Transcriptional response of endometrial cells to Insulin, cultured using microfluidics.

Soo Young Baik, Alisha Maini, Haidee Tinning, Dapeng Wang, Daman Adlam, Peter T Ruane, Niamh Forde
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Abstract

Obesity is a rapidly growing public health issue among women of reproductive age associated with decreased reproductive function including implantation failure. This can result from a myriad of factors including impaired gametes and endometrial dysfunction. The mechanisms of how obesity-related hyperinsulinaemia disrupts endometrial function are poorly understood. We investigated potential mechanisms by which insulin alters endometrial transcript expression. Ishikawa cells were seeded into a microfluidics device attached to a syringe pump to deliver a constant flow rate of 1uL/min of the following: 1) control 2) vehicle control (acetic acid) or, 3) Insulin (10 ng/ml) for 24 hours (n=3 biological replicates). Insulin-induced transcriptomic response of endometrial epithelial cells was determined via RNA sequencing, and DAVID and Webgestalt to identify Gene Ontology (GO) terms and signalling pathways. A Total of 29 transcripts showed differential expression levels across two comparison groups (control v vehicle control; vehicle control v insulin). Nine transcripts were differentially expressed in vehicle control v insulin comparison (p<0.05). Functional annotation analysis of transcripts altered by insulin (n=9) identified three significantly enriched GO terms: SRP-dependent cotranslational protein targeting to membrane, poly(A) binding, and RNA binding (p<0.05). Over-representation analysis found three significantly enriched signalling pathways relating to insulin-induced transcriptomic response: protein export, glutathione metabolism, and ribosome pathways (p<0.05). Transfection of siRNA for RASPN successfully knocked down expression (p<0.05) but this did not have any effect on cellular morphology. Insulin-induced dysregulation of biological functions and pathways highlight potential mechanisms by which high insulin concentrations within maternal circulation may perturb endometrial receptivity.

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子宫内膜细胞对胰岛素的转录反应,微流体培养。
肥胖是育龄妇女中一个迅速增长的公共健康问题,与生殖功能下降有关,包括植入失败。这可能是由多种因素造成的,包括配子受损和子宫内膜功能障碍。肥胖相关的高胰岛素血症如何破坏子宫内膜功能的机制尚不清楚。我们研究了胰岛素改变子宫内膜转录物表达的潜在机制。将Ishikawa细胞植入与注射泵相连的微流体装置中,以1uL/min的恒定流速输送以下物质:1)对照物2)载体对照物(醋酸)或3)胰岛素(10 ng/ml),持续24小时(n=3个生物重复)。通过RNA测序、DAVID和Webgestalt鉴定基因本体(Gene Ontology, GO)术语和信号通路,确定胰岛素诱导的子宫内膜上皮细胞转录组反应。共有29个转录本在两个对照组(对照v对照;车辆控制v胰岛素)。对照与胰岛素对照有9个转录本差异表达(p
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