Endocrine-Related Cancer (2004) 11 295–303 Gene expression profiling in human insulinoma tissue: genes involved in the insulin secretion pathway and cloning of novel full-length cDNAs X-C Wang, S-Y Xu1, X-Y Wu 2, H-D Song1, Y-F Mao1, H-Y Fan 2,FYu1, B Mou1, Y-Y Gu1, L-Q Xu1, X-O Zhou1, Z Chen 2, J-L Chen1 and R-M Hu State Key Laboratory of Genetics Engineering, Institute of Endocrinology and Diabetology, Department of Endocrinology, Huashan Hospital, Fu Dan University, 12 Middle Urumqi Road, Shanghai 200040, People’s Republic of China Shanghai Institute of 1Endocrinology and 2Hematology, Ruijin Hospital, Shanghai Second Medical University, 197 Ruijin Er Road, Shanghai 200025, People’s Republic of China (Requests for offprints should be addressed to R-M Hu; Email: [email protected]) Abstract Insulinoma is a clinically common cause of organic hypoglycemia. The prominent characteristic of insulinoma is endogenous hyperinsulinism. Until now, the molecular biology of human insulinoma has been little understood. In this study, gene expression profiling of human insulinoma was established by expressed sequence tag (EST) sequencing and cDNA array. A total of 2063 clones were obtained, of these, 1589 clones were derived from EST sequencing, 975 clones were derived from cDNA array and 501 clones were shared by the two methods. G protein a-stimulating activity polypeptide (Gsa) and carboxypeptidase E (CPE) were the most highly expressed genes in human insulinoma, as derived by EST sequencing and cDNA array respectively. The genes involved in the protein/insulin secretion pathway were strongly expressed in human insulinoma tissue. Meanwhile, eight full-length cDNAs of novel genes were cloned and sequenced. The results demonstrated the molecular biology of human insulinoma tissue at the level of transcript abundance and validated the efficacy of EST sequencing combined with cDNA array in the construction of gene expression profiling. In conclusion, the predominance of the genes participating in the secretory pathway suggested that regulation of secretion might be a major mechanism by which insulin release is abnormally increased in patients with insulinomas. It was also concluded that overexpression of the Gsa gene played an important role in the pathogenesis of insulinoma. Endocrine-Related Cancer (2004) 11 295–303 Introduction Diabetes is characterized by hyperglycemia resulting from dysfunction of b-cells, and/or insensitivity to insulin, of The maintenance of plasma glucose concentration is peripheral tissues. Hypoglycemia can be the result of critical to the survival of most organisms; this is due to drugs, critical illness including hepatic or renal failure, plasma glucose being the predominant fuel utilized by hormonal deficiencies, endogenous hyperinsulinism (insu- organic cells under most conditions, especially cells of the linoma), etc. (Larsen et al. 2002). nervous system. The b-cells of the pancreatic islets of An insulinoma is the most common cause of Langerhans play a central role in glucose homeostasis by hypoglycemia resulting from endogenous hyperinsulin- secreting the polypeptide hormone insulin. Under phy- ism. The most consistent insulin secretory abnormalities siological conditions, b-cells sense plasma glucose con- associated with insulinoma are oversecretion of insulin centration and maintain tight control over insulin and failure of the normal decrease in insulin secretion biosynthesis and secretion. Functional disorders of which occurs as plasma glucose levels decline in the b-cells could result in hyperglycemia or hypoglycemia. postabsorptive state. Insulinomas are rare, the estimated Endocrine-Related Cancer (2004) 11 295–303 Online version via http://www.endocrinology.org 1351-0088/04/011–295 # 2004 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/27/2021 07:35:41PM via free access Wang et al.: Gene expression profiling in insulinoma tissue incidence being one case per 250 000 patient-years in the package (Genetic Computer Version 9.0). The good- United States, but it is a curable cause of potentially lethal quality sequences were referred to those longer than hypoglycemia. Insulinomas almost always come to 100 bp and containing less than 3% ambiguous bases. clinical attention because of hypoglycemia (Larsen et al. After being compared with public databases such as 2002). Most insulinomas are derived from monoclonal GenBank, dbEST and Unigene by using Blast and Fasta proliferation of the b-cells of the pancreatic islets, which software, analyzable sequences were categorized into: means insulinomas are an optimal model for studying known genes that shared at least 95% homology with b-cell biology. Insulinoma cells offer several advantages certain genes over at least 100 bp of DNA sequences; over cultured b-cell lines and isolated islet material: (1) known ESTs that couldn’t meet the criteria of known studies of b-cell lines are conducted in vitro; (2) isolated genes but shared at least 95% homology over at least islet material is a complex tissue composed of varied cell 100 bp of ESTs in dbEST; novel ESTs that shared no types. Insulinoma cells offer not only a pure b-cell homology or little homology with known genes and population but also a study in vivo. known ESTs. ESTs corresponding to known genes were To illuminate the molecular biology of insulinoma/ divided into several categories according to the Gene b-cells, gene expression profiling of human insulinoma Ontology Consortium functions (Ashburner et al. 2000) tissue has been established using expressed sequence tag (http://www.geneontology.org). Clustering of the ESTs (EST) sequencing and cDNA array. As a result, the genes generated in the work was performed using CAT 3.2 from involved in the secretory pathway of protein/insulin are Pangea (Oakland, CA, USA) with default parameters. strongly expressed in insulinoma tissue and several new full-length cDNAs have been cloned. cDNA microarray A total of 14 000 clones were spotted onto nylon Materials and methods membrane-based cDNA arrays by using Spotter (Bio- RNA extraction Robotics, Cambridge, UK); these clones were derived from human liver and hemopoietic stem cells and Insulinoma and normal pancreatic tissues were obtained hypothalamus–pituitary–adrenal (HPA) libraries or pur- from patients with insulinomas and adult males who had chased from Research Genetics (Huntsville, AL, USA). died of accidents respectively. Total RNA was extracted Approximately 1–2 mg mRNA of human insulinoma from frozen tissues using TRIzol reagent (Invitrogen). tissue were labeled in a reverse transcription (RT) in the mRNA was separated and purified using oligo(dT) presence of 200 mCi [a-33P]deoxyadenosine 50-tripho- (Qiagen). sphate (DuPont NEN, Boston, MA, USA) using Moloney murine leukemia virus reverse transcriptase according to cDNA library construction the manufacturer’s instructions (Promega). Prehybridiza- tion was carried out in 20 ml prehybridization solution cDNA synthesis was performed with a CapFinder PCR (6ÂSSC, 0.5% SDS, 5ÂDenhardt’s solution, 100 mg/ml cDNA library construction kit (Clontech). denatured salmon sperm DNA) at 688C for 4 h. Overnight hybridization with the 33P-labeled cDNA in 10 ml cDNA sequencing hybridization solution (6ÂSSC, 0.5% SDS, 100 mg/ml salmon sperm DNA) was followed by stringent washing Bacteria growth and plasmid extraction were performed (0.1ÂSSC, 0.5% SDS, at 658C for 1 h). Membranes were in a 96-well format (Qiagen). Sequencing reactions were exposed to a phosphor screen overnight and scanned performed on a 9600 thermal reactor (Perkin-Elmer, using a FLA-3000A plate/fluorescent image analyzer (Fuji Shelton, CT, USA) using a dye primer cycle sequencing Photo Film, Tokyo, Japan). The data analyses of kit (Perkin-Elmer), and partial cDNA sequencing of each hybridized membranes were performed using Array clone was taken from the 50 end. The reaction products Gauge software (Fuji Photo Film). were analyzed using an ABI 3700 DNA sequencer (Perkin-Elmer). Semi-quantitative RT-PCR Total RNA (1 mg) was incubated with DNase I and Bioinformatics analysis and data management reverse-transcribed with oligo(dT) using the superscript II Sequencing data were transmitted to a Sun station (Sun RT-PCR system (Life Technologies). RT product (1 ml) Microsystems, Santa Clara, CA, USA). Analysis of was amplified with primer pairs specific for the genes to be sequences, including quality assessment and quality base studied. Semi-quantitative multiplex PCR assays were trimming of ESTs, was performed with a GCG software designed to compare the RT-PCR products of the genes 296 www.endocrinology.org Downloaded from Bioscientifica.com at 09/27/2021 07:35:41PM via free access Endocrine-Related Cancer (2004) 11 295–303 under study with b-actin transcript. Conditions and significance > 99.9%. As a result, a total of 975 clones primer sequences are available on request. Each RT- were found expressed in human insulinoma tissue: 616 PCR product was loaded on 1.5% agarose gel containing known genes accounted for 63.2%, 275 known ESTs 0.5 mg/ml ethidium bromide. Gel images were made and accounted for 29.2% and 74 novel ESTs for 7.6%. To saved using the Gel Documentation System (UVP, confirm the accuracy and validity of the results of cDNA Upland, CA, USA). array, several expressed clones, including the highest and lowest expressed, were selected for RT-PCRs. The outcomes of RT-PCRs proved the cDNA array to be Cloning of full-length cDNAs accurate and valid. ESTs from dbEST and other databases were selected out; The highly expressed genes in human insulinoma are these ESTs were highly homologous with known ESTs listed in Tables 1 and 2 and some of these genes were from our cDNA library. Assemblies were carried out chosen to undergo semi-quantitative RT-PCR, as shown among known ESTs and selected ESTs using Autoassem- in Fig. 1. Using ESTs sequencing, G protein a-stimulating bler software (PE Applied Biosystems, Foster City, CA, activity polypeptide Gsa (14 copies) and insulin (13 USA).