Engineering extracellular matrix factories to study how the cellular microenvironment regulates gene expression

点击翻译按钮获取中文摘要

Mammalian cells and tissues exist in complex three-dimensional (3D) microenvironments, in which diverse signalling molecules and support structures dynamically interact forming a complex network or extracellular matrix (ECM). Beyond providing structural support to cells, the matrix conveys environmental signals that direct many aspects of normal cell behaviour, including shape, survival and migration. We found rapid induction of the ECM glycoprotein tenascin-C upon tissue damage (Midwood et al. Nat Med 2009) and during infection (Goh et al. JI 2010) that enables effective host responses in the mouse in vivo (Midwood et al. Nat Med 2009; Piccinini et al. Cell Reports 2012). Notably, we discovered that tenascin-C can sustain pro-inflammatory cytokine synthesis by regulating macrophage microRNA expression (Piccinini et al. Cell Reports 2012). Thus, this ECM protein creates a cellular microenvironment that profoundly influences cell phenotype and behaviour by regulating microRNA levels and, in turn, gene expression profiles. However, we do not know how tenascin-C does this and whether this occurs also in humans. Replicating these signals in conventional cell culture systems is often challenging. Moreover, while artificially-created 3D cell culture models have shown potential in tissue engineering and stem cell research, they fail to closely mimic the in-vivo-like microenvironment. Some reproduce the three dimensionality of the matrix, but lack the biochemical cues; others contain non-physiologically relevant microenvironmental signals or do not allow studying the contribution of individual components to the cellular process of interest.The aim of this project is to engineer physiologically relevant human ECM factories that can be used to define whether specific components of the ECM, including tenascin-C, impact gene expression profiles by regulating microRNA levels of human macrophages and cancer cells. For this, CRISPR/Cas9 technology will be employed for genome editing of human fibroblast cell lines, which will be used to generate cell-free human fibroblast-derived 3D matrix models. These models with be biochemically, biophysically and functionally characterized, and used as substrate for the culture of cell types of interest, including primary human monocyte-derived macrophages. Effects of individual ECM molecule depletion on candidate target gene expression (e.g. early response inflammatory microRNAs and their targets) will be investigated by real-time PCR. If these models are validated, there is also the possibility to perform RNA sequencing to globally identify genes and pathways that are affected by the matrix. Contingency plans include stable shRNA or siRNA knockdown to deplete candidate ECM molecules. Collectively, this project develops a biotechnology tool for thoroughly dissecting the role of individual, human microenvironmental signals from the ECM in the posttranscriptional regulation of gene expression, presenting new opportunities for research on host responses to infection, and tumour microenvironments.