Exosomal circ_IFT80 Enhances Tumorigenesis and Suppresses Radiosensitivity in Colorectal Cancer by Regulating miR-296-5p/MSI1 Axis
Authors: Liang Li
Xiangcai Zou
Zhipeng Jiang
Tengfei Hao
Journal: Cancer Management and Research (2021)
Discipline: CLINICAL MEDICINE
Keywords: MSI1; circ_IFT80; colorectal cancer; exosomes; miR-296-5p.
Affiliations: Department of Digestive Medicine Center, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, People's Republic of China., Department of Gastrointestinal Surgery, The Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China., Department of Gastrointestinal Surgery, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, People's Republic of China.

Abstract:  Background: Exosomal circular RNAs (circRNAs) can act as biomarkers and play crucial roles in colorectal cancer (CRC) and radiosensitivity. The aim of this study was to explore the functions and regulatory mechanism of exosomal circRNA intraflagellar transport 80 (circ_IFT80) in tumorigenesis and radiosensitivity of CRC. Methods: Exosomes were detected using transmission electron microscopy (TEM). Protein levels were determined by Western blot assay. The expression of circ_IFT80, microRNA-296-5p (miR-296-5p) and musashi1 (MSI1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell cycle distribution, cell apoptosis, and cell proliferation were detected by flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. Colony formation assay was used to determine the radiosensitivity of cells. The interaction between miR-296-5p and circ_IFT80 or MSI1 was verified by dual-luciferase reporter assay. A xenograft tumor model was established to explore the role of exosomal circ_IFT80 in vivo. Results: Circ_IFT80 was upregulated in exosomes derived from CRC patient serum and CRC cells. Exosomal circ_IFT80 or circ_IFT80 overexpression facilitated tumorigenesis by increasing cell proliferation and reducing apoptosis, and inhibited radiosensitivity via promoting colony formation and inhibiting apoptosis. Additionally, circ_IFT80 acted as a sponge of miR-296-5p, and miR-296-5p reversed the effects of circ_IFT80 on tumorigenesis and radiosensitivity. Moreover, MSI1 was a direct target of miR-296-5p. Furthermore, miR-296-5p overexpression inhibited tumorigenesis and promoted radiosensitivity by downregulating MSI1. Exosomal circ_IFT80 also accelerated tumor growth in vivo. Conclusion: Exosomal circ_IFT80 promoted tumorigenesis and reduced radiosensitivity by regulating miR-296-5p/MSI1 axis, which might provide a novel avenue for treatment of CRC. 

Retracted: No

PubPeer comments:
 In this paper, miR-296-5p is said to be measured by qPCR and normalised to U6. The indicated primers for U6 bind perfectly to the U6 RNA. The indicated primers for miR-296-5p,  5u02b9-GTATCCAGTGCAGGGTCCGA-3u02b9 (sense) and 5u02b9-CGACGAGGGCCCCCCCT-3u02b9 (anti-sense) , include a reverse primer sharing some sequence with the mature 296-5p. But unless I am mistaken, a PCR primer set designed to amplify an RT product prepared from a mature microRNA would be expected instead to have a  forward primer that matches the mature miRNA (not a reverse primer, as here), and a  reverse primer that amplifies some artificial sequence that is introduced during RT. It seems that the forward and reverse primers may have been switched, and that the strategy was a stem-loop RT, although the RT primer is not disclosed. It is possible that I do not fully understand the amplification strategy, so I hope the authors can respond with a clear description of the RT and amplification strategy, which does not seem to be present in the publication. ,