Circular RNA NOX4 promotes the development of colorectal cancer via the microRNA‑485‑5p/CKS1B axis
Authors: Ximin Wang
Dongxu Zheng
Donghong Huang
Shuangyin Liang
Geng Tao
Journal: Oncology Reports (2020)
Discipline: CLINICAL MEDICINE
Keywords: colorectal cancer; circular RNA NADPH oxidase 4; microRNA-485-5p; CKS1B; glycolysis.
Affiliations: Department of Clinical Laboratory, The Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian 362000, P.R. China.

Abstract:  Colorectal cancer (CRC) is a common malignancy globally. The aim of the present study was to explore the role and the working mechanism of circular RNA NADPH oxidase 4 (circNOX4; circBase ID, hsa_circ_0023990) in CRC. Reverse transcription‑quantitative (RT‑q)PCR was used to examine the expression of circNOX4, NOX4 mRNA and microRNA (miR)‑485‑5p in CRC tissues and cell lines. 3‑(4,5‑Dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide and Transwell assays were performed to analyze CRC cell viability and motility. The glycolytic ability of CRC cells was assessed by measuring glucose consumption, lactate production, extracellular acidification and O2 consumption rates using commercial kits. The starBase database was used to predict the targets of circNOX4 and miR‑485‑5p, and the interaction was confirmed by dual‑luciferase reporter and RNA immunoprecipitation assays. A murine xenograft model was established to verify the role of circNOX4 in CRC in vivo. The results demonstrated that the expression of circNOX4 was upregulated in CRC tissues and cell lines compared with that in adjacent normal tissues and a normal colon epithelial cell line, respectively. The expression of circNOX4 was negatively associated with the prognosis of patients with CRC. CircNOX4 silencing suppressed the proliferation, migration, invasion and glycolysis of CRC cells. miR‑485‑5p was identified as a target of circNOX4. CircNOX4 promoted CRC progression by sponging miR‑485‑5p. miR‑485‑5p was demonstrated to bind to the 3' untranslated region (UTR) of CDC28 protein kinase regulatory subunit 1B (CKS1B). miR‑485‑5p overexpression‑mediated malignant properties of CRC cells were partly reversed by the transfection with the CKS1B overexpression plasmid. CircNOX4 silencing restrained the CRC xenograft growth in vivo. Collectively, the results of the present study demonstrated that circNOX4 may serve an oncogenic role in CRC by promoting the proliferation, migration, invasion and glycolysis of CRC cells via the miR‑485‑5p/CKS1B axis. 

Retracted: No

PubPeer comments:
 Fig 1.  (A) RT-qPCR was used to determine the expression levels of circNOX4 in CRC tissues and adjacent normal tissues. (B) The level of circNOX4 was determined in CRC tissues from patients grouped according to clinical staging by RT-qPCR. ![file] Values of circNOX4 range from 0.2 to 3.5 when the CRC tissues are grouped by clinical staging in (B), but from 1.5 to 3.8 when they are grouped together in (A). There seem to be two quite different circNOX4 distributions. Am I missing something? , 
 #1 I don t think you re missing anything. There were 46 patients, 20 in stages I and II and 26 in stages III and IV. They should all appear together as the  tumor group in Figure 1A, and then separated by stage (I,II vs III,IV) in Figure 1B. That is, the  red group in 1A is split into green and red in Figure 1B. However, although the numbers of points are consistent, the values are different, including many smaller values in Figure 1B vs 1A. ![file] However, perhaps the authors compared everything to the average of the  control group in each panel, defined as  1 , that is,  normal in 1A and I+II in 1B. , 
 I do have additional concerns about the microRNA part of this publication. microRNAs like miR-485-5p are too short to be amplified directly, so they require a special reverse transcription process for qPCR. No such procedures are indicated in the methods. The general reverse transcription kit described in the methods would not work. Even if an appropriate RT had been done, the primers described in the methods for miR-485-5p are difficult to reconcile with the sequence of mature miR-485-5p. Typically, the forward primer for a mature miRNA qPCR is identical to the whole miRNA or a part of it. The sequence of miR-485-5p is AGAGGCUGGCCGUGAUGAAUUC I do not understand how the reported miRNA primers, forward, 5u2032-CCAAGCTTCACCCATTCCTAACAGGAC-3u2032 and reverse, 5u2032-CGGGATCCGTAGGTCAGTTACATGCATC-3u2032, would recognize any part of mature miR-485-5p. Now it is my turn to ask if I am missing something. I hope that the authors can provide the missing details of the reverse transcription and amplification procedures to help us understand how miR-485-5p could be quantified. ,