Lentivirus-mediated microRNA-26a-modified neural stem cells improve brain injury in rats with cerebral palsy
Authors: Qi Guo
Jing Zhang
Songyan Liu
Zhaoshi Zheng
Xinxin Li
Fuli Wang
Journal: Journal of cellular physiology (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: apoptosis; astrocyte; cerebral palsy; gene modification; glial fibrillary acidic protein
Affiliations: Department of NeurologyChina–Japan Union Hospital of Jilin University Changchun Jilin Province China

Abstract:  This study is launched to investigate the effect of lentivirus-mediated microRNA-26a (miR-26a)-modified neural stem cells (NSCs) in brain injury in rats with cerebral palsy (CP). The successfully constructed miR-26a lentivirus expression vector and empty vector virus were used to modify NSCs. The model of CP with ischemia and anoxia was established in rats. NSCs and miR-26a-NSCs were stereoscopically injected into the cerebral cortex of the modeled rats, respectively. The survival and migration of NSCs infected with recombinant lentivirus expressing green fluorescence in vivo was observed under a light microscope. The neurobehavioral functions, morphology, and ultrastructure of cerebral cortex and hippocampus, apoptosis of brain cells, expression of apoptosis-related protein caspase-3 and Bax, together with the expression of the glial fibrillary acidic protein (GFAP) in cerebral cortex and hippocampus were determined. Expression of miR-26a in NSCs infected with plVTHM-miR-26a increased significantly. After NSCs transplantation, the neurobehavioral status of CP rats was improved, the degree of brain pathological injury was alleviated, the apoptotic index of cells in cerebral cortex and hippocampus and the expression of the apoptotic protein (caspase-3 and Bax) were decreased, the expression of GFAP were significantly decreased. After miR-26a-NSCs transplantation, these aforementioned results further improved or decreased. Our study suggests that miR-26a-modified NSCs mediated by lentivirus can improve brain injury, inhibit apoptosis of brain cells and activation of astrocytes in CP rats. 

Retracted: No

PubPeer comments:
 Figure 7. The two fugures have been cropped, stretched, and rotated, but they can still be identified as coming from the same result. ![file] , 
 I have several additional concerns about this study. 1. Could the authors please clarify the cloning strategy for the miR-26a precursor by giving the exact primer sequences? The rat precursor miR-26a sequence was reportedly cloned into an overexpression vector, which was then placed into rat NSCs that were injected into the brains of the subjects. However, the forward and reverse primer sequences in  Table 1 Primer sequence do not seem to recognize any amplicon in rat or human. 2. Could the authors also please clarify how miR-26a expression was verified? miR-26a expression is shown in Figure 2c and Figure 3g and h. Was this pre-miR-26a? Or one of the mature miRNAs? And how was it measured? The forward and reverse primer sequences for  miR-26a in  Table 1 Primer sequence would not amplify precursor or either mature miRNA. Also, no microRNA-specific reverse transcription procedure is mentioned. 3. It is concerning that the entire justification for this study of miR-26a in 96 rats was given in two lines and is based on three misinterpreted references.  As an important regulator changing the certain messenger RNAs (mRNAs) expression, miR-26a has been suggested to be vital in the central nervous system (Haapa-Paananen et al., 2013). In fact, Haapa-Paananen do not mention miR-26a in their study, which investigates  hsa-miR-129, -136, -145, -155, -181b, -342-5p, -342-3p, -376a b.  In addition, miR-2s (sic) is overexpressed in the brain, which also essential in neural cells specification (Rinaldi et al., 2010; Smirnova et al., 2005). Assuming  miR-26a is meant instead of  miR-2s, neither paper says that miR-26a is overexpressed in brain. Rinaldi et al say that miR-26a is upregulated during restraint-induced stress, and Smirnova et al say that miR-26 is more abundant in astrocytes than in neurons. That is, there was no clear justification for this study. Any random microRNA could have been chosen for any random disease model. Animal research is important, but it should be necessary and well designed. Could the authors please clarify their choice to do a large-scale animal study? , 
 Fig 5b. ![file] , 
 Fig 5a,  Pathological observation of left cerebral cortex in rats by HE staining . Many unexpected similarities. ![] , 
 Fig 3a,  At 7 days after transplantation, the CP + NCu2010NSC group could clearly see the transplanted cells by using frozen sections at the transplant site . ![file] I have concerns. , 
 Fig 5c,  Observation of ultrastructure of left cerebral cortex in rats by a TEM (one panel at random). ![file] , 
 Two panels of Fig 5d. This is just lazy. ![file] , 
 Figure 6C has some concerns. ![file] ,