<p>Long Noncoding RNA CCAT1 Functions as a Competing Endogenous RNA to Upregulate ITGA9 by Sponging MiR-296-3p in Melanoma</p>
Authors: Yan Zheng
Limin Zhao
Jinghua Fan
Jun Yang
Di Li
Xiaoxiao Kang
Journal: Cancer Management and Research (2020)
Discipline: CLINICAL MEDICINE
Keywords: ITGA9; lncRNA CCAT1; melanoma; miR-296-3p.
Affiliations: Department of Dermatology, Xi'an Central Hospital Affiliated to Xi'an Jiaotong University, Xi'an, Shaanxi, People's Republic of China., Department of Dermatology, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, People's Republic of China.

Abstract:  Background: Melanoma is aggressive and lethal melanocytic neoplasm, and its incidence has increased worldwide in recent decades. Accumulating evidence has showed that various long noncoding RNAs (lncRNAs) participated in occurrence of malignant tumors, including melanoma. The present study was designed to investigate function of lncRNA colon cancer-associated transcript-1 (CCAT1) in melanoma. Methods: The expression levels of CCAT1, miR-296-3p and Integrin alpha9 (ITGA9) in melanoma tissues or cells were measured using real-time quantitative polymerase chain reaction (RT-qPCR). The concentrations of glucose and lactate were measured for assessing glycolysis of melanoma cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT), flow cytometry, and transwell assays were conducted to assess proliferation, apoptosis, and migration of melanoma cells. Western blot assay was performed to measure the protein expression of ITGA9, hexokinase 2 (HK2), and epithelial-mesenchymal transition (EMT)-related proteins in melanoma tissues or cells. The relationship among CCAT1, miR-296-3p, and ITGA9 was predicted and confirmed by bioinformatics analysis, dual-luciferase reporter, and RNA immunoprecipitation (RIP) assay, respectively. A xenograft experiment was established to assess the effect of CCAT1 knockdown in vivo. Results: CCAT1 was effectively increased in melanoma tissues and cells compared with matched controls, and deficiency of CCAT1 impeded cell glycolysis, proliferation, migration while induced apoptosis, which were abrogated by knockdown of miR-296-3p in melanoma cells. In addition, our findings revealed that ITGA9 overexpression abolished miR-296-3p overexpression-induced effects on melanoma cells. Importantly, CCAT1 regulated ITGA9 expression by sponging miR-296-3p. The results of xenograft experiment suggested that CCAT1 silencing inhibited melanoma cell growth in vivo. Conclusion: LncRNA CCAT1 promoted ITGA9 expression by sponging miR-296-3p in melanoma. 

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. Here, the  microRNA Reverse Transcription Kit is said to have been used to reverse transcribe the -3p microRNA. Then  RT-qPCR was carried out using SYBR-Green PCR kit with what are described as miR-296-3p specific primers  (Forward, 5u02b9-TGCCTAATTCAGAGGGTTGG-3u02b9; Reverse, 5u02b9-CTCCACTCCTGGCACACAG-3u02b9) . No reliable results for miR-296-5p would have been obtained with these methods. The TaqMan MicroRNA Reverse Transcription Kit cannot convert miRNAs into cDNA without a microRNA-specific RT primer that is sold separately along with proprietary qPCR primers and a TaqMan probe. When the correct RT primer is used, the product is DNA 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 cDNA must then be amplified by the proprietary, miRNA-specific primer probe mixture sold along with the RT primer. Although the sequences are proprietary (or were), basically, there s the 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. You could not amplify this cDNA with anything other than these primers. You could not just use another portion of the miR-296 precursor or primary transcript, for example, because it would not be part of the reverse-transcribed cDNA. What about the primers published in this paper? Here is the precursor sequence for miR-296, with the miR-296-3p arm (the one that is supposed to be amplified in this paper) in bold: AGGACCCUUCCAGAGGGCCCCCCCUCAAUCCUGUUGUGCCUAAUUCA**GAGGGUUGGGUGGAGGCUCUCC**UGAAGGGCUCU And here in bold is where the published  forward primer sequence would bind: AGGACCCUUCCAGAGGGCCCCCCCUCAAUCCUGUUG**UGCCUAAUUCAGAGGGUUGG**GUGGAGGCUCUCCUGAAGGGCUCU It is apparent that the published primer would only partially overlap with the miR-296-3p sequence. Importantly, no 5 sequence (the first half of the primer) would be present after reverse transcription with the TaqMan MicroRNA Reverse Transcription Kit. Also, the published reverse primer does not recognize the special reverse transcription kit adapter, but instead a sequence adjacent to the miR-296 precursor in the genome. This sequence would not be present in the cDNA. At most, the forward and reverse primers might amplify genomic DNA, but they would not tell us anything about the mature miR-296-3p and they would not make any product from the TaqMan MicroRNA Reverse Transcription Kit, even if the correct RT primer had been used. , 
 Retracted [7 December 2021] . ----------------------------- The Editor and Publisher of *Cancer Management and Research* wish to retract the published article. Concerns were raised regarding the reliability of the miR-296-3p specific primers used to conduct the RT-qPCR experiments described in the study. Based on the primer details given in the article it is unlikely the results reported would have been obtained. The authors responded to our queries but were unable to provide a satisfactory explanation in regard to the RT-qPCR experimental concerns and could not provide satisfactory original data for their study. The Editor advised for the article to be retracted. 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. ,