mA mRNA methylation initiated by METTL3 directly promotes YAP translation and increases YAP activity by regulating the MALAT1-miR-1914-3p-YAP axis to induce NSCLC drug resistance and metastasis
Authors: Guoming Su
Baoguang Hu
Weihua Di
Lingqun Kong
Yan Wu
Jing Du
Dan Jin
Jiwei Guo
Jiajia An
Lijuan Yang
Xiaohong Wang
Lei Pan
Journal: Journal of Hematology & Oncology (2019)
Discipline: CLINICAL MEDICINE
Keywords: MALAT1; METTL3; YAP; YTHDF1/3; eIF3b
Affiliations: Clinical Medical Laboratory, Binzhou Medical University Hospital, Binzhou, 256603, People's Republic of China., Cancer research institute, Binzhou Medical University Hospital, Binzhou, 256603, People's Republic of China. ., Cancer research institute, Binzhou Medical University Hospital, Binzhou, 256603, People's Republic of China., Department of Thyroid and Breast Surgery, Binzhou Medical University Hospital, Binzhou, 256603, People's Republic of China., Department of Pain, Binzhou Medical University Hospital, Binzhou, 256603, People's Republic of China., Department of Gastrointestinal Surgery, Binzhou Medical University Hospital, Binzhou, 256603, People's Republic of China., Department of Clinical Laboratory, Binzhou Medical University Hospital, Binzhou, 256603, People's Republic of China., Department of Hepatobiliary Surgery, Binzhou Medical University Hospital, Binzhou, 256603, People's Republic of China., Department of Respiratory and Critical Care Medicine, Binzhou Medical University Hospital, Binzhou, 256603, People's Republic of China., Department of Nursing, Binzhou Polytechnic University, Binzhou, 256603, People's Republic of China.

Abstract:  Background: METTL3 is an RNA methyltransferase that mediates m6A modification and is implicated in mRNA biogenesis, decay, and translation. However, the biomechanism through which METTL3 regulates MALAT1-miR-1914-3p-YAP axis activity to induce NSCLC drug resistance and metastasis is not very clear. Methods: The expression of mRNA was analyzed by qPCR assays. Protein levels were analyzed by western blotting and immunofluorescent staining. Cellular proliferation was detected by CCK8 assays. Cell migration and invasion were analyzed by wound healing and transwell assays, respectively. Promoter activities and gene transcription were analyzed by luciferase reporter assays. Finally, m6A modification was analyzed by MeRIP. Results: METTL3 increased the m6A modification of YAP. METTL3, YTHDF3, YTHDF1, and eIF3b directly promoted YAP translation through an interaction with the translation initiation machinery. Moreover, the RNA level of MALAT1 was increased due to a higher level of m6A modification mediated by METTL3. Meanwhile, the stability of MALAT1 was increased by METTL3/YTHDF3 complex. Additionally, MALAT1 functions as a competing endogenous RNA that sponges miR-1914-3p to promote the invasion and metastasis of NSCLC via YAP. Furthermore, the reduction of YAP m6A modification by METTL3 knockdown inhibits tumor growth and enhances sensitivity to DDP in vivo. Conclusion: Results indicated that the m6A mRNA methylation initiated by METTL3 promotes YAP mRNA translation via recruiting YTHDF1/3 and eIF3b to the translation initiation complex and increases YAP mRNA stability through regulating the MALAT1-miR-1914-3p-YAP axis. The increased YAP expression and activity induce NSCLC drug resistance and metastasis. 

Retracted: No

PubPeer comments:
 Could the authors please comment on the apparent repeated band in Figure 2H? *** ![file] , 
 #1 Same question for Figure 2K. I will refrain from further thoughts until authors have had an opportunity to respond. *** ![file] , 
 Dear Dr Biskrensis: I appreciate your interest in our work and your comment on our article in the article. I have carefully re-examined our manuscript and experimental records. The quality for some WB bands in Figure 2K are low, leading to some confusion about the results. In fact, theses bands are the different bands with difference as indicated in the Figure2K. Thank you again for reading my article carefully, and I appreciates your work very much. ![file] Here are the original uncropped scans for Figure 2K. ![file] ![file] Moreover, for Figure 2H, the expression of YTHDF3 group was wrongly placed in the expression of Cyr61. I will contact the journal and Journal of Hematology Oncology magazine for correction as soon as possible. Thank you again for reading my article carefully, and I apologize for the mistake in my work. Here are the right Figure 2H. ![file] Here are the original uncropped scans for Figure 2H. ![file] , 
 #3 Thank you for your reply. I agree there appear to be some differences and I may be mistaken, but the images in the paper appear unexpectedly similar. The images you have provided here do show more differences but they appear to be already cropped, and perhaps printed on paper (?). An image of the uncropped gel from which these came could better address my concerns. I appreciate your patience and cooperation. , 
 Dear Actinopolyspora Biskrensis. when we performed western blot, the PDVF membrane was cut to the indecated size instructed by the protein size and number inorder to incubate with antibdoy, respectively. The images were obtained by the ChemiDoc TMTM XRS+ (Bio-Rad) instead of the developing the film to get the original uncropped scans. , 
 Another concern in this paper: every fourth smear in the Tubulin band in Figure 1A has a similar shadow underneath. See red arrows. *** ![file] , 
 Figure 2A inverse image. Some of the issues shown with like-colored boxes. *** ![file] , 
 Some unexpected similarities in figure 4e. *** Inverse image ![file] , 
 Figure 6k appears to have a repeated image. *** ![file] , 
 Dear Actinopolyspora Biskrensis, thank you very much for your comments on our research. It will be helpful for our future work. For Fig 1a, the smear, which was not similar, in the Tubulin band did not affect the result of the expression level of Tubulin. For Fig 2a and 4e, after carefully checking our original data, we found these bands are not similar and we used the arrows, oval (Fig 2a) and rectangle (Fig 4e) to show the differences in the backgrounds and bands in Fig 2a and 4e. For 6k, after carefully checking our original data, we found that the IHC image of ABCG2 was mistakenly duplicated into the IHC image of ERCC1 when organizing the results. The unintentional error has no bearing on the work s scientific conclusions. We would contact the editorial office for correction. , 
 ![file] , 
 #11 Dr. Guo: Although there may be differences in the images as you have indicated, it is the unexpected *similarities* which concern me. If you provide the original uncropped scans, Iu2019m sure this experimental data will reassure readers. , 
 There are also more duplications to be found among the pathology images. Here s an example from Figure 2. ![file] , 
 https: jhoonline.biomedcentral.com articles 10.1186 s13045-020-00942-x (Mega)Correction to: J Hematol Oncol 12:135 (2019) https: doi.org 10.1186 s13045-019-0830-6 u201cThe original article [1] contains errors in Fig. 2h, Fig. 2n and Fig. 6k: 1) In Fig. 2h, the protein band of YTHDF3 was mistakenly duplicated into the protein band of Cyr61. 2) In Fig. 2n, the image of Control+Vector treatment group was mistakenly duplicated into the siMETTL3u2009+u2009YTHDF3 treatment group, and the image of METTL3u2009+u2009siYTHDF3 treatment group was unintentionally duplicated into the METTL3u2009+u2009YTHDF3 treatment group which were determined by transwell assay. 3) In Fig. 6k, the IHC image of ABCG2 was unintentionally duplicated onto the IHC image of ERCC1. *** **Figure 2** ![file] YTHDF3 recognizes m6A modification and promotes cellular growth and migration via YAP upregulation. h The expressions of YAP and its target genes, CTGF and Cyr61, were analyzed by RT-PCR and western blot. n The cellular invasion and migration growths were analyzed by transwell assay. Results were presented as meanu2009u00b1u2009SD of three independent experiments. *Pu2009 u20090.05 or **Pu2009 u20090.01 indicates a significant difference between the indicated groups. NS, not significant *** **Figure 6** ![file] The reduction of YAP m6A modification inhibits tumor growth and enhances DDP sensitivity in vivo. k The tumor nodules from controlvector, METTL3Vector and METTL3shYap groups were treated with PBS or DDP every three days. The protein expression levels of Ki67, ABCG2 and ERCC1were analyzed immunohistochemical staining assays (nu2009=u20095). Results were presented as meanu2009u00b1u2009SD three independent experiments. **Pu2009 u20090.01 indicates a significant difference between the indicated groups. NS, not significant *** These errors were mistakenly introduced when organising the results; however, these errors have no bearing on the worku2019s scientific conclusions as the statistical results are based on the correct pictures. The authors would like to note the correct versions of each of the above-noted sub-figures ahead. The only changes are to the panels of Fig. 2h, Fig. 2n, and Fig. 6k, and the rest of the figures are identical to the published version; furthermore, no other errors were found after repeated checking of the published data. The authors apologise to the Editor of Journal of Hematology Oncology and to the readership for any inconvenience caused.u201d , 
 Regarding the published Erratum, where the authors claim that  no other errors were found after repeated checking of the published data : I think the authors should take another look at the paper. Figure 6i,h has a serious problem. ![file] , 
 Corrected figure 6k appears to still have an area of overlap. *** ![file] , 
 #15 If the scale rulers in Figs 6c and 6i are accurate, some of those xenograft tumours were larger than the mice they were growing in. , 
 In this publication, miR-1914-3p is reported to be involved in a lncRNA-miRNA-protein  axis. Why was miR-1914-3p chosen for this study? According to the paper, miR-1914-3p was chosen after consulting with three databases:  miRbase, miRanda, and TargetScan. We concluded that miR-1914-3p was the candidate miRNA that targets YAP. It is strange that miR-1914-3p was predicted to be the candidate miRNA. miRBase is not a target database. It links out to several miRNA-target prediction tools databases, but it does not make predictions. TargetScan predicts approximately 200 miRNAs miRNA families as possibly targeting YAP1. miR-1914-3p is not even on this list. There is one microRNA with the same seed, but it targets a  poorly conserved site. miR-1914-3p is also an extremely low-abundance miRNA according to miRBase and mirgenedb, suggesting a low likelihood of biological function. Its high GC content means that it is relatively difficult to work with and will be likely to be spuriously predicted as a targeting miRNA. It would be helpful if the authors could indicate why they passed over many abundant miRNAs with better-conserved target sites to choose a microRNA that seems to be a very poor candidate. Additionally, the methods do not indicate any microRNA-specific qPCR methods or primers used to RT and amplify miR-1914-3p. Could the authors please provide the missing methods and sequences? , 
 Two images in Figure 6e also appear to overlap, after rotation. I do not believe this was previously addressed. *** ![file] ,