LINC00459 sponging miR-218 to elevate DKK3 inhibits proliferation and invasion in melanoma
Authors: Zhihai Cao
Zhuojun Zheng
Wenxian Xu
Yuhua Yang
Journal: Scientific Reports (2019)
Discipline: Multidisciplinary
Keywords: lncRNA biomarkers; melanoma; microarray analysis; ceRNAs; bioinformatics analysis
Affiliations: Department of Dermatology, The Third Affiliated Hospital of Soochow University, Changzhou, China., Department of Pathology, The First Affiliated Hospital of Soochow University, Suzhou, China., Department of Hematology, The Third Affiliated Hospital of Soochow University, Changzhou, China. ., Department of Emergency, The Third Affiliated Hospital of Soochow University, Changzhou, China. .

Abstract:  The lncRNA biomarkers in melanoma remain to be further explored. The lncRNAs with different expression levels in melanoma tissue were identified by microarray analysis. To investigate the biological functions of target lncRNA, several in-vivo and in-vitro studies were performed. Potential mechanisms of competitive endogenous RNAs (ceRNAs) were predicted by using bioinformatics analysis and explored by western blot assay, fluorescence in situ hybridization assay, real-time quantitative PCR (RT-qPCR) array, RNA pull-down analysis, AGO2-RIP assay, and dual-luciferase reporter assay. The results demonstrated decreased LINC00459 in melanoma cell lines and tissues. According to the in-vitro and in-vivo experiments, up-regulated LINC00459 had inhibitory effect on cell proliferation and invasion. Bioinformatics analyses suggested that miR-218 could be a direct target of LINC00459. In addition, miR-218 was proved to be able to directly target the dickkopf-related protein 3 (DKK3) gene. In conclusion, our analysis suggested that the LINC00459 could sponge miR-218 and increase the expression of DKK3 gene, thus inhibiting the invasion and proliferation of melanoma cells, which indicated that the LINC00459 could be an effective biomarker for melanoma and its potential as the therapeutic target. 

Retracted: No

PubPeer comments:
 I am questioning the exosome data in this study. The microsopy data is not clear. The morphology and ultra structure is questionable and there is no immuno staining. The CD63 immunoblots do not show any smearing and is therefore most probably not representing CD63. ![file] , 
  The CD63 immunoblots do not show any smearing and is therefore most probably not representing CD63. I do not find this comment to be scientifically sound. I am puzzled as to why does Indigofera expects the CD63 bands to show smears. I am attaching two such papers where the CD63 bands are very clear. Paper 1.  Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages DOI: doi.org 10.1371 journal.pone.0073706 ![file] Paper 2. DNA in serum extracellular vesicles is stable under different storage conditions DOI: 10.1186 s12885-016-2783-2 ![file] The presence or absence of smears in a Western blot depends on many factors and questioning any study just on the basis of these superficial things is not appropriate. Science is done on the basis of evidence, not emotions. , 
 Figure 3B appears to have some concerning areas of sharp transition. Could the authors provide original uncropped scans? *** Enhanced contrast ![file] , 
 #3 Thank you for your question. The original image mentioned in your question was provided as below. ![file] , 
 #2 Thank you for your detailed and clear explanation! , 
 #1 Thank you for your questions. Clearer TEM images of exosomes were shown as below. ![file] , 
 Please note that quite many researchers are not very experienced with WB to detect CD63, and many publications unfortunately show wrong protein band when using anti-CD63. Even some antibody producers are not familiar with the fact that CD63 is detected under non reducing conditions and always have a smeared appearance. Unfortunately, many of the anti-CD63 that are available may show unspecific bands, especially when used under the usual reducing conditions! I have tested several antibodies against CD63 and the first anti-CD63 blots I ran was under reducing conditions, simply because the producer (Santa Cruz Biotechnology) did not give any information regarding this. When testing several anti-CD63 I noticed that they indicated some faint bands at different MW, but after reading the literature more carefully, I learned that CD63 should be detected under non reducing conditions. When I changed to non reducing conditions, I detected a smeared band that was consistent in all further WBs. I also tested another antibody from Thermo Fischer (the producer of the exosome isolation kit I used) and got the same smeared band under the recommended non-reducing conditions. Furthermore, to be sure that the band was specific, I compared isolated exosomes with and a negative control (where I removed the exosomes) and the band was not detectable. Please see: https: tools.thermofisher.com content sfs manuals exosome_hu_cd63_det_man.pdf https: www.scbt.com p cd63-antibody-mx-49-129-5 , 
 I am still not convinced with the provided EM data presented here. You need to run immunogold labeling with cryo-electron microscopy to be sure. We always do that with new batches of isolated exosomes. , 
 I concur with Indigofera Tanganyikensis that a complete lack of smearing in Western blots for CD63 is a strong indication that the band is unspecific and may not be related to CD63. I have personally run perhaps a hundred blots over the years for exosomes or extracellular vesicles from many different cell lines and bio fluids, using several different antibodies for CD63 and I have never gotten sharp bands. Always I observe the extensive smearing expected for CD63, an extensively modified protein. , 
 I have to concur about the CD63, especially from exosomes. Here s a page from a site that sells exosomes, using the same antibody as in this paper. https: www.biocat.com cell-biology exosomes purified-exosomes-from-human-and-mouse-cell-lines , 
 In addition to the issues raised above, the methodologies in this study are not specific to exosomes. Polymer precipitation, as used here, does not specifically isolate exosomes (extracellular vesicles, EVs, from the multivesicular body) or even EVs in general. A polymer precipitate of blood plasma will consist almost entirely of lipoprotein particles and protein aggregates. Although a relatively small number of EVs may also be present, exosomes cannot be separated from other EVs, and they will have little contribution to protein findings like those reported here. For more information on rigorous isolation and characterization of EVs, see https: www.tandfonline.com doi full 10.1080 20013078.2018.1535750 ,