METTL3 promotes the progression of nasopharyngeal carcinoma through mediating M6A modification of EZH2
Authors: F-W Chen
X Zhao
X-J Li
C-H Cong
Q-Z Meng
F Zhu
Journal: European review for medical and pharmacological sciences (2020)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: METTL3; nasopharyngeal carcinoma; CDKN1C; EZH2; tumor progression
Affiliations: Department of Head and Neck Surgery, Linyi Cancer Hospital, Linyi, China. .

Abstract:  Objective: The aim of this study was to investigate whether METTL3 promoted the progression of nasopharyngeal carcinoma (NPC) by silencing CDKN1C through EZH2. Patients and methods: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the expression level of METTL3 in 48 pairs of NPC tissues and adjacent normal tissues. METTL3 expression in patients with different tumor lymph node metastasis (TNM) stages was detected by qRT-PCR as well. The Kaplan-Meier method was used to analyze the interplay between METTL3 expression and the prognosis of patients with NPC. At the same time, METTL3 expression in normal epithelial cell line (BEAS-2B) and NPC cell lines (SUNE-1 and C666-1) was examined using qRT-PCR. After METTL3 was knocked down in SUNE-1 cells, cell viability and migration abilities were analyzed by cell counting kit-8 (CCK-8) test and wound healing assay, respectively. The mRNA and protein expressions of EZH2 were detected by qRT-PCR and Western blot, respectively. RNA immunoprecipitation (RIP) assay was applied to detect the binding of METTL3 to EZH2 mRNA and the m6A modification on EZH2 mRNA. After knockdown of EZH2 in SUNE-1 cells, qRT-PCR was used to detect the mRNA expression of CDKN1C. Meanwhile, chromatin immunoprecipitation (ChIP) assay was conducted to analyze the binding of EZH2 to the CDKN1C promoter region. After down-regulation of METTL3 in SUNE-1 cells, the protein expressions of EZH2 and CDKN1C were detected using Western blot. After simultaneous knockdown of METTL3 and CDKN1C in SUNE-1 cells, CCK8 assay and wound healing assay were applied to examine cell viability and migration abilities. Results: METTL3 expression in NPC tissues was remarkably higher than that of adjacent normal tissues. Meanwhile, METTL3 expression in T3 and T4 tumors was significantly higher than that of T1 and T2 tumors. In patients with lymph node metastasis, the expression of METTL3 was remarkably higher than those without metastasis. Survival analysis demonstrated that patients with higher expression of METTL3 exhibited significantly longer overall survival time than those with lower METTL3 expression. QRT-PCR revealed that METTL3 was highly expressed in NPC cell lines, including SUNE-1 and C666-1. After knock-down of METTL3 in SUNE-1 cells, cell viability and migration abilities were both markedly weakened. Meanwhile, the protein expression of EZH2 was remarkably reduced. However, no significant changes were observed in EZH2 mRNA level. RIP assay revealed that METTL3 could bind to EZH2 mRNA, and a m6A modification was verified on EZH2 mRNA. After knockdown of EZH2, the mRNA level of CDKN1C in SUNE-1 cells was significantly up-regulated. CHIP assay indicated that EZH2 could bind to CDKN1C. Western blot showed that, after interfering with METTL3 in SUNE-1 cells, the protein expression of EZH2 decreased significantly, while CDKN1C was up-regulated. In addition, simultaneous downregulation of METTL3 and CDKN1C in SUNE-1 cells reversed the influence of METTL3 on cell viability and migration abilities. Conclusions: METTL3 was highly expressed in NPC tissues, which might inhibit EZH2 expression by mediating M6A modification of EZH2 mRNA. Furthermore, CDKN1C could increase the malignancy of NPC cells and promote the progression of NPC. 

Retracted: No

PubPeer comments:
 This paper is one of a set of (as of now) five papers that all share unexpected similarities. The papers are from different groups of authors at different hospitals, describing different cancer types. Yet, they share several similar features. * All five papers describe a group of 48 patients * All five papers describe the same set of GAPDH primers. One of them (the Reverse primer) does not appear to match the GAPDH mRNA sequence. * Figures 1 of all five papers show black bar graphs with error bars and a Kaplan-Meier curve with a blue and a red line * Two papers (XX Ma et al. and JL Xu et al.) share nearly identical positive correlation curves * All five papers show photos of wound healing (scratch) assays with the same background, suggesting they were all taken with the same microscope. Here is the set of papers: 1. J.-L. Xu, T. Hua, J. Ding, Y. Fan, Z.-J. Liu, J.-W. Lian - FOXF2 aggravates the progression of non-small cell lung cancer through targeting lncRNA H19 to downregulate PTEN - Eur Rev Med Pharmacol Sci 2019; 23 (24): 10796-10802 - DOI: 10.26355 eurrev_201912_19782 - Department of Oncology, Xi an Chest Hospital, Xi an, China 2. Q.-Z. Meng, C.-H. Cong, X.-J. Li, F. Zhu, X. Zhao, F.-W. Chen - METTL3 promotes the progression of nasopharyngeal carcinoma through mediating M6A modification of EZH2 - Eur Rev Med Pharmacol Sci 2020; 24 (8): 4328-4336 - DOI: 10.26355 eurrev_202004_21014 - Department of Head and Neck Surgery, Linyi Cancer Hospital, Linyi, China 3. T. Chen, J. Li, M.-H. Zhou, L.-J. Xu, T.-C. Pan - IL-6 stimulates lncRNA ZEB2-AS1 to aggravate the progression of non-small cell lung cancer through activating STAT1 - Eur Rev Med Pharmacol Sci 2020; 24 (7): 3734-3740 - DOI: 10.26355 eurrev_202004_20837 - Department of Cardiovascular Surgery, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 4. T. Zhou, J.-J. Fang, Y.-X. Zhou, Z.-P. Li, L. Jiang, W.-W. Han, Z.-H. Zhu - Long non-coding RNA ANCR promotes progression of NSCLC by inhibiting E-Ca expression - Eur Rev Med Pharmacol Sci 2020; 24 (3): 1250-1257 - DOI: 10.26355 eurrev_202002_20178 - Department of Respiratory Medicine, Zhoushan Hospital of Zhejiang University, Zhoushan, China 5. X.-X. Ma, Z.-G. Cao, S.-L. Zhao - m6A methyltransferase METTL3 promotes the progression of prostate cancer via m6A-modified LEF1 - Eur Rev Med Pharmacol Sci 2020; 24 (7): 3565-3571 - DOI: 10.26355 eurrev_202004_20817 - Department of Urology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China Here is a comparison of the Figures 1 from these papers. The very similar looking correlation curves of XX Ma et al. and JL Xu et al. have been highlighted with red boxes. ![file] Here is a comparison of the wound healing assay photos, with similar background spots highlighted in green and yellow boxes. Contrast enhanced. These appear to be irregularities in the microscope, which by itself is not a problem. But it is unexpected to see the same similarities in photos obtained across different laboratories. ![file] ![file] Could the authors please clarify how these photos look so similar? , 
 the paper titled  Simvastatin is beneficial to lung cancer progression by inducing METTL3-induced m6A modification on EZH2 mRNA also shared similarities with papers above, with similar idea, the same journal and similar publish date. , 
  From **December 2017 to October 2018** in Linyi Cancer Hospital, 48 NPC patients who received surgical resection were enrolled in this study. Fig 1D.  Overall survival of patients with higher expression of METTL3 was significantly lower than that of patients with lower expression . ![file] If I understand correctly, at the time of this paper s publication in 2020, the survival of patients had been tracked for 10 years after recruitment, i.e. to the period **December 2027 to October 2028**. , 
 The 48 nasopharyngeal cancer patients were recruited from December 2017-October 2018, presumably directly after their diagnosis, because they had not received anti-tumor therapy. Yet, despite the paper being published in 2020, the survival curve in Figure 1D shows a 10-year (120 months) follow up. Something is not right here. HT: Hoya Camphorifolia For similar cases, see also: https: pubpeer.com publications B97218A01C913B80E7A89CDA87BDF7 and https: pubpeer.com publications 50C7E1823EED971D34431E8574D873 ![file] , 
 This paper is one of a set of (as of now) five papers that all share unexpected similarities. The papers are from different groups of authors at different hospitals, describing different cancer types. Yet, they share several similar features. * All five papers describe a group of 48 patients * All five papers describe the same set of GAPDH primers. One of them (the RV primer) does not appear to match the GAPDH mRNA sequence. * Figures 1 of all five papers show black bar graphs with error bars and a Kaplan-Meier curve with a blue and a red line * Two papers (XX Ma et al. and JL Xu et al.) share nearly identical positive correlation curves * All five papers show photos of wound healing (scratch) assays with the same background, suggesting they were all taken with the same microscope. Here is the set of papers: 1. J.-L. Xu, T. Hua, J. Ding, Y. Fan, Z.-J. Liu, J.-W. Lian - FOXF2 aggravates the progression of non-small cell lung cancer through targeting lncRNA H19 to downregulate PTEN - Eur Rev Med Pharmacol Sci 2019; 23 (24): 10796-10802 - DOI: 10.26355 eurrev_201912_19782 - Department of Oncology, Xi an Chest Hospital, Xi an, China 2. Q.-Z. Meng, C.-H. Cong, X.-J. Li, F. Zhu, X. Zhao, F.-W. Chen - METTL3 promotes the progression of nasopharyngeal carcinoma through mediating M6A modification of EZH2 - Eur Rev Med Pharmacol Sci 2020; 24 (8): 4328-4336 - DOI: 10.26355 eurrev_202004_21014 - Department of Head and Neck Surgery, Linyi Cancer Hospital, Linyi, China 3. T. Chen, J. Li, M.-H. Zhou, L.-J. Xu, T.-C. Pan - IL-6 stimulates lncRNA ZEB2-AS1 to aggravate the progression of non-small cell lung cancer through activating STAT1 - Eur Rev Med Pharmacol Sci 2020; 24 (7): 3734-3740 - DOI: 10.26355 eurrev_202004_20837 - Department of Cardiovascular Surgery, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 4. T. Zhou, J.-J. Fang, Y.-X. Zhou, Z.-P. Li, L. Jiang, W.-W. Han, Z.-H. Zhu - Long non-coding RNA ANCR promotes progression of NSCLC by inhibiting E-Ca expression - Eur Rev Med Pharmacol Sci 2020; 24 (3): 1250-1257 - DOI: 10.26355 eurrev_202002_20178 - Department of Respiratory Medicine, Zhoushan Hospital of Zhejiang University, Zhoushan, China 5. X.-X. Ma, Z.-G. Cao, S.-L. Zhao - m6A methyltransferase METTL3 promotes the progression of prostate cancer via m6A-modified LEF1 - Eur Rev Med Pharmacol Sci 2020; 24 (7): 3565-3571 - DOI: 10.26355 eurrev_202004_20817 - Department of Urology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China Here is a comparison of the Figures 1 from these papers. The very similar looking correlation curves of XX Ma et al. and JL Xu et al. have been highlighted with red boxes. ![file] Here is a comparison of the wound healing assay photos, with similar background spots highlighted in green and yellow boxes. Contrast enhanced. These appear to be irregularities in the microscope, which by itself is not a problem. But it is unexpected to see the same similarities in photos obtained across different laboratories. ![file] ![file] Could the authors please clarify how these photos look so similar? , 
 Here are four Figures 1D, with pairs of 10-year survival curves, to show the range of variation when time-travelling cases of cancer are subgrouped by different biomarkers. All ostensibly different research teams. Clockwise from upper left: - [Chen Wang (2019)] . 48 cases, Non-small-cell lung cancer, ZEB2-AS1. - [Zhou et al (2020)] . NSCLC, ANCR. - [Chen et al (2020)] . NSCLC, IL-6. - **[Meng et al (2020)] . METTL3. Nasopharangeal cancer, METTL3**. ![file] Note the same number of surviving cases in each high-level subgroup when tracking of those cases stopped prematurely after 96 months. , 
 Retraction, July 2022: https: www.europeanreview.org article 29277  *The article u201cMETTL3 promotes the progression of nasopharyngeal carcinoma through mediating M6A modification of EZH2, by Q.-Z. Meng, C.-H. Cong, X.-J. Li, F. Zhu, X. Zhao, F.-W. Chen, published in Eur Rev Med Pharmacol Sci 2020; 24 (8): 4328-4336u2013DOI: 10.26355 eurrev_202004_21014u2013PMID: 32373970u201d has been retracted by the authors. After publication, several issues were raised on PubPeer about the reliability of the published results. The same authors stated that the study was not performed in accordance with the standard procedures required. In particular, Figure 1 also presents some concerns as it does not reflect the experimental data reported in the study.* *The Publisher apologizes for any inconvenience this may cause.* , 
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