Circular RNA circ_0003204 inhibits proliferation, migration and tube formation of endothelial cell in atherosclerosis via miR-370-3p/TGFβR2/phosph-SMAD3 axis
Authors: Yang Yang
Guixiang Song
Shan Xu
Aihua Wang
Shan Qiao
Jing Yuan
Shanchao Zhang
Xuxu Xu
Zhihua Si
Journal: Journal of Biomedical Science (2020)
Discipline: CLINICAL MEDICINE
Keywords: Atherosclerosis; Endothelial cell; Hsa_circ_0003204; MiR-370-3p; TGFβR2.
Affiliations: Department of Neurology, the First Affiliated Hospital of Shandong, First Medical University, NO.16766 JingShi Road, Jinan, 250014, Shandong, China. ., Department of Neurology, the First Affiliated Hospital of Shandong, First Medical University, NO.16766 JingShi Road, Jinan, 250014, Shandong, China.

Abstract:  Background: Circular RNAs (circRNAs) represent a class of non-coding RNAs (ncRNAs) which are widely expressed in mammals and tissue-specific, of which some could act as critical regulators in the atherogenesis of cerebrovascular disease. However, the underlying mechanisms by which circRNA regulates the ectopic phenotype of endothelial cells (ECs) in atherosclerosis remain largely elusive. Methods: CCK-8, transwell, wound healing and Matrigel assays were used to assess cell viability, migration and tube formation. QRT-qPCR and Immunoblotting were used to examine targeted gene expression in different groups. The binding sites of miR-370-3p (miR-370) with TGFβR2 or hsa_circ_0003204 (circ_0003204) were predicted using a series of bioinformatic tools, and validated using dual luciferase assay and RNA immunoprecipitation (RIP) assay. The localization of circ_0003204 and miR-370 in ECs were investigated by fluorescence in situ hybridization (FISH). Gene function and pathways were enriched through Metascape and gene set enrichment analysis (GSEA). The association of circ_0003204 and miR-370 in extracellular vesicles (EVs) with clinical characteristics of patients were investigated using multiple statistical analysis. Results: Circ_0003204, mainly located in the cytoplasm of human aorta endothelial cells (HAECs), was upregulated in the ox-LDL-induced HAECs. Functionally, the ectopic expression of circ_0003204 inhibited proliferation, migration and tube formation of HAECs exposed to ox-LDL. Mechanically, circ_0003204 could promote protein expression of TGFβR2 and its downstream phosph-SMAD3 through sponging miR-370, and miR-370 targeted the 3' untranslated region (UTR) of TGFβR2. Furthermore, the expression of circ_0003204 in plasma EVs was upregulated in the patients with cerebral atherosclerosis, and represented a potential biomarker for diangnosis and prognosis of cerebrovascular atherogenesis. Conclusions: Circ_0003204 could act as a novel stimulator for ectopic endothelial inactivation in atherosclerosis and a potential biomarker for cerebral atherosclerosis. 

Retracted: Yes

PubPeer comments:
 Brought to my attention by Prakash Hande, I was enticed by this advertisement on Twitter to look at some u201c[high-impact publications in Life Sciences Biomedicine by Chinese researchers] ,u201d and this was the first paper I looked at. Boy am I sorry. A series of image concerns follows. ![file] , 
 Figure 2G. ![file] , 
 Figure 2E and 5G. ![file] , 
 Figure 5B *** ![file] , 
 Figure 5C and 5H seem to have been imaged with dirty equipment, but there appears to be at least one repeated image. I am not certain that this is intended. *** Enhanced contrast ![file] *** Showing location and caption ![file] , 
 #1 * Boy am I sorry. * While I - for one - am not in the least bit sorry that *A. biskrensis* reported issues in the figures of this paper before I could get around to it, I had likewise noted a few issues. So here is my second opinion, for what it is worth. In the Year of the Metal Ox, the season of the Mitch (as it was almost but not quite sung), Springer Nature now exhort us to enjoy their **u201cHappy Springu00a0Festival! Explore 300+ high impact publications by Chinese researchersu201d** which were published in that most recent Year of the Metal Rat. https: www.springernature.com gp researchers campaigns chinese-research *u201cWe have curated a collection of the most recent influential publications by Chinese researchers from across BMC, Nature Portfolio, Palgrave Macmillan and Springeru00a0including 300+ high impact journal articlesu00a0and most downloaded book chapters.u00a0We hope you enjoy reading this selection and find it helpful.u201d* This particular high impact article concerns a circular RNA. This is a topic much beloved of sundry PubPeer commentators. After exploring Fig. 5B, a duplicate control representing different experiments is hereby curated in the linked image. I hope you enjoy reading this selection and find it helpful. ![image] You know sometime you re bound to leave her But for now you re going to stay In the year of the rat , 
 #5 As the elephant fecal matter is to the dung beetle, so the circular RNA to the figure sleuth. Ditto for lncRNA and microRNA. Perhaps, in an idealised scientific literature from a galaxy far, far away, these research topics would not have this unusual attractiveness. I might not be a top Springer-Nature editor, but if I had edited this high impact manuscript, I would have complained about the poor quality of the wound assay images: It is not easy to see either the cells or their absences. Seeing is believing. And I surely canu2019t have refereed the manuscript because I just know that I would have sent back these images for the inconsistent scratch widths. Typically, wound healing or scratch assays are set up by scraping the cell monolayer with a pipette tip to create a ~0.5 mm wide cell-free zone into which the cells may migrate. At least if Wikipedia is to be believed. https: en.wikipedia.org wiki Wound_healing_assay Conventionally, lines may be drawn to indicate the boundaries of the scratch so that any cells inside will have moved back in. It would actually be better to show the field at zero hours after the scratch was made and then again after the allotted time, as in 24 hours here. Bizarrely, in three parts of Fig. 5, a single zero hour image is shown for each of the four 24 hour images, which are all different experimental conditions. Does not compute. As far as I can tell, the irregular spacing of the lines is being used here to indicate how far the cells have migrated back in. This is a seriously poor presentational approach because the cells do not advance uniformly into the scratch, so the lines have no actual meaning. What makes this worse is that in Fig. 2F, the classical line representation was already used. What is more, the zero hours are each supplied too. Just like it should be! Yay! Unfortunately, though, it turns out that one of the zero hour images in Fig. 2F then reappears as that arbitrary zero hour in Fig. 5C. I hope you enjoy reading this selection and find it helpful. ![image] , 
 Thanks so much for pointing out these errors for us. After checking the data, we found that in Figure 2G, the u201cCTLu201d image of u201csi-circ_0003204u201d had an error of overlap with u201cox-LDLu201d image of u201csi-circ_0003204u201d as  si-NC and  NC images of  ox-LDL , and the image about cell migration in Figure 2E had incorrect overlap with Figure 5G. The images were taken mistakenly due to filename confusion, and we are so sorry not to notice the duplicated images in Figure 5B at submission step, and this image will be updated with correct images. We are sorry not to have good quality of the wound assay images to see the cells clearly. We decided to make amends for these mistakes. We will ask for erratum to the journal as soon as possible. Thanks again for your comments. We apologize for these mistakes to the journal editors, reviewers and readers for any inconvenience, and will pay attention to this and not make the same mistakes in the future. Best Wishes. , 
 #8 Thank you for your reply. Please post the original data you intend to provide the journal here as well. Given the journalu2019s lackadaisical approach to editorial review, it seems that post publication peer review is the only way to prevent a recurrence of these mistakes in the revised manuscript. , 
 #8 Thank you for your quick reply. it turns out that circular RNA has become a surprisingly awkward area in which to conduct scientific research. Itu2019s entirely up to your team of course, but it might be worth reviewing the procedures for archiving and retrieving the data generated by your groupu2019s research activities. As the era of Open Science bears down upon upon us, it could be good to remember that u201cfortune favours the prepared mindu201d. And the converse. While you are checking your published images versus the research data that you have generated, might you be able to compare the two fields enclosed in the blue ellipse? They are much too similar to be unrelated but, at the same time, different enough not to be the same image repurposed. Confusingly, the scratch lines are differently positioned. Hopefully your archiving system can make this rather mystifying connection transparent. ![image] , 
 Retraction, October 2021: https: jbiomedsci.biomedcentral.com articles 10.1186 s12929-021-00761-3  The Editor-in-Chief has retracted this article. Concerns were raised regarding a number of figures, specifically: * Figure 2E: the CTL NC panel appears to partially overlap with the oxLDL SIS3 panel of Fig. 5G; * Figure 2F: the 0 h si-circ_0003204 panel appears to be identical with the 0 h panel of Fig. 5C; * Figure 2G: the CTL and ox-LLD panels for si-circ_0003204 appear to partially overlap; * Figure 2G: the ox-LDL panels for si-NC and NC appear to partially overlap; * Figure 5B: the inhibitor NC and miR-370 inhibitor panels for siRNA NC appear to be identical; * Figure 5C: the last panel appears to be identical to the second panel of Fig. 5H. The Editor-in-Chief therefore no longer has confidence in the reliability of the data reported in the article. Shanchao Zhang, Guixiang Song, Shan Qiao, Zhihua Si, Yang Yang, and Aihua Wang agree to this retraction. Jing Yan, Shan Xu, and Xuxu Xu have not responded to correspondence regarding this retraction. ,