MicroRNA‚Ä?32 inhibits the aggressiveness of glioblastoma multiforme by directly targeting IGF‚Ä?R
Authors: Meixiao Zhu
Xufei Zhang
Xudong Chen
Shunjin Sun
Journal: International Journal of Molecular Medicine (2019)
Discipline: CLINICAL MEDICINE
Keywords: glioblastoma multiforme; microRNA-432; IGF-1R.
Affiliations: Department of Neurosurgery, Sixth Affiliated Hospital of Wenzhou Medical University, Lishui, Zhejiang 323000, P.R. China., Laboratory Animal Center, Wenzhou Medical University, Wenzhou, Zhejiang 325035, P.R. China., Department of TCM Pharmacy, Sixth Affiliated Hospital of Wenzhou Medical University, Lishui, Zhejiang 323000, P.R. China.

Abstract:  MicroRNA‚Ä?32 (miR‚Ä?32) has been studied in multiple tumors, but the expression status, biological functions and the mechanism of action of miR‚Ä?32 in glioblastoma multiforme (GBM) are yet to be elucidated. In the present study, miR‚Ä?32 expression in GBM was determined and its clinical significance was evaluated among patients with GBM. The effects on the malignancy of GBM in vitro and in vivo were examined in detail and the interactions between miR‚Ä?32 and insulin‚Äëlike growth factor 1 receptor (IGF‚Ä?R) mRNA were then explored. miR‚Ä?32 expression in GBM tissue samples and cell lines was measured by reverse transcription‚Äëquantitative (RT‚Äëq)PCR. GBM cell proliferation, apoptosis, migration and invasion in vitro and tumor growth in vivo were evaluated by a Cell Counting Kit‚Ä? assay, flow‚Äëcytometric analysis, Transwell migration and invasion assays, and a tumor xenograft experiment, respectively. Bioinformatic analysis followed by a luciferase reporter assay, RT‚ÄëqPCR and western blotting was applied to demonstrate that IGF‚Ä?R is a direct target gene of miR‚Ä?32 in GBM cells. It was found that miR‚Ä?32 is downregulated in GBM tumors and cell lines. miR‚Ä?32 under expression obviously correlated with the Karnofsky Performance Status score and shorter overall survival among patients with GBM. Exogenous miR‚Ä?32 expression significantly reduced proliferation and induced apoptosis of GBM cells. In addition, miR‚Ä?32 overexpression impaired the migratory and invasive abilities of GBM cells in vitro and decreased their tumor growth in vivo. Furthermore, IGF‚Ä?R was validated as a direct target gene of miR‚Ä?32 in GBM cells. IGF‚Ä?R knockdown imitated the tumor‚Äësuppressive actions of miR‚Ä?32 overexpression in GBM cells. Rescue experiments proved IGF‚Ä?R downregulation to be essential for the effects of miR‚Ä?32 on GBM cells. The results of the present study revealed a tumor‚Äësuppressive role of the miR‚Ä?32‚ÄëIGF‚Ä?R axis in GBM cells and this axis may have implications for GBM therapy. 

Retracted: No

PubPeer comments:
 The Figure 5 panel I western blot looks very odd to me. I would appreciate others comments. Specifically, I tried to make clear what I mean, but the migration pattern of the proteins appears to not match up between the Tom20 protein and B-actin protein even though it is supposed to come from the same blot. In addition, there are black marks on the edges of the Tom20 protein blot, but these are absent on the B actin blot, which appears to make it seem like these are from different gels.  To try to be clearer, I drew a line comparison of how the blots migrations appear to me  The top line in the image I drew being Tom20 and the bottom corresponding to B-actin Do the authors have the original blot? , 
  In addition, there are black marks on the edges of the Tom20 protein blot, but these are absent on the B actin blot, which appears to make it seem like these are from different gels. The black marks are most likely from molecular weight standards on either side of the blot, which stand out because of the apparently long exposure time. In addition, the figure legend you helpfully posted states that the Actin bands are from a duplicate blot. , 
 #2 Is that an appropriate loading transfer control? , 
 #2 What happened to the loading control bands on the membrane Tom20 was probed on? Doesn t it make more sense to show those rather than the ones from a duplicate blot? , 
 #3 In this case: sure, why not? I cannot speak for the authors and their motivation not to reprobe the same membrane, but a) the actual loading control was Ponceau staining, as stated in the legend, b) there is a consistent qualitative difference across six different cell lines run in parallel, which apparently does not result from grossly altered protein content in detached cells, and c) the key findings were validated using another method (fig 5H). Would it be nice to also see the Ponceau staining? Certainly. Is there any reason to believe that this is anything other than a well-done experiment? In my opinion, no. , 
 Why would it typically be appropriate to perform a beta-actin blot on the same membrane as your protein of interest? This is not a trivial issue, and if we followed the arguments (more like rulings without proper evidence) set out by such people on here, including above, and Leonid Schneider, we would be calling for the retraction of tens of thousands of life sciences papers. But fortunately, and in fact, it is typically not appropriate to simply perform a beta-actin, GAPDH, HSP90 etc blot on each and every membrane. This is because these housekeeping controls are expressed at much higher levels in cells by several orders of magnitude relative to typical proteins of interest. At the level where you are able to detect your protein of interest, you are more often that not way outside of the linear range for the typical loading controls - therefore using a loading control in this way degrades the data! Large changes in loading will result in little or no change in your typical housekeeper loading control, therefore worsening the interpretation of your data. There is a lot of evidence for this:  Even just the LiCor website nicely explains it if you don t want to get in to the western blot methodology papers referenced in the above link. The only way to use beta-actin, GAPDH, HSP90 etc appropriately is to perform a gel using an amount of protein where these signals are linear i.e. as shown repeatedly in the figures above, loading maximum 1-5 ug. At this level of protein loading though you will not be able to detect many most proteins of interest, and so clearly these western blots cannot be performed on the same membrane. As eluded to above and in the paper methods (and nicely explained and fully referenced here https: pubpeer.com publications 2D084E91EDC85D6C23BC63ACF5E6A8#23 ), a total protein stain with a broad linear range, is the most appropriate way to verify the transfer and loading. The options, again with the relevant references, are explained here: https: pubpeer.com publications 2D084E91EDC85D6C23BC63ACF5E6A8#29 ,