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The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB Sasithorn Wanna-udom a, 1, Minoru Terashima a, 1, Hanbing Lyu a, Akihiko Ishimura a, Takahisa Takino b, Matomo Sakari c, Toshifumi Tsukahara c, Takeshi Suzuki a, * a
Division of Functional Genomics, Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Ishikawa, Japan Division of Education for Global Standard, Institute of Liberal Arts and Science, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Ishikawa, Japan c Area of Bioscience and Biotechnology, Japan Advanced Institute of Science and Technology, Nomi, 923-1292, Ishikawa, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 December 2019 Received in revised form 6 January 2020 Accepted 7 January 2020 Available online xxx
N6-Methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the role of m6A methyltransferase METTL3 in TGF-b-induced epithelial-mesenchymal transition (EMT) of lung cancer cell lines. The expression of METTL3 and m6A RNA modification were increased during TGF-b-induced EMT of A549 and LC2/ad lung cancer cells. Knockdown of METTL3 inhibited TGF-b-induced morphological conversion of the cells, enhanced cell migration potential and the expression changes of EMT-related marker genes such as CDH1/E-cadherin, FN1/Fibronectin and VIM/Vimentin. Mechanistic investigations revealed that METTL3 knockdown decreased the m6A modification, total mRNA level and mRNA stability of JUNB, one of the important transcriptional regulators of EMT. Over-expression of JUNB partially rescued the inhibitory effects of METTL3 knockdown in the EMT phenotypes. This study demonstrates that m6A methyltransferase METTL3 is indispensable for TGF-b-induced EMT of lung cancer cells through the regulation of JUNB. © 2020 Elsevier Inc. All rights reserved.
Keywords: Lung cancer Epithelial-mesenchymal transition RNA methylation METTL3 JUNB
1. Introduction N6-methyladenosine (m6A) is the most abundant internal chemical modification of mRNAs and long noncoding RNAs (lncRNAs) in eukaryotes [1]. Methyltransferase-like 3 (METTL3) is the catalytic subunit and forms the m6A methyltransferase complex with METTL14, WTAP and RBM15 [2]. On the other hand, m6A methylation is reversed by the demethylases, FTO and ALKBH5,
Abbreviations: EMT, epithelial-mesenchymal transition; TGF-b, Transforming Growth Factor-beta; m6A, N6-methyladenosine; TF, transcription factor; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; shRNA, small hairpin RNA; Me-RIP, methylated RNA immunoprecipitation; QRT-PCR, quantitative reverse transcribed polymerase chain reaction. * Corresponding author. Division of Functional Genomics, Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Ishikawa, Japan. E-mail address: [email protected] (T. Suzuki). 1 These authors contributed equally to this study.
which maintains a dynamic nature of this modification [1]. In mammalian cells, thousands of mRNAs are subject to m6A methylation, which has been implicated in pre-mRNA splicing, mRNA stability and translation efficiency [3e5]. It has been reported that m6A regulation plays important roles in many biological processes including development, metabolism, stemness maintenance and differentiation [6,7]. Recent studies have also revealed that METTL3 is involved in the development and progression of various types of cancer through the methylation of its target mRNAs [8,9]. Epithelial-mesenchymal transition (EMT) process plays pivotal roles in cancer metastasis [10]. EMT results in loss of cell-cell adhesion and cytoskeletal reorganization, thereby increasing motility, invasiveness and stem cell-like properties of cancer cells. Transforming Growth Factor-beta (TGF-b) has emerged as a major regulator of EMT along with cytokines and growth factors secreted by the tumor microenvironment [11]. One of the well-known hallmarks of EMT is the reversible changes in epithelial and mesenchymal gene expression. EMT-inducing transcription factors
https://doi.org/10.1016/j.bbrc.2020.01.042 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: S. Wanna-udom et al., The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.042
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(TFs) such as ZEB1, ZEB2, SNAI1, SNAI2 and TWIST can activate EMT program through the downregulation of E-cadherin, an epithelial cell marker. The phenotypic plasticity of EMT suggests that this process is controlled by epigenetic regulation including DNA methylation and histone modifications [12,13]. We have previously demonstrated that enzymatic regulation of histone H3 methylation is essential for TGF-b-induced EMT of lung cancer cells [14e16]. We have also discovered that some lncRNAs function as an “initiator” to recruit histone methyltransferase complex to the specific target genes during EMT process [17,18]. However, the functional role of m6A RNA methylation, another type of epigenetic regulation, has not been fully explored in the progression of EMT. Here we showed that the expression of METTL3 and m6A RNA modification were increased during TGF-b-induced EMT of A549 and LC2/ad lung cancer cell lines. METTL3 knockdown antagonized TGF-b-dependent EMT phenotypes and reduced the mRNA stability of JUNB, one of the critical transcriptional regulators of EMT. We also found that JUNB over-expression partially recovered the EMT phenotypes inhibited by METTL3 knockdown. This study demonstrates the function of METTL3 in facilitating TGF-b-induced EMT and identifies JUNB as the downstream target of METTL3.
immuno-fluorescence of E-cadherin and actin staining were performed as described previously [20]. Cell migration activities were measured in modified Boyden chambers consisting of Transwell membrane filter inserts (#3422, Corning) as described previously [17]. Cells on the lower surface of the filter were stained with 0.4% crystal violet and counted from at least four fields and three experiments. 2.4. m6A content analysis and m6A-RNA immunoprecipitation (Me-RIP) assay
2. Materials and methods
The content of m6A in total RNA was analyzed with the m6A RNA Methylation assay quantification kit according to the manufacturer’s protocol (ab185912, Abcam). Me-RIP was conducted according to previously described protocol [16] with a slight modification. Briefly, 100 mg of total RNA was incubated with antim6A antibody (ab151230, Abcam), in 500 ml of RIP buffer supplemented with protease inhibitors (#03969e21, Nakarai) and SUPERase-In (AM2694, Thermo Fisher). Then the immunocomplexes were recovered with Protein G-coupled Dynabeads (DB10003, Veritas). The precipitated RNAs were extracted with High Pure RNA Tissue Kit (#11828665001, Roche) and were quantified by QRT-PCR.
2.1. Plasmids, cell culture and virus infection
2.5. RNA stability assay
The small hairpin RNA (shRNA)-expressing lentivirus vectors were constructed as described previously [19]. The sequences of the oligonucleotides are listed in Supplementary Table S1. We confirmed that METTL3 transcripts and proteins were downregulated with the infection of both METTL3 shRNA-expressing lentiviruses (METTL3 sh#1 and sh#2) even in the presence of TGF-b by quantitative RT-PCR (QRT-PCR) and immunoblotting (Supplementary Fig. S1). We also confirmed that both METTL3 shRNAs caused the same effects in our EMT studies judged from cell morphologies and E-cadherin marker expression (Supplementary Fig. S1), and thus we presented the data of METTL3 shRNA#1 as a representative result. Human JUNB cDNA was tagged with FLAG6xHis-epitope, and then cloned into pDON-5 Neo plasmid (Takara) to produce retroviruses expressing JUNB. Human lung cancer cell lines, A549 and LC-2/ad, were purchased from ATCC and RIKEN BRC and cultured in DMEM and the mixture of RPMI1640 and HAMS F12 medium with 10% FBS, respectively. For EMT induction, A549 and LC-2/ad cells were treated with 1 ng/ml of TGF-b (R&D Systems) for 24e72 h. The protocols for the production and infection of cDNA or shRNA-expressing retroviruses were essentially the same as described previously [20].
To measure RNA stability, the cells were treated with actinomycin D (A9415, Sigma) at 5 mg/ml to block transcription. After incubation at the indicated periods, cells were collected, and RNA was isolated for QRT-PCR. GAPDH expression was used for normalization.
2.2. Quantitative PCR and immunoblotting RNA preparation and QRT-PCR analysis were performed as described previously [20]. QRT-PCR data were normalized with control human GAPDH expression. The averages from at least three independent experiments are shown with the standard deviations. P-values were calculated between control and the samples using Student’s t-test. Primers used for the QPCR were described previously [19,20] and listed in Supplementary Table S1. Immunoblotting was performed as described previously [17]. We used antiMETTL3 (A301-567A, Bethyl), anti-JUNB (#3753, CST) and antiGAPDH (6C5, Millipore) antibodies. 2.3. Cell staining, immuno-fluorescence and cell migration assay A549 or LC-2/ad cells were stained with 0.4% crystal violet to observe the changes of cell morphologies during EMT. Indirect
3. Results 3.1. m6A RNA modification and METTL3 expression were increased during TGF-b-induced EMT of lung cancer cells To investigate the potential role of m6A RNA modification in TGF-b-induced EMT process, we first examined the total m6A level of RNA in two lung cancer cell lines, A549 and LC-2/ad, with or without TGF-b treatment (Fig. 1A). The m6A methylated RNA level was significantly increased by TGF-b treatment in both cells, indicating that lung cancer cells undergoing EMT increased m6A RNA modification (Fig. 1A). Then we examined the expression of m6A methyltransferase, METTL3, during TGF-b-induced EMT of A549 and LC-2/ad cells (Fig. 1B). QRT-PCR analysis revealed that METTL3 expression was significantly upregulated in response to TGF-b in both cells (Fig. 1B). These results suggested that m6A RNA modification and its methyltransferase METTL3 might be involved in the EMT process of lung cancer cells. 3.2. Knockdown of METTL3 antagonized TGF-b-induced EMT phenotypes To explore the function of METTL3 in EMT, we knocked down the expression of METTL3 with lentivirus-mediated shRNA in A549 and LC2/ad cells. Then we examined the cell morphologies and the state of E-cadherin and actin with or without TGF-b treatment (Fig. 2A and B). Control cells revealed scattered, elongated or enlarged cell phenotypes characteristic of EMT in the presence of TGF-b. METTL3 knockdown seemed to enhance cell adhesion slightly, and obviously inhibited cell morphological changes mediated by TGF-b (Fig. 2A and B, upper panels). For the expression of an epithelial cell marker, E-cadherin, immuno-fluorescence staining showed that control cells almost lost E-cadherin signals
Please cite this article as: S. Wanna-udom et al., The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.042
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Fig. 1. The m6A RNA modification and METTL3 expression were increased during TGF-b-induced EMT of lung cancer cells. (A) The m6A methylated RNA level was elevated in A549 and LC2/ad cells after TGF-b treatment. m6A content was analyzed by ELISA kit in the cells with or without the treatment of 1 ng/ml of TGF-b for 48 h (n ¼ 3)(**, P < 0.01 comparing to control). (B) The expression of METTL3 gene was increased during TGF-b-induced EMT. QRT-PCR was performed in A549 and LC2/ad cells before and after TGF-b treatment (6 h, 12 h, 24 h, 48 h and 72 h)(n ¼ 3)(**, P < 0.01 comparing to control; *, P < 0.05 comparing to control).
Fig. 2. Knockdown of METTL3 antagonized TGF-b-induced EMT phenotypes. (A, B) METTL3 knockdown inhibited TGF-b-induced morphological changes of A549 (A) and LC2/ad (B) cells. The cells were infected with lentiviruses expressing control shRNA or METTL3 shRNA (sh#1) with or without TGF-b treatment for 48 h. The cells were stained with crystal violet (upper), with anti-E-cadherin antibody and DAPI (middle) or with TRITCphalloidin and DAPI (lower). Scale bars: 10 mm. (C, D) Knockdown of METTL3 affected the changes in expression of EMT-related marker genes induced by TGF-b. QRT-PCR was performed to detect the expression of CDH1/E-cadherin, FN1/Fibronectin and VIM/Vimentin in the control or METTL3 knockdown A549 (C) and LC2/ad (D) cells with or without TGF-b for 24 h (n ¼ 3)(**, P < 0.01; *, P < 0.05; ns, not significant). (E) METTL3 knockdown inhibited TGF-b-dependent increase of migrated cells through the filter. A549 cells that migrated through the filter within 24 h were fixed, stained and counted (n ¼ 12)(**, P < 0.01; ns, not significant). . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
after TGF-b treatment. METTL3 knockdown increased E-cadherin staining on the cell membrane compared to the control cells, which was still detected in the presence of TGF-b (Fig. 2A and B, middle panels). TGF-b caused actin stress fiber formation in control cells, but in the METTL3 knockdown cells we never detected actin stress fiber even after TGF-b treatment (Fig. 2A and B, lower panels). These results indicated that METTL3 knockdown antagonized TGF-
b-induced EMT phenotypes of A549 and LC-2/ad lung cancer cells such as morphological changes and cytoskeletal rearrangements. Since the changes in the expression of epithelial and mesenchymal genes are an characteristic feature of EMT, we examined the expression level of an epithelial cell marker, CDH1/E-cadherin, and mesenchymal cell markers, FN1/Fibronectin and VIM/Vimentin, in A549 (Fig. 2C) and LC-2/ad cells (Fig. 2D) with METTL3 knockdown.
Please cite this article as: S. Wanna-udom et al., The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.042
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QRT-PCR revealed that METTL3 knockdown significantly increased CDH1 expression and decreased the expression of FN1 and VIM. Moreover, it inhibited the expression changes of these EMT-related marker genes stimulated by TGF-b treatment (Fig. 2C and D). These results suggested the involvement of endogenous METTL3 in the transcriptional regulation of TGF-b-induced EMT program. To observe the effects of METTL3 knockdown in EMT-associated phenotypes, we measured cell motility by the transfilter assays. As shown in Fig. 2E, TGF-b treatment significantly increased the number of migrated A549 cells. METTL3 knockdown caused the reduction of migrated cells by itself, and also cancelled TGF-bdependent increase of migrated cells (Fig. 2E). This result indicated that METTL3 knockdown inhibited the enhanced cell migration potential of lung cancer cells associated with TGF-b-dependent EMT. 3.3. METTL3 regulated the expression of JUNB, one of the important transcription factors involved in EMT To further clarify the function of METTL3 in EMT process, we have performed a candidate gene approach to find potential targets regulated by METTL3. Since METTL3 was suggested to affect the transcriptional regulation of EMT (Fig. 2C and D), we focused on the EMT-related transcription factors (TFs) which were upregulated during TGF-b-induced EMT. Based on the previous studies [21], we selected 12 candidate TFs and examined the m6A methylation levels of their mRNAs by Me-RIP assay (Supplementary Fig. S2). The m6A levels of JUNB, ETS2, SNAI1, SNAI2, ZEB1 and ZEB2 transcripts were significantly increased by TGF-b treatment and decreased by METTL3 knockdown (Supplementary Fig. S2). Among them, we decided to examine JUNB as a candidate target TF regulated by METTL3 because of its remarkable changes of m6A level.
As shown in Fig. 3A, we confirmed that the m6A levels of JUNB mRNA were increased by TGF-b and decreased by METTL3 knockdown in A549 and LC2/ad lung cancer cells. METTL3 knockdown also cancelled the effect of TGF-b in the increase of m6A level. It has been reported that JUNB is one of the important TFs involved in TGF-b-induced EMT and its expression is upregulated by TGF-b [21,22]. Our results also indicated that the expression of JUNB mRNA and protein was increased after TGF-b treatment in A549 and LC2/ad cells (Fig. 3B and C). More importantly, METTL3 knockdown reduced the mRNA and protein level of JUNB in both cells and counteracted the effect of TGF-b (Fig. 3B and C). In the case of SOX4 mRNA, whose m6A levels were not significantly changed by TGF-b and METTL3 knockdown (Supplementary Fig. S2), its basal level expression and induction by TGF-b were not affected by METTL3 knockdown (Supplementary Fig. S3A). These results suggested that METTL3-mediated m6A RNA modification influenced the total expression level of its target JUNB mRNA. To examine the relationship between the m6A modification and stability of JUNB mRNA, we performed RNA stability assay after actinomycin D (ActD) treatment to block transcription in A549 and LC2/ad cells. QRT-PCR revealed that the levels of JUNB mRNA were decreased in the METTL3 knockdown cells compared to the control cells after ActD treatment, indicating the reduced stability of JUNB mRNA caused by METTL3 knockdown in both cells (Fig. 3D). However, METTL3 knockdown had no effect on the stability of SOX4 mRNA in A549 and LC2/ad cells, indicating the target specificity of METTL3 (Supplementary Fig. S3B). JUNB transcript was also significantly destabilized by METTL3 knockdown in the presence of TGF-b in both cells (Supplementary Fig. S3C). These results suggested that repressed expression of JUNB by METTL3 knockdown is explained by the decreased stability of JUNB mRNA due to its reduced m6A modification.
Fig. 3. Knockdown of METTL3 decreased the m6A modification, the expression and the stability of JUNB mRNA. (A) The m6A methylated JUNB mRNA level in the METTL3 knockdown cells. Me-RIP assay was performed in the control or METTL3 knockdown A549 and LC2/ad cells with or without TGF-b for 24 h (n ¼ 3)(**, P < 0.01; *, P < 0.05; ns, not significant). (B) The total JUNB mRNA level in the METTL3 knockdown cells. QRT-PCR for JUNB was performed in the cells similarly treated in (A) (n ¼ 3). (C) The JUNB protein level in the METTL3 knockdown cells. Immunoblotting was performed to detect JUNB protein in the cells shown in (A). As a control, anti-GAPDH antibody was used to show that equal amounts of proteins were loaded. (D) The mRNA stability of JUNB in the METTL3 knockdown cells. QRT-PCR was performed to detect JUNB mRNA in the control or METTL3 knockdown A549 and LC2/ad cells at the indicated period after actinomycin D (ActD) treatment (n ¼ 3)(**, P < 0.01; *, P < 0.05; compared to the control).
Please cite this article as: S. Wanna-udom et al., The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.042
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3.4. Over-expression of JUNB partially rescued TGF-b-dependent EMT phenotypes in the METTL3 knockdown cells
is one of the critical transcription factors regulated by METTL3 during TGF-b-induced EMT process.
To investigate whether JUNB is an important downstream target of METTL3 in TGF-b-induced EMT process, we examined the effects of JUNB over-expression in the METTL3 knockdown cells. A549 or LC2/ad cells were infected with control lentivirus or lentivirus expressing METTL3 shRNA and/or retrovirus expressing JUNB without or with treatment of TGF-b. The expression of METTL3 and JUNB in the cells was confirmed by QRT-PCR (Supplementary Fig. S4). Then we examined the expression of epithelial and mesenchymal marker genes to check the EMT phenotypes in A549 (Fig. 4A) and LC2/ad cells (Supplementary Fig. S5A). Overexpression of JUNB in the control cells showed little effects on gene expression except that FN1 expression was slightly upregulated as reported previously [22]. More importantly, in the METTL3 knockdown cells, JUNB over-expression partially recovered the TGF-b responses on gene expression such as repression of CDH1 and induction of FN1 and VIM (Fig. 4A and Supplementary Fig. S5A). As shown in Fig. 4B and Supplementary Fig. S5B, we confirmed that JUNB over-expression partially conferred TGF-b-dependent scattered, elongated or enlarged cell phenotype and E-cadherin downregulation to the METTL3 knockdown cells. The formation of actin stress fiber was also observed with JUNB over-expression and TGF-b in the METTL3 knockdown cells (Fig. 4B and Supplementary Fig. S5B). Furthermore, JUNB over-expression significantly increased the migratory activity of METTL3 knockdown A549 cells in the presence of TGF-b (Fig. 4C). These results indicated that JUNB over-expression partially rescued TGF-b-dependent EMT phenotypes in the METTL3 knockdown cells. Thus we conclude that JUNB
4. Discussion In this study, we found that the total m6A methylated RNA level and METTL3 expression were significantly elevated during TGF-binduced EMT of A549 and LC2/ad lung cancer cells. Knockdown of METTL3 itself strengthened the epithelial phenotypes of the cells, and antagonized TGF-b-induced EMT phenotypes, judged from the cell morphologies, cell migratory activities and the expression changes of epithelial and mesenchymal marker genes. Since m6A level of JUNB mRNA was dramatically increased by TGF-b and reduced by METTL3 knockdown, we focused on JUNB as the most important EMT-related TF regulated by METTL3 in lung cancer cells. The RNA stability assay revealed that the stability of JUNB mRNA was correlated with its m6A methylation level and enhanced by METTL3. Finally, we showed that JUNB over-expression partially recovered the inhibitory effects of METTL3 knockdown on TGF-binduced EMT phenotypes. Our results highlighted the critical roles of METTL3 RNA methyltransferase on the regulation of JUNB in TGFb-induced EMT of lung cancer cells. Although increasing evidences suggested that m6A modification participates in many pathological processes including cancer [8,9], its precise roles in controlling cancer progression have just started to be studied. Several papers have recently reported the involvement of METTL3 and its m6A methylated targets in various cancers. METTL3 was shown to regulate SOX2 mRNA in glioma stem cell maintenance and colorectal tumor progression [23,24], LEF1 in osteosarcoma progression [25], AFF4/NF-kB/MYC in bladder cancer
Fig. 4. Over-expression of JUNB partially rescued the TGF-b-dependent EMT phenotypes in the METTL3 knockdown cells. (A) The effects of JUNB over-expression in EMT-related marker gene expression. QRT-PCR was performed to detect the expression of CDH1/E-cadherin, FN1/Fibronectin and VIM/Vimentin in A549 cells infected with control lentivirus or lentivirus expressing METTL3 shRNA and/or retrovirus expressing JUNB with or without TGF-b treatment (n ¼ 3)(**, P < 0.01; *, P < 0.05; ns, not significant). (B, C) JUNB overexpression partially recovered TGF-b-induced cell morphological changes and cell migratory activities inhibited by METTL3 knockdown. A549 cells were infected with lentiviruses expressing METTL3 shRNA and/or retrovirus expressing JUNB with or without TGF-b. The cells were stained (B) with crystal violet (upper), with anti-E-cadherin antibody and DAPI (middle) or with TRITC-phalloidin and DAPI (lower). Scale bars: 10 mm. The cells similarly treated in (B) that migrated through the filter were fixed, stained and counted (C) (n ¼ 12)(**, P < 0.01; ns, not significant). . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: S. Wanna-udom et al., The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.042
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[26], and SNAI1 in EMT process of HeLa and HepG2 cells [27]. In this study, we found that METTL3 contributed to the progression of TGF-b-mediated EMT of A549 and LC2/ad lung cancer cells through the regulation of m6A level of JUNB for the first time. However, further in vivo experiments may be needed to provide direct evidence of METTL3 and JUNB function in lung cancer progression. The m6A methylation is observed on thousands of mRNAs and dynamically regulated in mammalian cells [1]. Our results indicated that the m6A levels of mRNAs for several EMT-related TFs including JUNB, ETS2, SNAI1, SNAI2, ZEB1 and ZEB2 were significantly increased by TGF-b treatment and decreased by METTL3 knockdown. Since JUNB mRNA showed the most remarkable changes of m6A level, we analyzed JUNB as a good candidate and proved JUNB as one of the critical TFs regulated by METTL3 in EMT. However, JUNB expression did not fully recovered TGF-b-dependent EMT phenotypes inhibited by METTL3 knockdown. This result suggested that other TFs such as ETS2, SNAIL and ZEB family and/or other cellular factors regulated by METTL3 might contribute to TGFb-induced EMT of lung cancer cells. Further studies will be necessary to clarify the precise mechanism for METTL3 function. JUNB was previously reported as one of the most important TFs in TGF-b-induced EMT of lung and breast cancer cells [21,22]. Knockdown of JUNB was shown to inhibit TGF-b-dependent cell morphology conversion, enhanced cell migration and the expression changes of epithelial and mesenchymal marker genes [21,22]. Especially, JUNB regulates the TGF-b-mediated induction of profibrotic factors such as FN1/Fibronectin ad TPM1/Tropomyosin, which play important roles in cell-matrix adhesion and actin stress fibers [22]. This is consistent with our observations that JUNB overexpression recovered the induction of FN1/Fibronectin and actin stress fiber formation preferentially among the TGF-b-dependent EMT phenotypes. In summary, we demonstrated the connection between the m6A RNA methylation by METTL3 and TGF-b-induced EMT of lung cancer cells. We discovered that JUNB is a critical TF regulated by METTL3 during EMT process. Our findings validated the significance of the regulation for m6A RNA modification in the important step of cancer progression and provided the possibility to develop novel therapeutic strategies for the treatment of cancer metastasis. Declaration of competing interest None. Acknowledgements We thank Mr. Hyuga Ajichi for his technical help and discussion. This work was supported in part by Grants-in-Aid for Scientific Research (grant numbers 19K07346 to T.S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the research grant from The Mitani Foundation for Research and Development, Japan. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.042. Transparency document
References [1] H. Shi, J. Wei, C. He, Where, when, and how: context-dependent functions of RNA methylation writers, readers, and erasers, Mol. Cell 74 (2019) 640e650. [2] J. Liu, Y. Yue, D. Han, X. Wang, Y. Fu, L. Zhang, G. Jia, M. Yu, Z. Lu, X. Deng, Q. Dai, W. Chen, C. He, A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation, Nat. Chem. Biol. 10 (2014) 93e95. [3] X. Wang, Z. Lu, A. Gomez, G.C. Hon, Y. Yue, D. Han, Y. Fu, M. Parisien, Q. Dai, G. Jia, B. Ren, T. Pan, C. He, N6-methyladenosine-dependent regulation of messenger RNA stability, Nature 505 (2014) 117e120. [4] X. Wang, B.S. Zhao, I.A. 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 (2015) 1388e1399. [5] S. Zaccara, R.J. Ries, S.R. Jaffrey, Reading, writing and erasing mRNA methylation, Nat. Rev. Mol. Cell Biol. 20 (2019) 608e624. [6] P.J. Hsu, H. Shi, C. He, Epitranscriptomic influences on development and disease, Genome Biol. 18 (2017) 197. [7] B.S. Zhao, I.A. Roundtree, C. He, Post-transcriptional gene regulation by mRNA modifications, Nat. Rev. Mol. Cell Biol. 18 (2017) 31e42. [8] J. Liu, B.T. Harada, C. He, Regulation of gene expression by N(6)methyladenosine in cancer, Trends Cell Biol. 29 (2019) 487e499. [9] Q. Lan, P.Y. Liu, J. Haase, J.L. Bell, S. Huttelmaier, T. Liu, The critical role of RNA m(6)A methylation in cancer, Cancer Res. 79 (2019) 1285e1292. [10] X. Ye, R.A. Weinberg, Epithelial-mesenchymal plasticity: a central regulator of cancer progression, Trends Cell Biol. 25 (2015) 675e686. [11] R. Derynck, B.P. Muthusamy, K.Y. Saeteurn, Signaling pathway cooperation in TGF-beta-induced epithelial-mesenchymal transition, Curr. Opin. Cell Biol. 31 (2014) 56e66. [12] W.L. Tam, R.A. Weinberg, The epigenetics of epithelial-mesenchymal plasticity in cancer, Nat. Med. 19 (2013) 1438e1449. [13] L. Sun, J. Fang, Epigenetic regulation of epithelial-mesenchymal transition, Cell. Mol. Life Sci. 73 (2016) 4493e4515. [14] T. Suzuki, M. Terashima, S. Tange, A. Ishimura, Roles of histone methylmodifying enzymes in development and progression of cancer, Cancer Sci. 104 (2013) 795e800. [15] S. Tange, D. Oktyabri, M. Terashima, A. Ishimura, T. Suzuki, JARID2 is involved in transforming growth factor-beta-induced epithelial-mesenchymal transition of lung and colon cancer cell lines, PLoS One 9 (2014), e115684. [16] M. Terashima, A. Ishimura, S. Wanna-Udom, T. Suzuki, Epigenetic regulation of epithelial-mesenchymal transition by KDM6A histone demethylase in lung cancer cells, Biochem. Biophys. Res. Commun. 490 (2017) 1407e1413. [17] M. Terashima, S. Tange, A. Ishimura, T. Suzuki, MEG3 long noncoding RNA contributes to the epigenetic regulation of epithelial-mesenchymal transition in lung cancer cell lines, J. Biol. Chem. 292 (2017) 82e99. [18] M. Terashima, A. Ishimura, S. Wanna-Udom, T. Suzuki, MEG8 long noncoding RNA contributes to epigenetic progression of the epithelial-mesenchymal transition of lung and pancreatic cancer cells, J. Biol. Chem. 293 (2018) 18016e18030. [19] M. Yoshida, A. Ishimura, M. Terashima, Z. Enkhbaatar, N. Nozaki, K. Satou, T. Suzuki, PLU1 histone demethylase decreases the expression of KAT5 and enhances the invasive activity of the cells, Biochem. J. 437 (2011) 555e564. [20] Z. Enkhbaatar, M. Terashima, D. Oktyabri, S. Tange, A. Ishimura, S. Yano, T. Suzuki, KDM5B histone demethylase controls epithelial-mesenchymal transition of cancer cells by regulating the expression of the microRNA-200 family, Cell Cycle 12 (2013) 2100e2112. [21] H. Chang, Y. Liu, M. Xue, H. Liu, S. Du, L. Zhang, P. Wang, Synergistic action of master transcription factors controls epithelial-to-mesenchymal transition, Nucleic Acids Res. 44 (2016) 2514e2527. [22] M. Gervasi, A. Bianchi-Smiraglia, M. Cummings, Q. Zheng, D. Wang, S. Liu, A.V. Bakin, JunB contributes to Id2 repression and the epithelial-mesenchymal transition in response to transforming growth factor-beta, J. Cell Biol. 196 (2012) 589e603. [23] A. Visvanathan, V. Patil, A. Arora, A.S. Hegde, A. Arivazhagan, V. Santosh, K. Somasundaram, Essential role of METTL3-mediated m(6)A modification in glioma stem-like cells maintenance and radioresistance, Oncogene 37 (2018) 522e533. [24] T. Li, P.S. Hu, Z. Zuo, J.F. Lin, X. Li, Q.N. Wu, Z.H. Chen, Z.L. Zeng, F. Wang, J. Zheng, D. Chen, B. Li, T.B. Kang, D. Xie, D. Lin, H.Q. Ju, R.H. Xu, METTL3 facilitates tumor progression via an m(6)A-IGF2BP2-dependent mechanism in colorectal carcinoma, Mol. Cancer 18 (2019) 112. [25] W. Miao, J. Chen, L. Jia, J. Ma, D. Song, The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1, Biochem. Biophys. Res. Commun. 516 (2019) 719e725. [26] M. Cheng, L. Sheng, Q. Gao, Q. Xiong, H. Zhang, M. Wu, Y. Liang, F. Zhu, Y. Zhang, X. Zhang, Q. Yuan, Y. Li, The m(6)A methyltransferase METTL3 promotes bladder cancer progression via AFF4/NF-kappaB/MYC signaling network, Oncogene 38 (2019) 3667e3680. [27] X. Lin, G. Chai, Y. Wu, J. Li, F. Chen, J. Liu, G. Luo, J. Tauler, J. Du, S. Lin, C. He, H. Wang, RNA m(6)A methylation regulates the epithelial mesenchymal transition of cancer cells and translation of Snail, Nat. Commun. 10 (2019) 2065.
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Please cite this article as: S. Wanna-udom et al., The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.042
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The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB Sasithorn Wanna-udom a, 1, Minoru Terashima a, 1, Hanbing Lyu a, Akihiko Ishimura a, Takahisa Takino b, Matomo Sakari c, Toshifumi Tsukahara c, Takeshi Suzuki a, * a
Division of Functional Genomics, Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Ishikawa, Japan Division of Education for Global Standard, Institute of Liberal Arts and Science, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Ishikawa, Japan c Area of Bioscience and Biotechnology, Japan Advanced Institute of Science and Technology, Nomi, 923-1292, Ishikawa, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 December 2019 Received in revised form 6 January 2020 Accepted 7 January 2020 Available online xxx
N6-Methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the role of m6A methyltransferase METTL3 in TGF-b-induced epithelial-mesenchymal transition (EMT) of lung cancer cell lines. The expression of METTL3 and m6A RNA modification were increased during TGF-b-induced EMT of A549 and LC2/ad lung cancer cells. Knockdown of METTL3 inhibited TGF-b-induced morphological conversion of the cells, enhanced cell migration potential and the expression changes of EMT-related marker genes such as CDH1/E-cadherin, FN1/Fibronectin and VIM/Vimentin. Mechanistic investigations revealed that METTL3 knockdown decreased the m6A modification, total mRNA level and mRNA stability of JUNB, one of the important transcriptional regulators of EMT. Over-expression of JUNB partially rescued the inhibitory effects of METTL3 knockdown in the EMT phenotypes. This study demonstrates that m6A methyltransferase METTL3 is indispensable for TGF-b-induced EMT of lung cancer cells through the regulation of JUNB. © 2020 Elsevier Inc. All rights reserved.
Keywords: Lung cancer Epithelial-mesenchymal transition RNA methylation METTL3 JUNB
1. Introduction N6-methyladenosine (m6A) is the most abundant internal chemical modification of mRNAs and long noncoding RNAs (lncRNAs) in eukaryotes [1]. Methyltransferase-like 3 (METTL3) is the catalytic subunit and forms the m6A methyltransferase complex with METTL14, WTAP and RBM15 [2]. On the other hand, m6A methylation is reversed by the demethylases, FTO and ALKBH5,
Abbreviations: EMT, epithelial-mesenchymal transition; TGF-b, Transforming Growth Factor-beta; m6A, N6-methyladenosine; TF, transcription factor; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; shRNA, small hairpin RNA; Me-RIP, methylated RNA immunoprecipitation; QRT-PCR, quantitative reverse transcribed polymerase chain reaction. * Corresponding author. Division of Functional Genomics, Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Ishikawa, Japan. E-mail address: [email protected] (T. Suzuki). 1 These authors contributed equally to this study.
which maintains a dynamic nature of this modification [1]. In mammalian cells, thousands of mRNAs are subject to m6A methylation, which has been implicated in pre-mRNA splicing, mRNA stability and translation efficiency [3e5]. It has been reported that m6A regulation plays important roles in many biological processes including development, metabolism, stemness maintenance and differentiation [6,7]. Recent studies have also revealed that METTL3 is involved in the development and progression of various types of cancer through the methylation of its target mRNAs [8,9]. Epithelial-mesenchymal transition (EMT) process plays pivotal roles in cancer metastasis [10]. EMT results in loss of cell-cell adhesion and cytoskeletal reorganization, thereby increasing motility, invasiveness and stem cell-like properties of cancer cells. Transforming Growth Factor-beta (TGF-b) has emerged as a major regulator of EMT along with cytokines and growth factors secreted by the tumor microenvironment [11]. One of the well-known hallmarks of EMT is the reversible changes in epithelial and mesenchymal gene expression. EMT-inducing transcription factors
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Please cite this article as: S. Wanna-udom et al., The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.042
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(TFs) such as ZEB1, ZEB2, SNAI1, SNAI2 and TWIST can activate EMT program through the downregulation of E-cadherin, an epithelial cell marker. The phenotypic plasticity of EMT suggests that this process is controlled by epigenetic regulation including DNA methylation and histone modifications [12,13]. We have previously demonstrated that enzymatic regulation of histone H3 methylation is essential for TGF-b-induced EMT of lung cancer cells [14e16]. We have also discovered that some lncRNAs function as an “initiator” to recruit histone methyltransferase complex to the specific target genes during EMT process [17,18]. However, the functional role of m6A RNA methylation, another type of epigenetic regulation, has not been fully explored in the progression of EMT. Here we showed that the expression of METTL3 and m6A RNA modification were increased during TGF-b-induced EMT of A549 and LC2/ad lung cancer cell lines. METTL3 knockdown antagonized TGF-b-dependent EMT phenotypes and reduced the mRNA stability of JUNB, one of the critical transcriptional regulators of EMT. We also found that JUNB over-expression partially recovered the EMT phenotypes inhibited by METTL3 knockdown. This study demonstrates the function of METTL3 in facilitating TGF-b-induced EMT and identifies JUNB as the downstream target of METTL3.
immuno-fluorescence of E-cadherin and actin staining were performed as described previously [20]. Cell migration activities were measured in modified Boyden chambers consisting of Transwell membrane filter inserts (#3422, Corning) as described previously [17]. Cells on the lower surface of the filter were stained with 0.4% crystal violet and counted from at least four fields and three experiments. 2.4. m6A content analysis and m6A-RNA immunoprecipitation (Me-RIP) assay
2. Materials and methods
The content of m6A in total RNA was analyzed with the m6A RNA Methylation assay quantification kit according to the manufacturer’s protocol (ab185912, Abcam). Me-RIP was conducted according to previously described protocol [16] with a slight modification. Briefly, 100 mg of total RNA was incubated with antim6A antibody (ab151230, Abcam), in 500 ml of RIP buffer supplemented with protease inhibitors (#03969e21, Nakarai) and SUPERase-In (AM2694, Thermo Fisher). Then the immunocomplexes were recovered with Protein G-coupled Dynabeads (DB10003, Veritas). The precipitated RNAs were extracted with High Pure RNA Tissue Kit (#11828665001, Roche) and were quantified by QRT-PCR.
2.1. Plasmids, cell culture and virus infection
2.5. RNA stability assay
The small hairpin RNA (shRNA)-expressing lentivirus vectors were constructed as described previously [19]. The sequences of the oligonucleotides are listed in Supplementary Table S1. We confirmed that METTL3 transcripts and proteins were downregulated with the infection of both METTL3 shRNA-expressing lentiviruses (METTL3 sh#1 and sh#2) even in the presence of TGF-b by quantitative RT-PCR (QRT-PCR) and immunoblotting (Supplementary Fig. S1). We also confirmed that both METTL3 shRNAs caused the same effects in our EMT studies judged from cell morphologies and E-cadherin marker expression (Supplementary Fig. S1), and thus we presented the data of METTL3 shRNA#1 as a representative result. Human JUNB cDNA was tagged with FLAG6xHis-epitope, and then cloned into pDON-5 Neo plasmid (Takara) to produce retroviruses expressing JUNB. Human lung cancer cell lines, A549 and LC-2/ad, were purchased from ATCC and RIKEN BRC and cultured in DMEM and the mixture of RPMI1640 and HAMS F12 medium with 10% FBS, respectively. For EMT induction, A549 and LC-2/ad cells were treated with 1 ng/ml of TGF-b (R&D Systems) for 24e72 h. The protocols for the production and infection of cDNA or shRNA-expressing retroviruses were essentially the same as described previously [20].
To measure RNA stability, the cells were treated with actinomycin D (A9415, Sigma) at 5 mg/ml to block transcription. After incubation at the indicated periods, cells were collected, and RNA was isolated for QRT-PCR. GAPDH expression was used for normalization.
2.2. Quantitative PCR and immunoblotting RNA preparation and QRT-PCR analysis were performed as described previously [20]. QRT-PCR data were normalized with control human GAPDH expression. The averages from at least three independent experiments are shown with the standard deviations. P-values were calculated between control and the samples using Student’s t-test. Primers used for the QPCR were described previously [19,20] and listed in Supplementary Table S1. Immunoblotting was performed as described previously [17]. We used antiMETTL3 (A301-567A, Bethyl), anti-JUNB (#3753, CST) and antiGAPDH (6C5, Millipore) antibodies. 2.3. Cell staining, immuno-fluorescence and cell migration assay A549 or LC-2/ad cells were stained with 0.4% crystal violet to observe the changes of cell morphologies during EMT. Indirect
3. Results 3.1. m6A RNA modification and METTL3 expression were increased during TGF-b-induced EMT of lung cancer cells To investigate the potential role of m6A RNA modification in TGF-b-induced EMT process, we first examined the total m6A level of RNA in two lung cancer cell lines, A549 and LC-2/ad, with or without TGF-b treatment (Fig. 1A). The m6A methylated RNA level was significantly increased by TGF-b treatment in both cells, indicating that lung cancer cells undergoing EMT increased m6A RNA modification (Fig. 1A). Then we examined the expression of m6A methyltransferase, METTL3, during TGF-b-induced EMT of A549 and LC-2/ad cells (Fig. 1B). QRT-PCR analysis revealed that METTL3 expression was significantly upregulated in response to TGF-b in both cells (Fig. 1B). These results suggested that m6A RNA modification and its methyltransferase METTL3 might be involved in the EMT process of lung cancer cells. 3.2. Knockdown of METTL3 antagonized TGF-b-induced EMT phenotypes To explore the function of METTL3 in EMT, we knocked down the expression of METTL3 with lentivirus-mediated shRNA in A549 and LC2/ad cells. Then we examined the cell morphologies and the state of E-cadherin and actin with or without TGF-b treatment (Fig. 2A and B). Control cells revealed scattered, elongated or enlarged cell phenotypes characteristic of EMT in the presence of TGF-b. METTL3 knockdown seemed to enhance cell adhesion slightly, and obviously inhibited cell morphological changes mediated by TGF-b (Fig. 2A and B, upper panels). For the expression of an epithelial cell marker, E-cadherin, immuno-fluorescence staining showed that control cells almost lost E-cadherin signals
Please cite this article as: S. Wanna-udom et al., The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.042
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Fig. 1. The m6A RNA modification and METTL3 expression were increased during TGF-b-induced EMT of lung cancer cells. (A) The m6A methylated RNA level was elevated in A549 and LC2/ad cells after TGF-b treatment. m6A content was analyzed by ELISA kit in the cells with or without the treatment of 1 ng/ml of TGF-b for 48 h (n ¼ 3)(**, P < 0.01 comparing to control). (B) The expression of METTL3 gene was increased during TGF-b-induced EMT. QRT-PCR was performed in A549 and LC2/ad cells before and after TGF-b treatment (6 h, 12 h, 24 h, 48 h and 72 h)(n ¼ 3)(**, P < 0.01 comparing to control; *, P < 0.05 comparing to control).
Fig. 2. Knockdown of METTL3 antagonized TGF-b-induced EMT phenotypes. (A, B) METTL3 knockdown inhibited TGF-b-induced morphological changes of A549 (A) and LC2/ad (B) cells. The cells were infected with lentiviruses expressing control shRNA or METTL3 shRNA (sh#1) with or without TGF-b treatment for 48 h. The cells were stained with crystal violet (upper), with anti-E-cadherin antibody and DAPI (middle) or with TRITCphalloidin and DAPI (lower). Scale bars: 10 mm. (C, D) Knockdown of METTL3 affected the changes in expression of EMT-related marker genes induced by TGF-b. QRT-PCR was performed to detect the expression of CDH1/E-cadherin, FN1/Fibronectin and VIM/Vimentin in the control or METTL3 knockdown A549 (C) and LC2/ad (D) cells with or without TGF-b for 24 h (n ¼ 3)(**, P < 0.01; *, P < 0.05; ns, not significant). (E) METTL3 knockdown inhibited TGF-b-dependent increase of migrated cells through the filter. A549 cells that migrated through the filter within 24 h were fixed, stained and counted (n ¼ 12)(**, P < 0.01; ns, not significant). . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
after TGF-b treatment. METTL3 knockdown increased E-cadherin staining on the cell membrane compared to the control cells, which was still detected in the presence of TGF-b (Fig. 2A and B, middle panels). TGF-b caused actin stress fiber formation in control cells, but in the METTL3 knockdown cells we never detected actin stress fiber even after TGF-b treatment (Fig. 2A and B, lower panels). These results indicated that METTL3 knockdown antagonized TGF-
b-induced EMT phenotypes of A549 and LC-2/ad lung cancer cells such as morphological changes and cytoskeletal rearrangements. Since the changes in the expression of epithelial and mesenchymal genes are an characteristic feature of EMT, we examined the expression level of an epithelial cell marker, CDH1/E-cadherin, and mesenchymal cell markers, FN1/Fibronectin and VIM/Vimentin, in A549 (Fig. 2C) and LC-2/ad cells (Fig. 2D) with METTL3 knockdown.
Please cite this article as: S. Wanna-udom et al., The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.042
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QRT-PCR revealed that METTL3 knockdown significantly increased CDH1 expression and decreased the expression of FN1 and VIM. Moreover, it inhibited the expression changes of these EMT-related marker genes stimulated by TGF-b treatment (Fig. 2C and D). These results suggested the involvement of endogenous METTL3 in the transcriptional regulation of TGF-b-induced EMT program. To observe the effects of METTL3 knockdown in EMT-associated phenotypes, we measured cell motility by the transfilter assays. As shown in Fig. 2E, TGF-b treatment significantly increased the number of migrated A549 cells. METTL3 knockdown caused the reduction of migrated cells by itself, and also cancelled TGF-bdependent increase of migrated cells (Fig. 2E). This result indicated that METTL3 knockdown inhibited the enhanced cell migration potential of lung cancer cells associated with TGF-b-dependent EMT. 3.3. METTL3 regulated the expression of JUNB, one of the important transcription factors involved in EMT To further clarify the function of METTL3 in EMT process, we have performed a candidate gene approach to find potential targets regulated by METTL3. Since METTL3 was suggested to affect the transcriptional regulation of EMT (Fig. 2C and D), we focused on the EMT-related transcription factors (TFs) which were upregulated during TGF-b-induced EMT. Based on the previous studies [21], we selected 12 candidate TFs and examined the m6A methylation levels of their mRNAs by Me-RIP assay (Supplementary Fig. S2). The m6A levels of JUNB, ETS2, SNAI1, SNAI2, ZEB1 and ZEB2 transcripts were significantly increased by TGF-b treatment and decreased by METTL3 knockdown (Supplementary Fig. S2). Among them, we decided to examine JUNB as a candidate target TF regulated by METTL3 because of its remarkable changes of m6A level.
As shown in Fig. 3A, we confirmed that the m6A levels of JUNB mRNA were increased by TGF-b and decreased by METTL3 knockdown in A549 and LC2/ad lung cancer cells. METTL3 knockdown also cancelled the effect of TGF-b in the increase of m6A level. It has been reported that JUNB is one of the important TFs involved in TGF-b-induced EMT and its expression is upregulated by TGF-b [21,22]. Our results also indicated that the expression of JUNB mRNA and protein was increased after TGF-b treatment in A549 and LC2/ad cells (Fig. 3B and C). More importantly, METTL3 knockdown reduced the mRNA and protein level of JUNB in both cells and counteracted the effect of TGF-b (Fig. 3B and C). In the case of SOX4 mRNA, whose m6A levels were not significantly changed by TGF-b and METTL3 knockdown (Supplementary Fig. S2), its basal level expression and induction by TGF-b were not affected by METTL3 knockdown (Supplementary Fig. S3A). These results suggested that METTL3-mediated m6A RNA modification influenced the total expression level of its target JUNB mRNA. To examine the relationship between the m6A modification and stability of JUNB mRNA, we performed RNA stability assay after actinomycin D (ActD) treatment to block transcription in A549 and LC2/ad cells. QRT-PCR revealed that the levels of JUNB mRNA were decreased in the METTL3 knockdown cells compared to the control cells after ActD treatment, indicating the reduced stability of JUNB mRNA caused by METTL3 knockdown in both cells (Fig. 3D). However, METTL3 knockdown had no effect on the stability of SOX4 mRNA in A549 and LC2/ad cells, indicating the target specificity of METTL3 (Supplementary Fig. S3B). JUNB transcript was also significantly destabilized by METTL3 knockdown in the presence of TGF-b in both cells (Supplementary Fig. S3C). These results suggested that repressed expression of JUNB by METTL3 knockdown is explained by the decreased stability of JUNB mRNA due to its reduced m6A modification.
Fig. 3. Knockdown of METTL3 decreased the m6A modification, the expression and the stability of JUNB mRNA. (A) The m6A methylated JUNB mRNA level in the METTL3 knockdown cells. Me-RIP assay was performed in the control or METTL3 knockdown A549 and LC2/ad cells with or without TGF-b for 24 h (n ¼ 3)(**, P < 0.01; *, P < 0.05; ns, not significant). (B) The total JUNB mRNA level in the METTL3 knockdown cells. QRT-PCR for JUNB was performed in the cells similarly treated in (A) (n ¼ 3). (C) The JUNB protein level in the METTL3 knockdown cells. Immunoblotting was performed to detect JUNB protein in the cells shown in (A). As a control, anti-GAPDH antibody was used to show that equal amounts of proteins were loaded. (D) The mRNA stability of JUNB in the METTL3 knockdown cells. QRT-PCR was performed to detect JUNB mRNA in the control or METTL3 knockdown A549 and LC2/ad cells at the indicated period after actinomycin D (ActD) treatment (n ¼ 3)(**, P < 0.01; *, P < 0.05; compared to the control).
Please cite this article as: S. Wanna-udom et al., The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.042
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3.4. Over-expression of JUNB partially rescued TGF-b-dependent EMT phenotypes in the METTL3 knockdown cells
is one of the critical transcription factors regulated by METTL3 during TGF-b-induced EMT process.
To investigate whether JUNB is an important downstream target of METTL3 in TGF-b-induced EMT process, we examined the effects of JUNB over-expression in the METTL3 knockdown cells. A549 or LC2/ad cells were infected with control lentivirus or lentivirus expressing METTL3 shRNA and/or retrovirus expressing JUNB without or with treatment of TGF-b. The expression of METTL3 and JUNB in the cells was confirmed by QRT-PCR (Supplementary Fig. S4). Then we examined the expression of epithelial and mesenchymal marker genes to check the EMT phenotypes in A549 (Fig. 4A) and LC2/ad cells (Supplementary Fig. S5A). Overexpression of JUNB in the control cells showed little effects on gene expression except that FN1 expression was slightly upregulated as reported previously [22]. More importantly, in the METTL3 knockdown cells, JUNB over-expression partially recovered the TGF-b responses on gene expression such as repression of CDH1 and induction of FN1 and VIM (Fig. 4A and Supplementary Fig. S5A). As shown in Fig. 4B and Supplementary Fig. S5B, we confirmed that JUNB over-expression partially conferred TGF-b-dependent scattered, elongated or enlarged cell phenotype and E-cadherin downregulation to the METTL3 knockdown cells. The formation of actin stress fiber was also observed with JUNB over-expression and TGF-b in the METTL3 knockdown cells (Fig. 4B and Supplementary Fig. S5B). Furthermore, JUNB over-expression significantly increased the migratory activity of METTL3 knockdown A549 cells in the presence of TGF-b (Fig. 4C). These results indicated that JUNB over-expression partially rescued TGF-b-dependent EMT phenotypes in the METTL3 knockdown cells. Thus we conclude that JUNB
4. Discussion In this study, we found that the total m6A methylated RNA level and METTL3 expression were significantly elevated during TGF-binduced EMT of A549 and LC2/ad lung cancer cells. Knockdown of METTL3 itself strengthened the epithelial phenotypes of the cells, and antagonized TGF-b-induced EMT phenotypes, judged from the cell morphologies, cell migratory activities and the expression changes of epithelial and mesenchymal marker genes. Since m6A level of JUNB mRNA was dramatically increased by TGF-b and reduced by METTL3 knockdown, we focused on JUNB as the most important EMT-related TF regulated by METTL3 in lung cancer cells. The RNA stability assay revealed that the stability of JUNB mRNA was correlated with its m6A methylation level and enhanced by METTL3. Finally, we showed that JUNB over-expression partially recovered the inhibitory effects of METTL3 knockdown on TGF-binduced EMT phenotypes. Our results highlighted the critical roles of METTL3 RNA methyltransferase on the regulation of JUNB in TGFb-induced EMT of lung cancer cells. Although increasing evidences suggested that m6A modification participates in many pathological processes including cancer [8,9], its precise roles in controlling cancer progression have just started to be studied. Several papers have recently reported the involvement of METTL3 and its m6A methylated targets in various cancers. METTL3 was shown to regulate SOX2 mRNA in glioma stem cell maintenance and colorectal tumor progression [23,24], LEF1 in osteosarcoma progression [25], AFF4/NF-kB/MYC in bladder cancer
Fig. 4. Over-expression of JUNB partially rescued the TGF-b-dependent EMT phenotypes in the METTL3 knockdown cells. (A) The effects of JUNB over-expression in EMT-related marker gene expression. QRT-PCR was performed to detect the expression of CDH1/E-cadherin, FN1/Fibronectin and VIM/Vimentin in A549 cells infected with control lentivirus or lentivirus expressing METTL3 shRNA and/or retrovirus expressing JUNB with or without TGF-b treatment (n ¼ 3)(**, P < 0.01; *, P < 0.05; ns, not significant). (B, C) JUNB overexpression partially recovered TGF-b-induced cell morphological changes and cell migratory activities inhibited by METTL3 knockdown. A549 cells were infected with lentiviruses expressing METTL3 shRNA and/or retrovirus expressing JUNB with or without TGF-b. The cells were stained (B) with crystal violet (upper), with anti-E-cadherin antibody and DAPI (middle) or with TRITC-phalloidin and DAPI (lower). Scale bars: 10 mm. The cells similarly treated in (B) that migrated through the filter were fixed, stained and counted (C) (n ¼ 12)(**, P < 0.01; ns, not significant). . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: S. Wanna-udom et al., The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.042
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[26], and SNAI1 in EMT process of HeLa and HepG2 cells [27]. In this study, we found that METTL3 contributed to the progression of TGF-b-mediated EMT of A549 and LC2/ad lung cancer cells through the regulation of m6A level of JUNB for the first time. However, further in vivo experiments may be needed to provide direct evidence of METTL3 and JUNB function in lung cancer progression. The m6A methylation is observed on thousands of mRNAs and dynamically regulated in mammalian cells [1]. Our results indicated that the m6A levels of mRNAs for several EMT-related TFs including JUNB, ETS2, SNAI1, SNAI2, ZEB1 and ZEB2 were significantly increased by TGF-b treatment and decreased by METTL3 knockdown. Since JUNB mRNA showed the most remarkable changes of m6A level, we analyzed JUNB as a good candidate and proved JUNB as one of the critical TFs regulated by METTL3 in EMT. However, JUNB expression did not fully recovered TGF-b-dependent EMT phenotypes inhibited by METTL3 knockdown. This result suggested that other TFs such as ETS2, SNAIL and ZEB family and/or other cellular factors regulated by METTL3 might contribute to TGFb-induced EMT of lung cancer cells. Further studies will be necessary to clarify the precise mechanism for METTL3 function. JUNB was previously reported as one of the most important TFs in TGF-b-induced EMT of lung and breast cancer cells [21,22]. Knockdown of JUNB was shown to inhibit TGF-b-dependent cell morphology conversion, enhanced cell migration and the expression changes of epithelial and mesenchymal marker genes [21,22]. Especially, JUNB regulates the TGF-b-mediated induction of profibrotic factors such as FN1/Fibronectin ad TPM1/Tropomyosin, which play important roles in cell-matrix adhesion and actin stress fibers [22]. This is consistent with our observations that JUNB overexpression recovered the induction of FN1/Fibronectin and actin stress fiber formation preferentially among the TGF-b-dependent EMT phenotypes. In summary, we demonstrated the connection between the m6A RNA methylation by METTL3 and TGF-b-induced EMT of lung cancer cells. We discovered that JUNB is a critical TF regulated by METTL3 during EMT process. Our findings validated the significance of the regulation for m6A RNA modification in the important step of cancer progression and provided the possibility to develop novel therapeutic strategies for the treatment of cancer metastasis. Declaration of competing interest None. Acknowledgements We thank Mr. Hyuga Ajichi for his technical help and discussion. This work was supported in part by Grants-in-Aid for Scientific Research (grant numbers 19K07346 to T.S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the research grant from The Mitani Foundation for Research and Development, Japan. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.042. Transparency document
References [1] H. Shi, J. Wei, C. He, Where, when, and how: context-dependent functions of RNA methylation writers, readers, and erasers, Mol. Cell 74 (2019) 640e650. [2] J. Liu, Y. Yue, D. Han, X. Wang, Y. Fu, L. Zhang, G. Jia, M. Yu, Z. Lu, X. Deng, Q. Dai, W. Chen, C. He, A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation, Nat. Chem. Biol. 10 (2014) 93e95. [3] X. Wang, Z. Lu, A. Gomez, G.C. Hon, Y. Yue, D. Han, Y. Fu, M. Parisien, Q. Dai, G. Jia, B. Ren, T. Pan, C. He, N6-methyladenosine-dependent regulation of messenger RNA stability, Nature 505 (2014) 117e120. [4] X. Wang, B.S. Zhao, I.A. 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 (2015) 1388e1399. [5] S. Zaccara, R.J. Ries, S.R. Jaffrey, Reading, writing and erasing mRNA methylation, Nat. Rev. Mol. Cell Biol. 20 (2019) 608e624. [6] P.J. Hsu, H. Shi, C. He, Epitranscriptomic influences on development and disease, Genome Biol. 18 (2017) 197. [7] B.S. Zhao, I.A. Roundtree, C. He, Post-transcriptional gene regulation by mRNA modifications, Nat. Rev. Mol. Cell Biol. 18 (2017) 31e42. [8] J. Liu, B.T. Harada, C. He, Regulation of gene expression by N(6)methyladenosine in cancer, Trends Cell Biol. 29 (2019) 487e499. [9] Q. Lan, P.Y. Liu, J. Haase, J.L. Bell, S. Huttelmaier, T. Liu, The critical role of RNA m(6)A methylation in cancer, Cancer Res. 79 (2019) 1285e1292. [10] X. Ye, R.A. Weinberg, Epithelial-mesenchymal plasticity: a central regulator of cancer progression, Trends Cell Biol. 25 (2015) 675e686. [11] R. Derynck, B.P. Muthusamy, K.Y. Saeteurn, Signaling pathway cooperation in TGF-beta-induced epithelial-mesenchymal transition, Curr. Opin. Cell Biol. 31 (2014) 56e66. [12] W.L. Tam, R.A. Weinberg, The epigenetics of epithelial-mesenchymal plasticity in cancer, Nat. Med. 19 (2013) 1438e1449. [13] L. Sun, J. Fang, Epigenetic regulation of epithelial-mesenchymal transition, Cell. Mol. Life Sci. 73 (2016) 4493e4515. [14] T. Suzuki, M. Terashima, S. Tange, A. Ishimura, Roles of histone methylmodifying enzymes in development and progression of cancer, Cancer Sci. 104 (2013) 795e800. [15] S. Tange, D. Oktyabri, M. Terashima, A. Ishimura, T. Suzuki, JARID2 is involved in transforming growth factor-beta-induced epithelial-mesenchymal transition of lung and colon cancer cell lines, PLoS One 9 (2014), e115684. [16] M. Terashima, A. Ishimura, S. Wanna-Udom, T. Suzuki, Epigenetic regulation of epithelial-mesenchymal transition by KDM6A histone demethylase in lung cancer cells, Biochem. Biophys. Res. Commun. 490 (2017) 1407e1413. [17] M. Terashima, S. Tange, A. Ishimura, T. Suzuki, MEG3 long noncoding RNA contributes to the epigenetic regulation of epithelial-mesenchymal transition in lung cancer cell lines, J. Biol. Chem. 292 (2017) 82e99. [18] M. Terashima, A. Ishimura, S. Wanna-Udom, T. Suzuki, MEG8 long noncoding RNA contributes to epigenetic progression of the epithelial-mesenchymal transition of lung and pancreatic cancer cells, J. Biol. Chem. 293 (2018) 18016e18030. [19] M. Yoshida, A. Ishimura, M. Terashima, Z. Enkhbaatar, N. Nozaki, K. Satou, T. Suzuki, PLU1 histone demethylase decreases the expression of KAT5 and enhances the invasive activity of the cells, Biochem. J. 437 (2011) 555e564. [20] Z. Enkhbaatar, M. Terashima, D. Oktyabri, S. Tange, A. Ishimura, S. Yano, T. Suzuki, KDM5B histone demethylase controls epithelial-mesenchymal transition of cancer cells by regulating the expression of the microRNA-200 family, Cell Cycle 12 (2013) 2100e2112. [21] H. Chang, Y. Liu, M. Xue, H. Liu, S. Du, L. Zhang, P. Wang, Synergistic action of master transcription factors controls epithelial-to-mesenchymal transition, Nucleic Acids Res. 44 (2016) 2514e2527. [22] M. Gervasi, A. Bianchi-Smiraglia, M. Cummings, Q. Zheng, D. Wang, S. Liu, A.V. Bakin, JunB contributes to Id2 repression and the epithelial-mesenchymal transition in response to transforming growth factor-beta, J. Cell Biol. 196 (2012) 589e603. [23] A. Visvanathan, V. Patil, A. Arora, A.S. Hegde, A. Arivazhagan, V. Santosh, K. Somasundaram, Essential role of METTL3-mediated m(6)A modification in glioma stem-like cells maintenance and radioresistance, Oncogene 37 (2018) 522e533. [24] T. Li, P.S. Hu, Z. Zuo, J.F. Lin, X. Li, Q.N. Wu, Z.H. Chen, Z.L. Zeng, F. Wang, J. Zheng, D. Chen, B. Li, T.B. Kang, D. Xie, D. Lin, H.Q. Ju, R.H. Xu, METTL3 facilitates tumor progression via an m(6)A-IGF2BP2-dependent mechanism in colorectal carcinoma, Mol. Cancer 18 (2019) 112. [25] W. Miao, J. Chen, L. Jia, J. Ma, D. Song, The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1, Biochem. Biophys. Res. Commun. 516 (2019) 719e725. [26] M. Cheng, L. Sheng, Q. Gao, Q. Xiong, H. Zhang, M. Wu, Y. Liang, F. Zhu, Y. Zhang, X. Zhang, Q. Yuan, Y. Li, The m(6)A methyltransferase METTL3 promotes bladder cancer progression via AFF4/NF-kappaB/MYC signaling network, Oncogene 38 (2019) 3667e3680. [27] X. Lin, G. Chai, Y. Wu, J. Li, F. Chen, J. Liu, G. Luo, J. Tauler, J. Du, S. Lin, C. He, H. Wang, RNA m(6)A methylation regulates the epithelial mesenchymal transition of cancer cells and translation of Snail, Nat. Commun. 10 (2019) 2065.
Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.042
Please cite this article as: S. Wanna-udom et al., The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.042