title: MicroRNAs in gastric and oesophageal cancer-associated myofibroblasts
Creators: name: Wang, Liyi
Publisher: name: University of Liverpool Repository
Year: 2013

description: MicroRNAs (miRNAs) are small RNAs of ~22 nucleotides in length that regulate gene expression post-transcriptionally. MicroRNAs regulate many biological processes, and may contribute to cancer initiation and progression. In the normal gastrointestinal mucosa, myofibroblasts are involved in wound healing, and in tumours, they play a role in cancer progression. The present study aimed to determine the miRNA expression profiles in different populations of myofibroblasts from gastric and oesophageal tissues, namely, cancer-associated myofibroblasts (CAMs), adjacent non-tumour tissue myofibroblasts (ATMs) and normal tissue myofibroblasts (NTMs), and to investigate the processes that are influenced by differential miRNA abundance in different myofibroblast populations. In addition, miRNA profiling of a putative myofibroblast precursor cell type, mesenchymal stromal cells (MSCs), was studied. Global miRNA expression profiling of a panel of previously characterised gastric and oesophageal myofibroblasts was determined using locked nucleic acid (LNA) miRNA arrays. The microarray data for hsa-miR-29b, hsa-miR-181d, hsa-miR-214 and hsa-miR-424 were validated by quantitative RT-PCR. Using unsupervised miRNA datasets, principal component analysis (PCA) segregated gastric and oesophageal NTMs, and CAMs from gastric and oesophageal carcinomas. Analysis of global miRNA expression showed distinct profile of MSCs in comparison to gastric and oesophageal NTMs. Hierarchical clustering analysis of miRNAs exhibiting significantly different abundance revealed distinct clusters between CAMs and ATMs, CAMs and NTMs and between ATMs and NTMs. In 12 paired samples of gastric CAMs and their corresponding ATMs, 12 miRNAs were significantly different with hsa-miR-181d being the most up-regulated miRNA in CAMs. Using already validated targets of 10 of these miRNAs, an analysis using MetaCore® indicated that the regulation of cell cycle progression and Wnt signalling were differentially affected. In a comparison of each CAM and its corresponding ATM, Wnt signalling was the only process that was significantly targeted by miRNAs that were different in abundance, in all 12 pairs. Using previously determined gene expression data, pairwise analysis of 20 transcripts associated with Wnt signalling showed a significant increase in WNT5A and TGFB2 mRNAs in CAMs compared to their ATMs. Additionally, Western blot analysis showed increased expression of Wnt-5a in CAMs. The results of migration and EdU incorporation assays indicated that Wnt-5a induced the migration and proliferation of both CAMs and ATMs. The abundance of hsa-miR-181d was significantly increased in CAMs by Wnt-5a. Migration of AGS (gastric cancer) cells was stimulated by Wnt-5a and was inhibited in the presence of TIMP-3, a validated target of the miR-181 family. After hsa-miR-181d knockdown, the migration and proliferation of CAMs were significantly decreased, and stimulation with Wnt-5a reversed the migratory inhibition of hsa-miR-181d knockdown CAMs. Conditioned medium from hsa-miR-181d knockdown CAMs inhibited the migration of AGS cells. The findings suggest that differential miRNA abundance is a feature of normal myofibroblasts from the stomach and oesophagus, and myofibroblasts from normal and cancer tissues. One consequence is increased Wnt-5a in gastric CAMs that is implicated in the regulation of cell migration, in health and disease, at least partly through modulation of hsa-miR-181d.