Long non-coding RNAs as surrogate indicators for chemical stress responses in human-induced pluripotent stem cells

Abstract

In this study, we focused on two biological products as ideal tools for toxicological assessment: long non-coding RNAs (lncRNAs) and human-induced pluripotent stem cells (hiPSCs). lncRNAs are an important class of pervasive non-protein-coding transcripts involved in the molecular mechanisms associated with responses to cellular stresses. hiPSCs possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical issues associated with human embryonic stem cells. Here, we identified six novel lncRNAs (CDKN2B-AS1, MIR22HG, GABPB1-AS1, FLJ33630, LINC00152, and LINC0541471_v2) that respond to model chemical stresses (cycloheximide, hydrogen peroxide, cadmium, or arsenic) in hiPSCs. Our results indicated that the lncRNAs responded to general and specific chemical stresses. Compared with typical mRNAs such as p53-related mRNAs, the lncRNAs highly and rapidly responded to chemical stresses. We propose that these lncRNAs have the potential to be surrogate indicators of chemical stress responses in hiPSCs.

Figure 1. Alterations in mRNA and lncRNA expression levels by four chemical stresses in hiPSCs.

Type A genes indicate pluripotency-related genes; Type B genes indicate p53-related genes; Type C genes indicate lncRNAs. hiPSCs were treated with (A) 100 µM cycloheximide, (B) 100 µM hydrogen peroxide, (C) 1 µM cadmium, or (D) 100 nM arsenic for 24 h. Expression levels of the indicated RNAs were determined by RT-qPCR. Quantitative values in response to vehicles alone were set to 1. GAPDH mRNA levels were used for normalization.

Figure 2. Alterations in lncRNA expression levels by two chemical stresses at various doses in hiPSCs.

hiPSCs were treated with (A) cycloheximide or (B) hydrogen peroxide for 24 h. Expression levels of the indicated RNAs were determined by RT-qPCR. Quantitative values in response to vehicles alone were set to 1. GAPDH mRNA levels were used for normalization. Values represent the mean ± standard error obtained from two independent experiments.

Figure 3. Novel lncRNAs highly and rapidly respond to chemical stresses compared with the responses of p53-related mRNAs in hiPSCs.

hiPSCs were treated with (A) 100 µM cycloheximide or (B) 100 µM hydrogen peroxide. The expression levels of the indicated RNAs were determined at various time points. Quantitative values at 0 h were set to 1. GAPDH mRNA levels were used for normalization. Values represent the mean ± standard error obtained from two independent experiments.