<p>LncRNA NEAT1 Promotes Proliferation, Migration And Invasion Via Regulating miR-296-5p/CNN2 Axis In Hepatocellular Carcinoma Cells</p>
Authors: Guobin Du
Yandong Li
Xiyan Ding
Shuqiu Xiu
Yahui Liu
Journal: OncoTargets and Therapy (2019)
Discipline: CLINICAL MEDICINE
Keywords: CNN2; HCC; NEAT1; miR-296-5p; progression.
Affiliations: Department of Hepatopancreatobiliary Surgery, The First Hospital of Jilin University, Changchun City, Jilin, People's Republic of China., Department of Neurology, Beihua University, Jilin City, Jilin, People's Republic of China., Department of Medical, Beihua University, Jilin City, Jilin, People's Republic of China., President's Office, The Sixth Peoples's Hospital of Jilin City, Jilin City, Jilin, People's Republic of China.

Abstract:  Background: Emerging evidence has revealed that long noncoding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) is implicated in the development of various cancers. However, the underlying molecular mechanisms of NEAT1 in hepatocellular carcinoma (HCC) remain unclear. Methods: The expression of NEAT1, miR-296-5p and Calponin 2 (CNN2) was detected by quantitative real-time polymerase chain reaction or Western blot, respectively. Cell proliferation and apoptosis were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay or flow cytometry, respectively. Transwell assay was used to determine cell migration and invasion. The interaction between miR-296-5p and NEAT1 or CNN2 was analyzed by dual-luciferase reporter assay and RIP assay. Huh7 cells transfected with sh-NEAT1 were used to establish the murine xenograft model. Results: NEAT1 was elevated in HCC tissues and cell lines. Knockdown of NEAT1 significantly inhibited proliferation, migration and invasion of HCC cells in vitro as well as tumor growth in vivo. NEAT1 was a sponge of miR-296-5p and remarkably reduced the level of miR-296-5p in HCC cells. Furthermore, NEAT1 silence significantly decreased the expression of CNN2, which was the direct target of miR-296-5p. Besides that, the tumor suppression caused by NEAT1 silence could be rescued by CNN2 restoration or miR-296-5p inhibition in vitro. Additionally, NEAT1 indirectly regulated CNN2 expression by competing to miR-296-5p in vitro and in vivo. Conclusion: LncRNA NEAT1 contributes to HCC progression by regulating miR-296-5p/CNN2 axis, providing a novel regulatory mechanism for HCC development and a promising therapeutic target for the HCC treatment. 

Retracted: Yes

PubPeer comments:
 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. In this case, it is miR-296-5p. Unfortunately, the methods and primers would not give any useful information about the mature miRNA, miR-296-5p.  For miRNA expression detection, SuperScript III First-Strand Synthesis System (Invitrogen) was used to generate cDNA. As specified in the instructions, this kit is for RNAs greater than 100 nucleotides in length. Mature miR-296-5p is 21 nt long. This kit is inappropriate for miRNA reverse transcription. Next, qPCR is done with what are described as miR-296-5p specific primers  (Forward, 5u02b9-TGCCTAATTCAGAGGGTTGG-3u02b9; Reverse, 5u02b9-CTCCACTCCTGGCACACAG-3u02b9) . Even if a usable cDNA could have been made using the SuperScript system (and this is impossible) no reliable results for miR-296-5p would have been obtained with these primers. Here is the precursor sequence for miR-296, with the miR-296-5p arm (the one that is supposed to be amplified in this paper) 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, possibly even a portion of a primary transcript, but they would not tell us anything about the mature miR-296-5p that is supposed to be the subject of this publication. , 
 Maybe I just didn t understand something, but shouldn t the numbers in each of the boxes under  low and  high expression add up to 15? Since there were 15 low and 15 high? ![Table1] , 
 Fig 7B. ![file] These images are vaguely biological in appearance, and could well be tumors, but the way that they are hovering above a featureless grey plane (with digital shadows) leaves one wondering whether their outlines are also digital, and what they looked like in their original contexts. , 
 Retracted [18 October 2021] . ------------------------------------------ The Editor and Publisher of *OncoTargets and Therapy* wish to retract the published article. Concerns were raised regarding the integrity of some of the data provided in the article. The concerns related specifically to qPCR sequence data, the data presented in Table 1 and the images provided in Figure 7B. The authors responded to our queries but were unable to provide the original data for their study. The authors decided it was best to retract the published article. Our decision-making was informed by our policy on publishing ethics and integrity and the COPE guidelines on retraction. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as u201cRetractedu201d. ,