Transcriptomic data of human adrenocortical NCI-H295R cells treated with cortisol biosynthesis inhibitors

Adrenal corticosteroid biosynthesis dysregulation can give rise to various pathological conditions, such as Cushing's syndrome, a disorder characterized by the sustained and excessive production of cortisol. Despite the development of several classes of steroidogenesis inhibitors to treat human diseases associated with cortisol overproduction, their use is limited by insufficient efficacy, adverse effects, and/or tolerability. Recently, we identified a series of benzimidazolylurea derivatives, including the representative compound CJ28, as novel cortisol biosynthesis inhibitors [1]. They significantly inhibited both basal and stimulated production of cortisol in NCI-H295R cells, a human adrenocarcinoma cell line. The inhibitory effects were attributed to both attenuated steroidogenesis and de novo cholesterol biosynthesis. Here, we provide transcriptomic (RNA-seq) data from adrenal cell cultures in response to treatment with either CJ28 or metyrapone (MET), an inhibitor of 11β-hydroxylase). Total RNA was extracted from the cells treated with vehicle (0.1% DMSO), CJ28 (30 µM), or MET (30 µM) for 24 h. Primary sequence data were acquired using paired-end sequencing on an Illumina NovaSeq 6000 platform. The raw RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database (GSE236435). This dataset is a useful resource for providing valuable information on the gene expression networks underlying adrenocortical steroidogenesis.


a b s t r a c t
Adrenal corticosteroid biosynthesis dysregulation can give rise to various pathological conditions, such as Cushing's syndrome, a disorder characterized by the sustained and excessive production of cortisol.Despite the development of several classes of steroidogenesis inhibitors to treat human diseases associated with cortisol overproduction, their use is limited by insufficient efficacy, adverse effects, and/or tolerability.Recently, we identified a series of benzimidazolylurea derivatives, including the representative compound CJ28, as novel cortisol biosynthesis inhibitors [1] .They significantly inhibited both basal and stimulated production of cortisol in NCI-H295R cells, a human adrenocarcinoma cell line.The Keywords: Adrenal gland NCI-H295R Cortisol Corticosteroid Steroidogenesis Benzimidazolylureas Metyrapone inhibitory effects were attributed to both attenuated steroidogenesis and de novo cholesterol biosynthesis.Here, we provide transcriptomic (RNA-seq) data from adrenal cell cultures in response to treatment with either CJ28 or metyrapone (MET), an inhibitor of 11 β-hydroxylase).Total RNA was extracted from the cells treated with vehicle (0.1% DMSO), CJ28 (30 μM), or MET (30 μM) for 24 h.Primary sequence data were acquired using paired-end sequencing on an Illumina NovaSeq 60 0 0 platform.The raw RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database (GSE236435).This dataset is a useful resource for providing valuable information on the gene expression networks underlying adrenocortical steroidogenesis. ©

Value of the Data
• These data provide valuable information for understanding the effects of the two cortisol biosynthesis inhibitors with distinct modes of action on genome-wide gene expression profiles in cultured human adrenocortical cells.• These data can be valuable for researchers interested in studying genes and biological pathways regulating adrenal steroidogenesis.Additionally, they may offer insights into therapeutic strategies for treating pathological conditions linked with excess cortisol production.
• The raw RNA-seq data can be re-analyzed using various workflows and bioinformatics tools, providing additional insights into the molecular mechanisms underlying adrenal corticosteroid biosynthesis.

Objective
Glucocorticoids (GCs), such as cortisol in primates and corticosterone in rodents, serve as adrenocortical steroid hormones that mediate adaptive responses to stress and coordinate circadian physiology [2] .Hypercortisolism, known as Cushing's syndrome (CS), encompasses pathological conditions characterized by prolonged elevations in circulating GC levels, leading to the development of various metabolic, cardiovascular, immune/inflammatory, or neuropsychiatric diseases [2][3][4] .Several steroidogenesis inhibitors, such as metyrapone (MET, an inhibitor of 11 β-hydroxylase), ketoconazole (an inhibitor of cytochrome P450-containing enzymes), and mitotane (an inhibitor of 3 β-hydroxysteroid dehydrogenase), have been used to treat patients with CS.However, these drugs, that target specific steps of the steroidogenic pathway, still exhibit widespread adverse effects and show unsatisfactory efficacy [3 , 4] .Recently, we identified a series of benzimidazolylurea derivatives as a novel class of cortisol synthesis inhibitors that simultaneously target both steroidogenesis and de novo cholesterol biosynthesis [1] .Our steroidogenesis inhibitors caused alterations in the mRNA expression of a broad spectrum of genes linked to steroidogenesis, indicating that their actions are evidently distinct from those of the steroidogenic enzyme(s) inhibitors.Therefore, we examined genome-wide RNA expression profiles in cultured human adrenocortical cells in response to treatment with CJ28, a representative benzimidazolylurea derivative, or MET.

Data Description
We provided RNA-seq data from human adrenocortical NCI-H295R cell cultures treated with two distinct cortisol biosynthesis inhibitors, CJ28 and MET, each with unique chemical structures and modes of action.These treatments were compared with vehicle (VEH: 0.1% DMSO)-treated control cells.The experimental procedures and chemical structures of the cortisol synthesis inhibitors used in the study are shown in Figs. 1 a and b, respectively.The inhibitory effects of the chemical compounds were validated by reduced cortisol release into the culture medium ( Fig. 1 c).The cells used for cortisol measurement were then subjected to RNA-seq experiments as described in the Materials and Methods section.The sequenced library sizes and mapping statistics are summarized in Table 1 .Fig. 2 a displays the correlation between the gene expression profiles for all pairwise combinations of samples, including CJ28, MET, and VEH.The hierarchical tree indicates that CJ28treated cells exhibited distinct RNA profiles compared to MET-or VEH-treated cells, which were similar to each other based on normalized gene expression values.The differentially expressed genes (DEGs) after treatment with CJ28 or MET in comparison with the VEH-treated controls are summarized in Fig. 2 b.Using the criteria of Log2 fold change (FC) > 1.5 and P < 0.01, 787 and 18 genes were identified as DEGs with significantly altered expression by CJ28 and MET, respectively.Fig. 2 c exemplifies the relative mRNA expression levels of several key steroidogenic genes, comparing the two cortisol biosynthesis inhibitors.Despite having similar effects on cortisol production, treatment with CJ28 only significantly altered the mRNA expression levels of steroidogenic genes such as NR5A1, NR0B1, STAR, CYP11A1, HSD3B2, CYP17A1 and CYP21A1 , whereas MET did not.The DEGs obtained after CJ28 treatment, were utilized for gene enrichment analyses to gain insight into the possible modes of action in the related research article [1] .In this study, we present a comprehensive set of RNA-seq data, including those from MET-treated H295R cells, in addition to previously reported data showing the effects of CJ28.

Measurement of cortisol production
To validate the inhibitory effects of the inhibitors used in the study, cortisol levels in the culture medium were determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Arbor Assays, Ann Arbor, MI, USA).The medium was centrifuged at 4 °C for 5 min (10,0 0 0 × g) and then subjected to ELISA for cortisol measurement.

Preparation of RNA samples and RNA-seq analysis
Total RNA was isolated from frozen H295R cells using the microRNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol.RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and ribosomal RNA depletion was evaluated using the Ribo-Zero TM reagent (Illumina, San Diego, CA, USA).The cDNA libraries were prepared using the TruSeq TM Stranded Total RNA Prep Kit (Illumina) as per the manufacturer's instructions.Primary sequence data were acquired using paired-end sequencing on an Illumina NovaSeq 60 0 0 platform (Illumina).Raw sequence reads were trimmed for adaptor sequences using Cutadapt software (version 2.8) [5] .The trimmed sequence reads were mapped to GRCh38 using STAR Aligner (version 2.7.10b) [6] .Read count extraction and normalization were carried out using RSEM (version 1.3.3)[7] .The correlation matrix and hierarchical

Fig. 1 .
Fig. 1.Experimental procedures and validation of the effects of cortisol biosynthesis inhibitors.(a) Schematic representation of experimental procedures.(b) Chemical structures of the cortisol biosynthesis inhibitors used in this study.(c) Inhibitory effects of the compounds on spontaneous cortisol productions from the H295R cell cultures.

Fig. 2 .
Fig. 2. Analyses of DEGs obtained from CJ28-or MET-treated H295R cells.(a) Correlation of gene expression profiles for all pairwise combinations of samples using Pearson's correlation coefficient.(b) Volcano plots showing the DEGs in CJ28-or MET-treated H295R cell cultures.Genes with Log2 fold change (FC) > 1.5 and P < 0.01 were highlighted by red circles.Gene symbols for the top 10 DEGs are indicated.(c) Relative mRNA expression levels of key steroidogenic genes calculated from RNA-seq results.Data are presented as mean ± SEM of arbitrary unit (A.U.), with mean values of VEH group set as 1 (n = 4 for each group; * * : p < 0.01 vs. VEH group by Student's t-test).

Table 1
Key QC metrics of RNA-seq library after alignment with STAR.