inhibited the activity of Wnt/b-catenin signaling pathway. Finally, LEF1 overexpression abrogates the repression effect of METTL3 silence on the proliferation, migration and invasion abilities of OS cells. Our results support the notion that promoting the m6A level of LEF1 is responsible for m6A methyltransferase METTL3-induced OS cell progression. However, further in vivo studies needed to be performed to provide direct genetic evidence of METTL3/LEF1 axis in OS pathogenesis. In summary, our results demonstrate that the m6A methyltransferase METTL3 contributes to the OS cell progression through
Please cite this article as: W. Miao et al., The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.128
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Fig. 4. LEF1 over-expression abrogates the repression effect of METTL3 silence on the proliferation, migration and invasion abilities of OS cells. (A) The efficiency of METTL3 silence and LEF1 over-expression in HOS and SAOS-2 cells were confirmed by quantitative real-time PCR. *P < 0.05. (B) The efficiency of METTL3 silence and LEF1 over-expression in HOS and SAOS-2 cells were confirmed by western blot. *P < 0.05. (C)The cell viability of HOS and SAOS-2 cells were analyzed by CCK8 assay. *P < 0.05 v. s. Lv-Scramble group; #P < 0.05 v. s. Lv-shMETTL3 group. (D) The migration ability of HOS and SAOS-2 cells were analyzed by wound healing assay. *P < 0.05. (E) The invasion ability of HOS and SAOS2 cells were analyzed by invasion chamber assays. *P < 0.05. (F) The metastatic bone tumor model was used to evaluate the bone-tumor formation ability of HOS in vivo. Bioluminescence images of HOS-luc tumor-bearing mice and relative luminescence intensities were presented.*P < 0.05.
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Please cite this article as: W. Miao et al., The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.128
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The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1 Wujun Miao a, 1, Jiajia Chen c, d, 1, Lianshun Jia a, *, Jun Ma a, **, Dianwen Song b, c, *** a
Department of Orthopedics, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai, 200003, China Department of Orthopedics, The First People's Hospital, Shanghai Jiao Tong University, 650 Xinsongjiang Road, Shanghai, 201620, China c Department of Orthopedics, Shanghai General Hospital of Nanjing Medical University, 650 Xinsongjiang Road, Shanghai, 201620, China d Department of Spine Surgery, The Second Affiliated Hospital of Nantong University, 6Haier Lane North Road, Nantong, Jiangsu, 226001, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 May 2019 Accepted 22 June 2019 Available online xxx
Osteosarcoma(OS) is the most common and aggressive malignant bone sarcoma,which occurs in rapidly growing bones in children and adolescents. However, the underlying molecular mechanisms of OS development have not been fully illustrated. N6-Methyladenosine (m6A) is the most prevalent internal chemical modification of mRNAs, which is involved in many pathological processes in cancer development. However, its role and regulatory mechanism in OS remain unknown. In this study, we aimed to investigate the roles of m6A and its methyltransferase METTL3 in OS development. The results showed that m6A level for RNA methylation and the expression level of METTL3 were up-regulated in human OS tissues and OS cell lines. Functionally, lentivirus-mediated METTL3 silence in HOS and SAOS-2 cells inhibited the cell proliferation, migration and invasion ability. Further mechanism analysis suggested that METTL3 silence decreased the m6A methylation and total mRNA level of lymphoid enhancerbinding factor 1 (LEF1), followed by inhibited the activity of Wnt/b-catenin signaling pathway. Moreover, LEF1 over-expression abrogates the repressive effects of METTL3 silence on the proliferation, migration and invasion abilities of OS cells. Together, these results revealed that the m6A methyltransferase METTL3 promotes osteosarcoma cell progression by regulating the m6A level of LEF1 and activating Wnt/b-catenin signaling pathway. © 2019 Published by Elsevier Inc.
Keywords: Osteosarcoma METTL3 m6A LEF1
1. Introduction As one of the most common cancers, human osteosarcoma (OS) has a high mortality and heterogeneous presentation [1,2]. Amputation was the most common treatment for osteosarcoma over the past decades [3]. Though the 5-year survival rate of patients with OS has already significantly improved, there are still many patients with poor prognosis [4]. Therefore, a more comprehensive understanding of the underlying molecular
* Corresponding author. Department of Orthopedics, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai, 200003, China. ** Corresponding author. Department of Orthopedics, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai, 200003, China. *** Corresponding author. Department of Orthopedics, The First People's Hospital, Shanghai Jiao Tong University, 650 Xinsongjiang Road, Shanghai, 201620, China. E-mail addresses: [email protected] (L. Jia), [email protected] (J. Ma), [email protected] (D. Song). 1 Wujun Miao and Jiajia Chen contributed equally.
mechanisms is urgently required for the treatment of OS. In eukaryotes, N6-Methyladenosine (m6A) is one of the most common internal chemical modifications of mRNAs [5], which is catalyzed by a methyltransferase complex, including of methyltransferase-like 3 and 14 (METTL3 and METTL14) and their cofactors, such as Wilms tumor 1-associated protein (WTAP), VIRMA (KIAA1429)and RBM15 [6]. On the other hand, this modification is reversed by two mammalian RNA demethylases, alkylation repair homolog protein 5(ALKBH5) [7] and the fat mass and obesity-associated protein(FTO) [8], which maintain the dynamic balance of m6A methylation and demethylation in mammalian cells. By methylating its target mRNAs, METTL3 is involved in a variety of pathological processes, including various cancers [9e12]. However, the roles and underlying molecular basis of METTL3 in the progress of human osteosarcoma remain unknown. Previous studies demonstrated that the Wnt signaling pathway plays critical roles in tissue homeostasis, stem cell biology, organogenesis as well as cancer progression [13,14]. The binding between LRP5/6 transmembrane receptors with secreted Wnt ligands to
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Please cite this article as: W. Miao et al., The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.128
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Frizzled hold back the degradation of the signaling pool of cytoplasmic b-catenin. Avoiding from degradation, b-catenin gradually accumulates in the nucleus followed by binding to transcription factors, the LEF/TCF (lymphoid enhancer-binding factor and T cell factor) family, to stimulate the downstream gene expression. Moreover, previous studies have shown that LEF1 contributes to metastasis and chemoresistance in several cancers including osteosarcoma [15,16]. However, the underlying mechanism by which LEF1 is regulated, especially m6A modification of mRNA, in human osteosarcoma is still largely unknown. Here, our data indicated that total m6A methylated RNA level and the expression level of METTL3 were elevated in human OS tissues and cell lines. METTL3 silence in HOS and SAOS-2 cells inhibited the cell proliferation, migration and invasion ability. Mechanism analysis further suggested that METTL3 silence decreased the m6A methylation and total mRNA level of lymphoid enhancer-binding factor 1 (LEF1), subsequently inhibited the activity of Wnt/b-catenin signaling pathway. Furthermore, LEF1 overexpression abrogates the repression effect of METTL3 silence on the abilities of proliferation, migration and invasion in OS cells. Taken together, these results reveal that the m6A methyltransferase METTL3 promotes osteosarcoma cell progression through regulating the m6A level of LEF1 and activating Wnt/b-catenin signaling. Our data indicate that METTL3 may be a therapeutic target which is potential to be used for the treatment of human OS. 2. Materials and methods 2.1. Patient and clinical samples with ethical approval A total of 40 pairs of patient specimens were used in this research. These human samples were obtained according to the principles of the Declaration of Helsinki and approved by Changzheng Hospital (Shanghai, China). Written informed consent was obtained from all patients between 2000 and 2014. 2.2. Quantitative real-time PCR (qRT-PCR)
antibody, followed by incubation with diaminobenzene as the chromogen, and hematoxylin as the nuclear counterstain. 2.5. Cell lines and cell culture Human OS cell lines (U2OS, MG-63, Saos-2 and HOS) and normal human osteoblast cells (NHOst) were obtained from the Chinese Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All these cell lines were maintained in DMEM supplemented with 10% fetal bovine serum and 1% PS (penicillin and streptomycin) under standard culture conditions (5% CO2, 37 C). 2.6. Cell proliferation assay Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China) was used to measure the cell proliferation as previously described [12]. 2.7. Migration and invasion assay The cell migration ability was detected by wound healing assay as previously reported [17]. The invasion assay was analyzed using transwell chamber (8-mm pores, BD Falcon, Franklin Lakes, NJ, USA). 2.8. TOP/FOP-Flash reporter assay TOP/FOP-Flash reporter assay was performed as previously reported [19]. Briefly, the OS cells were seeded in 24-well plates followed by transfection with TOP/FOP flash plasmids (Simo Biomedical Technology, Shanghai, China). The luciferase activity was examined by the Dual Luciferase Assay kit (Promega, Madison, WI). 2.9. m6A RNA methylation quantification The m6ARNA Methylation Assay Kit (Abcam, ab185912) was used to evaluate the content of m6A in total RNA as previously reported [12].
Total RNA was lysed using TRIzol reagent and used for the synthesis of the cDNA with One-Step RT-PCR Kit (Thermo Fisher). The real-time PCR was performed using the ABI Vii7 system (Applied Biosystems, USA). GAPDH was used as a housekeeping gene. Primers were synthesized as follows, the human METTL3 forward primer:50 -CAAGCTGCACTTCAGACGAA-30 ,and human METTL3 reverse primer: 50 -GCTTGGCGTGTGGTCTTT-30 ; the human LEF1 forward primer:50 -CTACCCATCCTCACTGTCAGTC-30 ,and human LEF1reverse primer: 50 -GGATGTTCCTGTTTGACCTGAGG-30 ; GAPDH forward: 50 -TGACTTCAACAGCGACACCCA-30 ,and GAPDH reverse: 50 -CACCCTGTTGCTGTAGCCAAA-30 . Relative gene expression was calculated by the comparative CT method (DDCT).
2.10. Me-RIP assay
2.3. Western blot Western blotting was performed with antibodies against METTL3, LEF1 and GAPDH (Abcam, Cambridge, MA, USA)as previously described [17]. Human GAPDH was used as an endogenous control to normalize the protein loading.
The Metastatic Bone Tumor Model was established via injecting HOS cells into the right tibia of each animal as described previously [21]. Two weeks after the injection, the bioluminescence images of bone tumors in the tibia of each animal were observed by an in vivo imaging system (Xenogen IVIS-200, Caliper Life Sciences, Hopkinton).
2.4. Immunohistochemistry
2.13. Statistical analysis
The protocol for the immunohistochemistry was performed as previously described [18]. Briefly, the tissue section was blocked with 5% BSA and then incubated with rabbit anti-human METTL3 antibody at 1:200 dilution at 4 C overnight. Finally, the tissue section was incubated with HRP-labeled goat anti rabbit secondary
Data were expressed as the means ± SEM. The unpaired, 2-tailed t-test was used for comparisons between 2 groups. For multiple comparisons, ANOVA or repeated ANOVA followed by the Bonferroni post hoc test was used with GraphPad Prism® version 6.0 software. A P value < 0.05 was considered statistically significant.
The methylated m6A RNA immunoprecipitation (me-RIP) was performed as described previously [12]. The methylated LEF1RNA was evaluated by qRT-PCR. 2.11. RNA stability assay The mRNA stability of LEF1 is analyzed as previously reported [20]. 2.12. Establishment of the metastatic bone tumor model
Please cite this article as: W. Miao et al., The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.128
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3. Results 3.1. The m6A methylated RNA level and its methyltransferase METTL3 is elevated in in human OS tissues and cell lines To explore the potential role of m6A in human OS, we firstly analyzed the total methylated RNA (m6A) level in human OS tissues. We collected 40pairs of fresh OS tissues and corresponding normal tissues and analyzed the total methylated RNA (m6A) level. As shown in the Fig. 1A, the m6A methylated RNA level of was remarkedly increased in human OS tissues compared with corresponding adjacent normal tissues. Similarly, the m6A methylated RNA level was elevated in the human OS cell lines (U2OS, MG-63, Saos-2 and HOS) compared with the normal osteoblast cells (NHOst) (Fig. 1B). Next, we screened the critical methyltransferase and found that the mRNA expression of METTL3 was consistently elevated in human OS tissues (Fig. 1C) and cell lines (Fig. 1D). The increased expression of METTL3 protein in human OS tissues was further confirmed by western blot (Fig. 1E) and immunohistochemistry (Fig. 1F). These results showed that METTL3 may be a cancer-promoting gene in the development of human OS via m6A modification. 3.2. Silence of METTL3 inhibits the abilities of proliferation, migration and invasion in OS cells To further explore the functional role of METTL3 in human OS, we silenced the expression of METTLL3 with lentivirus-mediated shRNA in human OS cells HOS and SAOS-2. The efficiency of METTL3 silence in HOS and SAOS-2 cells were confirmed by quantitative real-time PCR (Fig. 2A and F) as well as western blot (Fig. 2B and G). Cell proliferation assay was then performed and the data indicated that METTL3 silence obviously inhibited proliferation ability of HOS (Fig. 2C) and SAOS-2 (Fig. 2H) cells. To investigate its role in OS migration and invasion, wound healing and invasion
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assays were conducted. The results suggested that METTL3 silence dramatically repressed the abilities of migration (Fig. 2D and I) and invasion (Fig. 2E and J) in HOS and SAOS-2 cells. These results demonstrated that METTL3 silence inhibits the proliferation, migration and invasion abilities of OS cells in vitro.
3.3. Silence of METTL3 represses the expression and m6A methylated level of LEF1 and inhibits the activity of wnt/b-catenin signaling pathway To explore the underlying mechanisms of METTL3 silencemediated tumor suppression, we analyzed whether LEF1, a downstream component of the Wnt signaling pathway, was involved in oncogenic role of METTL3 in human OS. We found that LEF1 mRNA level was also obviously elevated in human OS tissues (Fig. 3A). Moreover, the level of LEF1 was positively correlated with the level of METTL3 in human OS tissues by linear regression analysis (Fig. 3B). We next examined whether METTL3 could regulate endogenous LEF1 mRNA expression in OS cells. The data showed that METTL3 silence reduced the mRNA (Fig. 3C) and protein(Fig. 3D) levels of LEF1 in HOS and SAOS-2 cells. Furthermore, Wnt reporter activity assay showed that the activity of Wnt/ b-catenin signaling pathway was dramatically decreased in METTL3 silenced HOS and SAOS-2 cells (Fig. 3E). Meanwhile, the methylated level of LEF1mRNAin HOS and SAOS-2 cells was reduced by METTL3 silence. To further analyze the effect of m6A level decrease on the stability of LEF1 mRNA, we conducted RNA stability assays and found that knockdown of METTL3 shortened the half-life of LEF1 mRNA transcript in HOS and SAOS-2 cells. Thus, METTL3 knockdown-induced repression of LEF1 expression is at least in part due to the decreased stability of LEF1 mRNA transcript upon METTL3-mediated m6A methylation of LEF1 mRNA (Fig. 3G). Collectively, these findings suggested that METTL3 silence represses the expression of LEF1 by methylating its mRNA.
Fig. 1. The m6A methylated RNA level ant its methyltransferase METTL3 is elevated in human osteosarcoma. (A)The methylated RNA (m6A) level in 40 paired human OS tissues and adjacent normal tissues. **P < 0.01. (B)The methylated RNA (m6A) level in the human OS cell lines (U2OS, MG-63, Saos-2 and HOS) and the normal osteoblast cells (NHOst). *P < 0.05; **P < 0.01. (C) The mRNA level of METTL3 in 40 paired human OS tissues and adjacent normal tissues were analyzed by qRT-PCR. **P < 0.01.(D) The mRNA level of METTL3 in the human OS cell lines (U2OS, MG-63, Saos-2 and HOS) and the normal osteoblast cells(NHOst) were analyzed by qRT-PCR. *P < 0.05; **P < 0.01. (E) The protein level of METTL3 in 4 paired human OS tissues and adjacent normal tissues were analyzed by western blot. **P < 0.01. (F) The protein level of METTL3 in 4 paired human OS tissues and adjacent normal tissues were analyzed by IHC.
Please cite this article as: W. Miao et al., The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.128
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Fig. 2. Silence of METTL3 inhibits the proliferation, migration and invasion abilities of OS cells in vitro. (A, F) The efficiency of METTL3 silence in HOS and SAOS-2 cells were confirmed by quantitative real-time PCR. *P < 0.05.(B, G) The efficiency of METTL3 silence in HOS and SAOS-2 cells were confirmed by western blot.*P < 0.05. (C, H) The cell viability of HOS and SAOS-2 cells were analyzed by CCK8 assay. (D, I) The migration ability of HOS and SAOS-2 cells were analyzed by wound healing assay. *P < 0.05. (E, J) The invasion ability of HOS and SAOS-2 cells were analyzed by invasion chamber assays. *P < 0.05.
3.4. LEF1over-expression abrogates the repression effect of METTL3 silence on the abilities of proliferation, migration and invasion in OS cells To further explore whether LEF1 is critical to the repressive effects of METTL3 silence on OS cells, we next overexpressed LEF1 in HOS and SAOS-2 cells. The efficiency of LEF1 over-expression in HOS and SAOS-2 cells were confirmed by quantitative real-time PCR (Fig. 4A) and western blot (Fig. 4B). Next, the cell proliferation, migration and invasion were examined. Our data showed that over-expression of LEF1 dramatically abrogated the tumorsuppressive effects of METTL3 silence on cell proliferation (Fig. 4C), migration (Fig. 4D) and invasion (Fig. 4E). We further evaluated these effects in a bone tumor model. HOS cells stably expressing the firefly luciferase gene and the indicated genes were injected into the tibia of nude mice to establish the metastatic bone tumor model. The mice were examined with an in vivo imaging
system two weeks later. The results showed that the animals in the METTL3 silence group have much lower luminescence intensities compare with the control group (Fig. 4F), suggesting decreased progression of the bone tumors' formation. In comparison, the animals injected with LEF1 overexpressed HOS cells showed similar luminescence intensities with control group. However, LEF1 overexpression significantly reversed the repression of METTL3 silence on the bone tumors’ formation (Fig. 4F). 4. Discussion In this study, our results provide insight into the biological role of METTL3 and m6A modification in OS progression for the first time. We found a significantly elevated expression of METTL3 in primary OS tissues and cell lines. In addition, our data showed that the total m6A methylated RNA level and the expression level of METTL3 were elevated in human OS tissues and cell lines.
Please cite this article as: W. Miao et al., The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.128
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Fig. 3. Silence of METTL3 inhibits the expression and m6A methylated level of LEF1 and inhibits the activity of wnt/b-catenin signaling pathway. (A) The mRNA level of LEF1in 40 paired human OS tissues and adjacent normal tissues were analyzed by qRT-PCR. **P < 0.01. (B)Linear regression analysis is used to evaluate the positive correlation between METTL3 expression and LEF1 mRNA expression. (C) The mRNA level of LEF1 in the METTL3 silenced HOS and SAOS-2 cell lines were analyzed by quantitative real-time PCR. *P < 0.05. (D) The protein level of LEF1 in the METTL3 silenced HOS and SAOS-2 cell lines were analyzed by western blot. *P < 0.05. (E) The activity of wnt/b-catenin signaling pathway in the METTL3 silenced HOS and SAOS-2 cell lines were analyzed by TOP/FOP-Flash reporter assay. *P < 0.05. (F) The methylated LEF1 mRNA level in the METTL3 silenced HOS and SAOS-2 cell lines were analyzed by Me-RIP assay. *P < 0.05.(G) The mRNA half-life (T1/2) of LEF1 in HOS and SAOS-2 cells transduced with METTL3 shRNA (Lv-shMETTL3) or control vector (Lv-NC)after transcription inhibition (TI).
As one of the most common chemical modifications of human mRNA, m6A modification is believed to play a crucial role in cancer progression. In this study, we found that the expression of METTL3 was elevated inhuman OS tissues compared with adjacent normal tissues and in OS cell lines compared with normal osteoblast cells. By silencing METTL3, we found that METTL3 silence repressed the cell abilities of proliferation, migration and invasion in vitro and in vivo. For the first time, these findings demonstrated that METTL3 serves as oncogenic role in human OS. Previous studies have shown that there are two distinct mechanisms of METTL3's involvement in tumor progression: m6Adependent as well as m6A-independent.For instance, knockdown of METTL3 in hepatocellular carcinoma (HCC) can suppress m6A methylation-mediated degradation of SOC2 followed by increased expression, thus hampering the progression of HCC [22]. Moreover, METTL3 heightens m6A modification of HBXIP and upregulates its expression, which further promotes the development of breast cancer [23]. However, METTL3 is also found to bind to gene
promoters and promote the expression of oncogenes [24]. Aberrant activation of Wnt/b-catenin pathway is recognized to plays a critical role in the development of OS. In this study, we analyzed the underlying mechanism of METTL3 in human osteosarcoma by focusing on the its effects on Wnt signaling pathway through regulating m6A modification of LEF1. Our results indicate that METTL3 silence decreased the m6A level and mRNA level of lymphoid enhancer-binding factor 1 (LEF1), subsequently inhibited the activity of Wnt/b-catenin signaling pathway. Finally, LEF1 overexpression abrogates the repression effect of METTL3 silence on the proliferation, migration and invasion abilities of OS cells. Our results support the notion that promoting the m6A level of LEF1 is responsible for m6A methyltransferase METTL3-induced OS cell progression. However, further in vivo studies needed to be performed to provide direct genetic evidence of METTL3/LEF1 axis in OS pathogenesis. In summary, our results demonstrate that the m6A methyltransferase METTL3 contributes to the OS cell progression through
Please cite this article as: W. Miao et al., The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.128
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Fig. 4. LEF1 over-expression abrogates the repression effect of METTL3 silence on the proliferation, migration and invasion abilities of OS cells. (A) The efficiency of METTL3 silence and LEF1 over-expression in HOS and SAOS-2 cells were confirmed by quantitative real-time PCR. *P < 0.05. (B) The efficiency of METTL3 silence and LEF1 over-expression in HOS and SAOS-2 cells were confirmed by western blot. *P < 0.05. (C)The cell viability of HOS and SAOS-2 cells were analyzed by CCK8 assay. *P < 0.05 v. s. Lv-Scramble group; #P < 0.05 v. s. Lv-shMETTL3 group. (D) The migration ability of HOS and SAOS-2 cells were analyzed by wound healing assay. *P < 0.05. (E) The invasion ability of HOS and SAOS2 cells were analyzed by invasion chamber assays. *P < 0.05. (F) The metastatic bone tumor model was used to evaluate the bone-tumor formation ability of HOS in vivo. Bioluminescence images of HOS-luc tumor-bearing mice and relative luminescence intensities were presented.*P < 0.05.
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Please cite this article as: W. Miao et al., The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.128