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The role of long intergenic non-coding RNA in intestina stem cells

Final Report Summary - LINCRNA (The role of long intergenic non-coding RNA in intestina stem cells)

Last decade, lincRNAs (long intergenic non-coding RNAs) emerged as novel regulators in multiple biological processes such as development, pluripotency and tumorigenesis. Although studies on lincRNAs have exponentially increased with thousands of lincRNAs identified in various cell types from different organisms, a vast majority of lincRNAs have not been functionally characterized. To understand the physiological relevance of lincRNAs, comprehensive functional studies including loss of function studies at the level of the organism are imperative.
In adult mammals, the intestinal epithelium is the most rapidly self-renewing tissue fueled by intestinal stem cells (ISC). The epithelium is formed into two distinct compartments; differentiated cells in villus and ISCs and transit amplifying cells in crypt. The unique structure and vigorous self-renewing property make intestinal epithelium one of the most accessible models for adult stem cell study. Recently in vitro culture system (so called organoids) has been developed from ISCs. The organoid resembles the three‐dimensional architecture of intestinal epithelium and faithfully recapitulates the self-renewal and differentiation processes.
This project aimed to study the physiological role of lincRNAs in ISC development by utilizing in vitro organoids culture system and in vivo mouse model. For this purpose, the lead researcher first identified lincRNAs which are specifically expressed in ISCs. About 100 ISC-specific lincRNAs have been identified by carrying out RNA-sequencing in Lgr5 (ISC marker gene) positive cells from mouse intestine. To narrow down the list, the lead researcher investigated whether the expression of ISC specific lincRNAs are regulated by transcription factors Ascl2, Tcf4, beta-catenin which are important for ISC maintenance and differentiation. By comparing the ISC-specific lincRNAs with available ChIP-seq data of the transcription factors, about 20 lincRNAs have been selected and their expression has been confirmed by performing RT-PCR and in situ hybridization.
To do functional analysis of the lincRNAs using organoid system, the lead researcher has established over-expression and knock-down strategy in organoids. Furthermore, cutting-edge genome editing method using CRISPR/Cas9 has been applied successfully to organoids. The lead researcher together with colleagues introduced CRISPR/Cas9 system into intestine organoids to modify genome sequence of the protein coding genes, which can be applied to lincRNA genes. This result has been published in Cell Stem Cell.
To investigate physiological role of those lincRNAs at the organism level, the lead researcher developed a new knock-out mouse strategy to block lincRNA expression in a tissue specific, timed manner in the adult mouse. Through the several steps of breeding, intestine specific lincRNA knock-out mice have been generated recently. The preliminary results with the knock-out mice suggested that one of the lincRNAs might play a role in the maintenance of ISC. It seemed that deletion of the lincRNA in intestine affected stem cell viability. This knock-out mouse needs to be analyzed further to confirm the phenotype and to define the detailed mechanism within several months. This study will become the first report on intestinal stem cell-specific lincRNAs. Therefore, this project will contribute to reveal unidentified aspect of adult stem cells, providing a novel regulation mechanism in adult stem cells. The outcome of this study will lay the foundation in understanding lincRNAs' roles in adult stem cells with respect to pluripotency and differentiation. This novel result will be also valuable for understanding lincRNAs in other adult stem cells. In addition, given that ISCs are the origin of colon cancer and the source of its propagation, the results will undoubtedly be important in the field of cancer biology and will be implicated in development of diagnostic markers and therapeutic targets for cancer.