CN109481682B - Application of CFTR (circulating fluid TR) inhibitor in preparation of reagent for regulating Hedgehog signal path - Google Patents

Abstract

The invention provides application of CFTR inhibitors in preparation of a reagent for regulating a Hedgehog signal pathway, relates to the technical field of biological engineering, and provides several CFTR inhibitors which can effectively inhibit expression of key proteins Ihh, SMO and Gli of the Hedgehog signal pathway and expression of β -catenin, so that the Hedgehog signal pathway is inhibited, a molecular regulation mechanism of the CFTR inhibitors for regulating the Hedgehog signal pathway is disclosed, and a new way is provided for research on physiological functions of CFTR, CF related diseases and intestinal cancer.

Description

Application of CFTR (circulating fluid TR) inhibitor in preparation of reagent for regulating Hedgehog signal path

Technical Field

The invention relates to the technical field of biological engineering, in particular to application of a CFTR (circulating fluid TR) inhibitor in preparation of a reagent for regulating a Hedgehog signal pathway.

Background

The gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) is called CFTR gene, and encodes a protein containing 1480 amino acid residues, which is a chloride channel located in the apical plasma membrane of epithelial cells, and which is the only ion channel protein in the ABC (ATP-bliningeasote) protein family. Contains two hydrophobic domains, both of which have a complex transmembrane portion, two ATP-binding domains and one intracellular domain. CFTR is a cystic fibrosis transmembrane conductance regulator that regulates the transport of electrolyte ions from epithelial membranes. The CFTR gene was originally recognized because mutations in this gene were found to cause Cystic Fibrosis (CF).

Cystic Fibrosis (CF) is an autosomal recessive genetic disease, which is most frequently found in caucasian populations, and is caused by mutations in the CFTR gene, which encodes a cAMP-regulated anion channel on the apical membrane of epithelial cells in various organs. Currently, over 2000 mutations have been identified in cystic fibrosis, with the majority of the mutation types being deletion of phenylalanine at position 508 (Δ F508), resulting in defective folding of the CFTR protein and degeneration of the endoplasmic reticulum, occurring in more than 80% of CF patients. CFTR gene mutations affect mucosal physiology of the respiratory, digestive and reproductive systems, leading to a wide range of clinical manifestations including pancreatic insufficiency, focal biliary cirrhosis, infertility and chronic airway obstruction.

Inhibitors of CFTR mainly include inhibitors obtained by screening from small molecule combinatorial chemistry libraries, and natural product inhibitors, the first discovered inhibitors of CFTR being aryl aminobenzoate-based compounds DPC and NPPB. Currently, inhibitors of CFTR are predominantly of class 2: thiazolidinone compounds and pyrimidineones. At present, chinese patent publication CN101668732 discloses a CFTR inhibitor compound and its use, the compound or composition is a hydrazone-containing compound of CFTR inhibitor, mainly used for treating diarrhea. Chinese patent publication CN104398509B discloses the application of CFTR inhibitor CFTRinh-172 in the preparation of drugs for preventing and treating leukemia cell-mediated diseases, wherein CFTRinh-172 effectively inhibits the proliferation of leukemia cells by inhibiting the high expression of CFTR in mononuclear cells. CFTR inhibitors are used clinically in many cases in secretory diarrhea and cystic fibrosis.

The Hedgehog signaling pathway mainly comprises 3 parts of Hh ligand proteins including Shh, Ihh and Dhh, membrane receptor proteins Patched (PTCH) and Smoothened (SMO), and downstream transcription factors Gli (Gli1, Gli2, Gli3), wherein Gli1 is a transcription factor in the Hedgehog signaling pathway, the expression level of which reflects the degree of activation of the Hedgehog signaling pathway, in the absence of Hh signal, the PTCH inhibits SMO, thereby inhibiting cleavage of Gli, when ligand binds to the PTCH, inhibition of SMO is released, Gli is cleaved and transported to the nucleus, resulting in increased risk of development of various cancers in Hh target genes including PTCH and Gli themselves and proliferation-related genes including cyclin D1, although CF mutation and CFTR mutations have been recently reported in patients, and in patients with increased risk of cancer development in various tissues, such as cystic fibrosis, CF proliferation, colorectal cancer, and colorectal cancer-related diseases.

The application aims at the vacancy of the prior art, researches the regulation effect of a CFTR inhibitor on a Hedgehog signal pathway, discovers the inhibition effect of the CFTR inhibitor on the Hedgehog signal pathway, and further provides an application subject of the application of the CFTR inhibitor in the preparation of a reagent for regulating the Hedgehog signal pathway. Because the Hedgehog signal channel is closely related to the formation of tumors, the application subject of the CFTR inhibitor in the preparation of the drugs for treating and preventing the tumors is further provided.

Disclosure of Invention

The invention aims to provide application of a CFTR (circulating fluid TR) inhibitor in preparation of a reagent for regulating a Hedgehog signaling pathway, and provides a new way for research and treatment or diagnosis of CFTR physiological functions, CF-related diseases and intestinal cancer.

The invention provides application of a CFTR (circulating fluid TR) inhibitor in preparation of a reagent for regulating a Hedgehog signal path.

Specifically, the application is the application of the CFTR inhibitor in preparing a reagent for inhibiting a Hedgehog signal path.

Specifically, the application is the application of the CFTR inhibitor in preparing a reagent for down-regulating the expression of β -catenin protein.

Specifically, the application is the application of the CFTR inhibitor in preparing a reagent for down-regulating the expression of Ihh, SMO and Gli1 proteins.

Preferably, the CFTR inhibitor is selected from the group consisting of siRNA, shRNA or Anti-CFTR antibody directed against CFTR.

Further preferably, the sequence of the siRNA is:

siRNA-1：5’-GUUAAGAAUCCCACCUGCUUUCAGCUU-3’；

siRNA-2：5’-GUGCAAAUUCAGAGCUUUGUGGAACAG-3’；

the sequence of the shRNA is as follows:

shRNA-1：5’-GCTTCTCTGGGACTTGTTACA-3’；

shRNA-2：5’-GCTGCAAAGATCAATGAAAGA-3’；

shRNA-3：5’-GGATCTACTGGAGCAGGAAAG-3’。

preferably, the CFTR Inhibitor is CFTRinh-172 and CFTR Inhibitor II (GlyH-101);

the chemical structural formula of the CFTRinh-172 is as follows:

the chemical structural formula of the CFTR Inhibitor II (GlyH-101) is as follows:

preferably, the CFTR inhibitor is an oligomer of resveratrol, such as resveratrol dimer (trans-e-viniferin, TV), resveratrol tetramer (r-2-viniferin, RV); licorice root extract, such as Isoliquiritigenin (Isooliquitiritigenin); stevioside compounds, such as steviol (steviol); oligomeric procyanidin compounds, such as Crofelemer (SP-303); rabdosia extract, such as oridonin (oridonin) or a Catechin compound, such as Catechin (Catechin, C), Epicatechin (EC), Epigallocatechin gallate (EGCG);

further preferably, the resveratrol oligomer is resveratrol dimer (trans-epsilon-viniferin, TV) or resveratrol tetramer (r-2-viniferin, RV);

and (3) resveratrol dimer:

a resveratrol tetramer:

more preferably, the catechin compound is catechin, epicatechin, or epigallocatechin gallate;

catechin:

epicatechin;

epigallocatechin gallate:

the invention has the following technical effects:

the invention reports a molecular regulation mechanism of a CFTR (circulating fluid TR) inhibitor for regulating a Hedgehog signal path for the first time, the provided CFTR inhibitors can effectively inhibit the expression of key proteins Ihh, SMO (small molecule oxidizing) and Gli (Gli) proteins of the Hedgehog signal path and the expression of β -catenin, so that the Hedgehog signal path is inhibited, and the provided CFTR inhibitor provides a new way for the research on the physiological function of the CFTR, CF (CF-related disease) and intestinal cancer.

Drawings

FIG. 1 is a view of the small intestine of the mouse in example 1 observed under an ordinary microscope by H.E. staining;

FIG. 2 is a graph showing the expression of PCNA protein in the small intestine of the mouse in example 1 under a fluorescence microscope;

FIG. 3 is a polyacrylamide gel electrophoresis chart and a histogram of expression of CFTR and PCNA proteins in example 2;

FIG. 4 is a polyacrylamide gel electrophoresis chart and a histogram of expression levels of the PTCH, Ihh, SMO and Gli proteins of example 3;

FIG. 5 is the polyacrylamide gel electrophoresis chart and the histogram of the expression amount of the Gli protein in example 4;

FIG. 6 is a graph showing the expression of PCNA protein observed under a fluorescence microscope in the small intestine of the mouse in example 4;

FIG. 7 is a histogram of the expression levels of CFTR, β -catenin, Ihh, SMO and Gli1 proteins in IEC-18 cells treated with siRNA and shRNA in example 5;

FIG. 8 is a histogram of the expression levels of CFTR, β -catenin, Ihh, SMO and Gli1 proteins in IEC-18 cells treated with Anti-CFTR antibody in example 5;

FIG. 9 is a histogram of the expression levels of CFTR, β -catenin, Ihh, SMO and Gli1 proteins in IEC-18 cells treated with a chemical CFTR-like inhibitor in example 5.

Detailed Description

The following description of the embodiments is only intended to aid in the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The following description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions.

The mice used in the examples were female genital tract Δ F508-CFTR (F508) mice, with a mutated CFTR gene, 3-bp deletion, eliminating phenylalanine, which corresponds to the most common CFTR mutation in humans.

Example 1

Hyperproliferation and crypt deepening of intestinal epithelial cells of F508 mice

The small intestine of the Wild Type (WT) and F508 mice, respectively, was sectioned from paraffin tissue, stained by h.e. (hematoxylin-eosin), and observed under a common microscope, and the results are shown in fig. 1.

The small intestines of the wild-type and F508 mice were sectioned, stained, and the expression of Proliferating Cell Nuclear Antigen (PCNA) was observed under a fluorescent microscope, and the results are shown in FIG. 2.

The results show that the depth of the intestinal crypts of the F508 mice is obviously increased compared with the wild type, and PCNA positive cells are obviously increased. Indicating that the increase in crypt depth is caused by an increase in proliferation of the cells.

Example 2

F508 mouse intestinal epithelial cell hyperproliferation and CFTR protein expression

The CFTR and PCNA proteins in the gut of wild-type mice and F508 mice were analyzed using Western blot.

The method comprises the following specific steps:

weighing small intestines of 7 wild-type mice and 6F 508 mice respectively 100mg, placing the small intestines in an EP tube, adding 1ml of RIPA strong lysis solution (RIPA + 1% PMSF) into each tube, shearing tissues, ultrasonically crushing for 3 times, standing for 30min on ice for full lysis, centrifuging for 25min at 12000rpm under the condition of 4 ℃, taking supernatant to be a protein sample, determining the protein content by using a BCA method, taking 20 mu g/porin sample according to the quantitative result, carrying out polyacrylamide gel electrophoresis (SDS-PAGE), using β -actin as an internal reference, sealing for 1h at room temperature after membrane conversion, washing the membrane for 3 times by using BCA method, adding corresponding primary antibody (BCA 1:3000) after dilution, placing the membrane on a shaking bed for overnight at 4 ℃, washing the membrane for 3 times by using the membrane containing 0.05% Tween-20, adding corresponding dilution (secondary antibody 1:3000) after dilution, incubating for 1h at room temperature, using the membrane containing 0.05% Tween-20% and detecting the result by using ECST 3. standard light-emitting method.

The results show that the expression of the CFTR protein is inhibited in the small intestine and the expression level of the PCNA protein is significantly increased in F508 mice compared to wild type mice. The mutation of CFTR gene and the inhibition or down regulation of CFTR can promote the proliferation of intestinal epithelial cells, and further the inhibition or down regulation of CFTR is involved in the proliferation of small intestine crypts.

Example 3

CFTR gene mutation for inhibiting intestinal epithelial cell Hedgehog signal path

Western blot analysis of key proteins in the Hedgehog signaling pathway, including PTCH, Ihh, SMO and Gli proteins, in the gut of 7 wild-type mice and 6F 508 mice in example 2 was used. The results are shown in FIG. 4.

The results show that PTCH is activated in the small intestine, expression is significantly up-regulated, and Ihh, SMO and Gli are significantly down-regulated in F508 mice compared to wild type mice. The mutation of the CFTR gene indicates that the suppression or down-regulation of CFTR inhibits the Ihh, SMO and Gli proteins in the Hedgehog signaling pathway.

Example 4

β -catenin signal activation inhibits intestinal epithelial cell proliferation

Wild-type mice and F508 mice were treated with LiCl to activate the β -catenin signal in the mice, and wild-type mice and F508 mice that were not treated with LiCl were used as controls (control, Con) to analyze the expression level of Gli1 using Western blot as described in example 2. the results are shown in FIG. 5.

The small intestine of the 4 groups of mice was sectioned and stained by the method described in example 1, and the expression of PCNA protein was observed under a fluorescent microscope, and the results are shown in FIG. 6.

The results in FIG. 5 indicate that activation of β -catenin can reverse down-regulation of Gli1, and the results in FIG. 6 indicate that increased PCNA positive cells in the small intestine of F508 mice can be reduced by activation of β -catenin, which indicates that activation of β -catenin signaling inhibits intestinal epithelial cell proliferation and promotes the Hedgehog pathway, i.e., mutation of CFTR gene, CFTR is inhibited or down-regulated, and that a decrease in β -catenin level inhibits the Hedgehog pathway.

Example 5

CFTR inhibitors inhibit the Hedgehog signaling pathway

Collecting rat ileum cell IEC-18 (purchased from Shanghai Pongjing industries, Ltd., product number: BJ-ATCC0007), recovering cells in 89% DMEM (high sugar) + 10% FBS + 1% double-resistant medium, subculturing, selecting IEC-18 cells with good growth state, digesting with pancreatin, counting cells, and counting by 1 × 10⁶The density of each hole is inoculated in a cell culture plate, when the experimental group is siRNA or shRNA slow virus transduction particles aiming at CFTR, the control group is siRNA-nc or shRNA-nc slow virus transduction particles, and then transfection and culture are carried out; when the experimental group is antibody or other chemical CFTR inhibitor, the control group is added with an equal amount of DMSO (dimethyl sulfoxide) and placed at 37 ℃ with 5% CO₂After 12h of culture in the cell culture chamber, the cells in the experimental group and the control group were counted, and the results are shown in Table 1 (P. ltoreq.0.05), and the expression levels of CFTR, β -catenin, Ihh, SMO and Gli1 proteins were analyzed by Western blot analysis in example 2, and the results are shown in FIGS. 7 to 9.

The CFTR inhibitor is siRNA slow virus transduction particle (purchased from Shanghai Jima pharmaceutical technology Co., Ltd., product number: D-series), shRNA slow virus transduction particle (purchased from Shanghai Jima pharmaceutical technology Co., Ltd., product number: E-S-1), Anti-CFTR antibody (purchased from Shanghai Riqi Biotech Co., Ltd., product number: Rs-1277R) and chemical inhibitor aiming at CFTR;

chemical classes of CFTR inhibitors include: CFTRinh-172 (from Sigma-Aldrich, cat # CAS number 307510-92-5), CFTR inhibinor II (trade name: GlyH-101, from Sigma-Aldrich, cat # CAS number 328541-79-3), isolated from wild grape vine (Viviaamurensis Rupr.) by the method of "dazzling resveratrol oligomer CFTR Inhibitor discovery and molecular pharmacology research [ D ]. Liaoning university, 2014.", isoliquiritigenin (from Shanghai Van bioscience, cat # BIO-0481), steviol (from Shanghai ancient Biotech, Inc., cat # GD-0029), Crofelder (from Salemix, trade name: Fulyzaq), oridonin (from Shanghai bioscience, cat # GD-125, Skogaku, Co., USA), the goods number is: GD-125-46), EC (from shanghai guduo biotechnology, cat #: GD-125-47) or EGCG (from shanghai guduo biotechnology, cat #: GD-125-50);

the sequence of the siRNA is:

siRNA-1：5’-GUUAAGAAUCCCACCUGCUUUCAGCUU-3’；

siRNA-2：5’-GUGCAAAUUCAGAGCUUUGUGGAACAG-3’；

siRNA-cn：5’-CUUCCUCUCUUUCUCUCCCUUGUGA-3’。

the sequence of the shRNA is:

shRNA-1：5’-GCTTCTCTGGGACTTGTTACA-3’；

shRNA-2：5’-GCTGCAAAGATCAATGAAAGA-3’；

shRNA-3：5’-GGATCTACTGGAGCAGGAAAG-3’；

shRNA-cn：5’-CGAAGAGACCCTGAACAATGT-3’。

table 1: cell number in Experimental and control groups

As can be seen from Table 1, the number of cells in the experimental group is significantly increased compared with the control group, and similarly, the chemical CFTR inhibitors TV, RV, isoliquiritigenin, steviol, Crofelemer, oridonin, C, EC and EGCG also have similar effects, which indicates that the CFTR inhibitors promote the proliferation of ileum cells of rats.

The results of fig. 7-9 show that these CFTR inhibitors significantly down-regulate the expression of CFTR, β -catenin, Ihh, SMO, and Gli1 proteins, indicating that these CFTR inhibitors inhibit the expression of CFTR, inhibit the expression of β -catenin, Ihh, SMO, and Gli1 proteins, and that CFTR inhibitors inhibit the Hedgehog pathway by inhibiting β -catenin expression and the key proteins Ihh, SMO, and Gli1 proteins in the Hedgehog signaling pathway.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (1)

1. Use of a CFTR inhibitor for the preparation of an agent for the treatment of intestinal cancer;

   the CFTR inhibitor is: resveratrol dimer, resveratrol tetramer, isoliquiritigenin, steviol, Crofelemer, oridonin, catechin, epicatechin, and epigallocatechin gallate;

   the resveratrol dimer is as follows:

   

   ；

   the resveratrol tetramer is as follows:

   

   ；

   the catechin is as follows:

   

   ；

   the epicatechin is as follows:

   

   ；

   the epigallocatechin gallate is characterized in that:

   

   。

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