JP2020164470A - Enhancer of expression level of il-8 gene or mhc class ii-associated gene containing a yap inhibitor as active ingredient - Google Patents

Abstract

To provide agents that enhance the expression level of IL-8 gene or MHC class II-associated gene.SOLUTION: An enhancer of the expression level of IL-8 gene or MHC class II-associated gene containing a YAP inhibitor as an active ingredient is prepared. Preferably, the YAP inhibitor is Verteporfin, Y27632, Blebbistatin, Latrunculin B, Cytochalasin D, Simvastatin, Botulinum C3 toxin, Statin, or Dobutamine.SELECTED DRAWING: None

Description

The present invention relates to an agent for enhancing the expression level of an IL-8 gene or an MHC class II-related gene containing a YAP inhibitor as an active ingredient.

The transcriptional conjugate protein YAP (Yes associated protein) is the target molecule of the Hippo signaling system. The present inventors focused on the Hippo signal transduction system that controls both cell proliferation and cell death, and that YAP is a gravity resistance gene essential for three-dimensional organ formation, and that YAP is a physical that occurs in cells. It has been reported that a three-dimensional organ that resists gravity is constructed by controlling the arrangement of a plurality of tissues as well as the three-dimensionalization of individual tissues through force (see Non-Patent Document 1). Therefore, YAP is attracting attention as a factor involved in three-dimensional organ formation.

On the other hand, in recent years, research on the relationship between YAP control and anti-tumor immune response has been advanced. For example, a method for treating cancer in a subject, including a step of administering a YAP inhibitor and an immunotherapeutic agent to the subject. (Refer to Patent Document 1), or a preventive or therapeutic agent for cancer having an abnormality in the Hippo pathway, which contains ivermectin or milbemycin D as an active ingredient, acts on the Hippo pathway, and suppresses the activity of YAP1 / TAZ (see Patent Document 2). ) Is disclosed.

Research on the relationship between YAP regulation and inflammatory enteritis is also underway. For example, GP130, which is a receptor for interleukin 6 (IL-6), binds to IL-6 to activate YAP and Notch. It is disclosed that it causes inflammation and promotes intestinal repair and regeneration (see Non-Patent Document 2).

Special Table 2018-505223 Japanese Unexamined Patent Publication No. 2016-088919

Porazinski et al., Nature 521, 2015 Taniguchi et al., Nature, 519:57 (2015)

As mentioned above, it has been clarified that YAP is involved in three-dimensional organs, cancer, inflammation and the like. By the way, interleukin 8 (IL-8) and MHC class II-related genes are known to be involved in inflammatory diseases. Controlling the production of IL-8 and MHC class II-related genes is important in therapeutic strategies for many diseases. Therefore, an object of the present invention is to provide an agent that enhances the expression level of an IL-8 gene or an MHC class II-related gene.

In order to solve the above problems, the present inventors comprehensively performed RNA-seq analysis between wild-type embryos and YAP mutant (hir) embryos. As a result, it was suggested that there is a relationship between the expression of the YAP gene and the expression of the immune system gene. Therefore, when the effect of each gene expression on the presence or absence of salmonella infection was investigated at the cellular level, the expression of binding tissue growth factor (CTGF) and cysteine-rich protein 61 (CYR61), which are genes related to YAP activity, was revealed by salmonella infection. We found that it decreased and that IL-8 expression was higher in YAP-deficient cells than in wild cells. Furthermore, they found that the expression of CIITA, which is an MHC class II-related molecule, was increased in YAP-deficient cells, and completed the present invention.

That is, the present invention is as follows.
[1] An agent for enhancing the expression level of an IL-8 gene or an MHC class II-related gene containing a YAP inhibitor as an active ingredient.
[2] The YAP inhibitor is characterized by being verteporfin, Y27632, brevistatin, latrunculin B, cytochalasin D, zymbastatin, botulinum C3 toxin, statin, or dobutamine. The enhancer according to the above [1].
[3] The enhancer according to the above [1], wherein the YAP inhibitor is a nucleic acid molecule that suppresses the expression of the YAP gene.
[4] The nucleic acid molecule that suppresses the expression of the YAP gene is at least one nucleic acid molecule selected from the group consisting of double-stranded siRNA, microRNA, shRNA, antisense nucleic acid, and ribozyme. [3] The enhancer according to the above.
[5] The enhancer according to the above [1], wherein the YAP inhibitor is an antibody that binds to a YAP protein.
[6] The enhancer according to any one of the above [1] to [5], wherein the MHC class II-related gene is a CIITA gene.

By using the YAP inhibitor of the present invention, it is possible to control the expression level of the IL-8 gene or the MHC class II-related gene.

This is the result of examining the expression level of the CTGF gene or the IL-8 gene in Caco2 cells before and after Salmonella infection in Example 1. The vertical axis is a relative value when the expression level at the time of No infection is 1. This is the result of examining the expression level of the CTGF gene in RPE1 cells (wild type) or RPE1 cells (YAP − / −) before and after Salmonella infection in Example 2. The vertical axis is the relative value when the expression level of RPE1 cells (wild type) at the time of No infection is 1. This is the result of examining the expression level of the CYR61 gene in RPE1 cells (wild type) or RPE1 cells (YAP − / −) before and after Salmonella infection in Example 3. The vertical axis is the relative value when the expression level of RPE1 cells (wild type) at the time of No infection is 1. This is the result of examining the expression level of the IL-8 gene in RPE1 cells (wild type) or RPE1 cells (YAP − / −) before and after Salmonella infection in Example 4. The vertical axis is the relative value when the expression level of RPE1 cells (wild type) at the time of NO infection is 1. This is the result of examining the expression level of the CIITA gene, which is an MHC class II-related gene, in RPE1 cells (wild type) or RPE1 cells (YAP − / −) in Example 5. The vertical axis is the relative value when the expression level of RPE1 cells (wild type) at the time of No infection is 1.

The agent for enhancing the expression level of the IL-8 gene or MHC class II-related gene of the present invention (hereinafter, also referred to as “inhibitor of the present invention”) is an IL-8 gene or MHC class II-related gene containing a YAP inhibitor as an active ingredient. The above-mentioned YAP inhibitor is not particularly limited as long as it is a substance that inhibits the function or activity of YAP, and is a substance that binds to YAP and inhibits interaction with TEAD, or YAP. Examples include substances that inhibit the nuclear translocation of YAP and substances that promote the phosphorylation of YAP. Specifically, actin polymerization inhibitors such as Verteporfin, Rho-binding kinase inhibitor Y27632, Blebbistatin, Latrunculin B, and Cytochalasin D, botulinum. C3 toxin (Botulinum toxin C3), statins (Statins), dobutamine (Dobutamine) can be mentioned.

As the YAP inhibitor, a nucleic acid molecule that suppresses the expression of the YAP gene can also be mentioned. The nucleic interfering RNA molecule that suppresses the expression of the YAP gene is not particularly limited as long as the expression of the YAP gene can be suppressed, and those that suppress the transcription of the YAP gene, those that disrupt the YAP gene, and those that suppress the translation of the YAP gene are suppressed. Examples thereof include double-stranded siRNA, microRNA, shRNA, guide RNA targeting the RAB3B gene, antisense nucleic acid, ribozyme, etc., which target the full-length or partial sequence of the YAP gene. The nucleotide sequence of the YAP gene can be obtained from the NCBI website (https://www.ncbi.nlm.nih.gov/).

As the YAP inhibitor, an antibody that binds to the YAP protein can also be mentioned. Examples of the antibody that binds to the YAP protein include an antibody that binds to the YAP protein shown in SEQ ID NO: 1, preferably an antibody that specifically binds to the YAP protein, a monoclonal antibody, a polyclonal antibody, and a human. It may be an antibody such as an antibody, a chimeric antibody, or a humanized antibody, and some of the antibodies such as F (ab') ₂ , Fab, diabody, Fv, ScFv, Sc (Fv) _{2 and the} like may be used. Also included is an antibody fragment consisting of.

The above-mentioned "agent for enhancing the expression level of IL-8 gene or MHC class II-related gene" refers to the IL-8 gene or MHC class II-related agent when the administration target is a human and the YAP inhibitor is absent in human cells. It means a substance that increases the expression level of IL-8 gene or MHC class II-related gene as compared with the expression level of the gene. Specifically, it contains a substance in which the mRNA expression level and transcriptional activity of the IL-8 gene or the MHC class II-related gene are increased as compared with the control (without YAP inhibitor) in human cells.

The degree of increase in the expression level of the IL-8 gene or the MHC class II-related gene is not particularly limited, but is preferably 1.2 times or more, more preferably 1.5 times or more in a predetermined time after the treatment with the YAP inhibitor, as compared with the control. As mentioned above, more preferably, the case where the amount is twice or more can be preferably mentioned.

IL-8 is an inflammatory chemokine produced by macrophages, epithelial cells, vascular endothelial cells, etc., and plays a role in suppressing infection when infected with pathogens such as Salmonella.

As the above-mentioned MHC class II-related gene, the CIITA gene can be preferably mentioned. MHC class II-related genes include foreign substances that cause allergies, non-self-antigens such as cancer / sarcoma antigens that are non-self-antigens and pathogens that cause infections, and the causes of many autoimmune diseases that are designated as intractable diseases. It is a gene involved in self-antigen recognition and antigen presentation, and plays a role in eliminating infection when infected with pathogens such as salmonella.

The enhancer of the present invention may contain a pharmaceutically acceptable additive, and the additive includes physiological saline, buffered physiological saline, cell culture medium, dextrose, water for injection, glycerol, ethanol. And combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonic agents, fillers, and lubricants.

The dose of the enhancer of the present invention is not particularly limited as long as the activity of enhancing the expression level of the IL-8 gene or the MHC class II-related gene can be obtained, and the dose can be appropriately adjusted according to the administration subject. .. The administration target is not particularly limited as long as the activity of enhancing the expression level of the IL-8 gene or the MHC class II-related gene can be obtained, but mammals can be exemplified, and humans and rats can be preferably exemplified. It is possible, and in particular, humans can be preferably exemplified.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.

Salmonella was infected by adding a culture solution of Salmonella expressing GFP protein to Caco2 cells, which is a cell line derived from human colon cancer. Then, RNA was extracted from Caco2 cells before the salmonella infection (No infection), 0.5 hours and 2 hours after the infection using TRIzol (invitrogen), and a connective tissue growth factor (CTGF) was extracted by a quantitative RT-PCR method. ) The expression levels of the gene and IL-8 gene were examined. The results are shown in FIG. The CTGF gene is a gene that functions downstream of YAP.

Due to Salmonella infection, the expression level of the CTGF gene, which is a downstream gene of YAP, was reduced. In addition, Salmonella infection increased the expression level of the IL-8 gene.

In the wild type of RPE1 cells (RPE1 cells (wild type)) and PRE1 cells in which the YAP gene was knocked out (RPE1 cells (YAP-/-)), before the salmonella infection (No infection), 0.5 hours after infection, The expression level of the CTGF gene after 2 hours was examined by quantitative RT-PCR in the same manner as above. The results are shown in FIG.

In the wild type, Salmonella infection reduced the expression of the CTGF gene, which is a downstream gene of YAP, even in RPE1 cells. On the other hand, in YAP knockout RPE cells, the expression level of the CTGF gene was suppressed to the same extent as 2 hours after Salmonella infection in the wild type of RPE1 cells regardless of the presence or absence of Salmonella infection. Therefore, it was clarified that Salmonella infection suppressed the expression level of the downstream gene CTGF of YAP to the same extent as when YAP was not expressed.

In the wild type of RPE1 cells (RPE1 cells (wild type)) and PRE1 cells in which the YAP gene was knocked out (RPE1 cells (YAP-/-)), the above-mentioned pre-infection (No infection), 0.5 hours after infection, 2 The expression level of the cysteine rich protein 61 (CYR61) gene after hours was examined by quantitative RT-PCR in the same manner as above. The results are shown in FIG. The CYR61 gene is a gene that functions downstream of YAP.

In the wild type, Salmonella infection reduced the expression level of the CYR61 gene, which is a downstream gene of YAP, in RPE1 cells. On the other hand, in YAP knockout RPE cells, the expression level of the CYR61 gene was suppressed in the wild type of RPE1 cells to the same extent as 2 hours after Salmonella infection, regardless of the presence or absence of Salmonella infection. Therefore, it was clarified that Salmonella infection suppressed the expression of the downstream gene CYR61 of YAP to the same extent as when YAP was not expressed.

In the wild type of RPE1 cells (RPE1 cells (wild type)) and PRE1 cells in which the YAP gene was knocked out (RPE1 cells (YAP-/-)), before the salmonella infection (No infection), 0.5 hours after infection, The expression level of the IL-8 gene after 2 hours was examined by quantitative RT-PCR in the same manner as above. The results are shown in FIG.

In the wild type, Salmonella infection increased the expression level of the IL-8 gene in RPE1 cells. On the other hand, in YAP knockout RPE cells, the expression level increased 4.9 times in 0.5 hours after infection and 1.5 times in 2 hours after infection due to Salmonella infection, compared with the wild type. It was. Therefore, although the expression level of IL-8 increases due to Salmonella infection, it is possible to further increase the expression of IL-8 by suppressing the expression level of YAP, in other words, YAP is used during Salmonella infection. It was revealed that it suppresses the induction of IL-8 expression.

From the above data so far, it was observed that the expression level of YAP-related genes decreased and the IL-8 gene expression level increased due to salmonella infection. Therefore, in order to investigate the relationship between the YAP gene and the immune response, the expression level of the CIITA gene, which is a MHC class II-related gene involved in antigen recognition, antigen presentation, and neutrophil immune response, was investigated.

The expression of the CIITA gene was examined by quantitative RT-PCR in the wild type of RPE1 cells (RPE1 cells (wild type)) and PRE1 cells in which the YAP gene was knocked out (RPE1 cells (YAP-/-)) in the same manner as above. The results are shown in FIG.

In PRE1 cells in which the YAP gene was knocked out, the expression of the CIITA gene was increased 4-fold as compared with the wild-type RPE1 cells. Therefore, it is considered that YAP may negatively regulate the gene expression of MHC class II-related molecules, and it was clarified that the immune response can be regulated by suppressing the expression of the YAP gene.

Claims (6)

An agent for enhancing the expression level of an IL-8 gene or an MHC class II-related gene containing a YAP inhibitor as an active ingredient. Claims characterized in that the YAP inhibitor is verteporfin, Y27632, brevistatin, Latrunculin B, cytochalasin D, zimbastatin, botulinum C3 toxin, statin, or dobutamine. 1. The enhancer according to 1. The enhancer according to claim 1, wherein the YAP inhibitor is a nucleic acid molecule that suppresses the expression of the YAP gene. 3. The invention according to claim 3, wherein the nucleic acid molecule that suppresses the expression of the YAP gene is at least one nucleic acid molecule selected from the group consisting of double-stranded siRNA, microRNA, shRNA, antisense nucleic acid, and ribozyme. Enhancer. The enhancer according to claim 1, wherein the YAP inhibitor is an antibody that binds to a YAP protein. The enhancer according to any one of claims 1 to 5, wherein the MHC class II-related gene is a CIITA gene.