Upregulation of miR-630 Induced by Oxidative Damage Resists Cell Migration Through Targeting ALCAM in Human Lens Epithelium Cells
Authors: Hong Yan
Xiaoliang Zheng
Bo Yan
Jing Zhao
Chenjun Guo
Song Wang
Angang Yang
Lin Mei
Journal: Current Eye Research (2020)
Discipline: CLINICAL MEDICINE
Keywords: Cataract; cell migration; microRNA; oxidative damage.
Affiliations: Department of Ophthalmology, Affiliated Guangren Hospital School of Medicine, Xi'an Jiaotong University, Xi'an No. 4 Hospital, Shaanxi Eye Hospital, Xi'an, Shaanxi Province, China., Department of Ophthalmology, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi Province, China., Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an, Shaanxi Province, China.

Abstract:  Purpose: To investigate the role of miRNAs in regulating oxidative damage during cataract formation.Methods: Microarray analysis and gene expression profiling assay were used to separately evaluate the miRNAs and mRNAs profiles in normal human lens epithelium cell line HLE-B3 treated by H2O2. The expression level of miR-630 was detected by RT-qPCR and the gene expression profiles were performed with gene ontology analysis using Bio Informatical database. The targets of miR-630 were predicted using miRecords and the results were used for screening targets of miR-630 combined with the GO analysis above. The mRNA levels of ALCAM, PCDH7, COL12A2, and EDIL3 in HLE-B3 cells after oxidative stimulation or miR-630 mimics transfection were measured by RT-qPCR, and the expression of ALCAM regulated by miR-630 was confirmed by Western blot and dual-luciferase reporter gene assay. The level of cell migration was measured by transwell assay and scratching test after transfection of miR-630 mimics and ALCAM siRNAs.Results: The microarray analysis demonstrated that miR-630 was significantly increased in HLE-B3 cells after oxidative stimulation. ALCAM, PCDH7, COL12A2, and EDIL3 were screened to be the possible targets of miR-630 by miRecords combined with GO analysis, but the results of RT-qPCR, Western blot and dual-luciferase reporter gene assay showed that only the expression of ALCAM was repressed by miR-630 transfection. Cell migration was inhibited through transfection of miR-630 mimics or ALCAM siRNAs and the upregulation of miR-630 partly reduced the cell migration increased by oxidative stimulation.Conclusion: miR-630 is one of the miRNAs increased by oxidative stimulation in human lens epithelium cells. Its upregulation may inhibit cell migration by targeting on ALCAM, which is important for HLECs to resist behavioral changes induced by oxidative damage and may delay the progression of cataract. 

Retracted: No

PubPeer comments:
 There appears to be some image overlap between Figures 5, 6, and 7, however they seem to be labeled differently. Could the authors explain. Also the Western blot in Figure 3 appears overexposed. Could the authors provide a copy of the original scan? Thank you. *** From Figure 5 ![file] *** From Figure 6 ![file] *** From Figure 7 ![file] , 
 Dear viewer: Thank you for reading our article. We have checked our paper after receiving your mail, and find the problems are caused by different reasons. For the two pictures labeled by red rectangles in Figure 5 and Figure 6, they are not the same picture, but two photos with overlapping visual field taken from negative control in our Transwell experiment. When we transfected miR-630 and ALCAM into cells to detect their influences, a nonsense RNA sequence purchased from RiboBio (Guangzhou, China) was used as negative control. In the Transwell migration assay, several spots were chosen to evaluate the migration ability of cells and the two pictures are two of the selected spots. However, when we took pictures, we didnu2019t notice they were so close that the visual fields of the two photos were partially overlapped. We are sorry for not discovering the flaw and we can dissolve the misunderstand by using additional photos from other selected spots of negative control group to replace. Revised Figure 6 For the picture labeled by green rectangle in Figure 7 which resembles the one in Figure 6, it was a careless mistake we made when we collected photos to make Figure 7. We will fix the problem by replacing the wrong photo with a right one. Revised Figure 7 For the Western blot in Figure 3, we can show you the photo of the bands. Bands of Western blot in Figure 3 We are grateful for your help to point out these flaws which should be found by ourselves and avoided before submission. Weu2019re sorry for the mistakes and will contact the journal to change them. Thank you again for viewing our research. With best wishes, Sincerely yours, Hong Yan , 
 ![file] , 
 #3 I suggest the authors provide the original uncropped scan as is expected by the journal. This is a very recent paper, so the original data would be expected to be still be available. u201dRegarding images of electrophoretic gels and immunoblots, where parts of the same gel or blot are spliced together, this should be indicated on the figures with a dividing line, making it clear where the image has been joined. Areas from different gels should not be spliced together. Where loading controls are present, these should always be included in the image; if spliced together, any modifications to the loading control and area of interest must be identical.u201d u201dImages of immunoblots must not be overexposed and must provide a true representation of the results without masking the presence of other bands which could lead to a different interpretation of the results. Where applying high contrast settings to the immunoblot is unavoidable, authors must be prepared to share images of the full, uncropped blots showing the original image (including molecular weight markers), and any other versions of the images at multiple exposures. Authors should be prepared to share the original, uncropped and unprocessed images of immunoblots with the journal editorial office upon request.u201d , 
 Dear Reviewer: Thank you for your response. I read the instruction of the journal about immunoblot again. Iu2019m so sorry that the Western blot in our reserch was performed in 2016 though our article was published in 2020, and the data of Western blot we had was limited to meet the demands of the journal, but we can repeat this part of our research and we will show you the updated result if is necessary. Thank you again for reviewing our article. Attached are the revised two figures (as previous one is oversize). With best wishes, Sincerely yours, Hong Yan![file] ![file] , 
 Dear reviewer: Weu2019re sorry for replying you after a long time because the repeat of the Western blot in our article takes a few days. Now, the experiment is finished and we would like to show you our latest result which we hope it could be acceptable. Thank you so much for your help and advice. With best wishes, Sincerely yours, Hong Yan![file] , 
 #6 Iu2019m confused. Are the authors unable to provide the original data for a paper published a year ago? If so, I would suggest the authors retract this paper and republish the new experiments. ,