Application of membrane protein CMTM3 as drug target for treating inflammatory bowel disease and related colorectal cancer

Abstract

The invention relates to the field of biological medicine, and particularly provides application of a membrane protein CMTM3 as a drug target for treating inflammatory bowel disease and related colorectal cancer. The invention discovers and verifies that deletion of Cmtm3 can protect mice from AOM / DSS induced colorectal cancer (CAC) for the first time. A further research result proves that the endothelial cell-derived CMTM3 promotes the development of enteritis. The Cmtm3 gene knockout and the imatinimex can regulate the vascular permeability in a targeted manner and have a considerable curative effect. The application also discloses that the CMTM3 promotes vascular permeability to aggravate enteritis by stabilizing CLTC and increasing gridding protein mediated endocytosis of VE-cadherin in endothelial cells, thereby promoting the development of inflammation-related colon cancer, which indicates that the CMTM3 can be used as a potential therapeutic target for enteritis and colon cancer (CAC).

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical biology. Specifically, the present invention relates to the use of the membrane protein CMTM3 as a drug target for the treatment of inflammatory bowel disease and its associated colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is the third most lethal cancer globally. Chronic inflammatory states, such as inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), increase the risk of developing inflammation-associated colon cancer (CAC). It has been reported that compared with patients with sporadic colorectal cancer, the prognosis of CAC patients is worse, leading to an increased mortality rate. In recent years, epigenetic alterations, particularly changes in DNA methylation, have been observed during inflammation and CAC. However, compared with sporadic CRC, the molecular mechanisms of CAC are less understood. Given the increasing incidence of IBD year by year, inhibiting the development of enteritis contributes to early intervention in CAC and may ultimately reduce the mortality rate of CRC.

[0003] Currently, the treatment of IBD mainly aims to induce and maintain basic remission, prevent disease recurrence and progression to colorectal cancer, and avoid surgery and complications. The application of biological agents targeting immune responses and inflammatory factors, such as anti-TNF-α antibodies, provides new treatment options for moderate to severe patients. However, many patients currently do not respond to this drug or some patients may develop immunogenicity after long-term use, subsequently resulting in drug resistance. Ustekinumab targeting interleukin (IL)-12 and the IL-23p40 subunit, vedolizumab specifically binding to α4β7 integrin, and Janus kinase (JAK) inhibitors, etc., which have been marketed in recent years, all inhibit the production of inflammatory factors or inhibit immune cells from entering the intestinal tissue at the inflammatory site. However, due to the inhibition of the immune system function, the risk of severe infections in patients increases, and it may also increase the risk of malignant tumors. The pathogenesis of enteritis is multi-factorial, including the immune system as well as the intestinal epithelium and endothelium. The intestinal vascular endothelium is the second important barrier of the intestine, and endothelial cells are its most important component. Studies have shown that the intestinal vascular endothelium is involved in the pathogenesis of DSS (dextran sulfate sodium)-induced enteritis and CAC in mice. Due to the key role of vascular endothelium in the development of enteritis, its potential regulatory mechanisms need to be further elucidated.

[0004] CMTM (CKLF-like MARVEL transmembrane domain-containing family) is a chemokine-like factor superfamily containing the MARVEL transmembrane domain, encoded by 9 genes (CKLF and CMTM1-8) in humans. It is a gene family that has been studied by the inventor's research group for a long time. The CKLF and CMTM1-CMTM8 genes are located on human chromosomes 3 (CMTM6, CMTM7, CMTM8), 14 (CMTM5), and 16 (CMTM1, CMTM2, CMTM3, CMTM4), and on mouse chromosomes 8, 9, and 14. CMTM plays a key role in many biological processes, such as regulating the stability of epidermal growth factor receptor (EGFR), VE-cadherin, PD-L1, NEMO, CD58, β-catenin, etc., and is involved in tumorigenesis. CMTM has different expression profiles in human tumors and normal tissues, and different CMTM members play different roles in tumorigenesis and development. CMTM3 is a member of the CMTM family and is located at the key tumor suppressor gene locus 16q22.1. In some cancer cell lines and primary tumors, including gastric cancer, breast cancer, and colorectal cancer, CMTM3 is silenced or downregulated due to CpG methylation. CMTM3 inhibits the proliferation and migration of various tumor cells. However, there is currently no relevant report on the relationship between CMTM3 and inflammatory bowel disease and its related colorectal cancer. Summary of the Invention

[0005] Aiming at the deficiencies in existing treatments, the present invention discovers that the membrane protein CMTM3 promotes vascular permeability by disrupting VE-cadherin in endothelial cells, thus becoming a potential therapeutic target for enteritis and colorectal cancer (CAC). Therefore, the object of the present invention is to provide the application of the membrane protein CMTM3 as a drug target for treating inflammatory bowel disease and its related colorectal cancer. To achieve this object of the invention, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, there is provided the application of a membrane protein CMTM3 and / or CLTC as a target in screening drugs for treating inflammatory bowel disease and / or related colorectal cancer.

[0007] In one embodiment, the inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

[0008] In one embodiment, under inflammatory stimulation, CMTM3 promotes the occurrence of inflammatory bowel disease by disrupting VE-cadherin in endothelial cells.

[0009] The second aspect of the present invention provides an application of a membrane protein CMTM3 inhibitor in the preparation of a drug for treating inflammatory bowel disease and / or related colorectal cancer, wherein the membrane protein CMTM3 inhibitor can reduce the expression of the gene encoding membrane protein CMTM3 in a patient.

[0010] In one embodiment, the inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

[0011] The third aspect of the present invention provides an application of a reagent for regulating the expression of membrane protein CMTM3 and / or CLTC gene in the preparation of a drug for inhibiting protein molecules related to intestinal inflammatory response, wherein the reagent can reduce the expression of membrane protein CMTM3 gene in a patient.

[0012] The fourth aspect of the present invention provides a drug for treating inflammatory bowel disease, wherein the drug includes a membrane protein CMTM3 inhibitor and a pharmaceutically acceptable pharmaceutical excipient, and the membrane protein CMTM3 inhibitor can reduce the expression of the gene encoding membrane protein CMTM3 in a patient.

[0013] In one embodiment, the inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

[0014] The fifth aspect of the present invention provides an application of membrane protein CMTM3 and / or CLTC in the preparation of a drug for regulating vascular permeability.

[0015] The sixth aspect of the present invention provides an application of membrane protein CMTM3 and / or CLTC as a drug target for regulating VE-cadherin and vascular permeability in endothelial cells. Description of the Drawings

[0016] Figure 1 . Construction of Cmtm3 gene knockout mice, wherein: (A) Using guide RNA to target the first exon of the mouse Cmtm3 gene, Cmtm3 gene knockout mice were constructed. (B) PCR genotype identification of mouse tails. (C) Western blot analysis of the expression of CMTM3 protein in the colon tissues of WT and Cmtm3 - / - mice after 4 days of treatment with 3% DSS.

[0017] Figure 2 . Cmtm3 deficiency inhibits AOM / DSS-induced CAC in mice, wherein: (A) Schematic diagram of the CAC model. 7 days after the first injection of AOM (10 mg / kg), 2% DSS was given in drinking water for 5 days, and then normal drinking water was restored for 14 days. This cycle was repeated three times. The analysis was performed on the 100th day. 7 WT mice and 6 Cmtm3 - / - mice were treated. (B, C) Analysis of WT and Cmtm3- / - Colon length of mice. (D-F) Tumor number and tumor burden were measured. Red arrows indicate obvious tumors. (G) Tumors developed in the colon of WT and Cmtm3 mice were examined by hematoxylin and eosin staining - / - mice. Scale bar = 100 μm (left panel); inset scale bar = 20 μm (right panel). Data represent two independent experiments with similar results. Data are presented as mean ± standard deviation, and statistical significance was determined by two-tailed Student's t-test in (C, E, F). *P < 0.05.

[0018] Figure 3 .CMTM3 was significantly upregulated in UC patients and DSS-induced murine colitis, where: (A-C) Genome-wide transcriptional analysis data from patient colon biopsies. Datasets: GSE38713; GSE9452; GSE13367. (D, E) Expression data from DSS-induced murine colitis models. Datasets: GSE22307; GSE34874. (F) Tissue sections from normal individuals and UC patients (n = 5 per group) were stained with CMTM3 (red) antibody and DAPI (blue) and observed by confocal microscopy. Scale bar = 50 μm. (G) Positive cells were counted in 130 high-power fields of normal or colitic tissues. (H) CMTM3 (red) protein levels in Cmtm3 mice and WT mice treated with water or DSS were detected by immunofluorescence staining - / - Positive cells were counted in 15 high-power fields per mouse. n = 3, 4 per group. Scale bar = 50 μm. (I) Quantitative evaluation is shown on the right side of the representative pictures. Data are presented as mean ± standard deviation, and statistical significance was determined by two-tailed Student's t-test in (A-E, G, I). *P < 0.05, **P < 0.01, ****P < 0.0001; ns indicates no significant difference.

[0019] Figure 4 .Deletion of Cmtm3 protected mice from colitis, where: (A) Body weight loss of WT and Cmtm3 mice treated with water or DSS was measured; n = 3, 4. (B) DAI scores of WT and Cmtm3 mice treated with water or DSS; n = 3, 4. (C, D) Colon length of WT and Cmtm3 mice treated with water or DSS; n = 3, 4. (E) Representative micrographs of hematoxylin and eosin staining in the colon. Scale bar = 100 μm. (F) WT and Cmtm3 mice treated with DSS - / - mice. (F) WT and Cmtm3 mice treated with DSS - / - mice. (G) Representative micrographs of hematoxylin and eosin staining in the colon. Scale bar = 100 μm. (H) WT and Cmtm3 mice treated with DSS - / - mice. Scale bar = 100 μm. (I) WT and Cmtm3 mice treated with DSS - / -Histological scores of mice; n = 3, 4. (G-J) Representative images of MPO (G) and F4 / 80 (I) staining in the colon. Scale bar = 20 μm. The number of MPO-positive (H) and F4 / 80-positive (J) cells was analyzed in 5 regions of each colon tissue section; n = 3. (K) Appearance of Evans blue in the mouse colon. (L) Evans blue dye content extracted from colon tissue normalized to colon tissue weight; n = 3, 4. (M) WT and Cmtm3 treated with drinking water or DSS - / - Mice were intravenously injected with 70 kDa FITC-dextran (50 mg / kg); n = 3. Accumulation in intestinal crypts indicates vascular permeability. Scale bar = 50 μm. (N) Quantitative assessment, calculating the ratio of FITC signal within crypts to the total FITC signal. 15 crypts were analyzed per mouse. Data represent three independent experiments with similar results. Data are presented as mean ± standard deviation, and statistical significance was determined by one-way ANOVA (Tukey's multiple comparison test) for (A, D, F, H, J, L, N) and two-tailed Student's t-test for (B). *P < 0.05, ***P < 0.001, ****P < 0.0001; ns indicates no significant difference.

[0020] Figure 5 . CMTM3 derived from non-hematopoietic cells plays an important role in DSS-induced colitis, where: (A) Four groups of mice generated by bone marrow transplantation (WT-WT, WT-KO, KO-WT, and KO-KO; n = 3, 4 per group) were treated with 3% DSS for 4 days. (B) DAI scores of the four groups of mice were monitored daily. (C) Representative images of hematoxylin and eosin staining of colon tissue. Scale bar = 100 μm. Data represent two independent experiments with similar results. Data are presented as mean ± standard deviation, and statistical significance was determined by one-way ANOVA (Tukey's multiple comparison test) for (B). **P < 0.01, ***P < 0.001; ns indicates no significant difference.

[0021] Figure 6 . CMTM3 expression is upregulated in endothelial cells during colitis, where: (A) Immunofluorescence staining of CMTM3 (green), EpCAM (red), and DAPI (blue) in colon tissue of WT mice treated with water or DSS. Scale bar = 25 μm. (B) The number of cells expressing CMTM3 and EpCAM; n = 5. (C) Immunofluorescence staining of CD31 (green), CMTM3 (red), and DAPI (blue) in colon tissue of WT mice treated with water or DSS. Yellow arrows indicate CD31 +CMTM3 expression detected in cells. Data represent one experiment, repeated three times. Scale bar = 25 μm. (D) Number of cells expressing CMTM3 and CD31; n = 3. (E) CD31 (green), CMTM3 (red), and DAPI (blue) staining in colon sections from normal individuals and UC patients. Yellow arrows indicate CD31 + CMTM3 expression detected in cells. Scale bar = 25 μm. (F) Number of cells expressing CMTM3 and CD31; n = 5. (G) Expression of CMTM3 in healthy and inflamed human colonic endothelial cells based on scRNA-Seq analysis. The size of each circle reflects the percentage of cells in which CMTM3 was detected, and the color reflects the average expression level of CMTM3 in each cluster. Data are presented as mean ± standard deviation, and statistical significance was determined by two-tailed Student's t-test in (B, D, F). ***P < 0.001, ****P < 0.0001; ns indicates no significant difference.

[0022] Figure 7 . Generation of endothelial cell-specific Cmtm3-deficient mice, where: (A) Restriction map of the wild-type Cmtm3 allele targeting vector and the Cmtm3 allele after Cre-mediated deletion of exons 2-4. (B) PCR analysis of Cmtm3 floxed alleles and Cdh5-Cre transgene. Numbered from left to right, lane 1: DNA ladder; lane 2: negative control. The upper part of panel B shows the sizes of the PCR products for wild-type (+ / +) (lane 3) and homozygous floxed Cmtm3 gene (fl / fl) (lanes 4 and 5). The lower part of panel B shows the sizes of the PCR products without (lanes 3 and 5) and with (lane 4) the Cdh5-Cre transgene. All experimental mice were analyzed by tail genomic DNA. Cmtm3 fl / fl -Cdh5-Cre and Cmtm3 fl / fl Littermate mice were treated with acute or chronic DSS-induced colitis. (C) mRNA expression of Cmtm3 in endothelial cells sorted from the colon tissues of Cmtm3 fl / fl and Cmtm3 fl / fl -Cdh5-Cre mice. Data were normalized to β-Actin and fold change was calculated relative to the Cmtm3 fl / fl -Cdh5-Cre group. (D) Cmtm3 fl / fl and Cmtm3 ΔECImmunofluorescence staining of CMTM3 (red) and VE-cadherin (green) in mouse colon tissues. Counterstained with DAPI (blue). Scale bar = 25 μm. n = 3. Data are presented as mean ± standard deviation, and statistical significance was determined by (C) two-tailed Student's t-test. ***p < 0.001.

[0023] Figure 8 . Deletion of Cmtm3 in endothelial cells inhibits DSS-induced acute colitis, where: (A) Body weight loss measurements of Cmtm3 fl / fl and Cmtm3 ΔEC mice treated with water or DSS for 7 days; n = 7, 4 per group. (B) DAI scores of Cmtm3 fl / fl and Cmtm3 ΔEC mice treated with water or DSS. (C, D) Colon lengths of Cmtm3 fl / fl and Cmtm3 ΔEC mice treated with DSS. (E) Representative micrographs of hematoxylin and eosin staining in the colon. Scale bar = 200 μm (upper panel); scale bar = 100 μm (lower panel). (F) Histological scores of Cmtm3 fl / fl and Cmtm3 ΔEC mice treated with DSS. (G-J) Representative images of MPO (G) and F4 / 80 (I) staining in the colon. Scale bar = 100 μm (upper panel); scale bar = 20 μm (lower panel). The number of MPO-positive (H) and F4 / 80-positive (J) cells was analyzed in 8 regions of each colon tissue section; n = 3. (K) Cmtm3 fl / fl and Cmtm3 ΔEC mice were intravenously injected with 70 kDa FITC-dextran (50 mg / kg) after drinking water or DSS treatment; n = 3. Accumulation in intestinal crypts indicates vascular permeability. Scale bar = 30 μm. (L) Quantitative evaluation, calculating the ratio of FITC signal in crypts to the total FITC signal. Eight crypts were analyzed per mouse; n = 3. Data represent three independent experiments with similar results. Data are presented as mean ± standard deviation, and statistical significance was determined by (A, B, D, F, H, J) two-tailed Student's t-test and (L) one-way ANOVA (Tukey's multiple comparison test). *P < 0.05, **P < 0.01, ****P < 0.0001; ns indicates no significant difference.

[0024] Figure 9. Deletion of Cmtm3 in endothelial cells inhibits DSS-induced chronic colitis, where: (A) Schematic diagram of the chronic colitis model. After treatment with 2% DSS for 7 days, the mice were restored to normal drinking water for 14 days, and then treated with 2% DSS again for 7 days; n = 7, 5. (B) Cmtm3 fl / fl and Cmtm3 ΔEC Measurement of weight loss in mice. (C) DAI scores of Cmtm3 fl / fl and Cmtm3 ΔEC mice treated with DSS. (D, E) Colonic lengths of Cmtm3 fl / fl and Cmtm3 ΔEC mice treated with DSS. (F) Representative micrographs of hematoxylin and eosin staining in the colon. Scale bar = 200 μm (upper panel); scale bar = 100 μm (lower panel). (G) Histological scores of Cmtm3 fl / fl and Cmtm3 ΔEC mice treated with DSS. Data represent three independent experiments with similar results. Data are presented as mean ± standard deviation, and statistical significance was determined by two-tailed Student's t-test in (B, C, E, G). *P < 0.05, **P < 0.01.

[0025] Figure 10 . CMTM3 disrupts VE-cadherin in endothelial cells of colonic tissue, where: (A) Representative staining of CD31 (green), VE-cadherin (red), and DAPI (blue) in colonic tissues of WT and Cmtm3 - / - mice treated with drinking water or DSS. Scale bar = 50 μm (left panel); inset scale bar = 10 μm (right panel). Data represent three independent experiments with similar results. (B) The co-localization of CD31 and VE-cadherin was classified as high, medium, or negative / weak; n = 3. (C) Representative immunofluorescence images of CD31 (green), phosphorylated VE-cadherin-Y731 (red), and DAPI (blue) in colonic sections of WT and Cmtm3 - / - mice treated with drinking water or DSS. Yellow signals indicate the co-localization of CD31 and p-VE-cadherin-Y731. Scale bar = 50 μm (left panel); inset scale bar = 10 μm (right panel). Data represent three independent experiments with similar results. (D) Statistical graph showing the number of VE-cadherin-Y731 phosphorylation-positive cells per field of view (n = 3, 4; 12 fields of view per mouse). (E) Cmtm3 fl / fl and Cmtm3 ΔECWestern blot detection of the expression of p-VE-cadherin (Y731) and VE-cadherin in mouse colon tissues. β-Actin was used as an internal reference. Three mice were included in each group. (F) Quantification of VE-cadherin expression normalized to β-Actin. (G) Quantification of p-VE-cadherin (Y731) expression normalized to VE-cadherin. The data were repeated three times. The data are presented as the mean ± standard deviation, and statistical significance was determined by one-way ANOVA (Tukey's multiple comparison test) in (D, F, G) and chi-square test (Fisher's exact test) in (B). *P < 0.1, **P < 0.01, ****P < 0.0001; ns indicates no significant difference.

[0026] Figure 11 . CMTM3 disrupts VE-cadherin in HUVECs, where: (A) The delivery efficiency of fluorescently labeled siRNA (FAM-siRNA) in HUVECs was analyzed by flow cytometry. (B) HUVECs were transfected with Scr siRNA, siCMTM3-1# or siCMTM3-2#. The expression of CMTM3 in HUVECs stimulated with LPS (1 μg / mL) for 24 hours or unstimulated was analyzed by real-time fluorescence quantitative PCR (RT-PCR). (C) Representative immunofluorescence images of VE-cadherin (green) and DAPI (blue) in HUVECs transfected with Scr or siCMTM3-1#. Stimulated with LPS (1 μg / mL) for 24 hours or unstimulated. Yellow arrows indicate VE-cadherin detected in the plasma membrane. Scale bar = 10 μm. The data represent three independent experiments with similar results. (D) Immunofluorescence images of VE-cadherin-Y731 phosphorylation (green) and DAPI (blue) in HUVECs transfected with Scr or siCMTM3-1#. Stimulated with LPS (1 μg / mL) for 24 hours or unstimulated. Scale bar = 50 μm. (E) Fluorescence intensity of VE-cadherin-Y731 phosphorylation (green) per cell was quantified by normalizing to the lowest expression level. The data represent three independent experiments with similar results. The data are presented as the mean ± standard deviation, and statistical significance was determined by one-way ANOVA (Tukey's multiple comparison test) in (E). ****P < 0.0001; ns indicates no significant difference.

[0027] Figure 12 . CMTM3 promotes clathrin-mediated endocytosis (CME) in endothelial cells by upregulating the expression of CLTC, where: (A) WT and Cmtm3 treated with water or DSS - / -Immunofluorescence staining of CD31 (red), CLTC (green), and DAPI (blue) in mouse colon tissues. Data represent one experiment, repeated three times. Scale bar = 50 μm. (B) Statistical graph showing the percentage of CLTC expression (high, medium, or negative) in CD31-positive cells. n = 3; 10 fields of view per mouse. (C) HUVECs were transfected with an adenovirus encoding internal ribosome entry site (IRES)-EGFP (Mock) and an adenovirus encoding CMTM3-IRES-EGFP (ad-CMTM3) at a multiplicity of infection (MOI) of 100. CMTM3 expression was detected by Western blot. (D) HUVECs were infected with adenoviruses at MOIs of 0, 10, 50, and 100, and EGFP expression (green) was detected 24 or 48 hours later. Scale bar = 200 μm. (E) Representative immunofluorescence images of CLTC (red) and DAPI (blue) in HUVECs transfected with Mock and ad-CMTM3 (MOI 10). Data represent one experiment, repeated three times. Scale bar = 50 μm. (F) Quantification of the fluorescence intensity of CLTC (red) per cell. Quantitative results of immunofluorescence images are shown. (G) Images of siRNA-mediated CLTC knockdown in HUVECs, stained for CLTC (green) and DAPI (blue). Scale bar = 50 μm. (H) HUVECs were transfected with Scr or siCLTC and then transfected with an adenovirus at an MOI of 10 24 hours later. EGFP (green), VE-cadherin (red), and DAPI (blue) were observed by confocal microscopy. Yellow arrows indicate VE-cadherin detected in the plasma membrane. Scale bar = 50 μm. Data represent three independent experiments with similar results. (I) Expression of CLTC in healthy and inflamed human colonic endothelial cells based on single-cell RNA-sequencing (scRNA-Seq) analysis. The size of each circle reflects the percentage of endothelial cells in which CLTC was detected, and the color reflects the mean expression level of CLTC. Data are presented as mean ± standard deviation, and statistical significance was determined by (B) chi-square test (Fisher's exact test) and (F) two-tailed Student's t-test. ****p < 0.0001; ns indicates no significant difference.

[0028] Figure 13. CMTM3 interacts with CLTC and inhibits the ubiquitination and proteasome-dependent degradation of CLTC in HEK293T cells, where: (A) HEK293T cells transfected with Flag or Flag-CMTM3 expression plasmids were treated with 25 μg / mL cycloheximide (CHX) for 0, 6, and 12 hours. The expression of CLTC was detected by Western blot. (B) The data were normalized to β-Actin and the relative fold change compared to the control group was calculated. (C) HEK293T cells transfected with Flag-CMTM3 expression plasmid were treated with 25 μg / mL CHX alone, or with 25 μg / mL CHX plus 10 μM MG132 or 25 μg / mL CHX plus 20 μM chloroquine (CQ) for 12 hours. Subsequently, cell lysates were subjected to Western blot analysis using anti-Flag, anti-CLTC, and anti-β-Actin antibodies. (D) The data were normalized to β-Actin and the relative fold change compared to the control group was calculated. (E) HEK293T cells transfected with Flag or Flag-CMTM3 and HA-ubiquitin were treated with MG132 (10 μM) for 12 hours and then lysed with IP lysis buffer containing protease inhibitors. CLTC was immunoprecipitated and the precipitate was detected using anti-HA, anti-Flag, anti-CLTC, and anti-β-Actin antibodies. (F) HEK293T cells transfected with Flag or Flag-CMTM3 were immunoprecipitated with anti-Flag beads. Subsequently, cell lysates were subjected to Western blot analysis using anti-Flag, anti-CLTC, and anti-β-Actin antibodies. (G) HEK293T cells transfected with Flag or Flag-CMTM3 were immunoprecipitated with anti-CLTC antibody. Subsequently, cell lysates were subjected to Western blot analysis using anti-Flag, anti-CLTC, and anti-β-Actin antibodies.

[0029] (H) Representative immunofluorescence images of CLTC (green), Flag-CMTM3 (red), and DAPI (blue) in HEK293T cells transfected with Flag-CMTM3 for 48 hours. Isotype antibodies were used as controls. Scale bar = 5 μm. (I, J) In HEK293T cells transfected with Flag-CMTM3, CLTC was labeled with FITC (donor) and Flag-CMTM3 was labeled with TRITC (acceptor). The fluorescence intensity (I) and lifetime (J) of FITC-labeled CLTC in the donor alone group and the donor-acceptor group are shown. (K, L) GST and GST-CMTM3 proteins expressed in Escherichia coli BL21(DE3) and purified with glutathione agarose beads were used Incubation of HA-CLTC protein produced in the rapid coupled transcription / translation system. Binding proteins were identified by Western blot with anti-HA and anti-GST antibodies. Data are representative of three experiments with similar results. Data are expressed as mean ± standard deviation (SD). Statistical significance was determined by two-tailed Student's t-test (B, J) and one-way analysis of variance (ANOVA) combined with Tukey's multiple comparison test (D). *p < 0.1; **p < 0.01; ****p < 0.0001; ns indicates no significant difference.

[0030] Figure 14 . CMTM3 interacts with CLTC and inhibits the proteasome-dependent degradation of CLTC in HUVECs, where: (A) HUVECs transfected with Flag or CMTM3-Flag expressing adenovirus were treated with 25 μg / mL cycloheximide (CHX) for 0, 6, 12 hours. The expression of CLTC, Flag, and β-Actin was detected by Western blot. (B) Data were normalized to β-Actin and the relative fold change compared to the control group was calculated. (C) HUVECs transfected with CMTM3-Flag expressing adenovirus were treated with 25 μg / mL CHX alone, or with 25 μg / mL CHX plus 10 μM MG132 or 25 μg / mL CHX plus 20 μM chloroquine (CQ) for 12 hours. Subsequently, cell lysates were detected by Western blot with anti-Flag, anti-CLTC, and anti-β-Actin antibodies. (D) Data were normalized to β-Actin and the relative fold change compared to the control group was calculated. (E) HUVECs transfected with Flag or CMTM3-Flag were immunoprecipitated with anti-Flag beads. Subsequently, cell lysates were detected by Western blot with anti-Flag, anti-CLTC, and anti-β-Actin antibodies. (F) HUVECs transfected with Flag or CMTM3-Flag were immunoprecipitated with anti-CLTC antibody. Subsequently, cell lysates were detected by Western blot with anti-Flag, anti-CLTC, and anti-β-Actin antibodies. Data are representative of three experiments with similar results. Data are expressed as mean ± standard deviation (SD). Statistical significance was determined by two-tailed Student's t-test (B) and one-way analysis of variance (ANOVA) combined with Tukey's multiple comparison test (D). *p < 0.1; ***p < 0.001; ****p < 0.0001; ns indicates no significant difference.

[0031] Figure 15.CMTM3 is a potential therapeutic target for ulcerative colitis (UC), where: (A) Schematic of imatinib treatment. Starting from the first day of DSS treatment, imatinib mesylate (50 mg / kg) was dissolved in phosphate-buffered saline (PBS) and injected intraperitoneally once a day until the last day of DSS treatment. PBS was used as a control. (B) Percentage of body weight loss. Initial body weight was measured (day 0), and body weight was monitored daily throughout the experiment. (C) Disease activity index (DAI) scores for four groups. (D) Representative images of hematoxylin-eosin staining of colon tissues. (E) Histological scores. Scale bar = 100 μm. n = 3 - 5. Data are representative results of two experiments with similar results. Data are expressed as mean ± standard deviation (SD), and statistical significance was determined by one-way analysis of variance (ANOVA) combined with Tukey's multiple comparison test. **P <.01 and ***P <.001; ns indicates no significant difference. Detailed implementation manners

[0032] The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the examples regarding specific techniques or conditions, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained commercially.

[0033] Example 1 Role of CMTM3 in colon cancer and colitis

[0034] 1. Cmtm3 deletion inhibits azoxymethane / dextran sulfate sodium (AOM / DSS)-induced colon cancer (CAC)

[0035] To clarify the exact role of CMTM3 in CAC, Cmtm3 knockout (Cmtm3 - / - ) mice ( Figure 1 ) were generated by CRISPR-Cas9 gene editing technology. Deletion of Cmtm3 did not affect the reproduction and development of mice and had no effect on major organs. Subsequently, we established an inflammation-driven colon cancer model by injecting AOM and then performing three rounds of DSS treatment, with wild-type (WT) and Cmtm3 - / - mice ( Figure 2 A) as experimental subjects. As shown in Figure 2 B to G, Cmtm3 - / - mice showed less severe colon shortening ( Figure 2 B and C), reduced tumor number and volume ( Figure 2 D to F). Histologically, compared with tumor-bearing WT mice, Cmtm3 - / - mice had a lower degree of carcinogenesis ( Figure 2G). These results confirmed that CMTM3 promoted the development of CAC.

[0036] 2. CMTM3 was significantly upregulated in inflamed intestinal tissues of humans and mice

[0037] Since inflammation is a major driver of CAC, we further determined whether CMTM3 was involved in the enteritis stage. First, we analyzed the expression of CMTM3 in colon biopsy samples using the genome-wide transcriptional analysis data in the Gene Expression Omnibus (GEO) database (https: / / www.ncbi.nlm.nih.gov / ). As Figure 3 shown in A to C, CMTM3 was significantly higher in the biopsy samples of active UC patients compared with healthy control groups, non-lesional active ulcerative colitis (UC), and remission phase of UC. Similarly, murine Cmtm3 also increased during the development of DSS-induced enteritis ( Figure 3 D and E).

[0038] We also detected the protein level of CMTM3 in human samples and experimental enteritis mice by immunofluorescence staining. As Figure 3 shown in F and G, CMTM3 was significantly increased in the colon tissues of UC patients compared with healthy control groups. In addition, CMTM3 upregulation was also detected in the colon tissues of DSS-treated mice, while no CMTM3 expression was detected in untreated mice ( Figure 3 H and I), indicating the specificity of CMTM3 staining. These results suggest that CMTM3 was significantly upregulated in inflamed intestinal tissues of UC patients and DSS-induced enteritis mouse models.

[0039] 3. Cmtm3 deficiency protected mice from enteritis

[0040] Expression analysis indicated that CMTM3 might be involved in the development of CAC from the enteritis stage. Therefore, we used the DSS-induced enteritis model to study its role in enteritis. First, there were no differences in body weight, disease activity index (DAI), colon length, tissue structure, and immune cell infiltration between WT and Cmtm3 - / - mice ( Figure 4 ). Interestingly, compared with WT littermates, the body weight ( - / - A) and DAI ( Figure 4 B) of Cmtm3 Figure 4 mice were significantly reduced after DSS treatment. Cmtm3 - / - mice also showed a longer colon length ( Figure 4 C and D), less epithelial damage, and less inflammatory cell infiltration ( Figure 4E and F). In addition, compared with WT mice, Cmtm3 - / - mice had significantly fewer myeloperoxidase (MPO)-positive ( Figure 4 G and H) and F4 / 80-positive ( Figure 4 I and J) cells in DSS-induced colonic tissues. These results indicate that Cmtm3 deficiency has a protective effect in enteritis.

[0041] To determine whether CMTM3 regulates the intestinal vascular barrier, we treated WT and Cmtm3 - / - mice by intravenous injection of Evans blue (EB) dye or 70 kDa fluorescein isothiocyanate (FITC)-dextran. At rest, Cmtm3 deficiency did not alter the integrity of the vascular barrier. As reported, DSS promoted colonic vascular permeability in WT mice ( Figure 4 K to N). However, after DSS treatment, leakage of Evans blue dye ( - / - K and L) or FITC-dextran ( Figure 4 M and N) was significantly reduced in Cmtm3 Figure 4 mice, indicating decreased vascular permeability. Collectively, these data suggest that CMTM3 promotes the development of enteritis and regulates vascular permeability.

[0042] 4. CMTM3 from non-hematopoietic cells is the cause of DSS-induced enteritis

[0043] To further clarify the cell source responsible for the alleviation of enteritis in the absence of Cmtm3, we generated chimeric mice ( - / - A) by transplanting bone marrow cells from WT or Cmtm3 - / - donor mice into irradiated WT or Cmtm3 Figure 5 recipient mice. After 8 weeks of hematopoietic reconstitution, the mice were challenged with 3% DSS. As Figure 5 shown in B, the disease activity index (DAI) of Cmtm3 - / - recipient mice (red line) was lower than that of WT recipient mice transplanted with bone marrow cells from WT mice (black line). However, WT recipient mice transplanted with bone marrow cells from WT mice (black line) and Cmtm3 - / - donor mice (blue line) showed comparable DAI. In addition, the damage to the crypt structure and inflammatory cell infiltration were consistent with the DAI results ( Figure 5 C). These data indicate that CMTM3 from non-hematopoietic cells plays a pro-inflammatory role in the progression of enteritis.

[0044] 5. Deletion of CMTM3 in endothelial cells inhibits DSS-induced acute and chronic enteritis

[0045] Both intestinal epithelium and endothelium play important roles in enteritis. Since CMTM3 derived from non-hematopoietic cells exerts a pro-inflammatory effect, we first detected the expression of CMTM3 in epithelial cells and endothelial cells. As Figure 6 shown in A and B, CMTM3 was hardly expressed in epithelial cells of normal and DSS-induced enteritis mice. Interestingly, we detected an increase in CMTM3 in endothelial cells of DSS-treated mice ( Figure 6 C and D). In addition, compared with tissues of normal individuals, the protein level of CMTM3 in endothelial cells of colon tissues from patients with enteritis was also up-regulated ( Figure 6 E and F). We further verified the expression of CMTM3 in colon endothelial cells using a single-cell transcriptome atlas (http: / / cells.ucsc.edu / ?bp=colon&ds=human-colon). As Figure 6 shown in G, the expression level of CMTM3 in endothelial cells of UC patients was increased compared with that of healthy individuals. These results suggest that CMTM3 derived from endothelial cells may promote the development of enteritis.

[0046] To verify this hypothesis, we specifically knocked out Cmtm3 in endothelial cells

[0047] (Cmtm3 fl / fl Cdh5-Cre, Cmtm3 ΔEC ), and used Cmtm3 fl / fl mice as controls ( Figure 7 A to C). Cmtm3 ΔEC and Cmtm3 fl / fl mice developed normally and showed no macroscopic differences in major organs. Interestingly, compared with Cmtm3 fl / fl mice (black line), after treatment with DSS, Cmtm3 ΔEC mice (red line) showed significantly reduced weight loss ( Figure 8 A), lower disease activity index (DAI) ( Figure 8 B), and longer colon length ( Figure 8 C and D). Histological analysis of the colon showed that compared with Cmtm3 fl / fl mice, Cmtm3 ΔEC mice had significantly reduced crypt destruction and inflammatory cell infiltration ( Figure 8 E and F). In addition, in colon tissues treated with DSS, Cmtm3 ΔEC mice had significantly fewer myeloperoxidase (MPO)-positive cells ( Figure 8 G and H) and F4 / 80-positive cells ( Figure 8 I and J). To verify the regulatory effect of CMTM3 on the intestinal vascular barrier, we administered Cmtm3ΔEC and Cmtm3 fl / fl Mice were intravenously injected with 70 kDa FITC-dextran ( Figure 8 K and 8L). At rest, deletion of Cmtm3 in endothelial cells did not alter the integrity of the vascular barrier. As observed in Cmtm3 - / - mice, after treatment with DSS, leakage of FITC-dextran was significantly reduced in Cmtm3 ΔEC mice, indicating decreased vascular permeability. Collectively, these data confirm that endothelial cell-derived CMTM3 regulates vascular permeability and promotes the development of colitis.

[0048] In addition, we also verified the role of CMTM3 in a chronic colitis model ( Figure 9 A). First, mice were treated with 2% DSS for 7 days; 2 weeks later, they were treated with 2% DSS again for 7 days, followed by analysis. Compared with Cmtm3 fl / fl mice, Cmtm3 ΔEC mice showed significantly less weight loss ( Figure 9 B), lower DAI ( Figure 9 C), and longer colon length ( Figure 9 D and E). Cmtm3 ΔEC mice had a protective effect on the colonic epithelium and reduced inflammatory cell infiltration in the crypts ( Figure 9 F and G). Collectively, our data indicate that Cmtm3 ΔEC mice are less prone to severe inflammation. These results suggest that deletion of Cmtm3 in endothelial cells inhibits the development of acute and chronic colitis, which may in turn affect the occurrence of colorectal cancer (CAC).

[0049] Example 2 Investigation of the mechanism of action of CMTM3

[0050] 1. CMTM3 disrupts VE-cadherin in endothelial cells

[0051] VE-cadherin is an important adhesion molecule that maintains the stability of endothelial junctions. To determine whether endothelial cell-derived CMTM3 regulates the intestinal vascular barrier through VE-cadherin, we examined the distribution of VE-cadherin in endothelial cells of wild-type (WT) and Cmtm3 - / - mice. As shown in Figure 10 A and B, compared with WT mice, the co-localization of VE-cadherin with platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) was more obvious in DSS-treated Cmtm3 - / - mice, indicating that Cmtm3 - / -More continuous adherens junctions were formed in the colon tissues of mice, which may lead to reduced vascular permeability. Therefore, CMTM3 may disrupt VE-cadherin in endothelial cells.

[0052] Since the disruption of VE-cadherin begins with its tyrosine phosphorylation, we further examined the phosphorylation levels of tyrosine residues. In the DSS-induced colitis tissues of WT mice, the phosphorylation of VE-cadherin Y731 was significantly increased, while it was decreased in Cmtm3 knockout mice ( Figure 10 C and D), indicating that the upregulation of VE-cadherin Y731 phosphorylation depends on the presence of CMTM3. We also examined Cmtm3 ΔEC and Cmtm3 fl / fl expression of VE-cadherin and its phosphorylation level at Y731 site in the colon tissues of mice. In the absence of DSS treatment, knockout of Cmtm3 in endothelial cells had no effect on the expression of VE-cadherin and its phosphorylation level at Y731 site. However, knockout of Cmtm3 inhibited the downregulation of VE-cadherin and reduced the upregulation of VE-cadherin-Y731 phosphorylation level ( Figure 10 E to 11G).

[0053] Next, we investigated the effect of CMTM3 on VE-cadherin in vitro using human umbilical vein endothelial cells (HUVECs). We verified the efficient silencing of CMTM3 in HUVECs ( Figure 11 A and B). Under untreated conditions, CMTM3 deficiency did not affect VE-cadherin at adherens junctions. Since lipopolysaccharide (LPS)-induced inflammatory responses are associated with the pathology of ulcerative colitis (UC), we treated HUVECs with LPS. Interestingly, knockdown of CMTM3 inhibited the LPS-induced loss of VE-cadherin on the cell membrane ( Figure 11 C). Consistent with the in vivo results, the phosphorylation level of VE-cadherin Y731 was upregulated after LPS treatment, but CMTM3 inhibition significantly reduced its phosphorylation response to LPS stimulation ( Figure 11 D and E). Therefore, CMTM3 disrupts VE-cadherin in endothelial cells under inflammatory stimulation.

[0054] 2. CMTM3 promotes clathrin-mediated endocytosis of VE-cadherin in endothelial cells by upregulating CLTC expression

[0055] Previous studies have shown that CMTM3 regulates the endocytosis and trafficking of VE-cadherin, but there is no interaction between them and the mechanism is unclear. Since VE-cadherin is mainly internalized through the clathrin-dependent pathway, we hypothesized that CMTM3 may promote clathrin-mediated endocytosis. AsFigure 12 As shown in A and B, after DSS treatment, the expression of CLTC in CD31⁺ endothelial cells in mouse colon tissues increased significantly, while it decreased in Cmtm3 knockout mice. We further confirmed that overexpression of CMTM3 ( Figure 12 C and D) enhanced the expression level of CLTC in HUVECs ( Figure 12 E and F), while knockdown of CLTC ( Figure 12 G) rescued the downregulation of VE-cadherin on the cell membrane caused by CMTM3 ( Figure 12 H). In addition, we analyzed the single-cell transcriptome atlas and found that the expression of CLTC was upregulated in endothelial cells of UC patients ( Figure 12 I), which further confirmed the involvement of endothelial cell-derived CLTC in enteritis. These results indicate that CMTM3 promotes clathrin-mediated endocytosis of VE-cadherin in endothelial cells by upregulating CLTC expression, thus exacerbating enteritis.

[0056] 3. CMTM3 interacts with CLTC and inhibits the ubiquitination and proteasome-dependent degradation of CLTC

[0057] CMTM3 stabilizes NEMO by inhibiting the ubiquitination of NEMO. To our knowledge, there is currently no report on the regulation of the protein stability of CLTC. To explore the mechanism by which CMTM3 upregulates CLTC, we treated control and CMTM3-overexpressing cells with the protein synthesis inhibitor cycloheximide (CHX). As Figure 13 shown in A, after the cells were treated with CHX for 12 hours, the protein level of CLTC decreased significantly, and this effect could be reversed by the proteasome inhibitor MG132, but not by the lysosome inhibitor chloroquine (CQ) ( Figure 13 B). These results indicate that CLTC is degraded through the proteasome pathway.

[0058] Next, we verified whether CMTM3 could inhibit the ubiquitination of CLTC. As Figure 13 shown in C, CMTM3 significantly reduced the ubiquitination level of CLTC. These data confirm that CMTM3 stabilizes CLTC by antagonizing the ubiquitination and proteasome-dependent degradation of CLTC.

[0059] In addition, we further determined whether CMTM3 interacts with CLTC. As Figure 13 shown in D to F, co-immunoprecipitation (IP) ( Figure 13 D and E) and high-resolution imaging ( Figure 13 F) confirmed the interaction between CMTM3 and CLTC proteins. Fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) ( Figure 13G and H), and glutathione S-transferase (GST) pull-down assays ( Figure 13 I and J) further confirmed the direct interaction between CMTM3 and CLTC. Thus, CMTM3 interacts with CLTC and inhibits the ubiquitination and proteasome-dependent degradation of CLTC. We further verified the above conclusion in HUVECs. As Figure 14 shown in A and 14B, when cells were treated with CHX, the protein level of CLTC in the control group decreased significantly compared with that in the CMTM3 overexpression group. The degradation of CLTC could be reversed by MG132, but not by CQ ( Figure 14 C and 14D). Subsequently, we confirmed the interaction between CLTC and CMTM3 in HUVECs ( Figure 14 E and F), which was consistent with the results we observed in HEK293T cells.

[0060] Example 3 CMTM3 is a potential therapeutic target for ulcerative colitis

[0061] Imatinib is a tyrosine kinase inhibitor, and increasing reports have shown that imatinib can restore VE-cadherin junctions. To clarify whether CMTM3 can be used as a therapeutic target and compare its efficacy with imatinib, we treated WT and Cmtm3 - / - mice treated with DSS with daily PBS or imatinib ( Figure 15 A). During colitis, compared with PBS treatment, imatinib significantly alleviated the weight loss ( Figure 15 B), DAI ( Figure 15 C), and histological damage ( Figure 15 D and E) of WT mice, which was consistent with previous reports. Interestingly, compared with WT mice (black line), Cmtm3 knockout (red line) significantly alleviated colitis, while the weight loss, DAI, and histological damage of imatinib-treated WT mice (blue line) and Cmtm3 knockout mice (green line) were comparable, indicating that imatinib was ineffective in Cmtm3 knockout mice. These results suggest that both CMTM3 and imatinib target vascular permeability and have comparable efficacy. Given that imatinib mainly acts by restoring VE-cadherin junctions, our study further confirmed that CMTM3 promotes the development of colitis by disrupting VE-cadherin junctions in endothelial cells and promoting vascular permeability.

[0062] Therefore, CMTM3 may promote vascular permeability by disrupting VE-cadherin in endothelial cells, thus becoming a potential therapeutic target for colitis and colorectal cancer (CAC).

[0063] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. Use of membrane protein CMTM3 and / or CLTC as a target in screening for drugs for treating inflammatory bowel disease and / or related colorectal cancer.

2. The application according to claim 1, characterized in that, The inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

3. The application according to claim 1 or 2, characterized in that, Under inflammatory stimulation, the CMTM3 promotes the occurrence of inflammatory bowel disease by disrupting VE-cadherin in endothelial cells.

4. Use of a membrane protein CMTM3 inhibitor in the preparation of a drug for treating inflammatory bowel disease and / or related colorectal cancer, characterized in that, The membrane protein CMTM3 inhibitor can reduce the expression of the gene encoding membrane protein CMTM3 in patients.

5. The drug according to claim 4, wherein The inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

6. Use of a reagent for regulating the expression of membrane protein CMTM3 and / or CLTC gene in the preparation of a drug for inhibiting proteins related to intestinal inflammatory response, characterized in that, The reagent can reduce the expression of the membrane protein CMTM3 gene in patients.

7. A drug for treating inflammatory bowel disease, characterized in that, The drug includes a membrane protein CMTM3 inhibitor and a pharmaceutically acceptable pharmaceutical excipient, wherein the membrane protein CMTM3 inhibitor can reduce the expression of the gene encoding membrane protein CMTM3 in patients.

8. The drug according to claim 7, wherein, The inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

9. Use of membrane protein CMTM3 and / or CLTC in the preparation of drugs for regulating vascular permeability.

10. Use of membrane protein CMTM3 and / or CLTC as a drug target for regulating VE-cadherin and vascular permeability in endothelial cells.

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