Abstract
Intestinal epithelial cells are constantly exposed to physiologically hypoxic environment. The further reduction of tissue oxygen delivery may result in the intestinal epithelial cells function impairment, being a sign of active inflammation. The cultivation conditions are important when performing in vitro studies, since those may affect the cells’ properties. The study was aimed to assess the integrin receptor expression in the human colon adenocarcinoma Caco-2 cell line when simulating both hypoxic condition using the cobalt chloride and microcirculation. Transcriptome analysis revealed the significantly increased expression of the integrin receptors ITGA2 and ITGA5 α2- and α5-subunit genes under hypoxic conditions, as well as the reduction of ITGA5 during incubation in the microfluidic chip. The expression of β-subunits did not change. Analysis of microRNA transcriptomes revealed the decreased expression of hsa-miR-766-3p and hsa-miR-23b-5p microRNA. One of the validated targets for both microRNAs is mRNA of gene ITGA5. It has been shown that microcirculation makes it possible to bring the intestinal epithelial cells cultivation conditions closer to physiological conditions. The possible biological significance of the detected integrin expression profile alterations and the role of microcirculation have been discussed.
Highlights
Author contribution: Maltseva DV — working with cultured cells, molecular biology research, data analysis, manuscript writing; Poloznikov AA — proteomic and transcriptomic analysis data processing, bioinformatics analysis, functional analysis of genes, statistical analysis, manuscript writing, study management; Artyushenko VG — interpreting the study results, manuscript reviewing
Mass spectrometry of proteome for the treated and reference cells failed to detect the significant uncrease in the level of the following four proteins encoded by the HIF-1 signalling pathway genes: ENO2 by 19.6, HMOX1 by 29.1, PDK1 by 2.8, and SLC2A1 by 2.2 times respectively
Proteome analysis revealed the total of 120 proteins, the expression of which significantly changed under hypoxia-mimicking conditions
Summary
Author contribution: Maltseva DV — working with cultured cells, molecular biology research, data analysis, manuscript writing; Poloznikov AA — proteomic and transcriptomic analysis data processing, bioinformatics analysis, functional analysis of genes, statistical analysis, manuscript writing, study management; Artyushenko VG — interpreting the study results, manuscript reviewing. Acknowledgement: the authors express their appreciation to the Center for Collective Use "Human Proteome" (IBMC) for the opportunity to use the equipment for proteome analysis, and to the Center for Precision Genome Editing and Genetic Technologies for Biomedicine of the Pirogov Russian National Research Medical University (Moscow, Russia) for the opportunity to use the molecular biology technologies. Целью работы было провести оценку экспрессии интегриновых рецепторов в клетках аденокарциномы толстого кишечника человека Caco-2 при моделировании условий гипоксии хлоридом кобальта (II) и микроциркуляции. С помощью транскриптомного анализа обнаружено значимое увеличение экспрессии генов Ƚ2- и Ƚ5-субъединиц интегриновых рецепторов ITGA2 и ITGA5 в условиях гипоксии и уменьшение ITGA5 при инкубировании в микрофлюидном чипе. Ключевые слова: гипоксия, интегрины, микроРНК, мРНК, микрофлюидный чип, протеом, кишечная микробиота, Caco-2.
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