Long non-coding RNA LINC00470 in serum derived exosome: a critical regulator for proliferation and autophagy in glioma cells
Authors: Yu Zhou
Qilin Qin
Wenjia Ma
Yan Cui
Min Liu
Journal: Cancer Cell International (2021)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Autophagy; Exosome; Glioma; LINC00470; PI3K/AKT/mTOR
Affiliations: Department of Neurosurgery, The Second Xiangya Hospital, Central South University, No. 139, Renmin Road, Changsha, 410011, Hunan, People's Republic of China., Department of Neurosurgery, The Second Xiangya Hospital, Central South University, No. 139, Renmin Road, Changsha, 410011, Hunan, People's Republic of China. .

Abstract:  Background: To explore the mechanism of LINC00470 in serum exosomes from glioma patients regulating the autophagy and proliferation of glioma cells. Methods: Exosomes were extracted from glioma patients (GBM-exo). Expression of LINC00470 in exosomes was analyzed with the clinicopathological characteristics of glioma patients. Glioma mouse model was established. The effects of LINC00470, miR-580-3p and WEE1 on cell autophagy and proliferation, as well as the activation of PI3K/AKT/mTOR pathway were measured. Dual luciferase reporter assay and RNA immunoprecipitation (RIP) were conducted to validate the binding of LINC00470 and miR-580-3p and of miR-580-3p and WEE1. Results: LINC00470 overexpressed in GBM-exo and associated with disease severity and postoperative survival time of glioma patients. GBM-exo deteriorated tumor progression in nude mice. Cells incubated with GBM-exo or transfected with pcDNA3.1-LINC00470/miR-580-3p inhibitor/pcDNA3.1-WEE1 had less autophagosome, downregulated LC3-II/LC3-I and Beclin1 expression levels and increased expression of p62 as well as strengthened proliferation ability. The PI3K/AKT/mTOR pathway was activated. LINC00470 competitively bound to miR-580-3p with WEE1. Conclusion: LINC00470 in GBM-exo can bind to miR-580-3p in glioma cells to regulate WEE1 expression and activate the PI3K/AKT/mTOR pathway, thereby inhibiting autophagy and enhancing the proliferation of glioma cells. 

Retracted: No

PubPeer comments:
 The methodologies used in this paper do not support conclusions about exosomes. 1. The method for exosome extraction is polymer based precipitation. This method does not specifically isolate exosomes or other extracellular vesicles (EVs). In serum, EVs are vastly outnumbered by lipoprotein particles, ribonucleoprotein particles and protein aggregates, and most of these particles are precipitated by polymer-based kits. 2. To show a specific role for exosomes, one must compare the relative presence and contributions of exosomes that come from the multivesicular body with other EVs like microvesicles from the plasma membrane, and also analyze other particles that commonly co isolate with EVs like lipoproteins and protein aggregates. Single particle characterization might identify exosomes and microvesicles in cell culture after rigorous characterisation of cellular compartments, but this cannot easily be done with bulk characterisation and not in serum where EVs come from many different cell types. The tetraspanins for example are not specific to exosomes even though some can be enriched in some membrane types. NTA is also not specific to exosomes or EVs. From serum, there might be 1000 precipitated lipoprotein particles per EV. 3. For uptake experiments lipid dyes are now well known to label serum proteins and obviously lipoproteins, and most can also form self aggregates. Lipid dye experiments are not really acceptable used as evidence of EV uptake. This is a problem with many papers, not just this one. 4. Because serum already contains RNase, addition of RNases alone will not digest RNA that is protected by proteins or lipids. One must add protease and or detergents to expose the RNA. Here, detergent is used, but crucial details like concentration and exposure time are not given so these experiments cannot be interpreted. Because of these issues, the study is not about exosomes and also not really about EVs more generally. It is a study of polymer precipitated particles from serum. The measurement of the lncRNA in serum precipitates is arbitrarily divided into  high and  low expression, and survival plots are then generated. The method of categorisation into low and high is not given. Would it not be better to assign absolute levels and plot against survival for all patients. This is a problem I see in many papers. There are several issues and discrepancies around the animal model experiments and their relationship with the in vitro experiments. 1. The results section mentions four treatment groups:  U251-GFP-Luc cells or U251-GFP-Luc cells transfected with sh-LINC00470 were inoculated into nude mice after 24 h of incubation with serum exosomes from HCs or glioma patients. However, results for the condition of sh-LINC00470-transfected cells exposed to HC serum precipitates are not described or shown, and the methods state that only three treatment groups were done. Maybe this is just a mistake in the text? But wouldn t it be an important control? 2. The number of mice in each condition is never explicitly stated. The methods say that 24 mice were purchased, but not how they were assigned or used. The figure and figure legend also do not state this. Images are shown for only one mouse per condition. 3. Results and methods state that the U251 cells were exposed for 24 hours to serum precipitates from patients or controls and then the CELLS were injected into the mouse model and monitored for 28 days. However, the figure legend states that the mice were  injected with serum exosomes of glioma patients. 4. No consideration is given to the possibility that the shRNA treatment has altered something besides the lncRNA. 5. It is difficult to understand how the minute quantities of lncRNA found in precipitated serum particles would enter U251 cells during a 24 hour incubation and then have effects in those cells after implantation into mice that become apparent only by 21 and 28 days post-implantation. There is no in vivo control to show that this is feasible and the in vivo experiments go for only 96 hours maximum. 6. For most experiments, the basic details of treatments and conditions are not provided. How many  exosomes were used to treat cells? How were they measured? How much RNA was transfected? For how long? Most of the work is not described in enough detail to be reproduced. ,