MicroRNA-126 Targeting PIK3R2 Inhibits NSCLC A549 Cell Proliferation, Migration, and Invasion by Regulation of PTEN/PI3K/AKT Pathway
Authors: Lei Song
Yue Gu
Jing Jie
Dan Li
Li-Ping Peng
Dan Zhao
Zhong-Mei Wen
Journal: Clinical Lung Cancer (2016)
Discipline: CLINICAL MEDICINE
Keywords: MicroRNA-126; Non–small-cell lung cancer; Phosphatase and tensin homology deleted on chromosome 10; Phosphatidylinositol 3-hydroxy kinase; Phosphatidylinositol 3-kinase regulatory subunit-β.
Affiliations: Department of Respiratory Medicine, First Hospital of Jilin University, Changchun, People's Republic of China., Department of Respiratory Medicine, First Hospital of Jilin University, Changchun, People's Republic of China. Electronic address: .

Abstract:  Background: Our study explored whether the microRNA-126 (miR-126)-mediated PTEN/PI3K/AKT (phosphatase and tensin homology deleted on chromosome 10/phosphatidylinositol 3-kinase regulatory subunit-β/AKT) signaling pathway by targeting PIK3R2 affects the proliferation, migration, and invasion of non-small-cell lung cancer (NSCLC) A549 cells. Materials and methods: Quantitative real-time polymerase chain reaction was used to measure the expression of miR-126 in A549 cells. The MTT (methyl thiazolyl tetrazolium) assay, cell scratch test, Transwell assay, and Western blot were used to detect the proliferation, migration, and invasion of A549 cells and protein expression in A549 cells, respectively. Results: The expression of miR-126 decreased and the expression of PIK3R2 increased in A549 cells (P &lt; .05, for both). Upregulation of miR-126 resulted in the decrease of the proliferation, migration, and invasive abilities of A549 cells, the downregulation of the expression of PIK3R2, PI3K, and phosphorylated Akt (p-Akt) protein, and the upregulation of PTEN expression (P &lt; .05 for all). Also, these abilities of A549 cells increased, and the expression of these 3 proteins was upregulated with downregulation of miR-126 (P &lt; .05 for all). The results of the dual luciferase reporter gene assay showed that PIK3R2 was the target gene of miR-126. PIK3R2, PI3K, and p-Akt proteins were downregulated, but PTEN protein was upregulated as PIK3R2 was silenced or the inhibitor of the PTEN/PI3K/AKT signaling pathway increased. Also, downregulation of miR-126 with silencing of PIK3R2 or increasing the inhibitor of the pathway caused increased PI3K and p-Akt protein expression and increased active proliferation, migration, and invasive abilities of A549 cells (P &lt; .05 for all). Conclusion: The upregulation of miR-126 in NSCLC A549 cells can reduce the expression of the target gene PIK3R2 and influence the PTEN/PI3K/AKT signaling pathway, suppressing the proliferation, migration, and invasive abilities of A549 cells. 

Retracted: No

PubPeer comments:
 Fig 4A.B above:  (A) The Invasive Ability of A549 Cells Was Inhibited After Transfection With miR-126 Mimics. (B) The Invasive Ability of A549 Cells Was Enhanced After Transfection With miR-126 Inhibitor . Overlapping details of the same Blank image have been used in A and B. Note also that the Mimics panel matches one panel of Fig 8B below,  The Transwell Assay and Cell Scratch Test Were Used to Detect the Invasion and Migration of A549 Cells (the Inhibitor + PIK3R2 siRNA condition). ![file] In fact it matches **two** panels of Fig 8B, in which two pairs of panels (for different conditions) appear to contain the same cells, differently arranged. ![file] Two pairs of scratch-test panels are also the same, as shown at bottom with enhanced contrast. , 
 Thank you for your concern about our research. One years ago, we have found the mistakes and then thoroughly checked the primary data. We admit that we have uploaded wrong pictures. And we have immediately contacted with editors to make a eratum. The link is listed below. https: pubmed.ncbi.nlm.nih.gov 32741637 . Many thanks for your comments and we apologize to the readership for the inconvenience caused. , 
 #1 Journal doesnu2019t make this easy to find: https: doi.org 10.1016 j.cllc.2020.06.009 To The Editor, The email is regarding a paper titled u201cMicroRNA-126 Targeting PIK3R2 Inhibits NSCLC A549 Cell Proliferation, Migration, and In- vasion by Regulation of PTEN PI3K AKT Pathwayu201d that we submitted to Clinical Lung Cancer previously. This paper has been online since April 6, 2016. Recently, we checked on the primary data thoroughly and found that some technical errors existed in the results. In addition, some primary pictures are not at a high quality. After full consideration, we decided to make a major revision on the figures published based on these reasons and the rigorous attitude of scholarship. We have re-run the experiments involved in Figure 1, Figure 2, Figure 3, Figure 4, and Figure 5. The results confirmed the conclusion that miR-126 could suppress the development in A549 cells. The specific indications are listed below: In Figure 1, the new results from the cell scratch test showed that miR-126 could inhibit the migration in A549 cells, which is consistent with the previous study. In Figure 2, the new results show that upregulation of miR-126 could inhibit the invasion of A549 cells in vitro, which is consistent with the previous study. In Figure 3, the new results show that the protein levels of PIK3R2 in A549 cells are higher than those in 16HBE cells, which is consistent with the previous study. In Figure 4, our new results showed that miR-126 could regulate the expression of PIK3R2, PTEN, PI3K, and p-Akt in A549 cells but not alter the expression of Akt. The new results also verified the role of PIK3R2 in miR-126 inhibiting the PTEN PI3K Akt signaling pathway. The new results are consistent with the previous study. In Figure 5, our new results confirmed that PIK3R2 and PTEN PI3K Akt signaling pathways were involved in the inhibitory effect of miR-126 on migration and invasion of A549 cells. The new results are consistent with the previous study. Above all, our new results in experiments conducted in A549 cells are consistent with the previous study and confirmed the previous conclusion. We promise this revision does not change any of the conclusions drawn in the study, and apologize to the editorial board, reviewers, and the readership for the inconvenience caused. , 
 #2  We admit that we have uploaded wrong pictures But how did those pictures come to exist in the first place? Pictures with the same cells positioned in different arrangements? That doesn t happen by accident. , 
 #4 We have declared that we have made some technical mistakes about the figures in the eratum letter. Some primary pictures were twisted and overlapped. Best regards! , 
 #4 Besides, we have noticed the mistakes a year before your kind comments. We had no motives to upload wrong pictures purposely and then corrected it ourselves only for a joy. , 
 #4 I agree that this set of images appears more likely to have been manipulated than to have occurred by u201cmistake.u201d #5 I thank the authors for replying here. I am unaware of any software bug or operator error that could cause the u201cprimary picturesu201d to become u201ctwistedu201d in a way that would rearrange cells in an image. Could the authors explain how the (presumably digital) images below cane to be affected by this process? At a minimum, other researchers need to be warned to avoid similar errors in their manuscripts. ![file] , 
 #7 Images in low resolution in this article were photographed from eyepiece lenses because the microscope was not equipped with a camera. Hence we think the mistakes may be from the repeated and spliced fields. It is indeed an error should be warned to us and other researchers and we will avoid recurrence of similar incidents in the future work. However, we believe the mistakes do not influence the conclusion if corrected and try tou00a0find waysu00a0tou00a0makeu00a0amends and apologizeu00a0to readers. Best regards! , 
 Figure 7A, B.  MicroRNA-126 (miR-126) Targeting PIK3R2 to Inhibit the Expression of the PTEN PI3K AKT (Phosphatase and Tensin Homology Deleted on Chromosome 10 Phosphatidylinositol 3-Kinase Regulatory Subunit-b AKT) Signaling Pathway. (A) Western Blot Showed That miR-126 Mimics Could Inhibit the Expression of PIK3R2 Protein and Downregulate the Expression of PI3K and phosphorylated-Akt (p-Akt) Proteins. (B) Western Blot Showed That miR-126 Inhibitor Could Upregulate the Expression of PIK3R2 Protein and Upregulate Expression of PI3K and p-Akt Proteins. Some unexpected rotational symmetries between the PTEN and p-Akt bands. ![file] ------------------------------- UPDATE. *[left]* Figure 7A, B again. *[right]* Figure 6A,B from [Chen et al (2019)] .  Effect of transfection with miR-126 mimics or inhibitor on related protein levels in U87MG cells. A, changes in protein levels after transfection with miR-126 mimics, B, changes in protein levels after transfection with miR-126 inhibitor . ![file] ,