UBE2C, Directly Targeted by miR-548e-5p, Increases the Cellular Growth and Invasive Abilities of Cancer Cells Interacting with the EMT Marker Protein Zinc Finger E-box Binding Homeobox 1/2 in NSCLC
Authors: Baoguang Hu
Weihua Di
Lingqun Kong
Yan Wu
Jing Du
Dan Jin
Jiwei Guo
Jiajia An
Wansheng Wang
Xiaohong Wang
Journal: Theranostics (2019)
Discipline: CLINICAL MEDICINE
Keywords: EMT; UBE2C; ZEB1/2; cellular growth; lung cancer metastasis
Affiliations: Clinical Medical Laboratory, Binzhou Medical University Hospital, Binzhou, 256603, P.R. China., Cancer research institute, Binzhou Medical University Hospital, Binzhou, 256603, P.R. China., Department of Thyroid and Breast Surgery, Binzhou Medical University Hospital, Binzhou, 256603, P.R. China., Department of Clinical Laboratory, Binzhou Medical University Hospital, Binzhou, 256603, P.R. China., Department of Gastrointestinal Surgery, Binzhou Medical University Hospital, Binzhou, 256603, P.R. China., Department of Hepatobiliary Surgery, Binzhou Medical University Hospital, Binzhou, 256603, P.R. China.

Abstract:  Background: Recent evidence indicates that UBE2C participates in carcinogenesis by regulating the cell cycle, apoptosis, metastasis, and transcriptional processes. Additionally, miR-548e-5p dysregulation plays a vital role in tumor progression. However, the molecular mechanism via which UBE2C is directly targeted by miR-548-5p, resulting in increase in cellular growth and invasiveness of cancer cells, and its interactions with the epithelial-mesenchymal transition (EMT) marker protein ZEB1/2 in non-small cell lung cancer (NSCLC) is not understood. Methods: Expression of UBE2C and miR-548e-5p was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The protein level of UBE2C and ZEB1/2 was analyzed using western blotting and immunofluorescence staining. Cellular proliferation was detected using the cell counting kit 8 (CCK8) and 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell migration, invasion, and growth were analyzed using the wound healing and transwell assay. Promoter activity and transcription was analyzed using the luciferase reporter assay. Chromatin immunoprecipitation was used to detect binding of UBE2C to 5'UTR-ZEB1/2. Results: We observed that 4,5-ubiquitin-conjugating enzyme E2C (UBE2C) expression was higher in NSCLC tissue than in the adjacent normal tissue and was associated with increased cell proliferation and invasion. UBE2C enhanced NSCLC progression and metastasis by affecting the cell cycle and inhibiting apoptosis. We also observed that miR-548e-5p was significantly downregulated in lung cancer tissue specimens, which decreased the expression of its direct substrate, UBE2C. Moreover, miR-548e-5p overexpression and UBE2C under-expression significantly suppressed lung cancer cell proliferation, migration, and invasion. Luciferase reporter and chromatin immunoprecipitation assays indicated that miR-548e-5p directly binds to the 3'-UTR of UBE2C and decreases UBE2C mRNA expression. Furthermore, UBE2C knockdown downregulated the mesenchymal marker vimentin and upregulated the epithelial marker E-cadherin. Bioinformatics assays, coupled with western blotting and luciferase assays, revealed that UBE2C directly binds to the 5'-untranslated region (UTR) of the transcript of the E-cadherin repressor ZEB1/2 and promotes EMT in lung cancer cells. Conclusion: miR-548e-5p directly binds to the 3'-UTR of UBE2C and decreases UBE2C mRNA expression. UBE2C is an oncogene that promotes EMT in lung cancer cells by directly targeting the 5'-UTR of the transcript encoding the E-cadherin repressor ZEB1/2. miR-548e-5p, UBE2C, and ZEB1/2 constitute the miR-548e-5p-UBE2C-ZEB1/2 signal axis, which enhances cancer cell invasiveness by directly interacting with e EMT marker proteins. We believe that the miR-548e-5p-UBE2C-ZEB1/2 signal axis may be a suitable diagnostic marker and a potential target for lung cancer therapy. 

Retracted: Yes

PubPeer comments:
 Figure 1 seems confused. Some of the Tubulin bands in 1D seem to reappear in 1H as Lamin bands, but only after a horizontal flip. Could the authors review and comment? *** ![file] , 
 #1 Similar issues with figure 3A and 3B. There are some other background features which repeat, but Iu2019m not certain this isnu2019t a dirty scanner. *** Contrast enhanced ![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 Figures are low, leading to some confusion about the results. In fact, theses bands are the different bands with difference as indicated in the Figures. Thank you again for reading my article carefully, and I appreciates your work very much agian! ![file] The original uncropped scans for Figure 1D and 1H were following: Figure 1H ![file] Figure 1D ![file] ![file] The original uncropped scans for Figure 3A and 3B were following: Figure 3A ![file] Figure 3B ![file] ![file] , 
 #3 Thank you for responding. By uncropped, I mean a full gel scan similar to those below: ![file] Regarding 1H and 1D: As before, I agree there appear to be some differences and I may be mistaken, but the images in the paper appear unexpectedly similar, including some background features which do not appear in the u201coriginalsu201d you provided. An image of the uncropped gel from which these came could address my concerns. , 
 #3 For the  original scans you provided for Fig. 3A B, I see the striking similarities between the control of the 109 bp GAPDH (Fig. 3A) and that of the 112 bp ZEB2 (Fig. 3B) ![] , 
 #3 Dear Actinopolyspora Biskrensis. when we performed western blot, the PDVF membrane was cut to the indecated size instructed by the protein size and number. The images were obtained by the ChemiDoc $TM$ XRS+ (Bio-Rad) instead of the developing the film. Therefore original uncropped scans were showed as Figure 1D and 1H. , 
 #5 Dear Oeneis Buddha, although they are similar but they are actually different as indicated as follow: ![file] , 
 There appears to be a concerning duplication in figure 8. The contents of the highlighted green boxes appear near-identical. However, the surrounding parts of the image deviate suggesting this is not a simple case of the same image having accidentally been uploaded twice when preparing the manuscript. Could the authors perhaps shine a light on this? ![file] , 
 After inverting the images in Figures 2A and 2B, several areas of abrupt transition appear, suggesting the figures may have been spliced. Some areas of concern noted with arrows. *** Inverse image ![file] *** Original image ![file] , 
 Retracted [25 July 2020] .  The Editor-In-Chief of Theranostics, in consultation and agreement with the editorial board members, retracts the article u201cUBE2C, Directly Targeted by miR-548e-5p, Increases the Cellular Growth and Invasive Abilities of Cancer Cells Interacting with the EMT Marker Protein Zinc Finger E-box Binding Homeobox 1 2 in NSCLCu201d [1] on the basis of questions related to irregularities within several figures. The questions about data irregularities also raise questions about the conclusions within the paper. ,