Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa Cells

Abstract

Apoptosis is a form of regulated cell death that plays a critical role in survival and developmental homeostasis. There are numerous reports on regulation of apoptosis by protein-coding genes as well as small non-coding RNAs, such as microRNAs. However, there is no comprehensive investigation of circular RNAs (circRNA) that are differentially expressed under apoptotic conditions. We have performed a transcriptomics study in which we first triggered apoptosis in HeLa cells through treatment with four different agents, namely cisplatin, doxorubicin, TNF-α and anti-Fas mAb. Total RNAs isolated from control as well as treated cells were treated with RNAse R to eliminate the linear RNAs. The remaining RNAs were then subjected to deep-sequencing to identify differentially expressed circRNAs. Interestingly, some of the dys-regulated circRNAs were found to originate from protein-coding genes well-documented to regulate apoptosis. A number of candidate circRNAs were validated with qPCR with or without RNAse R treatment as well. We then took advantage of bioinformatics tools to investigate the coding potential of differentially expressed RNAs. Additionally, we examined the candidate circRNAs for the putative miRNA-binding sites and their putative target mRNAs. Our analyses point to a potential for circRNA-mediated sponging of miRNAs known to regulate apoptosis. In conclusion, this is the first transcriptomics study that provides a complete circRNA profile of apoptotic cells that might shed light onto the potential role of circRNAs in apoptosis.

Identification of circRNAs in the Liver of Whitespotted Bamboo Shark (Chiloscyllium plagiosum)

“…CircRNAs, a novel type of non-coding RNAs (ncRNAs) (Jeck and Sharpless, 2014;Hsiao et al, 2017), form covalent-closed continuous loops without 5 to 3 polarities and poly(A) tails (Liang and Wilusz, 2014;Yaylak et al, 2019). Large amounts of circRNAs have been successfully identified in a variety of organisms, including plants, animals, and humans (Rybak-Wolf et al, 2015;Maass et al, 2017;Ye et al, 2017).…”

Genomewide m6A Mapping Uncovers Dynamic Changes in the m6A Epitranscriptome of Cisplatin-Treated Apoptotic HeLa Cells

“…HeLa cells, purchased from DSMZ GmbH (Leibniz, Germany), and an ME-180 (HTB-33) cell line, purchased from ATCC (Manassas, VA, USA), were cultured in RPMI 1640 (Gibco, ThermoFischer, Waltham, MA, USA) and McCoy’s 5A (Lonza, Switzerland) supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS) (Gibco, ThermoFischer, Waltham, MA, USA) in a humidified atmosphere of 5% CO 2 at 37 °C. Cisplatin (Santa Cruz Biotechnology, Dallas, TX, USA) treatments were carried out in triplicates essentially as described previously [ 12 ]. 0.1% ( v / v ) DMSO was used as a negative control for cisplatin.…”

Genomewide m6A mapping uncovers dynamic changes in the m6A epitranscriptome of cisplatin-treated apoptotic HeLa cells

“…HeLa cells, purchased from DKFZ GmbH (Germany), were cultured in RPMI 1640 (with 2 mM L-Glutamine, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, United States) in a humidified atmosphere of 5% CO 2 at 37°C. Cisplatin (Santa Cruz Biotechnology, United States) treatments were carried out in triplicates essentially as described previously (12). 0.1% DMSO was used as negative control for cisplatin.…”