Quantitative expression data of human estrogen receptor α variants in non-functioning pituitary adenomas obtained by reverse transcription-digital polymerase chain reaction analysis

Expression profiles of gonadal steroid receptor variants have been reportedly associated with malignancy in breast and prostate cancers [1,2]. However, such associations with pituitary tumors remain unclear. Therefore, the expression levels of the wild-type ESR1 (ERα66) and the ESR1 variants (ERαi34, ERαi45c, and ERαΔ5) transcripts encoding constitutively active ERα proteins with C-terminal truncation in non-functioning pituitary adenomas (NFPAs) were evaluated using reverse transcription-digital polymerase chain reaction. The results revealed that the expression levels of the variants were approximately two orders of magnitude lower than that of ERα66 in NFPAs. These data were based on our previous article entitled “Accurate assessment of estrogen receptor profiles in non-functioning pituitary adenomas using RT-digital PCR and immunohistochemistry” [3].


Value of the Data
• Expression profiles of gonadal steroid receptor variants have been reportedly associated with malignancy in breast and prostate cancers. In these tumors, the expression levels of the variants that are not normally detected were increased as compared to the wild-type. • To date, several studies have reported that the wild-type full-length estrogen receptor (ER) is involved in the development of non-functioning pituitary adenomas, but there have been no such reports for ER variants. • The data obtained in this study will be valuable to researchers with interests in endocrinology and steroid hormone receptors, especially ERs. • In this study, a quantitative method was established to evaluate the expression levels of human ESR1 . Furthermore, this method can be used not only for pituitary tumors but also for tumors originating in other organs.

Data Description
The human ESR1 gene contains eight conventional coding exons (exons 1-8) and several cryptic exons. Alternative splicing of the exons generates multiple ESR1 variants with distinct structures and functions [4 , 5] . ER αi34 [5] , ER αi45c [4 , 6] , and ER α 5 [4 , 7] variant transcripts encode C-terminally truncated ER α proteins with strong constitutive activation. The mRNA structures of wild-type ESR1 (ER α66) and the ESR1 variants are represented schematically in Fig. 1 . To the best of our knowledge, this is the first report examining the expression levels of ESR1 variants in non-functioning pituitary adenomas (NFPAs). In the present study, the expression levels of the variant transcripts and wild-type ESR1 were compared.
The expression levels of the ER α66 and variant transcripts in NFPAs were quantified using reverse transcription-digital polymerase chain reaction (RT-dPCR) ( Fig. 2 , Table 1 ). The expression values were determined by dividing the copy number of the target gene by the geometric mean of the copy numbers of the internal control genes, GAPDH and ALAS1 . The normalized values (mean ± SEM) of ER α66, ER αi34, ER αi45c, and ER α 5 were 0.063 ± 0.014, 0.001 ± 0.0 0 02,  Source: mRNA expression levels of wild-type ESR1 (ER α66) and the ESR1 variants (ER αi34, ER αi45c, and ER α 5) in NFPA tissues were quantified by RT-dPCR analysis. Data are expressed as mean ± SEM. The expression levels were normalized against the combination of the internal control genes, GAPDH and ALAS1 . The columns with different letters indicate significant differences. Raw data are shown in Table 1 . 0.0 0 03 ± 0.0 0 0 08, and 0.0 01 ± 0.0 0 02, respectively. In NFPA tissues, the expression levels of the variants were approximately two orders of magnitude lower than that of ER α66.

Sample preparation
NFPA specimens were collected from 20 patients with a definitive pathological diagnosis. Detailed patient information is reported in our related research article [3] . Total RNA was extracted from the NFPAs immediately after resection using NucleoSpin® RNA Plus kits (Macherey-Nagel GmbH & Co. KG, Düren, Germany) and reverse-transcribed using ReverTra Ace® reverse transcriptase (Toyobo Co., Ltd., Osaka, Japan). The reverse transcription reaction was conducted at 42 °C for 60 min and then terminated by heating at 75 °C for 15 min.

RT-dPCR analysis
For expression analyses, dPCR was performed with the QuantStudio 3D Digital PCR System platform and a GeneAmp® PCR System 9700 (Thermo Fisher Scientific, Waltham, MA, USA). The dPCR primers were synthesized by Thermo Fisher Scientific. The TaqMan Gene Expression Assay identification codes of the primers were AI1RXEC for ESR1 , AI39TQS for ER αi34, AI20VKK for ER αi45c, AI0IY74 for ER α 5, Hs99999905_m1 for GAPDH , and Hs00963537_m1 for ALAS1 . Each dPCR reaction comprised cDNA corresponding to 150 ng of total RNA and 0.75 μl of Taq-Man Genotyping Master Mix in accordance with the manufacturer's protocol. The PCR mixture (14.5 μl) was loaded onto each QuantStudio 3D digital PCR chip (Thermo Fisher Scientific). The cycling condition of the dPCR reaction comprised an initial denaturing step at 96 °C for 10 min, followed by 39 cycles at 60 °C for 2 min and 98 °C for 30 s, and a final extension step at 60 °C for 2 min, as described previously [3] .

Statistical Analysis
All statistical analyses were performed using IBM SPSS Statistics for Windows, version 25.0 (IBM Corporation, Armonk, NY, USA). The data were assessed using one-way analysis of variance followed by Tukey's post-hoc test. A P -value of less than < 0.05 was considered statistically significant.

Ethics Statement
The study design and protocol were approved by the Ethics Review Committee of Nippon Medical School (approval number 29-06-767) and written informed consent was obtained from all patients.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.