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Computational investigation of epithelial cell dynamic phenotype in vitro.

ABSTRACT: When grown in three-dimensional (3D) cultures, epithelial cells typically form cystic organoids that recapitulate cardinal features of in vivo epithelial structures. Characterizing essential cell actions and their roles, which constitute the system's dynamic phenotype, is critical to gaining deeper insight into the cystogenesis phenomena.Starting with an earlier in silico epithelial analogue (ISEA1) that validated for several Madin-Darby canine kidney (MDCK) epithelial cell culture attributes, we built a revised analogue (ISEA2) to increase overlap between analogue and cell culture traits. Both analogues used agent-based, discrete event methods. A set of axioms determined ISEA behaviors; together, they specified the analogue's operating principles. A new experimentation framework enabled tracking relative axiom use and roles during simulated cystogenesis along with establishment of the consequences of their disruption.ISEA2 consistently produced convex cystic structures in a simulated embedded culture. Axiom use measures provided detailed descriptions of the analogue's dynamic phenotype. Dysregulating key cell death and division axioms led to disorganized structures. Adhering to either axiom less than 80% of the time caused ISEA1 to form easily identified morphological changes. ISEA2 was more robust to identical dysregulation. Both dysregulated analogues exhibited characteristics that resembled those associated with an in vitro model of early glandular epithelial cancer.We documented the causal chains of events, and their relative roles, responsible for simulated cystogenesis. The results stand as an early hypothesis--a theory--of how individual MDCK cell actions give rise to consistently roundish, cystic organoids.

SUBMITTER: Kim SH

PROVIDER: S-EPMC2696420 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Computational investigation of epithelial cell dynamic phenotype in vitro.

Kim Sean H J SH Park Sunwoo S Mostov Keith K Debnath Jayanta J Hunt C Anthony CA

Theoretical biology & medical modelling 20090528

<h4>Background</h4>When grown in three-dimensional (3D) cultures, epithelial cells typically form cystic organoids that recapitulate cardinal features of in vivo epithelial structures. Characterizing essential cell actions and their roles, which constitute the system's dynamic phenotype, is critical to gaining deeper insight into the cystogenesis phenomena.<h4>Methods</h4>Starting with an earlier in silico epithelial analogue (ISEA1) that validated for several Madin-Darby canine kidney (MDCK) ep ...[more]

PMID: 19476639

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Project description:PURPOSE:Retinoblastoma (RB), a primary pediatric intraocular tumor, arises from primitive retinal layers. Several novel molecular strategies are being developed for the clinical management of RB. miRNAs are known to regulate cancer-relevant biological processes. Here, the role of selected miRNAs, namely, miR-532-5p and miR-486-3p, has been analyzed for potential therapeutic targeting in RB. METHODS:A comprehensive bioinformatic analysis was performed to predict the posttranscriptional regulators (miRNAs) of the select panel of genes [Group 1: oncogenes (HMGA2, MYCN, SYK, FASN); Group 2: cancer stem cell markers (TACSTD, ABCG2, CD133, CD44, CD24) and Group 3: cell cycle regulatory proteins (p53, MDM2)] using Microcosm, DIANALAB, miRBase v 18, and REFSEQ database, and RNA hybrid. The expressions of five miRNAs, namely, miR-146b-5p, miR-532-5p, miR-142-5p, miR-328, and miR-486-3p, were analyzed by qRT-PCR on primary RB tumor samples (n = 30; including 17 invasive RB tumors and 13 noninvasive RB tumors). Detailed complementary alignment between 5' seed sequence of differentially expressed miRNAs and the sequence of target genes was determined. Based on minimum energy level and piCTAR scores, the gene targets were selected. Functional roles of these miRNA clusters were studied by using mimics in cultured RB (Y79, Weri Rb-1) cells in vitro. The gene targets (SYK and FASN) of the studied miRNAs were confirmed by qRT-PCR and western blot analysis. Cell proliferation and apoptotic studies were performed. RESULTS:Nearly 1948 miRNAs were identified in the in silico analysis, From this list, only 9 upregulated miRNAs (miR-146b-5p, miR-305, miR-663b, miR-299, miR-532-5p, miR-892b, miR-501, miR-142-5p, and miR-513b) and 10 downregulated miRNAs (miR-1254, miR-328, miR-133a, miR-1287, miR-1299, miR-375, miR-486-3p, miR-720, miR-98, and miR-122*) were found to be common with the RB serum miRNA profile. Downregulation of five miRNAs (miR-146b-5p, miR-532-5p, miR-142-5p, miR-328, and miR-486-3p) was confirmed experimentally. Predicted common oncogene targets (SYK and FASN) of miR-486-3p and miR-532-5p were evaluated for their mRNA and protein expression in these miRNA mimic-treated RB cells. Experimental overexpression of these miRNAs mediated apoptotic cell death without significantly altering the cell cycle in RB cells. CONCLUSION:Key miRNAs in RB pathogenesis were identified by an in silico approach. Downregulation of miR-486-3p and miR-532-5p in primary retinoblastoma tissues implicates their role in tumorigenesis. Prognostic and therapeutic potential of these miRNA was established by the miRNA mimic strategy.

Project description:How do individual epithelial cells (ECs) organize into multicellular structures? ECs are studied in vitro to help answer that question. Characteristic growth features include stable cyst formation in embedded culture, inverted cyst formation in suspension culture, and lumen formation in overlay culture. Formation of these characteristic structures is believed to be a consequence of an intrinsic program of differentiation and de-differentiation. To help discover how such a program may function, we developed an in silico analogue in which space, events, and time are discretized. Software agents and objects represent cells and components of the environment. "Cells" act independently. The "program" governing their behavior is embedded within each in the form of axioms and an inflexible decisional process. Relationships between the axioms and recognized cell functions are specified. Interactions between "cells" and environment components during simulation give rise to a complex in silico phenotype characterized by context-dependent structures that mimic counterparts observed in four different in vitro culture conditions: a targeted set of in vitro phenotypic attributes was matched by in silico attributes. However, for a particular growth condition, the analogue failed to exhibit behaviors characteristic of functionally polarized ECs. We solved this problem by following an iterative refinement method that improved the first analogue and led to a second: it exhibited characteristic differentiation and growth properties in all simulated growth conditions. It is the first model to simultaneously provide a representation of nonpolarized and structurally polarized cell types, and a mechanism for their interconversion. The second analogue also uses an inflexible axiomatic program. When specific axioms are relaxed, growths strikingly characteristic of cancerous and precancerous lesions are observed. In one case, the simulated cause is aberrant matrix production. Analogue design facilitates gaining deeper insight into such phenomena by making it easy to replace low-resolution components with increasingly detailed and realistic components.

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Computational investigation of epithelial cell dynamic phenotype in vitro.

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Computational investigation of epithelial cell dynamic phenotype in vitro.

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