FTO-Mediated N6-Methyladenosine Modification of KLF3 Promotes Osteosarcoma Progression

FTO-Mediated N6-Methyladenosine Modification of KLF3 Promotes Osteosarcoma Progression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article FTO-Mediated N6-Methyladenosine Modification of KLF3 Promotes Osteosarcoma Progression Hong-Jian Shan, Wen-Xiang Gu, Gang Duan, Zhi-Man Zhu, Hong-Liang Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-983314/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract N6-methyladenosine (m6A) methylation is the most common and abundant methylation modification of eukaryotic mRNAs, which is involved in tumor initiation and progression. This study aims to explore the functional role and regulatory mechanism of FTO in osteosarcoma progression. In this study, we detected the expressions of KLF3 in OS cells and tissues by qRT-PCR and western blot, and found that the mRNA and protein expressions of KLF3 were increased in OS cells and tissues and significantly related to tumor size, metastasis, and TNM stage and poor prognosis of OS patients. FTO promoted the proliferation and invasion and suppressed apoptosis of OS cells through CCK-8, Transwell and apoptosis assays. Further mechanism dissection revealed that FTO and YTHDF2 enforced the decay of KLF3 mRNAs and decreased its expression. Knockdown of FTO led to elevate m6A and mRNA levels. FTO-mediated m6A modification regulated KLF3 expression in the YTHDF2-dependent manner. Moreover, KLF3 overexpression abrogated FTO-induced oncogenic effects on the proliferation and invasion of OS cells. Overall, our findings showed that FTO-mediated m6A modification of KLF3 promoted OS progression, which may provide a therapeutic target for osteosarcoma. Drug Discovery, Design, & Development m6A methylation FTO osteosarcoma KLF3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Osteosarcoma (OS), as the most common primary malignant bone tumor, originates from mesenchymal cells, mostly in long bone, and can also be seen in distal femur or proximal tibia. Osteosarcoma is often accompanied by micrometastasis before operation and characterized by recurrent possibility and suboptimal prognosis [ 1 ]. 15%-20% of patients with OS are diagnosed with distant metastases at the first diagnosis [ 2 ]. Even after radiotherapy and chemotherapy, the survival rate of 5-year is still less than 20% for OS complicated with lung metastasis [ 3 ]. The combination of surgery and radiotherapy and chemotherapy has made some progress in improving the prognosis of patients with OS, but the curative effect is still not ideal [ 4 ]. Therefore, we urgently need to explore the mechanism of osteosarcoma progression, find key molecular targets and improve the prognosis of osteosarcoma. m6A methylation is a dynamic and reversible epigenetic modification of RNAs, which mainly affects the splicing, translation and stability of mRNAs and the epigenetic effects of non-coding RNAs [ 5 , 6 ]. The biological process of m6A is mainly regulated by three kinds of key-related kinases, m6A methyltransferase (Writers), m6A demethylas (Erasers) and methylation reading protein (Readers) [ 5 , 6 ]. Methyltransferase complexes composed of various writers, such as METTL3/14, WTAP and KIAA1429, catalyze the m6A modification of adenylate on mRNAs. Demethylase includes FTO and ALKHB5, and its function is to demethylate the bases modified by m6A. The main function of readers is to recognize the m6A-modified RNA, thus activating downstream regulatory pathways such as RNA processing, mRNA degradation, translation and decay [ 7 , 8 ]. m6A RNA methylation plays multiple roles in mammalian growth and development, including embryonic development, circadian rhythm and DNA damage response. More and more evidence shows that m6A methylation is closely related to tumorigenesis and development [ 6 , 9 , 10 ]. METTL3 promotes cell proliferation, migration and invasion of and activates Wnt/β-catenin pathway in OS through regulating m6A methylation and LEF1 [ 11 ]. Blockade of METTL3 inversely inhibits growth and invasion of OS cells by suppressing ATAD2 [ 12 ]. ALKBH5 augments lncRNA PVT1 expression in an m6A dependent manner and facilitate OS tumorigenesis [ 13 ]. These studies show that m6A methylation plays an important role in the occurrence and development of OS, but the specific mechanism and role of m6A reader FTO has not been reported. Krüppel-like factor 3 (KLF3), a member of the KLF family, was originally a KLFI homolog cloned from human hematopoietic tissue. KLF3 can recruit transcriptional corepressor C-terminal binding protein and complete fusion in the target gene promoter, regulating transcription of target genes [ 14 , 15 ]. KLF3 plays an important role in B cell development, erythrocyte and fat production, nervous system and muscle gene regulation, cell differentiation and proliferation [ 14 , 15 ]. Aberrant expression of KLF3 has been detected in several cancers, such as lung cancer [ 16 ], acute leukemia [ 17 ]s, colorectal cancer [ 18 ], sarcoma [ 19 ]. Silencing KLF3 contributes to lung cancer metastasis and the EMT process by regulating the STAT3 signaling pathway [ 16 ]. Decreased KLF3 is associated with aggressive phenotypes and poor prognosis for colorectal cancer patients [ 18 ]. KLF3 inhibits the pro-metastatic miR-182 expression and sarcoma metastasis [ 19 ]. All these support the potential tumor-suppressor gene of KLF3. However, the underlying mechanism by which KLF3 is affected, especially m6A methylation, is virtually unknown in OS. In the study, we initially investigated the expression of FTO and found that increased expression of FTO was related to clinicopathological characteristics and poor prognosis of OS patients. FTO elicited potent oncogenic functions in promoting the proliferation and migration of OS cells. Moreover, we determined the effect of FTO-mediated m6A modification on KLF3 expression. Overall, FTO-KLF3 may serve as a potential therapeutic target for osteosarcoma. Methods Detailed procedures are provided in Supplementary Materials and methods. Results FTO is increased expression in OS To evaluate the expression of FTO, we investigate the expression pattern of FTO in OS cells and the normal osteoblast cells (Nhost) by qPCR. And results showed that the mRNA levels of FTO in OS cells were elevated (Fig. 1a). We also detected the mRNA levels of FTO in OS and adjacent tissues. Similarly, mRNA levels of FTO were frequently increased in cancer tissues compared with matched normal tissues (Fig. 1b). IHC staining was also used to detect the FTO expression in OS and matched normal tissues (Fig. 1c). According to the quantitative analysis of IHC staining of tissue samples, we divided the FTO expression into high expression and low expression. 64.3% of OS tissues showed high expression, whereas the low expression rate of FTO in normal tissues was only 26.2% (Fig. 1d). We further analyzed the relationship between the FTO expression and clinicopathological parameters and postoperative survival of OS. Results from the IHC assay showed that there was a significant correlation between the high expression of FTO and several clinicopathological features (Table 1), such as tumor size, metastasis, and TNM stage, but not with the gender and age. In order to further understand the relationship between FTO and survival of OS, according to the FTO expression, we performed Kaplan-Meier survival curve analysis. The FTO expression was negatively correlated with the 5-year overall survival of patients with OS (Fig. 1e). FTO promotes OS cell proliferation, migration and invasion In order to further study the effect of FTO expression on the proliferation of OS cells, FTO knockdown or overexpression was used to detect the proliferation ability of OS cells at 24h, 48h, 72h, respectively. Transfection efficiencies of FTO knockdown or overexpression were analyzed by qPCR in OS cells (Fig. 2a). Results showed that overexpression of FTO in OS cells promoted cell proliferation (Fig. 2b), while knockdown of FTO inhibited cell proliferation (Fig. 2b). As shown in (Fig. 2c), Clone formation also supported the role of FTO in contributing to proliferation of OS cells, and FTO downregulation weakened the cell proliferation. In addition, enforced expression of FTO inhibited OS cell apoptosis, however, blockade of FTO expedited apoptosis (Fig. 2d). Scratch healing assay was used to verify the migration ability of FTO to OS cells. Amplification of FTO accelerated scratch healing of OS cells (Fig. 2e). Transwell chamber assay showed that FTO attenuates the invasion of OS cells; inversely, silencing FTO attenuated the invasive ability (Fig. 2f). These findings indicated FTO promoted proliferation, migration and invasion of OS cells. FTO inhibits KLF3 expression in OS As the first discovered demethylase FTO, its discovery reveals the dynamic and reversible process of m6A modification, and it can reverse the m6A modification on RNAs [ 22 ]. Previous results indicated that KLF3 is decreased expression in sarcomas [ 19 ]. It has been reported that the tumor suppressor KLF4, another member of the KLF family, could be regulated in an m6A dependent manner bladder cancer [ 23 ]. METTL3-mediated m6A methylation directly promotes the mRNA decay of KLF4 through m6A binding protein YTHDF2 [ 23 ]. Similarly, we identify whether the role of KLF3 in OS is regulated by m6A eraser FTO. Therefore, we subsequently analyzed the profile of m6A content in OS tissues. Results from m6A methylation quantification showed that m6A modification is significantly decreased in the total RNAs of OS tissues compared to adjacent normal tissues (Fig. 3a). m6A methylation quantification and dot blot assays were employed to detect the effect of FTO on m6A methylation in OS cells. As expected, m6A level was down-regulated in OS cells expressing FTO (Fig. 3b-c). Western blotting confirmed that FTO contributed to decrease KLF3 expression in OS cells, and silencing FTO conferred by siRNA inversely increased KLF3 (Fig. 3d), which were consistent with results from qRT-PCR (Fig. 3e). IHC staining also supported the decreased expression of KLF3 in OS tissues compared with normal tissues (Fig. 3f). Correlation analysis revealed that there was a significant negative correlation between FTO and KLF3 mRNA levels in OS tissues (Fig. 3g). Thus, we speculated that FTO can negatively regulate the expression of KLF3. FTO regulates KLF3 expression in an m6A-dependent manner The functional interaction between m6A methylase and demethylase determines the dynamic and reversible regulation of m6A modification. And m6A binding proteins can bind to mRNA containing m6A, thus affecting the fate of target mRNAs [ 24 ]. Me-RIP qPCR suggested that knockdown of FTO augmented the m6A enrichment on KLF3 mRNAs in OS cells (Fig. 3h). FTO can negatively regulate the content of KLF3 mRNAs (Fig. 3e). We carried out the stability assay of KLF3 mRNAs. The results suggested that, after treatment with Actinomycin D (Transcription inhibitor), the residual percentage of KLF3 mRNA in FTO group was significantly lower than that in control group (Fig. 3i). Previous studies have confirmed that m6A binding protein YTHDF2 affects the stability of m6A modified RNA by locating them at mRNA decay sites [ 24 ]. Transfection efficiencies of YTHDF2 knockdown or overexpression were analyzed by qPCR in OS cells (Fig. 3j). YTHDF2 attenuated the mRNA and protein levels of KLF3 (Fig. 3k-l). And YTHDF2 reduced the half-life of KLF3 mRNA (Fig. 3m). RIP-qPCR assay proved the interaction between YTHDF2 protein and KLF3 mRNA in OS cells, which was enhanced in OS cells expressing FTO (Fig. 3n). Silencing YTHDF2 abrogated FTO-mediated the decrease of KLF3 mRNA and protein levels in OS cells (Fig. 3o-p). qRT-PCR results also verified that YTHDF2 knockdown reversed FTO-induced mRNA degrading of KLF3 (Fig. 3q). Overall, Silencing FTO accelerated the YTHDF2-involved decay of KLF3 mRNA in OS cells. KLF3 impairs the FTO-induced proliferation and invasion of OS cells To ascertain whether KLF3 is involved in FTO function, OS cells transfected with FTO plasmids showed lower expression of KLF3, which could be reversed by overexpressing KLF3 (Fig. 4a). Moreover, FTO dramatically accelerated the proliferation of OS cells, whereas enforced expression of KLF3 reversed the FTO-induced promotion effects (Fig. 4b). Cloning formation efficiencies were significantly lower in OS cells expressing FTO and KLF3 than that of OS cells expressing FTO (Fig. 4c). FTO inhibited apoptosis, which was severely impeded in OS cells with ectopic expression of KLF3 (Fig. 4d). Analogously, KLF3 dramatically suppressed FTO-induced cell invasion (Fig. 4e). Cyclin D1 and p21 is the key protein of cell cycle regulation. FTO increased Cyclin D1 expression and decreased p21 expression in OS cells (Fig. 4f), and KLF3 could reverse the alternation. EMT also plays an important role in the process of tumor metastasis. FTO could enhance N-cadherin and Vimentin expression and suppress E-cadherin expression (Fig. 4f), which were compromised by KLF3 amplification. Thus, our findings indicated that FTO promoted cell proliferation and invasion by abating KLF3 in OS cells. Discussion m6A methylation, as the most important RNA internal modification in eukaryotes, was first discovered in 1974 and attracted great attention recently [ 6 , 7 ]. m6A methylation occurs in all stages of the RNA life cycle, from RNA synthesis to nucleation, translation regulation to RNA degradation [ 24 ]. m6A modification is closely related to the occurrence and development of cancer and participates in cell differentiation, proliferation, migration, immunity and drug resistance. Deregulation of m6A regulators in OS is inevitable [ 11 , 13 , 25 , 26 ]. Miao et al. reported that METTL3 contributes to proliferation, migration and invasion of OS cells and activates Wnt/β-catenin signaling pathway by regulating m6A modification of LEF1 mRNAs [ 11 ]. Analogously, METTL3 also promotes proliferation and invasion of OS cells by increasing ATAD2 expression via an m6A-dependent manner [ 12 ]. ALKBH5 attenuates the m6A modification of PVT1 mRNA and impedes the binding of YTHDF2 to PVT1 mRNA, which increases PVT1 expression and accelerates OS growth in vivo [ 13 ]. In addition, m6A-related enzymes are involved in regulating the pluripotency of osteosarcoma stem cells [ 26 , 27 ]. All these implies potential roles of m6A methylation in tumorigenesis and progression of OS. And the relationship between FTO and OS has not been reported. In the study, our results showed that the protein and mRNA levels of FTO were increased in OS tissues and cells. Through IHC staining of OS samples, we also found that FTO expression in OS tissues was significantly higher than that in paracancerous normal tissues, and increased expression of FTO was associated with poor prognosis and aggressive phenotypes in OS. Ectopic expression of FTO promoted the proliferation, migration and invasion of OS cells and inhibited the apoptosis, and knockdown of FTO shows the opposite effects. These findings supported that FTO promotes OS progression. KLFs are one of the key components of the mammalian Sp/KLFSp zinc finger protein family. KLF3, as a member of KLFs, can recruit transcriptional corepressor C-terminal-binding protein to promoters of downstream molecules and primarily effectuate transcriptional inhibition [ 15 , 17 ]. Similar to other Sp/KLFSp zinc finger protein members, KLF3 contains conserved binding sites of GC-rich motif or CACCC box, which causes the regulation diversity for target genes [ 28 ]. Therefore, KLF3 has multiple biological functions, including erythropoiesis, adipogenesis, muscle regulation, B-cell development, differentiation and apoptosis [ 17 , 29 ]. Aberrant expression of KLF3 is related to the occurrence and progress of various tumors, such as acute leukemia [ 17 ], sarcoma [ 19 ], lung cancer [ 16 ], colorectal cancer [ 18 ], melanoma cancer [ 30 ]. Loss of KLF3 potentiates lung cancer metastasis and EMT process through controlling STAT3 pathway [ 16 ]. DNA methylation-mediated KLF3 silencing augments the pro-metastatic miR-182 in human sarcoma cells [ 19 ]. Decreased KLF3 is associated with poor prognosis and may work as a promising predictor in colorectal cancer [ 18 ]. These suggest that KLF3 may be a potential tumor suppressor gene. It has been reported that m6A-mediated modification could regulate the methylation of other members of KLFs, such as KLF1 [ 31 ], KLF4 [ 32 ], and KLF5 [ 33 ]. RNA demethylation FTO promotes the phenotype conversion of human aortic vascular smooth muscle cells through increasing KLF5 expression in an m6A-dependent manner [ 33 ]. However, the potential mechanism of whether KLF3 is affected by m6A-mediated methylation modification have never been reported. Subsequently, we further explore whether FTO-mediated m6A modification regulates the expression of KLF3. We quantitatively detected the m6A modification of total mRNAs in OS and adjacent tissues and found that m6A content in OS samples was lower than that in adjacent samples, It is consistent with previous studies that total m6A level is decreased in OS [ 34 ]. After knocking down and overexpressing FTO, we detected the changes of KLF3 expression and found that FTO negatively regulated the mRNA and protein expressions of KLF3 in OS cells. IHC staining showed that KLF3 was down-regulated in OS tissues compared with normal tissues. And decreasing FTO can potentiate m6A modification of KLF3 mRNAs. As m6A “reader”, the C-terminal of YTHDF2 can specifically recognize m6A-modified mRNAs to reduce the half-life of target mRNAs and decrease the stability of mRNAs [ 35 ]. We also proved the interaction between YTHDF2 protein and KLF3 mRNA in OS cells. Given that FTO positively regulates the content of KLF3 mRNA in cells, we investigated the effect of FTO-mediated m6A on the stability of KLF3 mRNAs. Our results showed that knockdown of FTO decreases the stability of KLF3 mRNA in OS cells, and deletion YTHDF2 impedes the KLF3 mRNA decay induced by silencing FTO. There was also a negative correlation between FTO and KLF3 expression in OS tissues. We concluded that FTO regulates the level of KLF3 mRNAs in an m6A-dependent manner in OS. In vitro studies demonstrated that FTO promoted the proliferation and invasion of OS cells. However, KLF3 protected against FTO-induced promotion effects on cell proliferation and invasion. Cyclin D1 promotes cell proliferation by binding to and activating cyclin dependent kinase CDK4 unique to G1 phase. And p21 can form a complex with cyclin D/CDK and arrest the cell cycle in G1 phase. EMT concomitant with the down-regulation of epithelial markers and up-regulation of mesenchymal phenotype markers is an important process of invasion and distant metastasis of cancer. FTO up-regulated Cyclin D1 and N-cadherin level and down-regulated p21 and E-cadherin level in OS cells, and expressing KLF3 compromised the FTO-induced alternations. Collectively, FTO enhances the cell proliferation and invasion by silencing KLF3 expression in OS cells. This study focused on the effects of FTO on the proliferation and invasion of OS cells by regulating the m6A methylation of KLF3 mRNAs. Our results also supported the view that m6A modification is involved in the progression of OS. However, further researches are needed to provide direct epigenetic evidence of the FTO/KLF3 pathway in the pathogenesis of OS. Conclusions The expression of FTO is increased in OS and affects the prognosis survival of OS patients. FTO promotes the proliferation and invasion, and inhibits apoptosis of OS cells through regulating KLF3 expression via an m6A-dependent manner. And by revealing the previously unknown regulatory mechanisms of FTO and KLF3, it provides a new direction for the molecular mechanism of occurrence and development of OS. In addition, considering the importance of m6A modification in human cancers, it is possible to explore new strategies for the diagnosis and treatment of OS by targeting FTO with selective inhibitors. Declarations Ethics approval and consent to participate The study was approved by the Review Board of the Affiliated Hospital of Xuzhou Medical University. All participants provided informed consent as outlined in the manuscript. Consent for publication All authors approved the final version of this manuscript. Availability of data and materials All data and materials are available for verification as needed. Competing interests The authors confirm that there are no conflicts of interest. Funding This work was supported by the China’s 62 nd Postdoctoral Fund (2017M621826), Project of Jiangsu Postdoctoral Fund (2018K261C) in 2018, and the General Program of Natural Science Foundation of Jiangsu Province (BK20201154). Authors' contributions Conceived and designed the project: Hong-Jian Shan, Hong-Liang Chen and Zhi-Man Zhu; Performed the project: Hong-Jian Shan, Wen-Xiang Gu; Analyzed the data: Hong-Jian Shan, Gang Duan; Contributed reagents/materials/analysis tools: Hong-Jian Shan, Wen-Xiang Gu, Gang Duan, Zhi-Man Zhu, Hong-Liang Chen; Wrote the paper: Hong-Jian Shan. All authors read and approved the final manuscript. Acknowledgements This work was supported by the China’s 62 nd Postdoctoral Fund (2017M621826), Project of Jiangsu Postdoctoral Fund (2018K261C) in 2018, and the General Program of Natural Science Foundation of Jiangsu Province (BK20201154). Compliance with Ethical Standards Disclosure of potential conflicts of interest The authors confirm that there are no conflicts of interest. Research involving Human Participants and/or Animals The study involving human specimens was approved by the Review Board of the Affiliated Hospital of Xuzhou Medical University. This article does not contain any studies with animals performed. Informed consent All participants provided informed consent as outlined in the manuscript. References Lu KH, Lin RC, Yang JS, Yang WE, Reiter RJ, Yang SF Molecular and Cellular Mechanisms of Melatonin in Osteosarcoma. Cells 2019, 8, doi: 10.3390/cells8121618 Bielack SS, Kempf-Bielack B, Delling G, Exner GU, Flege S, Helmke K, Kotz R, Salzer-Kuntschik M, Werner M, Winkelmann W et al (2002) Prognostic factors in high-grade osteosarcoma of the extremities or trunk: an analysis of 1,702 patients treated on neoadjuvant cooperative osteosarcoma study group protocols. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 20:776–790. doi: 10.1200/JCO.2002.20.3.776 Mialou V, Philip T, Kalifa C, Perol D, Gentet JC, Marec-Berard P, Pacquement H, Chastagner P, Defaschelles AS, Hartmann O (2005) Metastatic osteosarcoma at diagnosis: prognostic factors and long-term outcome--the French pediatric experience. Cancer 104:1100–1109. doi: 10.1002/cncr.21263 Harrison DJ, Geller DS, Gill JD, Lewis VO, Gorlick R (2018) Current and future therapeutic approaches for osteosarcoma. Expert Rev Anticancer Ther 18:39–50. doi: 10.1080/14737140.2018.1413939 Huo FC, Zhu ZM, Pei DS (2020) N(6) -methyladenosine (m(6) A) RNA modification in human cancer. Cell proliferation 53:e12921. doi: 10.1111/cpr.12921 Huang W, Chen TQ, Fang K, Zeng ZC, Ye H, Chen YQ (2021) N6-methyladenosine methyltransferases: functions, regulation, and clinical potential. J Hematol Oncol 14:117. doi: 10.1186/s13045-021-01129-8 Zhu ZM, Huo FC, Pei DS (2020) Function and evolution of RNA N6-methyladenosine modification. Int J Biol Sci 16:1929–1940. doi: 10.7150/ijbs.45231 Jiang X, Liu B, Nie Z, Duan L, Xiong Q, Jin Z, Yang C, Chen Y (2021) The role of m6A modification in the biological functions and diseases. Signal transduction targeted therapy 6:74. doi: 10.1038/s41392-020-00450-x Lan Q, Liu PY, Bell JL, Wang JY, Huttelmaier S, Zhang XD, Zhang L, Liu T (2021) The Emerging Roles of RNA m(6)A Methylation and Demethylation as Critical Regulators of Tumorigenesis, Drug Sensitivity, and Resistance. Cancer research 81:3431–3440. doi: 10.1158/0008-5472.CAN-20-4107 Uddin MB, Wang Z, Yang C (2021) The m(6)A RNA methylation regulates oncogenic signaling pathways driving cell malignant transformation and carcinogenesis. Mol Cancer 20:61. doi: 10.1186/s12943-021-01356-0 Miao W, Chen J, Jia L, Ma J, Song D (2019) The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1. Biochem Biophys Res Commun 516:719–725. doi: 10.1016/j.bbrc.2019.06.128 Zhou L, Yang C, Zhang N, Zhang X, Zhao T, Yu J (2020) Silencing METTL3 inhibits the proliferation and invasion of osteosarcoma by regulating ATAD2. Biomedicine pharmacotherapy = Biomedecine pharmacotherapie 125:109964. doi: 10.1016/j.biopha.2020.109964 Chen S, Zhou L, Wang Y (2020) ALKBH5-mediated m(6)A demethylation of lncRNA PVT1 plays an oncogenic role in osteosarcoma. Cancer cell international 20:34. doi: 10.1186/s12935-020-1105-6 Khan K, Safi S, Abbas A, Badshah Y, Dilshad E, Rafiq M, Zahra K, Shabbir M Unravelling Structure, Localization, and Genetic Crosstalk of KLF3 in Human Breast Cancer. BioMed research international 2020, 2020 , 1354381, doi: 10.1155/2020/1354381 Pearson RC, Funnell AP, Crossley M (2011) The mammalian zinc finger transcription factor Kruppel-like factor 3 (KLF3/BKLF). IUBMB Life 63:86–93. doi: 10.1002/iub.422 Sun W, Hu S, Zu Y, Deng Y (2019) KLF3 is a crucial regulator of metastasis by controlling STAT3 expression in lung cancer. Molecular carcinogenesis 58:1933–1945. doi: 10.1002/mc.23072 Yan M, Liu H, Xu J, Cen X, Wang Q, Xu W, Wang W, Qiu Z, Ou J, Dong Y et al (2020) Expression of human Kruppel-like factor 3 in peripheral blood as a promising biomarker for acute leukemia. Cancer medicine 9:2803–2811. doi: 10.1002/cam4.2911 Wang X, Jiang Z, Zhang Y, Wang X, Liu L, Fan Z (2017) RNA sequencing analysis reveals protective role of kruppel-like factor 3 in colorectal cancer. Oncotarget 8:21984–21993. doi: 10.18632/oncotarget.15766 Sachdeva M, Dodd RD, Huang Z, Grenier C, Ma Y, Lev DC, Cardona DM, Murphy SK, Kirsch DG (2015) Epigenetic silencing of Kruppel like factor-3 increases expression of pro-metastatic miR-182. Cancer letters 369:202–211. doi: 10.1016/j.canlet.2015.08.016 Wu R, Liu Y, Yao Y, Zhao Y, Bi Z, Jiang Q, Liu Q, Cai M, Wang F, Wang Y et al (2018) FTO regulates adipogenesis by controlling cell cycle progression via m(6)A-YTHDF2 dependent mechanism. Biochimica et biophysica acta Molecular cell biology of lipids 1863:1323–1330. doi: 10.1016/j.bbalip.2018.08.008 Huo FC, Zhu ZM, Zhu WT, Du QY, Liang J, Mou J (2021) METTL3-mediated m(6)A methylation of SPHK2 promotes gastric cancer progression by targeting KLF2. Oncogene 40:2968–2981. doi: 10.1038/s41388-021-01753-1 Mauer J, Jaffrey SR, FTO (2018) m(6) Am, and the hypothesis of reversible epitranscriptomic mRNA modifications. FEBS Lett 592:2012–2022. doi: 10.1002/1873-3468.13092 Xie H, Li J, Ying Y, Yan H, Jin K, Ma X, He L, Xu X, Liu B, Wang X et al (2020) METTL3/YTHDF2 m(6) A axis promotes tumorigenesis by degrading SETD7 and KLF4 mRNAs in bladder cancer. J Cell Mol Med 24:4092–4104. doi: 10.1111/jcmm.15063 Chen J, Fang X, Zhong P, Song Z, Hu X (2019) N6-methyladenosine modifications: interactions with novel RNA-binding proteins and roles in signal transduction. RNA Biol 16:991–1000. doi: 10.1080/15476286.2019.1620060 Hu Y, Zhao X Role of m6A in osteoporosis, arthritis and osteosarcoma (Review). Experimental and therapeutic medicine 2021 , 22 , 926, doi: 10.3892/etm.2021.10358 Zhang Y, Wang Y, Ying L, Tao S, Shi M, Lin P, Wang Y, Han B (2021) Regulatory Role of N6-methyladenosine (m(6)A) Modification in Osteosarcoma. Frontiers in oncology 11:683768. doi: 10.3389/fonc.2021.683768 Liu H, Qin G, Ji Y, Wang X, Bao H, Guan X, Wei A, Cai Z (2021) Potential role of m6A RNA methylation regulators in osteosarcoma and its clinical prognostic value. J Orthop Surg Res 16:294. doi: 10.1186/s13018-021-02422-5 Dang DT, Pevsner J, Yang VW (2000) The biology of the mammalian Kruppel-like family of transcription factors. Int J Biochem Cell Biol 32:1103–1121. doi: 10.1016/s1357-2725(00)00059-5 Zhang Y, Hao J, Zheng Y, Jing D, Shen Y, Wang J, Zhao Z (2015) Role of Kruppel-like factors in cancer stem cells. Journal of physiology biochemistry 71:155–164. doi: 10.1007/s13105-015-0381-4 Ding F, Lai J, Gao Y, Wang G, Shang J, Zhang D, Zheng S (2019) NEAT1/miR-23a-3p/KLF3: a novel regulatory axis in melanoma cancer progression. Cancer cell international 19:217. doi: 10.1186/s12935-019-0927-6 Kuppers DA, Arora S, Lim Y, Lim AR, Carter LM, Corrin PD, Plaisier CL, Basom R, Delrow JJ, Wang S et al (2019) N(6)-methyladenosine mRNA marking promotes selective translation of regulons required for human erythropoiesis. Nature communications 10:4596. doi: 10.1038/s41467-019-12518-6 Chien CS, Li JY, Chien Y, Wang ML, Yarmishyn AA, Tsai PH, Juan CC, Nguyen P, Cheng HM, Huo TI et al. METTL3-dependent N(6)-methyladenosine RNA modification mediates the atherogenic inflammatory cascades in vascular endothelium. Proc Natl Acad Sci USA 2021, 118, doi: 10.1073/pnas.2025070118 Ma D, Liu X, Zhang JJ, Zhao JJ, Xiong YJ, Chang Q, Wang HY, Su P, Meng J, Zhao YB (2020) Vascular Smooth Muscle FTO Promotes Aortic Dissecting Aneurysms via m6A Modification of Klf5. Frontiers in cardiovascular medicine 7:592550. doi: 10.3389/fcvm.2020.592550 Zhou C, Zhang Z, Zhu X, Qian G, Zhou Y, Sun Y, Yu W, Wang J, Lu H, Lin F et al. N6-Methyladenosine modification of the TRIM7 positively regulates tumorigenesis and chemoresistance in osteosarcoma through ubiquitination of BRMS1. EBioMedicine 2020, 59 , 102955, doi: 10.1016/j.ebiom.2020.102955 Wang JY, Lu AQ (2021) The biological function of m6A reader YTHDF2 and its role in human disease. Cancer cell international 21:109. doi: 10.1186/s12935-021-01807-0 Tables Due to technical limitations, table 1 is only available as a download in the Supplemental Files section. Supplementary Files Table1.xlsx SupplementaryMaterialsandmethods.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-983314","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":57641723,"identity":"6df9da66-5d17-4e1b-8818-884504cb44ec","order_by":0,"name":"Hong-Jian Shan","email":"","orcid":"","institution":"Affiliated Hospital of Xuzhou Medical University; the Affiliated Jiangning Hospital with Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Hong-Jian","middleName":"","lastName":"Shan","suffix":""},{"id":57641724,"identity":"ce7e69b8-8763-4721-831c-cf881f9f8839","order_by":1,"name":"Wen-Xiang Gu","email":"","orcid":"","institution":"Affiliated Hospital of Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wen-Xiang","middleName":"","lastName":"Gu","suffix":""},{"id":57641725,"identity":"3abc3a66-47b9-40c3-b180-24758ad26ce7","order_by":2,"name":"Gang Duan","email":"","orcid":"","institution":"second Affiliated Hospital of Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Gang","middleName":"","lastName":"Duan","suffix":""},{"id":57641726,"identity":"2aa36080-c9d8-40d3-8650-b140e2fa9b56","order_by":3,"name":"Zhi-Man Zhu","email":"","orcid":"","institution":"Jiangsu College of Nursing","correspondingAuthor":false,"prefix":"","firstName":"Zhi-Man","middleName":"","lastName":"Zhu","suffix":""},{"id":57641727,"identity":"92dab7d8-7fc0-4d48-be4b-3a0b2b55c862","order_by":4,"name":"Hong-Liang Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYBACA3YGNiBlw8AgAeYzE6GFGawljXQth0nQYs7M/OzBxx3nE+fP7k6TYKiwTmxgP3sArxbLZjZzw5lnbhszzjm7TYLhTHpiA09eAn6HHeZhk+Ztuy3HLJG7TYKx7XBigwSPAWEtf9vO8bCBtfwjVgtj2wE5HrCWBqK0sJlJ9rYlG0tI5G62SDiWbtzGk0NAy/HmZxI/2+wS58/I3XjjQ421bD/7GfxaUEECELORoH4UjIJRMApGAQ4AAPmuPQFA2zKgAAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated Hospital of Xuzhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Hong-Liang","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2021-10-16 14:53:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-983314/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-983314/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":14770423,"identity":"85949bae-46e5-45fb-9916-d62683ca287c","added_by":"auto","created_at":"2021-10-21 19:01:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":468718,"visible":true,"origin":"","legend":"FTO is increased in OS tissues. \nThe mRNA levels of FTO were detected in OS cells and normal osteoblast Nhost cells (a). The mRNA levels of FTO in 9 paired OS tissues and the corresponding normal tissues (b). Representative images of IHC staining were used to analyze the protein levels of FTO in OS tissues adjacent and normal tissues (c). FTO expression was calculated in OS and normal tissues via quantitative analysis of IHC staining of FTO protein (d). Kaplan–Meier and log-rank test were used to analyze the 5-year overall survival OS patients with high or low FTO expression (e). Data represents the means ± SD. **, P \u003c 0.01; ***, P \u003c 0.001.\n","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-983314/v1/9b64243d9ee47f822cb61cf4.png"},{"id":14770428,"identity":"d5e2c755-11c5-40cf-b718-5a703bfccc6f","added_by":"auto","created_at":"2021-10-21 19:01:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1155101,"visible":true,"origin":"","legend":"FTO promotes proliferation, invasion of OS cells and inhibits cell apoptosis. \nTransfection efficiencies of FTO knockdown or overexpression were analyzed by qPCR in OS cells (a). The cell viability of OS cells was measured by CCK8 assay after FTO knockdown or overexpression (b-c). The effects of FTO in OS cells on the clone formation (c). FTO-knockdown or -overexpression on cell apoptosis was determined by flow cytometry assays (d). OS cell motilities were analyzed by measuring the wound closure after FTO knockdown or overexpression (e). Transwell chamber assays were performed to detect the invasive ability of OS cells after FTO knockdown or overexpression (f). Data represents the means ± SD. *, P \u003c 0.05; **, P \u003c 0.01; ***, P \u003c 0.001.\n","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-983314/v1/10787afbe61c9af75f3a6b27.png"},{"id":14770426,"identity":"bcca1adf-44a6-44ff-b593-c591f43a0346","added_by":"auto","created_at":"2021-10-21 19:01:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":620487,"visible":true,"origin":"","legend":"FTO regulates KLF3 expression in an m6A-dependent manner in OS cells.\nm6A methylation quantification was performed in OS and adjacent normal tissues (a). m6A quantification and dot blot assays were employed to detect the effect of FTO on m6A methylation in OS cells (b-c). The effects of FTO knockdown or overexpression on the protein and mRNA levels were determined by western blotting and qRT-PCR (d-e). Representative images of IHC staining were used to analyze the protein levels of FTO in OS tissues adjacent and normal tissues (f). Correlation analysis of FTO and KLF3 mRNA expressions in OS tissues (Pearson correlation test (g). Me-RIP qPCR was performed to measure m6A enrichment on KLF3 mRNAs in OS cells after knocking down FTO (h). RNA stability assay was used to detect the mRNA stability of KLF3 after FTO overexpression in cells with the treatment of actinomycin D (0.2 mM) for the indicated time (i). Transfection efficiencies of YTHDF2 knockdown or overexpression were analyzed by qPCR in OS cells (j). YTHDF2 attenuated the mRNA and protein levels of KLF3 in OS cells (k-j). The effect of YTHDF2 on the mRNA stability of KLF3 was performed (m). RIP-qPCR assay was performed to analyze the interaction between YTHDF2 protein and KLF3 mRNA in OS cells (n). The effect of silencing YTHDF2 on FTO-mediated the decrease of KLF3 mRNA and protein was performed by qRT-PCR and Western blotting (o-p). The role of FTO-induced mRNA degrading of KLF3 in OS cells was analyzed (q). Data represents the means ± SD. **, P \u003c 0.01; ***, P \u003c 0.001.\n","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-983314/v1/7ebb8e10bd70385b83bf1958.png"},{"id":14770759,"identity":"b6158fb3-eebc-4a96-9c38-719f76274b53","added_by":"auto","created_at":"2021-10-21 19:04:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":675193,"visible":true,"origin":"","legend":"KLF3 impairs the FTO-induced proliferation and invasion of OS cells. \nKLF3 could reverse the FTO-induced decrease of KLF3 protein (a). KLF3 impaired the FTO-induced promotion effect on OS cell proliferation and clone formation (b-c). Flow cytometry assay was performed to investigate the role of KLF3 in FTO-involved apoptosis in OS cells (d). The effect of KLF3 on FTO-induced cell invasion (e). The effects of FTO and (or) KLF3 on the cycle regulator and EMT markers in OS cells (f). Data represents the means ± SD. **, P \u003c 0.01; ***, P \u003c 0.001.\n","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-983314/v1/3524e75d1680479eeb9a426c.png"},{"id":14770760,"identity":"432fcec2-5dab-463f-9f61-5aff9dc5982e","added_by":"auto","created_at":"2021-10-21 19:04:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2678236,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-983314/v1/09f8aafb-8360-4f60-97ae-e7526cb3372c.pdf"},{"id":14770425,"identity":"9b4dfa19-8e16-4947-b35c-8e030157565d","added_by":"auto","created_at":"2021-10-21 19:01:17","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12320,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-983314/v1/e33889780dc1dad6f1f98145.xlsx"},{"id":14770424,"identity":"414f35a6-3386-4fa5-9f53-eab9f9221e14","added_by":"auto","created_at":"2021-10-21 19:01:17","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22356,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialsandmethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-983314/v1/90202494380ff617e615ee6b.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eFTO-Mediated N6-Methyladenosine Modification of KLF3 Promotes Osteosarcoma Progression\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteosarcoma (OS), as the most common primary malignant bone tumor, originates from mesenchymal cells, mostly in long bone, and can also be seen in distal femur or proximal tibia. Osteosarcoma is often accompanied by micrometastasis before operation and characterized by recurrent possibility and suboptimal prognosis\u0026nbsp;[\u003ca href=\"#_ENREF_1\" title=\"Lu, 2019 #798\"\u003e1\u003c/a\u003e]. 15%-20% of patients with OS are diagnosed with distant metastases at the first diagnosis\u0026nbsp;[\u003ca href=\"#_ENREF_2\" title=\"Bielack, 2002 #799\"\u003e2\u003c/a\u003e]. Even after radiotherapy and chemotherapy, the survival rate of 5-year is still less than 20% for OS complicated with lung metastasis\u0026nbsp;[\u003ca href=\"#_ENREF_3\" title=\"Mialou, 2005 #800\"\u003e3\u003c/a\u003e]. The combination of surgery and radiotherapy and chemotherapy has made some progress in improving the prognosis of patients with OS, but the curative effect is still not ideal\u0026nbsp;[\u003ca href=\"#_ENREF_4\" title=\"Harrison, 2018 #119\"\u003e4\u003c/a\u003e]. Therefore, we urgently need to explore the mechanism of osteosarcoma progression, find key molecular targets and improve the prognosis of osteosarcoma.\u003c/p\u003e\n\u003cp\u003em6A methylation is a dynamic and reversible epigenetic modification of RNAs, which mainly affects the splicing, translation and stability of mRNAs and the epigenetic effects of non-coding RNAs\u0026nbsp;[\u003ca href=\"#_ENREF_5\" title=\"Huo, 2020 #801\"\u003e5\u003c/a\u003e,\u003ca href=\"#_ENREF_6\" title=\"Huang, 2021 #802\"\u003e6\u003c/a\u003e]. The biological process of m6A is mainly regulated by three kinds of key-related kinases, m6A methyltransferase (Writers), m6A demethylas (Erasers) and methylation reading protein (Readers)\u0026nbsp;[\u003ca href=\"#_ENREF_5\" title=\"Huo, 2020 #801\"\u003e5\u003c/a\u003e,\u003ca href=\"#_ENREF_6\" title=\"Huang, 2021 #802\"\u003e6\u003c/a\u003e]. Methyltransferase complexes composed of various writers, such as METTL3/14, WTAP and KIAA1429, catalyze the m6A modification of adenylate on mRNAs. Demethylase includes FTO and ALKHB5, and its function is to demethylate the bases modified by m6A. The main function of readers is to recognize the m6A-modified RNA, thus activating downstream regulatory pathways such as RNA processing, mRNA degradation, translation and decay\u0026nbsp;[\u003ca href=\"#_ENREF_7\" title=\"Zhu, 2020 #804\"\u003e7\u003c/a\u003e,\u003ca href=\"#_ENREF_8\" title=\"Jiang, 2021 #806\"\u003e8\u003c/a\u003e]. m6A RNA methylation plays multiple roles in mammalian growth and development, including embryonic development, circadian rhythm and DNA damage response. More and more evidence shows that m6A methylation is closely related to tumorigenesis and development\u0026nbsp;[\u003ca href=\"#_ENREF_6\" title=\"Huang, 2021 #802\"\u003e6\u003c/a\u003e,\u003ca href=\"#_ENREF_9\" title=\"Lan, 2021 #803\"\u003e9\u003c/a\u003e,\u003ca href=\"#_ENREF_10\" title=\"Uddin, 2021 #805\"\u003e10\u003c/a\u003e]. METTL3 promotes cell proliferation, migration and invasion of and activates Wnt/\u0026beta;-catenin pathway in OS through regulating m6A methylation and LEF1\u0026nbsp;[\u003ca href=\"#_ENREF_11\" title=\"Miao, 2019 #807\"\u003e11\u003c/a\u003e].\u0026nbsp;Blockade of METTL3 inversely inhibits growth and invasion of OS cells by suppressing ATAD2\u0026nbsp;[\u003ca href=\"#_ENREF_12\" title=\"Zhou, 2020 #808\"\u003e12\u003c/a\u003e]. ALKBH5 augments lncRNA PVT1 expression in an m6A dependent manner and facilitate OS tumorigenesis\u0026nbsp;[\u003ca href=\"#_ENREF_13\" title=\"Chen, 2020 #809\"\u003e13\u003c/a\u003e]. These studies show that m6A methylation plays an important role in the occurrence and development of OS, but the specific mechanism and role of m6A reader FTO has not been reported.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKr\u0026uuml;ppel-like factor 3 (KLF3), a member of the KLF family, was originally a KLFI homolog cloned from human hematopoietic tissue. KLF3 can recruit transcriptional corepressor C-terminal binding protein and complete fusion in the target gene promoter, regulating transcription of target genes\u0026nbsp;[\u003ca href=\"#_ENREF_14\" title=\"Khan, 2020 #810\"\u003e14\u003c/a\u003e,\u003ca href=\"#_ENREF_15\" title=\"Pearson, 2011 #811\"\u003e15\u003c/a\u003e]. KLF3 plays an important role in B cell development, erythrocyte and fat production, nervous system and muscle gene regulation, cell differentiation and proliferation\u0026nbsp;[\u003ca href=\"#_ENREF_14\" title=\"Khan, 2020 #810\"\u003e14\u003c/a\u003e,\u003ca href=\"#_ENREF_15\" title=\"Pearson, 2011 #811\"\u003e15\u003c/a\u003e]. Aberrant expression of KLF3 has been detected in several cancers, such as lung cancer\u0026nbsp;[\u003ca href=\"#_ENREF_16\" title=\"Sun, 2019 #812\"\u003e16\u003c/a\u003e], acute leukemia\u0026nbsp;[\u003ca href=\"#_ENREF_17\" title=\"Yan, 2020 #813\"\u003e17\u003c/a\u003e]s, colorectal cancer\u0026nbsp;[\u003ca href=\"#_ENREF_18\" title=\"Wang, 2017 #814\"\u003e18\u003c/a\u003e], sarcoma\u0026nbsp;[\u003ca href=\"#_ENREF_19\" title=\"Sachdeva, 2015 #824\"\u003e19\u003c/a\u003e]. Silencing KLF3 contributes to lung cancer metastasis and the EMT process by regulating the STAT3 signaling pathway\u0026nbsp;[\u003ca href=\"#_ENREF_16\" title=\"Sun, 2019 #812\"\u003e16\u003c/a\u003e]. Decreased KLF3 is associated with aggressive phenotypes and poor prognosis for colorectal cancer patients\u0026nbsp;[\u003ca href=\"#_ENREF_18\" title=\"Wang, 2017 #814\"\u003e18\u003c/a\u003e]. KLF3 inhibits the pro-metastatic miR-182 expression and sarcoma metastasis\u0026nbsp;[\u003ca href=\"#_ENREF_19\" title=\"Sachdeva, 2015 #824\"\u003e19\u003c/a\u003e]. All these support the potential tumor-suppressor gene of KLF3. However, the underlying mechanism by which KLF3 is affected, especially m6A methylation, is virtually unknown in OS.\u003c/p\u003e\n\u003cp\u003eIn the study, we initially investigated the expression of FTO and found that increased expression of FTO was related to clinicopathological characteristics and poor prognosis of OS patients. FTO elicited potent oncogenic functions in promoting the proliferation and migration of OS cells. Moreover, we determined the effect of FTO-mediated m6A modification on KLF3 expression. Overall, FTO-KLF3 may serve as a potential therapeutic target for osteosarcoma.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eDetailed procedures are provided in Supplementary Materials and methods.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eFTO is increased expression in OS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the expression of FTO, we investigate the expression pattern of FTO in OS cells and the normal osteoblast cells (Nhost) by qPCR. And results showed that the mRNA levels of FTO in OS cells were elevated (Fig. 1a). We also detected the mRNA levels of FTO in OS and adjacent tissues. Similarly, mRNA levels of FTO were frequently increased in cancer tissues compared with matched normal tissues (Fig. 1b). IHC staining was also used to detect the FTO expression in OS and matched normal tissues (Fig. 1c). According to the quantitative analysis of IHC staining of tissue samples, we divided the FTO expression into high expression and low expression. 64.3% of OS tissues showed high expression, whereas the low expression rate of FTO in normal tissues was only 26.2% (Fig. 1d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe further analyzed the relationship between the FTO expression and clinicopathological parameters and postoperative survival of OS. Results from the IHC assay showed that there was a significant correlation between the high expression of FTO and several clinicopathological features (Table 1), such as tumor size, metastasis, and TNM stage, but not with the gender and age. In order to further understand the relationship between FTO and survival of OS, according to the FTO expression, we performed Kaplan-Meier survival curve analysis. The FTO expression was negatively correlated with the 5-year overall survival of patients with OS (Fig. 1e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFTO promotes OS cell proliferation, migration and invasion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to further study the effect of FTO expression on the proliferation of OS cells, FTO knockdown or overexpression was used to detect the proliferation ability of OS cells at 24h, 48h, 72h, respectively. Transfection efficiencies of FTO knockdown or overexpression were analyzed by qPCR in OS cells (Fig. 2a). Results showed that overexpression of FTO in OS cells promoted cell proliferation (Fig. 2b), while knockdown of FTO inhibited cell proliferation (Fig. 2b). As shown in (Fig. 2c), Clone formation also supported the role of FTO in contributing to proliferation of OS cells, and FTO downregulation weakened the cell proliferation. In addition, enforced expression of FTO inhibited OS cell apoptosis, however, blockade of FTO expedited apoptosis (Fig. 2d).\u003c/p\u003e\n\u003cp\u003eScratch healing assay was used to verify the migration ability of FTO to OS cells. Amplification of FTO accelerated scratch healing of OS cells (Fig. 2e). Transwell chamber assay showed that FTO attenuates the invasion of OS cells; inversely, silencing FTO attenuated the invasive ability (Fig. 2f). These findings indicated FTO promoted proliferation, migration and invasion of OS cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFTO inhibits KLF3 expression in OS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs the first discovered demethylase FTO, its discovery reveals the dynamic and reversible process of m6A modification, and it can reverse the m6A modification on RNAs\u0026nbsp;[\u003ca href=\"#_ENREF_22\" title=\"Mauer, 2018 #818\"\u003e22\u003c/a\u003e]. Previous results indicated that KLF3 is decreased expression in sarcomas\u0026nbsp;[\u003ca href=\"#_ENREF_19\" title=\"Sachdeva, 2015 #824\"\u003e19\u003c/a\u003e]. It has been reported that the tumor suppressor KLF4, another member of the KLF family, could be regulated in an m6A dependent manner bladder cancer\u0026nbsp;[\u003ca href=\"#_ENREF_23\" title=\"Xie, 2020 #825\"\u003e23\u003c/a\u003e]. METTL3-mediated m6A methylation directly promotes the mRNA decay of KLF4 through m6A binding protein YTHDF2\u0026nbsp;[\u003ca href=\"#_ENREF_23\" title=\"Xie, 2020 #825\"\u003e23\u003c/a\u003e]. Similarly, we identify whether the role of KLF3 in OS is regulated by m6A eraser FTO. Therefore, we subsequently analyzed the profile of m6A content in OS tissues. Results from m6A methylation quantification showed that m6A modification is significantly decreased in the total RNAs of OS tissues compared to adjacent normal tissues (Fig. 3a). m6A methylation quantification and dot blot assays were employed to detect the effect of FTO on m6A methylation in OS cells. As expected, m6A level was down-regulated in OS cells expressing FTO (Fig. 3b-c). Western blotting confirmed that FTO contributed to decrease KLF3 expression in OS cells, and silencing FTO conferred by siRNA inversely increased KLF3 (Fig. 3d), which were consistent with results from qRT-PCR (Fig. 3e). IHC staining also supported the decreased expression of KLF3 in OS tissues compared with normal tissues (Fig. 3f). Correlation analysis revealed that there was a significant negative correlation between FTO and KLF3 mRNA levels in OS tissues (Fig. 3g). Thus, we speculated that FTO can negatively regulate the expression of KLF3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFTO regulates KLF3 expression in an m6A-dependent manner\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe functional interaction between m6A methylase and demethylase determines the dynamic and reversible regulation of m6A modification. And m6A binding proteins can bind to mRNA containing m6A, thus affecting the fate of target mRNAs\u0026nbsp;[\u003ca href=\"#_ENREF_24\" title=\"Chen, 2019 #819\"\u003e24\u003c/a\u003e]. Me-RIP qPCR suggested that knockdown of FTO augmented the m6A enrichment on KLF3 mRNAs in OS cells (Fig. 3h).\u0026nbsp;FTO can negatively regulate the content of KLF3 mRNAs\u0026nbsp;(Fig. 3e).\u0026nbsp;We carried out the stability assay of KLF3 mRNAs. The results suggested that, after treatment with Actinomycin D (Transcription inhibitor), the residual percentage of KLF3 mRNA in FTO group was significantly lower than that in control group (Fig. 3i). Previous studies have confirmed that m6A binding protein YTHDF2 affects the stability of m6A modified RNA by locating them at mRNA decay sites\u0026nbsp;[\u003ca href=\"#_ENREF_24\" title=\"Chen, 2019 #819\"\u003e24\u003c/a\u003e]. Transfection efficiencies of YTHDF2 knockdown or overexpression were analyzed by qPCR in OS cells (Fig. 3j). YTHDF2 attenuated the mRNA and protein levels of KLF3 (Fig. 3k-l). And YTHDF2 reduced the half-life of KLF3 mRNA (Fig. 3m). RIP-qPCR assay proved the interaction between YTHDF2 protein and KLF3 mRNA in OS cells, which was enhanced in OS cells expressing FTO (Fig. 3n). Silencing YTHDF2 abrogated FTO-mediated the decrease of KLF3 mRNA and protein levels in OS cells (Fig. 3o-p). qRT-PCR results also verified that YTHDF2 knockdown reversed FTO-induced mRNA degrading of KLF3 (Fig. 3q). Overall, Silencing FTO accelerated the YTHDF2-involved decay of KLF3 mRNA in OS cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKLF3 impairs the FTO-induced proliferation and invasion of OS cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo ascertain whether KLF3 is involved in FTO function, OS cells transfected with FTO plasmids showed lower expression of KLF3, which could be reversed by overexpressing KLF3 (Fig. 4a). Moreover, FTO dramatically accelerated the proliferation of OS cells, whereas enforced expression of KLF3 reversed the FTO-induced promotion effects (Fig. 4b). Cloning formation efficiencies were significantly lower in OS cells expressing FTO and KLF3 than that of OS cells expressing FTO (Fig. 4c). FTO inhibited apoptosis, which was severely impeded in OS cells with ectopic expression of KLF3 (Fig. 4d). Analogously, KLF3 dramatically suppressed FTO-induced cell invasion (Fig. 4e). Cyclin D1 and p21 is the key protein of cell cycle regulation. FTO increased Cyclin D1 expression and decreased p21 expression in OS cells (Fig. 4f), and KLF3 could reverse the alternation. EMT also plays an important role in the process of tumor metastasis. FTO could enhance N-cadherin and Vimentin expression and suppress E-cadherin expression (Fig. 4f), which were compromised by KLF3 amplification. Thus, our findings indicated that FTO promoted cell proliferation and invasion by abating KLF3 in OS cells.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003em6A methylation, as the most important RNA internal modification in eukaryotes, was first discovered in 1974 and attracted great attention recently\u0026nbsp;[\u003ca href=\"#_ENREF_6\" title=\"Huang, 2021 #802\"\u003e6\u003c/a\u003e,\u003ca href=\"#_ENREF_7\" title=\"Zhu, 2020 #804\"\u003e7\u003c/a\u003e]. m6A methylation occurs in all stages of the RNA life cycle, from RNA synthesis to nucleation, translation regulation to RNA degradation\u0026nbsp;[\u003ca href=\"#_ENREF_24\" title=\"Chen, 2019 #819\"\u003e24\u003c/a\u003e]. m6A modification is closely related to the occurrence and development of cancer and participates in cell differentiation, proliferation, migration, immunity and drug resistance. Deregulation of m6A regulators in OS is inevitable\u0026nbsp;[\u003ca href=\"#_ENREF_11\" title=\"Miao, 2019 #807\"\u003e11\u003c/a\u003e,\u003ca href=\"#_ENREF_13\" title=\"Chen, 2020 #809\"\u003e13\u003c/a\u003e,\u003ca href=\"#_ENREF_25\" title=\"Hu, 2021 #820\"\u003e25\u003c/a\u003e,\u003ca href=\"#_ENREF_26\" title=\"Zhang, 2021 #822\"\u003e26\u003c/a\u003e]. Miao et al. reported that METTL3 contributes to proliferation, migration and invasion of OS cells and activates Wnt/\u0026beta;-catenin signaling pathway by regulating m6A modification of LEF1 mRNAs\u0026nbsp;[\u003ca href=\"#_ENREF_11\" title=\"Miao, 2019 #807\"\u003e11\u003c/a\u003e]. Analogously, METTL3 also promotes proliferation and invasion of OS cells by increasing ATAD2 expression via an m6A-dependent manner\u0026nbsp;[\u003ca href=\"#_ENREF_12\" title=\"Zhou, 2020 #808\"\u003e12\u003c/a\u003e]. ALKBH5 attenuates the m6A modification of PVT1 mRNA and impedes the binding of YTHDF2 to PVT1 mRNA, which increases PVT1 expression and accelerates OS growth in vivo\u0026nbsp;[\u003ca href=\"#_ENREF_13\" title=\"Chen, 2020 #809\"\u003e13\u003c/a\u003e]. In addition, m6A-related enzymes are involved in regulating the pluripotency of osteosarcoma stem cells\u0026nbsp;[\u003ca href=\"#_ENREF_26\" title=\"Zhang, 2021 #822\"\u003e26\u003c/a\u003e,\u003ca href=\"#_ENREF_27\" title=\"Liu, 2021 #832\"\u003e27\u003c/a\u003e]. All these implies potential roles of m6A methylation in tumorigenesis and progression of OS. And the relationship between FTO and OS has not been reported.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the study, our results showed that the protein and mRNA levels of FTO were increased in OS tissues and cells. Through IHC staining of OS samples, we also found that FTO expression in OS tissues was significantly higher than that in paracancerous normal tissues, and increased expression of FTO was associated with poor prognosis and aggressive phenotypes in OS. Ectopic expression of FTO promoted the proliferation, migration and invasion of OS cells and inhibited the apoptosis, and knockdown of FTO shows the opposite effects. These findings supported that FTO promotes OS progression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKLFs are one of the key components of the mammalian Sp/KLFSp zinc finger protein family. KLF3, as a member of KLFs, can recruit transcriptional corepressor C-terminal-binding protein to promoters of downstream molecules and primarily effectuate transcriptional inhibition\u0026nbsp;[\u003ca href=\"#_ENREF_15\" title=\"Pearson, 2011 #811\"\u003e15\u003c/a\u003e,\u003ca href=\"#_ENREF_17\" title=\"Yan, 2020 #813\"\u003e17\u003c/a\u003e]. Similar to other Sp/KLFSp zinc finger protein members, KLF3 contains conserved binding sites of GC-rich motif or CACCC box, which causes the regulation diversity for target genes\u0026nbsp;[\u003ca href=\"#_ENREF_28\" title=\"Dang, 2000 #827\"\u003e28\u003c/a\u003e]. Therefore, KLF3 has multiple biological functions, including erythropoiesis, adipogenesis, muscle regulation, B-cell development, differentiation and apoptosis\u0026nbsp;[\u003ca href=\"#_ENREF_17\" title=\"Yan, 2020 #813\"\u003e17\u003c/a\u003e,\u003ca href=\"#_ENREF_29\" title=\"Zhang, 2015 #828\"\u003e29\u003c/a\u003e]. Aberrant expression of KLF3 is related to the occurrence and progress of various tumors, such as acute leukemia\u0026nbsp;[\u003ca href=\"#_ENREF_17\" title=\"Yan, 2020 #813\"\u003e17\u003c/a\u003e], sarcoma\u0026nbsp;[\u003ca href=\"#_ENREF_19\" title=\"Sachdeva, 2015 #824\"\u003e19\u003c/a\u003e], lung cancer\u0026nbsp;[\u003ca href=\"#_ENREF_16\" title=\"Sun, 2019 #812\"\u003e16\u003c/a\u003e], colorectal cancer\u0026nbsp;[\u003ca href=\"#_ENREF_18\" title=\"Wang, 2017 #814\"\u003e18\u003c/a\u003e], melanoma cancer\u0026nbsp;[\u003ca href=\"#_ENREF_30\" title=\"Ding, 2019 #829\"\u003e30\u003c/a\u003e]. Loss of KLF3 potentiates lung cancer metastasis and EMT process through controlling STAT3 pathway\u0026nbsp;[\u003ca href=\"#_ENREF_16\" title=\"Sun, 2019 #812\"\u003e16\u003c/a\u003e]. DNA methylation-mediated KLF3 silencing augments the pro-metastatic miR-182 in human sarcoma cells\u0026nbsp;[\u003ca href=\"#_ENREF_19\" title=\"Sachdeva, 2015 #824\"\u003e19\u003c/a\u003e]. Decreased KLF3 is associated with poor prognosis and may work as a promising predictor in colorectal cancer\u0026nbsp;[\u003ca href=\"#_ENREF_18\" title=\"Wang, 2017 #814\"\u003e18\u003c/a\u003e]. These suggest that KLF3 may be a potential tumor suppressor gene. It has been reported that m6A-mediated modification could regulate the methylation of other members of KLFs, such as KLF1\u0026nbsp;[\u003ca href=\"#_ENREF_31\" title=\"Kuppers, 2019 #834\"\u003e31\u003c/a\u003e], KLF4\u0026nbsp;[\u003ca href=\"#_ENREF_32\" title=\"Chien, 2021 #835\"\u003e32\u003c/a\u003e], and KLF5\u0026nbsp;[\u003ca href=\"#_ENREF_33\" title=\"Ma, 2020 #836\"\u003e33\u003c/a\u003e]. RNA demethylation FTO promotes the phenotype conversion of human aortic vascular smooth muscle cells through increasing KLF5 expression in an m6A-dependent manner\u0026nbsp;[\u003ca href=\"#_ENREF_33\" title=\"Ma, 2020 #836\"\u003e33\u003c/a\u003e]. However, the potential mechanism of whether KLF3 is affected by m6A-mediated methylation modification have never been reported. Subsequently, we further explore whether FTO-mediated m6A modification regulates the expression of KLF3. We quantitatively detected the m6A modification of total mRNAs in OS and adjacent tissues and found that m6A content in OS samples was lower than that in adjacent samples, It is consistent with previous studies that total m6A level is decreased in OS\u0026nbsp;[\u003ca href=\"#_ENREF_34\" title=\"Zhou, 2020 #830\"\u003e34\u003c/a\u003e]. After knocking down and overexpressing FTO, we detected the changes of KLF3 expression and found that FTO negatively regulated the mRNA and protein expressions of KLF3 in OS cells. IHC staining showed that KLF3 was down-regulated in OS tissues compared with normal tissues.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnd decreasing FTO can potentiate m6A modification of KLF3 mRNAs. As m6A \u0026ldquo;reader\u0026rdquo;, the C-terminal of YTHDF2 can specifically recognize m6A-modified mRNAs to reduce the half-life of target mRNAs and decrease the stability of mRNAs\u0026nbsp;[\u003ca href=\"#_ENREF_35\" title=\"Wang, 2021 #831\"\u003e35\u003c/a\u003e]. We also proved the interaction between YTHDF2 protein and KLF3 mRNA in OS cells. Given that FTO positively regulates the content of KLF3 mRNA in cells, we investigated the effect of FTO-mediated m6A on the stability of KLF3 mRNAs. Our results showed that knockdown of FTO decreases the stability of KLF3 mRNA in OS cells, and deletion YTHDF2 impedes the KLF3 mRNA decay induced by silencing FTO. There was also a negative correlation between FTO and KLF3 expression in OS tissues. We concluded that FTO regulates the level of KLF3 mRNAs in an m6A-dependent manner in OS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn vitro studies demonstrated that FTO promoted the proliferation and invasion of OS cells. However, KLF3 protected against FTO-induced promotion effects on cell proliferation and invasion. Cyclin D1 promotes cell proliferation by binding to and activating cyclin dependent kinase CDK4 unique to G1 phase. And p21 can form a complex with cyclin D/CDK and arrest the cell cycle in G1 phase. EMT concomitant with the down-regulation of epithelial markers and up-regulation of mesenchymal phenotype markers is an important process of invasion and distant metastasis of cancer. FTO up-regulated Cyclin D1 and N-cadherin level and down-regulated p21 and E-cadherin level in OS cells, and expressing KLF3 compromised the FTO-induced alternations. Collectively, FTO enhances the cell proliferation and invasion by silencing KLF3 expression in OS cells. This study focused on the effects of FTO on the proliferation and invasion of OS cells by regulating the m6A methylation of KLF3 mRNAs. Our results also supported the view that m6A modification is involved in the progression of OS. However, further researches are needed to provide direct epigenetic evidence of the FTO/KLF3 pathway in the pathogenesis of OS.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe expression of FTO is increased in OS and affects the prognosis survival of OS patients. FTO promotes the proliferation and invasion, and inhibits apoptosis of OS cells through regulating KLF3 expression via an m6A-dependent manner. And by revealing the previously unknown regulatory mechanisms of FTO and KLF3, it provides a new direction for the molecular mechanism of occurrence and development of OS. In addition, considering the importance of m6A modification in human cancers, it is possible to explore new strategies for the diagnosis and treatment of OS by targeting FTO with selective inhibitors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e The study was approved by the Review Board of the Affiliated Hospital of Xuzhou Medical University.\u0026ensp;All participants provided informed consent as outlined in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e All authors approved the final version of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003eAll data and materials are available for verification as needed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors confirm that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was supported by the China\u0026rsquo;s 62\u003csup\u003end\u003c/sup\u003e Postdoctoral Fund (2017M621826), Project of Jiangsu Postdoctoral Fund (2018K261C) in 2018, and the General Program of Natural Science Foundation of Jiangsu Province (BK20201154).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e Conceived and designed the project: Hong-Jian Shan, Hong-Liang Chen and Zhi-Man Zhu; Performed the project: Hong-Jian Shan, Wen-Xiang Gu; Analyzed the data: Hong-Jian Shan, Gang Duan; Contributed reagents/materials/analysis tools: Hong-Jian Shan, Wen-Xiang Gu, Gang Duan, Zhi-Man Zhu, Hong-Liang Chen; Wrote the paper: Hong-Jian Shan. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eThis work was supported by the China\u0026rsquo;s 62\u003csup\u003end\u003c/sup\u003e Postdoctoral Fund (2017M621826), Project of Jiangsu Postdoctoral Fund (2018K261C) in 2018, and the General Program of Natural Science Foundation of Jiangsu Province (BK20201154).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure of potential conflicts of interest\u0026nbsp;\u003c/strong\u003eThe authors confirm that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch involving Human Participants and/or Animals\u003c/strong\u003e The study involving human specimens was approved by the Review Board of the Affiliated Hospital of Xuzhou Medical University. This article does not contain any studies with animals performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u0026nbsp;\u003c/strong\u003eAll participants provided informed consent as outlined in the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLu KH, Lin RC, Yang JS, Yang WE, Reiter RJ, Yang SF Molecular and Cellular Mechanisms of Melatonin in Osteosarcoma. Cells 2019, 8, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cells8121618\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBielack SS, Kempf-Bielack B, Delling G, Exner GU, Flege S, Helmke K, Kotz R, Salzer-Kuntschik M, Werner M, Winkelmann W et al (2002) Prognostic factors in high-grade osteosarcoma of the extremities or trunk: an analysis of 1,702 patients treated on neoadjuvant cooperative osteosarcoma study group protocols. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 20:776\u0026ndash;790. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1200/JCO.2002.20.3.776\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMialou V, Philip T, Kalifa C, Perol D, Gentet JC, Marec-Berard P, Pacquement H, Chastagner P, Defaschelles AS, Hartmann O (2005) Metastatic osteosarcoma at diagnosis: prognostic factors and long-term outcome--the French pediatric experience. Cancer 104:1100\u0026ndash;1109. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/cncr.21263\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarrison DJ, Geller DS, Gill JD, Lewis VO, Gorlick R (2018) Current and future therapeutic approaches for osteosarcoma. Expert Rev Anticancer Ther 18:39\u0026ndash;50. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/14737140.2018.1413939\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuo FC, Zhu ZM, Pei DS (2020) N(6) -methyladenosine (m(6) A) RNA modification in human cancer. Cell proliferation 53:e12921. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/cpr.12921\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang W, Chen TQ, Fang K, Zeng ZC, Ye H, Chen YQ (2021) N6-methyladenosine methyltransferases: functions, regulation, and clinical potential. J Hematol Oncol 14:117. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13045-021-01129-8\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu ZM, Huo FC, Pei DS (2020) Function and evolution of RNA N6-methyladenosine modification. Int J Biol Sci 16:1929\u0026ndash;1940. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7150/ijbs.45231\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang X, Liu B, Nie Z, Duan L, Xiong Q, Jin Z, Yang C, Chen Y (2021) The role of m6A modification in the biological functions and diseases. Signal transduction targeted therapy 6:74. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41392-020-00450-x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLan Q, Liu PY, Bell JL, Wang JY, Huttelmaier S, Zhang XD, Zhang L, Liu T (2021) The Emerging Roles of RNA m(6)A Methylation and Demethylation as Critical Regulators of Tumorigenesis, Drug Sensitivity, and Resistance. Cancer research 81:3431\u0026ndash;3440. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/0008-5472.CAN-20-4107\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUddin MB, Wang Z, Yang C (2021) The m(6)A RNA methylation regulates oncogenic signaling pathways driving cell malignant transformation and carcinogenesis. Mol Cancer 20:61. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12943-021-01356-0\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiao W, Chen J, Jia L, Ma J, Song D (2019) The m6A methyltransferase METTL3 promotes osteosarcoma progression by regulating the m6A level of LEF1. Biochem Biophys Res Commun 516:719\u0026ndash;725. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbrc.2019.06.128\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou L, Yang C, Zhang N, Zhang X, Zhao T, Yu J (2020) Silencing METTL3 inhibits the proliferation and invasion of osteosarcoma by regulating ATAD2. Biomedicine pharmacotherapy = Biomedecine pharmacotherapie 125:109964. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biopha.2020.109964\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Zhou L, Wang Y (2020) ALKBH5-mediated m(6)A demethylation of lncRNA PVT1 plays an oncogenic role in osteosarcoma. Cancer cell international 20:34. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12935-020-1105-6\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan K, Safi S, Abbas A, Badshah Y, Dilshad E, Rafiq M, Zahra K, Shabbir M Unravelling Structure, Localization, and Genetic Crosstalk of KLF3 in Human Breast Cancer. \u003cem\u003eBioMed research international\u003c/em\u003e 2020, \u003cem\u003e2020\u003c/em\u003e, 1354381, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2020/1354381\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePearson RC, Funnell AP, Crossley M (2011) The mammalian zinc finger transcription factor Kruppel-like factor 3 (KLF3/BKLF). IUBMB Life 63:86\u0026ndash;93. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/iub.422\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun W, Hu S, Zu Y, Deng Y (2019) KLF3 is a crucial regulator of metastasis by controlling STAT3 expression in lung cancer. Molecular carcinogenesis 58:1933\u0026ndash;1945. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/mc.23072\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan M, Liu H, Xu J, Cen X, Wang Q, Xu W, Wang W, Qiu Z, Ou J, Dong Y et al (2020) Expression of human Kruppel-like factor 3 in peripheral blood as a promising biomarker for acute leukemia. Cancer medicine 9:2803\u0026ndash;2811. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/cam4.2911\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Jiang Z, Zhang Y, Wang X, Liu L, Fan Z (2017) RNA sequencing analysis reveals protective role of kruppel-like factor 3 in colorectal cancer. Oncotarget 8:21984\u0026ndash;21993. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/oncotarget.15766\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSachdeva M, Dodd RD, Huang Z, Grenier C, Ma Y, Lev DC, Cardona DM, Murphy SK, Kirsch DG (2015) Epigenetic silencing of Kruppel like factor-3 increases expression of pro-metastatic miR-182. Cancer letters 369:202\u0026ndash;211. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.canlet.2015.08.016\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu R, Liu Y, Yao Y, Zhao Y, Bi Z, Jiang Q, Liu Q, Cai M, Wang F, Wang Y et al (2018) FTO regulates adipogenesis by controlling cell cycle progression via m(6)A-YTHDF2 dependent mechanism. Biochimica et biophysica acta Molecular cell biology of lipids 1863:1323\u0026ndash;1330. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbalip.2018.08.008\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuo FC, Zhu ZM, Zhu WT, Du QY, Liang J, Mou J (2021) METTL3-mediated m(6)A methylation of SPHK2 promotes gastric cancer progression by targeting KLF2. Oncogene 40:2968\u0026ndash;2981. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41388-021-01753-1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMauer J, Jaffrey SR, FTO (2018) m(6) Am, and the hypothesis of reversible epitranscriptomic mRNA modifications. FEBS Lett 592:2012\u0026ndash;2022. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/1873-3468.13092\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie H, Li J, Ying Y, Yan H, Jin K, Ma X, He L, Xu X, Liu B, Wang X et al (2020) METTL3/YTHDF2 m(6) A axis promotes tumorigenesis by degrading SETD7 and KLF4 mRNAs in bladder cancer. J Cell Mol Med 24:4092\u0026ndash;4104. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/jcmm.15063\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen J, Fang X, Zhong P, Song Z, Hu X (2019) N6-methyladenosine modifications: interactions with novel RNA-binding proteins and roles in signal transduction. RNA Biol 16:991\u0026ndash;1000. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/15476286.2019.1620060\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu Y, Zhao X Role of m6A in osteoporosis, arthritis and osteosarcoma (Review). \u003cem\u003eExperimental and therapeutic medicine\u003c/em\u003e \u003cb\u003e2021\u003c/b\u003e, \u003cem\u003e22\u003c/em\u003e, 926, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/etm.2021.10358\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Wang Y, Ying L, Tao S, Shi M, Lin P, Wang Y, Han B (2021) Regulatory Role of N6-methyladenosine (m(6)A) Modification in Osteosarcoma. Frontiers in oncology 11:683768. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fonc.2021.683768\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu H, Qin G, Ji Y, Wang X, Bao H, Guan X, Wei A, Cai Z (2021) Potential role of m6A RNA methylation regulators in osteosarcoma and its clinical prognostic value. J Orthop Surg Res 16:294. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13018-021-02422-5\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDang DT, Pevsner J, Yang VW (2000) The biology of the mammalian Kruppel-like family of transcription factors. Int J Biochem Cell Biol 32:1103\u0026ndash;1121. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s1357-2725(00)00059-5\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Hao J, Zheng Y, Jing D, Shen Y, Wang J, Zhao Z (2015) Role of Kruppel-like factors in cancer stem cells. Journal of physiology biochemistry 71:155\u0026ndash;164. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s13105-015-0381-4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing F, Lai J, Gao Y, Wang G, Shang J, Zhang D, Zheng S (2019) NEAT1/miR-23a-3p/KLF3: a novel regulatory axis in melanoma cancer progression. Cancer cell international 19:217. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12935-019-0927-6\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuppers DA, Arora S, Lim Y, Lim AR, Carter LM, Corrin PD, Plaisier CL, Basom R, Delrow JJ, Wang S et al (2019) N(6)-methyladenosine mRNA marking promotes selective translation of regulons required for human erythropoiesis. Nature communications 10:4596. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41467-019-12518-6\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChien CS, Li JY, Chien Y, Wang ML, Yarmishyn AA, Tsai PH, Juan CC, Nguyen P, Cheng HM, Huo TI et al. METTL3-dependent N(6)-methyladenosine RNA modification mediates the atherogenic inflammatory cascades in vascular endothelium. Proc Natl Acad Sci USA 2021, 118, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.2025070118\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa D, Liu X, Zhang JJ, Zhao JJ, Xiong YJ, Chang Q, Wang HY, Su P, Meng J, Zhao YB (2020) Vascular Smooth Muscle FTO Promotes Aortic Dissecting Aneurysms via m6A Modification of Klf5. Frontiers in cardiovascular medicine 7:592550. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fcvm.2020.592550\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou C, Zhang Z, Zhu X, Qian G, Zhou Y, Sun Y, Yu W, Wang J, Lu H, Lin F et al. N6-Methyladenosine modification of the TRIM7 positively regulates tumorigenesis and chemoresistance in osteosarcoma through ubiquitination of BRMS1. \u003cem\u003eEBioMedicine\u003c/em\u003e 2020, \u003cem\u003e59\u003c/em\u003e, 102955, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ebiom.2020.102955\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang JY, Lu AQ (2021) The biological function of m6A reader YTHDF2 and its role in human disease. Cancer cell international 21:109. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12935-021-01807-0\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eDue to technical limitations, table 1 is only available as a download in the Supplemental Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"m6A methylation, FTO, osteosarcoma, KLF3 ","lastPublishedDoi":"10.21203/rs.3.rs-983314/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-983314/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eN6-methyladenosine (m6A) methylation is the most common and abundant methylation modification of eukaryotic mRNAs, which is involved in tumor initiation and progression. This study aims to explore the functional role and regulatory mechanism of FTO in osteosarcoma progression. In this study, we detected the expressions of KLF3 in OS cells and tissues by qRT-PCR and western blot, and found that the mRNA and protein expressions of KLF3 were increased in OS cells and tissues and significantly related to tumor size, metastasis, and TNM stage and poor prognosis of OS patients. FTO promoted the proliferation and invasion and suppressed apoptosis of OS cells through CCK-8, Transwell and apoptosis assays. Further mechanism dissection revealed that FTO and YTHDF2 enforced the decay of KLF3 mRNAs and decreased its expression. Knockdown of FTO led to elevate m6A and mRNA levels. FTO-mediated m6A modification regulated KLF3 expression in the YTHDF2-dependent manner. Moreover, KLF3 overexpression abrogated FTO-induced oncogenic effects on the proliferation and invasion of OS cells. Overall,\u003cstrong\u003e \u003c/strong\u003eour findings showed that FTO-mediated m6A modification of KLF3 promoted OS progression, which may provide a therapeutic target for osteosarcoma. \u003c/p\u003e","manuscriptTitle":"FTO-Mediated N6-Methyladenosine Modification of KLF3 Promotes Osteosarcoma Progression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-10-21 19:01:15","doi":"10.21203/rs.3.rs-983314/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c30733f5-8b0a-4983-9ccb-72683d6e0a74","owner":[],"postedDate":"October 21st, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":8021728,"name":"Drug Discovery, Design, \u0026 Development"}],"tags":[],"updatedAt":"2021-10-21T19:01:17+00:00","versionOfRecord":[],"versionCreatedAt":"2021-10-21 19:01:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-983314","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-983314","identity":"rs-983314","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}