Project Title

RNA-Seq to analyze two related rat Growth Hormone-producing somatotroph cell lines for tissue-specific transcript expression

Authors' Affiliations

Pharm.D. students in the Concentration in Pharmacy Research; and Dept. of Pharmaceutical Sciences, Bill Gatton College of Pharmacy

Location

Clinch Mtn

Start Date

4-12-2019 9:00 AM

End Date

4-12-2019 2:30 PM

Poster Number

176

Faculty Sponsor’s Department

Pharmaceutical Sciences

Name of Project's Faculty Sponsor

Dr. David Hurley

Type

Poster: Competitive

Classification of First Author

Pharmacy Student

Project's Category

RNA

Abstract Text

Growth Hormone (GH), also known as somatotropin, is a protein hormone secreted from the anterior pituitary gland. GH action results in longitudinal bone growth in children and adolescents and continues later to control a variety of metabolic reactions in adults. DNA polymorphisms leading to a decrease in GH expression result in persons of short stature, whereas those leading to overexpression of GH produce either acromegaly or gigantism. In order to diagnose and treat patients with altered GH production, understanding the control of GH expression is thus clinically relevant. In order to understand how transcription of GH is determined by selective regulatory modulators, two rat somatotroph cell lines were studied that differed in their relative differentiation: MtT/S cells are considered as fully differentiated somatotrophs that express GH exclusively; MtT/Se cells are related somatotrophs with GH expression that is responsive to estrogen treatment. These cell lines were obtained from the Riken Cell Bank (Japan) and then grown and cultured in accordance with established protocols. After harvesting of cells from each line, total RNA was extracted using a rapid affinity method (Qiagen). After validation of the RNA integrity index of each RNA isolate (Affymetrix), they were sent to an off-site laboratory (NovoGene) for RNA-Seq analysis. Samples were examined in triplicate for comparison using standard procedures for adapter ligation, library construction, amplification and sequencing. In our previous work with single gene quantitative RT-PCR, we measured expression of 9 separate targets, primarily transcriptional controllers, in these cell lines. Now, the use of RNA-Seq quantifies the levels of all mRNAs in each sample without the need for specific primers or probes. Therefore, it is possible to find nearly all of the mRNAs that demonstrate changing abundance without requiring prior evidence of their role in the terminal differentiation of the MtT/S from MtT/Se cells. After extensive QC for validation, initial analysis of the data shows more than 36 million validated sequenced reads from each replicate sample with >96.39% mapping to the reference rat genome sequence. Basic analysis shows that 13,012 expressed genes were 87% similar, with 1,175 (9.0%) unique to MtT/Se cells and 484 (3.7%) genes selectively present in MtT/S cells. Of these, 329 genes were upregulated in expression while only 57 were reduced. For comparison with our previous study, we are now confirming differences in GH transcripts and the transcription factors/regulators previously measured, particularly the GH regulator Zn16, a protein with 16 zinc fingers known to bind to the GH promoter DNA. Further analysis of the RNA-Seq profile is focused on the unique and unidentified changes in expression that differentiate these cell lines. Analysis of the differentiation of MtT/S and MtT/Se cells will further understanding of GH regulation in the body.

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Apr 12th, 9:00 AM Apr 12th, 2:30 PM

RNA-Seq to analyze two related rat Growth Hormone-producing somatotroph cell lines for tissue-specific transcript expression

Clinch Mtn

Growth Hormone (GH), also known as somatotropin, is a protein hormone secreted from the anterior pituitary gland. GH action results in longitudinal bone growth in children and adolescents and continues later to control a variety of metabolic reactions in adults. DNA polymorphisms leading to a decrease in GH expression result in persons of short stature, whereas those leading to overexpression of GH produce either acromegaly or gigantism. In order to diagnose and treat patients with altered GH production, understanding the control of GH expression is thus clinically relevant. In order to understand how transcription of GH is determined by selective regulatory modulators, two rat somatotroph cell lines were studied that differed in their relative differentiation: MtT/S cells are considered as fully differentiated somatotrophs that express GH exclusively; MtT/Se cells are related somatotrophs with GH expression that is responsive to estrogen treatment. These cell lines were obtained from the Riken Cell Bank (Japan) and then grown and cultured in accordance with established protocols. After harvesting of cells from each line, total RNA was extracted using a rapid affinity method (Qiagen). After validation of the RNA integrity index of each RNA isolate (Affymetrix), they were sent to an off-site laboratory (NovoGene) for RNA-Seq analysis. Samples were examined in triplicate for comparison using standard procedures for adapter ligation, library construction, amplification and sequencing. In our previous work with single gene quantitative RT-PCR, we measured expression of 9 separate targets, primarily transcriptional controllers, in these cell lines. Now, the use of RNA-Seq quantifies the levels of all mRNAs in each sample without the need for specific primers or probes. Therefore, it is possible to find nearly all of the mRNAs that demonstrate changing abundance without requiring prior evidence of their role in the terminal differentiation of the MtT/S from MtT/Se cells. After extensive QC for validation, initial analysis of the data shows more than 36 million validated sequenced reads from each replicate sample with >96.39% mapping to the reference rat genome sequence. Basic analysis shows that 13,012 expressed genes were 87% similar, with 1,175 (9.0%) unique to MtT/Se cells and 484 (3.7%) genes selectively present in MtT/S cells. Of these, 329 genes were upregulated in expression while only 57 were reduced. For comparison with our previous study, we are now confirming differences in GH transcripts and the transcription factors/regulators previously measured, particularly the GH regulator Zn16, a protein with 16 zinc fingers known to bind to the GH promoter DNA. Further analysis of the RNA-Seq profile is focused on the unique and unidentified changes in expression that differentiate these cell lines. Analysis of the differentiation of MtT/S and MtT/Se cells will further understanding of GH regulation in the body.