Identification of RNA biomarkers for chemical safety screening in mouse embryonic stem cells using RNA deep sequencing analysis

Although it is not yet possible to replace in vivo animal testing completely, the need for a more efficient method for toxicity testing, such as an in vitro cell-based assay, has been widely acknowledged. Previous studies have focused on mRNAs as biomarkers; however, recent studies have revealed that non-coding RNAs (ncRNAs) are also efficient novel biomarkers for toxicity testing. Here, we used deep sequencing analysis (RNA-seq) to identify novel RNA biomarkers, including ncRNAs, that exhibited a substantial response to general chemical toxicity from nine chemicals, and to benzene toxicity specifically. The nine chemicals are listed in the Japan Pollutant Release and Transfer Register as class I designated chemical substances. We used undifferentiated mouse embryonic stem cells (mESCs) as a simplified cell-based toxicity assay. RNA-seq revealed that many mRNAs and ncRNAs responded substantially to the chemical compounds in mESCs. This finding indicates that ncRNAs can be used as novel RNA biomarkers for chemical safety screening

流れ系を利用した生体酸化還元反応の研究手法の開発

京都大学0048新制・課程博士博士(農学)甲第7875号農博第1033号新制||農||776(附属図書館)学位論文||H11||N3238(農学部図書室)UT51-99-G469京都大学大学院農学研究科農芸化学専攻(主査)教授 池田 篤治, 教授 上野 民夫, 教授 加藤 暢夫学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDA

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

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

The RNA Degradation Pathway Regulates the Function of GAS5 a Non-Coding RNA in Mammalian Cells

<div><p>Studies of various mRNAs have revealed that changes in the abundance of transcripts, through mRNA degradation, act as a critical step in the control of various biological pathways. Similarly, the regulation of non-coding RNA (ncRNA) levels is also considered to be important for their biological functions; however, far less is known about the mechanisms and biological importance of ncRNA turnover for the regulation of ncRNA functions. The growth arrest-specific 5 (GAS5) ncRNA accumulates during growth arrest induced by serum starvation and its transcript is degraded by the well characterized nonsense-mediated RNA decay (NMD) pathway. Historically, NMD was discovered as a RNA quality control system to eliminate aberrant transcripts; however, accumulating evidence shows that NMD also regulates the abundance of physiological transcripts. Interestingly, the GAS5 transcript has the ability to bind the glucocorticoid receptor (GR), resulting in the inhibition of its ligand-dependent association with DNA. The GR binds the promoters of various glucocorticoid-responsive genes, including apoptosis-related genes. In this study, we examined whether the RNA degradation pathway can regulate this function of GAS5. We measured the steady-state abundance and the decay rate of GAS5 in UPF1-depleted human cells using the 5′-bromo-uridine immunoprecipitation chase (BRIC) method, an inhibitor-free method for directly measuring RNA stability. We found that levels of the GAS5 transcript were elevated owing to prolonged decay rates in response to UPF1 depletion, and consequently the apoptosis-related genes, cIAP2 and SGK1, were down-regulated. In addition, serum starvation also increased the transcript levels of GAS5 because of prolonged decay rates, and conversely decreased levels of cIAP2 and SGK1 mRNA. Taken together, we found that the RNA degradation pathway can regulate the function of the GAS5 ncRNA in mammalian cells.</p> </div

Serum starvation increases the expression level and prolongs the decay rate of GAS5, and decreases the expression of glucocorticoid-responsive genes.

<p>HEK293 cells were cultured with 0, 0.5, and 10% serum as indicated. (A) The relative expression levels of GAS5 in cells with 0 and 0.5% serum relative to control cells (cultured with 10% serum) were determined by RT-qPCR. (B) The decay rates of GAS5 were determined using the Click-iT Nascent RNA Capture Kit (Invitrogen) in control cells (solid circle and black bar) and in serum-starved cells (open circle and grey bar). Relative quantitative values at 0 h were set to 100%. (C) The relative expression levels of glucocorticoid-responsive genes, cIAP2 and SGK1, and the PPARδ-responsive gene, ADRP, in cells with 0 and 0.5% serum relative to control cells (cultured with 10% serum) were determined by RT-qPCR. (A–C) The levels of GAPDH and ACTB were used for normalization. Values represent mean±SD obtained from two (in B) or three (in A and C) independent experiments (*<i>P</i><0.05, **<i>P</i><0.01, Student's t test).</p