miRNA-296-5p functions as a potential tumor suppressor in human osteosarcoma by targeting SND1
Authors: You-Jia Xu
Jian-Jian Shen
Ya-Zeng Huang
Ting-Xiao Zhao
Jun Zhang
Journal: Chinese Medical Journal (2021)
Discipline: CLINICAL MEDICINE
Keywords: Osteosarcoma; miR-296-5p; pathogenesis; anti-OS effects; proliferation
Affiliations: Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Soochow, Jiangsu 215004, China., Department of Orthopedics, Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang 315000, China., Department of Orthopedics, People's Hospital of Hangzhou Medical College, Hangzhou, Zhejiang 310014, China., Department of Orthopedics, Cixi People's Hospital, Ningbo, Zhejiang 310014, China., Department of Clinical Medicine, Bengbu Medical College, Bengbu, Anhui 233017, China., Osteoporosis Institute, Osteoporosis Institute of Soochow University, Soochow, Jiangsu 215004, China.

Abstract:  Background: The pathogenesis of osteosarcoma (OS) is still unclear, and it is still necessary to find new targets and drugs for anti-OS. This study aimed to investigate the role and mechanism of the anti-OS effects of miR-296-5p. Methods: We measured the expression of miR-296-5p in human OS cell lines and tissues. The effect of miR-296-5p and its target gene staphylococcal nuclease and tudor domain containing 1 on proliferation, migration, and invasion of human OS lines was examined. The Student's t test was used for statistical analysis. Results: We found that microRNA (miR)-296-5p was significantly downregulated in OS cell lines and tissues (control vs. OS, 1.802 ¬± 0.313 vs. 0.618 ¬± 0.235, t = 6.402, P &lt; 0.01). Overexpression of miR-296-5p suppressed proliferation, migration, and invasion of OA cells. SND1 was identified as a target of miR-296-5p by bioinformatic analysis and dual-luciferase reporter assay. Overexpression of SND1 abrogated the effects induced by miR-296-5p upregulation (miRNA-296-5p vs. miRNA-296-5p + SND1, 0.294 ¬± 0.159 vs. 2.300 ¬± 0.277, t = 12.68, P = 0.003). Conclusion: Our study indicates that miR-296-5p may function as a tumor suppressor by targeting SND1 in OS. 

Retracted: No

PubPeer comments:
 Figure 2D appears to have a repeated image. *** Closeup  , 
 This is one of several papers from different author teams that report using the same forward and reverse primers for quantitative PCR of miR-296-5p or miR-296-3p. Here, the  Taqman miRNA reverse transcription kit is said to have been used to reverse transcribe the -5p microRNA. Then  PCR amplification of the primers was analyzed with Taqman miR-296-5p microRNA assay. However, miR-296-5p specific primers are then listed as  miR-296-5p forward, 5u2032-TGC CTA ATT CAG AGG GTT GG-3u2032; miR-296-5p reverse, 5u2032-CTC CAC TCC TGG CAC ACA G-3u2032 . No reliable results for miR-296-5p would have been obtained with these methods. The reason for this is that this specific reverse transcription kit produces a cDNA in which the mature miRNA sequence is attached to a longer proprietary sequence that was introduced by the RT primer. This sequence is needed for qPCR because the miRNA is otherwise not long enough for two primers and a probe to attach. The qPCR is then done with a forward primer that covers the 5 part of the mature miRNA, a probe with several nucleotides corresponding to the 3 part of the mature miRNA and adjacent nucleotides in the RT-introduced sequence, and a reverse primer that recognizes another portion of the RT-introduced sequence. The primer sequences specified in the paper would not amplify the RT product. Here is the precursor sequence for miR-296, with the miR-296-5p arm (the one that is supposed to be amplified) in bold: AGGACCCUUCCAG**AGGGCCCCCCCUCAAUCCUGU**UGUGCCUAAUUCAGAGGGUUGGGUGGAGGCUCUCCUGAAGGGCUCU And here in bold is where the published  forward primer sequence would bind: AGGACCCUUCCAGAGGGCCCCCCCUCAAUCCUGUUG**UGCCUAAUUCAGAGGGUUGG**GUGGAGGCUCUCCUGAAGGGCUCU Obviously, this primer does not capture miR-296-5p at all. Also, the reverse primer recognizes a sequence adjacent to the miR-296 precursor in the genome, but this is not part of the precursor RNA. These forward and reverse primers might amplify genomic DNA, but they would not tell us anything about miR-296-5p and they would not make any product from the cDNA product of the TaqMan MicroRNA Reverse Transcription Kit. , 
 The papers that use these primers and methods incorrectly include the following: 1. [Zhang et al, 2019] 2. [Huang et al, 2021] 3. [Fan et al, 2020] 4. [Wang et al, 2020] 5. [Wang et al, 2020] 6. [Li et al, 2019] 7. [Zhang et al, 2021] 8. [Li et al, 2017] 9. [Ni et al, 2020] It appears to me that the first use of these primers in the literature was by a group that used them correctly, to examine the levels of the **primary** miR-296-containing transcript (Lee, et al, Mol Cells, 2017, and I want to stress that I find no problems with this paper.) It would seem that the methods and primers were subsequently copied without an understanding of the difference between primary, precursor, and mature miRNAs, or of the fact that precursor can give rise to different mature miRNAs. It is sobering for me to realize that entire author teams could be involved in putative microRNA research without understanding the fundamentals. , 
 COrrigendum, [5 July 2021] . ---------------------------------------- Following the original article s publication, the authors informed us of the following errors: The Figure 2 is submitted incorrectly, while correct Figure 2 should submit as following:  In the method part miRNA qRT-PCR analysis method should be corrected as follows: For Quantitative Real-time polymerase chain reaction (qRT-PCR) analysis of mature miR-296-5p, total RNA was reverse transcribed using specific RT primers and reagents from the Taqman miRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Then, the expression of miR-296-5p was analyzed using the Taqman miR-296-5p microRNA assay (Assay ID 000527, Applied Biosystems, Foster City, CA) with RNU6B used as an internal control, and measured using a prism 7900HT system (Applied Biosystems, Thermo Fisher, Waltham, MA, USA). For the quantification of SND1 mRNA, qRT-PCR was performed using the rapid quantitative SYBR-Green RT-PCR kit (Takara, Japan). GAPDH was used as an internal control. All reactions were performed in triplicate. The following primer sequences were used: SND1 forward, 5u2019-GGATGTGGAGTGAGGGGA-3u2019; SND1 reverse,5u2019- GTTTCACTGCCATCTGCTTC -3u2019; GAPDH forward 5u2019-GGT CTC TGA CTT CAA CA-3u2019; GAPDH reverse, 5u2019-GTGAGG GTC TCT CTC TTC CT-3u2019. The authors apologize for any inconvenience caused. ,