Lnc-PICSAR contributes to cisplatin resistance by miR-485-5p/REV3L axis in cutaneous squamous cell carcinoma
Authors: Dan Wang
Jing Yin
Xiaoqiang Zhou
Yang Zhou
Journal: Open Life Sciences (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: CSCC; DDP resistance; REV3L; exosome; lnc-PICSAR
Affiliations: Plastic Surgery Center, Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, 214000, China, Department of Dermatology, Medical College of Shaoguan University, Shaoguan, 512026, China, Department of Interventional Vascular, Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, 214000, China, Department of Dermatology, Yancheng First People’s Hospital, Jiangsu Province, No.166 yulong west road, Yancheng, Jiangsu, 224001, China

Abstract:  Background: Dysregulation of long noncoding RNAs (lncRNAs) is associated with drug resistance in multiple cancers. We explored the roles of lncRNA p38 inhibited cutaneous squamous cell carcinoma-associated lincRNA (PICSAR) in cisplatin (DDP) resistance of cutaneous squamous cell carcinoma (CSCC). Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure the expression of lnc-PICSAR, miR-485-5p and reversionless 3-like (REV3L) mRNA. The cell counting kit-8 (CCK-8) assay was conducted to evaluate DDP resistance and cell viability. The transwell assay was performed to determine cell migration and invasion. Western blot assay and immunohistochemistry (IHC) staining assay were carried out to measure protein levels. The dual-luciferase reporter assay was used to investigate the association between miR-485-5p and lnc-PICSAR or REV3L. Murine xenograft model was constructed to explore the function of lnc-PICSAR in vivo. The morphology of exosomes was analyzed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Results: Lnc-PICSAR was elevated in DDP-resistant CSCC cells. Lnc-PICSAR silencing suppressed cell viability, DDP resistance, migration and invasion in DDP-resistant CSCC cells. MiR-485-5p acted as a target of lnc-PICSAR, and miR-485-5p inhibition reversed the impacts of lnc-PICSAR silencing on DDP resistance and cell progression in DDP-resistant CSCC cells. Lnc-PICSAR promoted REV3L expression via sponging miR-485-5p. Moreover, REV3L overexpression overturned the effects of lnc-PICSAR on cell progression and DDP resistance. Lnc-PICSAR knockdown suppressed DDP resistance in vivo. In addition, lnc-PICSAR was increased in the exosomes derived from CSCC patients' serum and CSCC cells. Conclusion: Lnc-PICSAR enhanced DDP resistance via miR-485-5p/REV3L axis in DDP-resistant CSCC cells. Besides, exosome-mediated lnc-PICSAR might be involved in the regulation of drug resistance in CSCC. 

Retracted: No

PubPeer comments:
 Several questions and comments arise. The forward primer for a mature miRNA qPCR is usually identical to the whole miRNA or a part of it. The sequence of miR-485-5p is AGAGGCUGGCCGUGAUGAAUUC. Here, the primers are F: 5u2032-CCAAGCTTCACCCATTCCTAACAGGAC-3u2032 and R: 5u2032-CGGGATCCGTAGGTCAGTTACATGCATC-3u2032. I do not understand how they would recognize any part of mature miR-485-5p. 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 was quantified. I did not check the other primer sets, but this might be useful to do. Isolation of  exosomes was performed using a kit. Unfortunately, the kit described in the methods section relies on polymer precipitation. Using complex sources like serum, polymer precipitation does not isolate exosomes or the broader class of extracellular vesicles (EVs). Almost all of the material collected will be lipoprotein particles and protein aggregates. Also, two tetraspanins are measured, but they are not specific for exosomes, and they do not give any idea of purity. The CD63 band is curiously devoid of the customary smear that most antibodies detect. No contaminants are assayed. I would recommend that the authors correct the manuscript to refer to  extracellular particles instead of  exosomes. The methods section incorrectly states that  Nanoparticle tracking analysis (NTA) was performed. The indicated instrument is not an NTA instrument. It is a dynamic light scattering device. Also, NTA cannot indicate the  size distribution of exosomes . NTA and DLS detect particles, not EVs or exosomes, and the majority of exosomes and other small EVs are too small to be detected reliably by NTA. Could the authors please correct this mistake? ,