scholarly journals Development of real-time reverse transcription polymerase chain reaction assays to quantify insulin-like growth factor receptor and insulin receptor expression in equine tissue

2013 ◽  
Vol 80 (1) ◽  
Author(s):  
Stephen B. Hughes ◽  
Melvyn Quan ◽  
Alan Guthrie ◽  
Martin Schulman
Keyword(s):  
Polymerase Chain Reaction ◽  
Growth Factor ◽  
Insulin Receptor ◽  
Ribonucleic Acid ◽  
Muscle Metabolism ◽  
Insulin Like Growth Factor ◽  
Chain Reaction ◽  
Messenger Ribonucleic Acid ◽  
Receptor Assay ◽  
Polymerase Chain

The insulin-like growth factor system (insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor and six insulin-like growth factor-binding proteins) and insulin are essential to muscle metabolism and most aspects of male and female reproduction. Insulin-like growth factor and insulin play important roles in the regulation of cell growth, differentiation and the maintenance of cell differentiation in mammals. In order to better understand the local factors that regulate equine physiology, such as muscle metabolism and reproduction (e.g., germ cell development and fertilisation), real-time reverse transcription polymerase chain reaction assays for quantification of equine insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were developed. The assays were sensitive: 192 copies/µLand 891 copies/µL for insulin-like growth factor 1 receptor, messenger ribonucleic acid and insulin receptor respectively (95%limit of detection), and efficient: 1.01 for the insulin-like growth factor 1 receptor assay and 0.95 for the insulin receptor assay. The assays had a broad linear range of detection (seven logs for insulin-like growth factor 1 receptor and six logs for insulin receptor). This allowed for analysis of very small amounts of messenger ribonucleic acid. Low concentrations of both insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were detected in endometrium, lung and spleen samples, whilst high concentrations were detected in heart, muscle and kidney samples, this was most likely due to the high level of glucose metabolism and glucose utilisation by these tissues. The assays developed for insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid expression have been shown to work on equine tissue and will contribute to the understanding of insulin and insulin-like growth factor 1 receptor physiology in the horse.

scholarly journals MiR-384 Inhibits Malignant Biological Behavior Such as Proliferation and Invasion of Osteosarcoma by Regulating IGFBP3

2020 ◽  
Vol 19 ◽  
pp. 153303382090912
Author(s):  
Yuelong Tan ◽  
Linlin Chen ◽  
Siwei Li ◽  
He Hao ◽  
Delong Zhang
Keyword(s):  
Polymerase Chain Reaction ◽  
Growth Factor ◽  
Cell Lines ◽  
Binding Protein ◽  
Osteosarcoma Cell ◽  
Insulin Like Growth Factor ◽  
Chain Reaction ◽  
Factor Binding ◽  
Polymerase Chain ◽  
Proliferation And Invasion

Osteosarcoma is the most common primary malignant bone tumor in the clinic. It is more common in children and adolescents. It has high malignancy, early metastasis rate, rapid disease progression, and high mortality. Although past years have witnessed the great improvement in the treatments of osteosarcoma, there remains a long way to go. MicroRNAs affect the malignant biological behaviors such as tumor proliferation and metastasis by regulating their target genes. In this study, we investigated the role and mechanism of miR-384 in osteosarcoma. Quantitative real-time polymerase chain reaction assay was performed to detect the expression of miR-384 and insulin-like growth factor binding protein 3 in osteosarcoma tissues and cell lines and established its correlation with osteosarcoma tumor progression and metastasis. To probe whether miR-384 played a tumor suppression role in osteosarcoma, we carried out gain-of-function and loss-of-function assays. Cell Counting Kit-8, cell colony formation, and transwell assays were carried out to determine the cells proliferation and invasion, respectively. Western blot was used to detect the changes of epithelial–mesenchymal transition marker proteins and insulin-like growth factor binding protein 3. MiR-384 was downregulated in osteosarcoma tissues and cell lines. MiR-384 was overexpressed in G292 cells transfected with miR-384 mimics and knocked down in Saos-2 cells with small hairpin RNA targeting miR-384. Ectopic expression of miR-384 inhibited osteosarcoma cell proliferation, colony formation, and invasion. E-cadherin was brought to a decrease whereas N-cadherin and Snail to an increase under the silent expression of miR-384, while overexpression of miR-384 led to an opposite result. MiR-384 could regulate insulin-like growth factor binding protein 3 expression in osteosarcoma. Quantitative polymerase chain reaction and Western blotting results validated that miR-384 knockdown downgrades both messenger RNA and protein levels of insulin-like growth factor binding protein 3 in G292 cells, while miR-384 upregulation exerted an opposite effect in Saos-2 cells. Insulin-like growth factor binding protein 3 was upregulated in osteosarcoma tissues and osteosarcoma cell lines compared with normal ones. Through the bioinformatics database found that the upstream transcriptional regulator of insulin-like growth factor binding protein 3 is MECP2. So miR-384 can directly inhibit MECP2 and then promote the expression of insulin-like growth factor binding protein 3. These results suggested that miR-384 might be a potential therapeutic targets and biomarker in osteosarcoma.

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