Down-regulating microRNA-20a regulates CDH1 to protect against cerebral ischemia/reperfusion injury in rats
Authors: Xin-Gang Li
Xiang-Pin Wei
Xian-Ming Fu
Chun-Chun Yang
Lan-Jun Xie
Gang Li
Ling-Tao Qian
Hong-Bo Liu
Xian-Wei Zeng
Journal: Cell Cycle (2021)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Cerebral ischemia/reperfusion injury; cadherin-1; microRNA-20a; nerve injury; oxidative stress
Affiliations: Department of Neurosurgery, Qilu Hospital, Cheeloo College of Medicine, Shandong University , Jinan, Shandong, China., School of Medicine, Shandong University , Jinan, Shandong, China., Department of Neurosurgery, Fuyang People's Hospital , Fuyang, Anhui, China., Department of Neurosurgery, AnHui Provincial Hospital, Shandong University , Anhui, China., Department of Neurosurgery, Fuyang People's Hospital , Fuyang, China., Department of Stroke Center, Fuyang People's Hospital , Fuyang, China.

Abstract:  Studies have extensively focused on the involvement of microRNAs (miRNAs) in cerebral ischemia/reperfusion (I/R) injury but not much on the specific role of miR-20a. Hence, this study is purposed to decipher whether miR-20a could regulate cadherin 1 (CDH1) to affect cerebral I/R injury in rats. Rat transient middle cerebral artery occlusion model (MCAO) was established. Rats were injected with lentiviral solution containing miR-20a inhibitor, or overexpressed CDH1 or combined depleted miR-20a and CDH1 to explore their roles in cerebral I/R injury. Oxidative stress-related factors, miR-20a, CDH1, nuclear factor-kappaB (NF-Œ∫B) and Nestin expression in brain tissues were detected by RT-qPCR and western blot assay. The target relation between miR-20a and CDH1 was predicted by online website and further confirmed by luciferase activity assay. In rats with cerebral I/R injury, increased miR-20a and decreased CDH1 were found in brain tissues. Reduction of miR-20a or elevation of CDH1 attenuated behavior function in MCAO rats. Inhibiting miR-20a or restoring CDH1 restrained oxidative stress, attenuated pathological damage of neurons, promoted neuron survival, and down-regulated NF-Œ∫B and Nestin expression in brain tissues of MCAO rats. CDH1 was determined to a target gene of miR-20a. This study elucidates that down-regulating miR-20a elevates CDH1 to protect neurons from cerebral I/R injury, which paves a new way for treatment of cerebral I/R injury. 

Retracted: No

PubPeer comments:
 Fig 1b, 7a,  Changes in CDH1 protein content in rat brain tissues were determined by western blot analysis ....  Protein bands of NF-u03baB and Nestin in rat brain tissues .  These are curiously stylised Western Blots, devoid of background, with little progressive change in the shape of the protein blobs as the bands progress along the gel. Is it possible for the authors to upload a scan of the original? For comparison, here are Figs 5b,d and 7b from [Tang Hu (2021)] .  b HDAC1 and NPY protein expression in hippocampal tissues after inhibition of HDAC1 by western blot analysis... d NPY protein expression in hippocampal tissues after inhibition of miR-124-5p by western blot analysis ;  NPY protein expression in hippocampal tissues after inhibition of both miR-124-5p and NPY by western blot analysis . They seem to come from the same little region within  morphology space .  , 
 This article shares several characteristics with others in the eyebrow family of papers identified by Hoya Camphorifolia. 1. Table 1 is  Primer sequence 2. Primer problems: Although investigated miRNA is miR-20a-3p (Figure 1C), the fwd primer is for miR-20a-5p. Reverse primer unclear. 3. miRNA RT: miRNA-specific RT method, but no regular RT is mentioned. Unclear how non-miRNAs were RT ed 4. miRNA normalizer: U6 and GAPDH 5. miRNA-target alignment figure is mis-aligned , 
 Fig 4e,  Nissl staining of the integrity of neurons in the cortex .  Colored boxes mark overlaps with panels of Fig 5B of [Jim et al (2021)] ,  Nissl staining detected the survival of neurons in brain tissues .  , 
 Thanks for your comment. The western blots of Fig 1b, 7a in our paper (pubmed: 33345691 doi: 10.1080 15384101.2020.1856498) were simply processed by PS software, not scan of the raw western blots. Regarding your question about Fig 1b, 7a, we have contacted the author who did the western blots experiment to provide the raw western blots. However, due to improper storage of the tape during the relocation of the laboratory, the raw western blots severely damaged. In order to solve this problem, we have repeated western blots experiment (attachment for the raw western blots) and the results are consistent with the conclusion of the paper. Later, we will make errata on this issue.      , 
 Because the basic research on miRNA and rat brain tissue ischemia-reperfusion injury are certain routines, it is inevitable that there are similarities. During the research, we have ordered rat miR-20a-5p primers (forward: 5 -GCTGCCGTAAAGTGCTTATAGTG-3 , reverse: 5 -AGAGCAGGGTCCGAGGTA-3 ) and rat miR-20a-3p primers (forward: 5 -ACTGCATTACGAGCACTTACA-3 , reverse: 5 -AGAGCAGGGTCCGAGGTA-3 ). Due to carelessness, we have provided rat miR-20a-5p primers in Table 1 of the paper. We will make errata later. For miRNA RT reaction, we used ToYoBo reverse transcription reagent. The amount of experimental reagents were as follows: 4x DN Master Mix 2 u03bcl, RNA template 0.5pguff5e0.5u03bcg, 5x RT Master Mix II 2 u03bcl, add Nuclease-free Water to 10 u03bcl . The reaction steps were as follows: 37u00b0C 15 min, 50u00b0C 5 min, 98u00b0C 5 min, 4u00b0C 30 min. Since these experimental reagents and procedures are very common, only a brief overview is given in the paper. The miR-20a and CDH1 3 -UTR sequence binding site prediction is a direct screenshot from the TargetScan (http: www.targets can.orgd) database. Due to the misalignment of the magnification, there is no alignment. Later, we will make errata on this issue. , 
 The reason for this error is that due to insufficient magnification during the microscope observation process, the cortical neurons appear to be basically the same in shape, which leads to the misuse of the images. We have re-provided the experimental images of Fig 4e, and we will make an errata on this issue later.  ,