KR-102963940-B1 - A Method for Screening Compositions for Preventing or Treating Epithelial Barrier Dysfunction

Abstract

The present invention provides a screening method for compositions for the prevention or treatment of epithelial barrier dysfunction. The screening method of the present invention can rapidly and reliably identify candidate substances for fundamental therapeutic agents capable of restoring degenerated cytoskeletons and recovering cell-to-cell contact in individuals with impaired epithelial barrier function. The present invention can also be usefully applied to the search for auxiliary components capable of efficiently delivering pharmacological components by improving the epithelial barrier of target tissues or the cell membrane permeability of target cells. Furthermore, the present invention can be applied to the search for efficient compositions for inhibiting cancer metastasis that can suppress the progression of epithelial-mesenchymal transition (EMT) by reinforcing the epitheliality of tumor epithelial cells within cancer tissues.

Inventors

• 박보연
• 강수진

Assignees

• 연세대학교 산학협력단

Dates

Publication Date

20260512

Application Date

20200812

Claims (6)

1. A screening method for a composition for enhancing epithelial barrier or cell membrane permeability comprising the following steps: (a) a step of contacting a candidate substance with a biological sample containing cells expressing TRIM40 (Tripartite Motif Containing 40) protein; (b) a step of measuring the activity or expression level of TRIM40 protein in the sample, If the activity or expression amount of the TRIM40 protein above increases, the candidate substance is determined to be a composition for enhancing epithelial barrier or cell membrane permeability.
2. A method according to claim 1, characterized in that the candidate substance determined to be a composition for enhancing epithelial barrier or cell membrane permeability is applied as a drug delivery system that assists in the efficient delivery of pharmacological components.
3. A method according to claim 1, wherein the candidate substance determined to be a composition for enhancing epithelial barrier or cell membrane permeability promotes RhoA degradation to reduce LIMK1 phosphorylation and induces F-actin destabilization-mediated cortical actin breakdown.
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Description

A Method for Screening Compositions for Preventing or Treating Epithelial Barrier Dysfunction The present invention relates to a method for searching for candidate substances for the treatment of epithelial barrier dysfunction using TRIM40 protein as a screening target. Epithelial tissue is found in both vertebrates and invertebrates and plays a crucial role in the physiology of organisms. The epithelial cells that make up epithelial tissue form epithelial barriers by enveloping both the outer and inner surfaces of the body's cavities and lumens like membranes and forming rigid intercellular connections. This isolates the body's internal and external cavities and blocks the entry of microorganisms. Epithelial barriers function not only as a defensive mechanism but also as a means of controlling material transport, allowing the body to selectively absorb and excrete specific substances. As epithelial barriers and epithelial cells perform various physiological functions such as chemical and nutrient absorption, intercellular transport, sensory perception, waste elimination, and the blockage of pathogen entry, a loss of these functions can lead to the progression of various pathological conditions, including infectious diseases. Meanwhile, the intestine is an organ where the host and microorganisms maintain homeostasis, and intestinal homeostasis is achieved through intestinal epithelial cells. The intestinal epithelial barrier, formed by intestinal epithelial cells connecting to one another through the cytoskeleton, prevents excessive immune responses by blocking intestinal microorganisms from entering the lamina propria, where intestinal immune cells are located. Inflammatory bowel disease (IBD) is a representative disease caused by an excessive intestinal immune response. Although many studies have proposed risk factors associated with IBD, the specific genetic factors affecting epithelial barrier function and their mechanisms of action are largely unknown. Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention. Figure 1 illustrates that TRIM40 is involved in the pathogenesis of IBD through changes in actin cytoskeleton signaling. Figure 1a shows the results of RNA-seq analysis on UC-related gene expression profiles obtained from a publicly available RNA-sequencing (RNA-seq) dataset of UC patients. Green, yellow, blue, brown, and black dots represent REG, motility, MMP, chemokine, and TRIM protein genes, respectively. Figure 1b illustrates that TRIM40 is increased in UC and CD patients. TRIM40 expression was analyzed by RT-PCR, and quantitative values of TRIM40 mRNA levels were normalized to GAPDH. Figure 1c illustrates that TRIM40 expression is rarely detected in colon and intestinal epithelial cell lines. The expression of TRIM40 in various human cell lines, including SW480 (large intestine), HT-29 (large intestine) and Caco-2 (intestine), HeLa (cervix), U937 (lymphocyte), MDA-MB-231 (breast), and 293T (embryonic kidney), was analyzed by RT-PCR. Figure 2 illustrates that TRIM40 overexpression promotes the expression of inflammatory genes or cytoskeleton-related genes. Figure 2a shows the results of Venn diagram analysis from microarray and RNA-seq dataset analysis, revealing significant overlap between increased and decreased genes. Figure 2b shows the results of heat map analysis, illustrating significant differences in mRNA expression levels of cytokines, chemokines, IFN, or cytoskeleton-related genes within Myc-TRIM40-expressing HT-29 cells. Figure 2c illustrates that TRIM40 overexpression induces mRNA expression of chemokines, cytokines, IFN, or cytoskeleton-related proteins. mRNA expression of each gene was analyzed by qPCR. *P<0.05, **P<0.01 (Student's t-test), ISP40 (intestine specific protein 40) refers to TRIM40. Figure 3 is a figure showing that TRIM40 overexpression inhibits cell-to-cell contact. Figure 3a is Shows the fibrous pattern of TRIM40 within the cytoplasm. HT-29 or Caco-2 cells stably expressing the blank vector or Myc-TRIM40 were stained with an anti-Myc antibody. Nuclei were stained with DAPI (blue). Scale bar, 10 μM. Arrowheads indicate the fibrous pattern. Figure 3b analyzes HT-29 or Caco-2 cells expressing the blank vector or Myc-TRIM40 using differential interference (DIC). Scale bar, 10 μM. The graph on the right shows the results of quantifying intercellular distances. Edge-to-edge distances were measured by obtaining surface reflection interference DIC images that clearly show cell boundaries. The red, white, and yellow lines represent the boundaries of each cell. **P<0.01 (Student's t-test). Figure 4 is a figure showing that TRIM40 overexpression promotes the destabilization of cortical