Application of CUL7 as target in preparation of medicine for preventing and / or treating colitis

Abstract

The invention discloses application of CUL7 as a target spot in preparation of a medicine for preventing and / or treating colitis, and belongs to the technical field of medicine and pharmacology. The invention provides application of a CUL7 target spot in preparation of a medicine for preventing and / or treating colitis. The invention also provides application of CUL7 as a target spot in preparation of a preparation for diagnosing colitis, and application of a CUL7 gene inhibitor or a CUL7 protein inhibitor in preparation of a medicine for preventing and / or treating colitis. The invention proves the relationship between the CUL7 and the colitis, the CUL7 can be used as a target for preventing and treating the colitis, the CUL7 gene expression or the CUL7 intervention effect can be realized by utilizing any technology, the colitis can be effectively inhibited, and a basis is provided for the prevention, diagnosis and treatment of diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to the use of CUL7 as a target in the preparation of drugs for preventing and / or treating colitis. Background Art

[0002] Colitis is a group of intestinal diseases characterized by chronic inflammation of the colonic mucosa, primarily including inflammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn's disease (CD). The incidence of IBD in my country is increasing, and the disease is often prolonged and carries the risk of developing colon cancer. Therefore, there is an urgent need to explore key signaling pathways in the development of colitis, identify potential targets, design potential drugs targeting these targets, and promote effective diagnosis, prevention, and intervention of colitis.

[0003] The ubiquitin-proteasome system (UPS) is an important type of post-translational modification (PTM) of proteins, playing an important role in cellular processes such as signal transduction, metabolic regulation, development, apoptosis, cell cycle, and protein quality control. Abnormalities in the UPS can lead to serious diseases such as neurodegeneration, inflammation, and cancer. Currently, anticancer drugs targeting the UPS are an effective treatment, especially E3 ligases. Inhibitors targeting E1 or E2 enzymes have no clinical efficacy or are used in clinical research. Cullin (CUL) proteins include CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, and CUL7, which are part of the E3 ubiquitin ligase complex. The CUL-RING E3 ubiquitin ligase (CRL) accounts for approximately 20% of protein ubiquitination and degradation in cells. Prior art has disclosed the functional regulation and disease treatment options of Cullin proteins, including: inhibiting CUL7 can enhance the sensitivity of tumor cells to apoptosis inducers; pseudoinhibitors targeting the CRL complex (such as mLN4924) can interfere with CUL protein activity, regulate metabolism and inhibit tumor growth. The CUL5 complex regulates viral protein degradation, and HSP90 inhibitors can inhibit the replication of the new coronavirus by disrupting the stability of this complex. However, the prior art only discloses the therapeutic effects of CUL7 on tumor diseases and does not provide any guidance on the role of CUL7 in colitis.

[0004] The applicants discovered that CUL7 exhibits unique characteristics compared to typical CUL family members. These characteristics stem from its distinct motifs: the DOC domain and the CPH domain. The inventors' research has shown that CUL7 is highly expressed in patients with colitis. Interference with CUL7 gene expression using gene knockout or siRNA can effectively suppress colitis inflammation, suggesting the development of CUL7-targeted drugs for the prevention and treatment of colitis. Summary of the Invention

[0005] In response to the above-mentioned problems in the prior art, the present invention aims to design and provide a new target for anti-colitis to better prevent and / or treat colitis. In their research, the inventors found that using gene knockdown technology or siRNA to interfere with the gene expression of CUL7 can effectively inhibit colitis.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides the use of CUL7 as a target in the preparation of a drug for preventing and / or treating colitis.

[0008] In a second aspect, the present invention provides the use of CUL7 as a target in the preparation of a preparation for diagnosing colitis.

[0009] In a third aspect, the present invention provides a use of a CUL7 inhibitor in the preparation of a medicament for preventing and / or treating colitis.

[0010] According to any one of the uses, the colitis is LPS-induced colitis.

[0011] In the application, the CUL7 inhibitor is an agent that can reduce the expression level of CUL7 or inhibit the effect of CUL7.

[0012] In the application, the nucleic acid sequence of the siRNAs targeting the CUL7 gene sequence is shown as SEQ ID NO.1.

[0013] In the application, the CUL7 inhibitor is selected from at least one of CUL7 antibodies, modified CUL7, partial peptides of CUL7, siRNAs targeting CUL7 gene sequences, shRNAs targeting CUL7 gene sequences, antisense molecules targeting CUL7 gene sequences, DNA enzymes targeting CUL7 gene sequences, or expression vectors targeting CUL7 gene sequences containing siRNAs, shRNAs, or antisense molecules.

[0014] In the application, the CUL7 inhibitor can knock out or weaken the CUL7 gene, or reduce the expression level of CUL7.

[0015] In a fourth aspect, the present invention provides a pharmaceutical composition for preventing and / or treating colitis, wherein the pharmaceutical composition comprises a CUL7 inhibitor or a pharmaceutically acceptable carrier.

[0016] In the pharmaceutical composition for preventing and / or treating colitis, the pharmaceutically acceptable carrier comprises a lentivirus or a vesicle.

[0017] The present invention provides a method for preventing and / or treating colitis, comprising: targeting CUL7, reducing CUL7 expression and / or inhibiting the effects of CUL7, to prevent and / or treat colitis. Specifically, the method may be to use an inhibitor of CUL7 to reduce CUL7 expression (knockout or attenuation of CUL7 gene expression) and / or inhibit the effects of CUL7.

[0018] In the present invention, the prevention and treatment of colitis refers to the prevention or treatment of further aggravation of colitis.

[0019] In the present invention, an inhibitor refers to any agent that can reduce the expression level of CUL7 or inhibit the effects of CUL7. This inhibition can be achieved in a variety of ways. One class of inhibitors, such as antibodies and antibody fragments, binds to the CUL7 protein with sufficient affinity and specificity to neutralize its biological effects. Another class of CUL7 inhibitors includes siRNAs, shRNAs, antisense molecules, and DNA enzymes that target the CUL7 gene sequence, as known in the art. This agent can be any inhibitor known in the art that reduces the expression level of CUL7 and / or inhibits the effects of CUL7 itself, or it can be an agent based on this molecular formula that has been modified or restructured to still function as a CUL7 inhibitor.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] In this study, siRNA sequences specifically targeting the CUL7 gene were used to knock down CUL7 expression in colon cells HCT116 and DLD1. Real-time fluorescence quantitative PCR revealed that, compared with the control group, the transcriptional expression of inflammatory factors in CUL7 knockdown colon cells was suppressed. Furthermore, Western blotting revealed that, compared with the control group, the expression of key inflammatory regulatory molecules in CUL7 knockdown colon cells was significantly suppressed. This demonstrates the relationship between CUL7 and colitis, suggesting that CUL7 could serve as a target for the prevention and treatment of colitis. Using any technique to interfere with CUL7 expression or interfere with its function can effectively inhibit colitis, providing a basis for the prevention, diagnosis, and treatment of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Figures 1 and 2 show the results of GEO database analysis, where A is a comparison of CUL7 mRNA levels between healthy people and patients with inflammatory bowel disease, and B is a comparison of CUL7 mRNA levels between healthy people, patients with ulcerative colitis, and patients with Crohn's disease.

[0023] Figure 2 Comparison of mRNA levels of inflammatory factors CUL7, IL-1α, IL-1β, IL-6, and TNF-α in HCT116 and DLD1 colon cells with CUL7 knockdown treated with LPS, wherein A is a comparison of mRNA levels of inflammatory factors CUL7, IL-1α, IL-1β, IL-6, and TNF-α in HCT116 colon cells with CUL7 knockdown treated with LPS, and B is a comparison of mRNA levels of inflammatory factors CUL7, IL-1α, IL-1β, IL-6, and TNF-α in DLD1 colon cells with CUL7 knockdown treated with LPS;

[0024] Figure 3 Figure 3 is a comparison of the levels of key inflammatory regulatory molecules CUL7, ERK p42 / p44, ERK p-p42 / p44, NF-κB p65, and NF-κB p-p65 in HCT116 and DLD1 colon cells with CUL7 knockdown treated with LPS. A is a comparison of the levels of key inflammatory regulatory molecules CUL7, ERK p42 / p44, ERK p-p42 / p44, NF-κB p65, and NF-κBp-p65 in HCT116 colon cells with CUL7 knockdown treated with LPS. B is a statistical graph of CUL7 protein expression in Figure A. C is a statistical graph of ERK p-p42 / p44 protein expression in Figure A. D is a statistical graph of NF-κB p-p65 protein expression in Figure A. E is a statistical graph of CUL7, ERK p42 / p44, ERK p-p42 / p44, and NF-κB in DLD1 colon cells with CUL7 knockdown treated with LPS. Comparison of protein levels of p65 and NF-κB p-p65, key molecules regulating inflammation. F is the statistical graph of CUL7 protein expression in Figure E, G is the statistical graph of ERK p-p42 / p44 protein expression in Figure E, and H is the statistical graph of NF-κB p-p65 protein expression in Figure E. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below with reference to the embodiments and the accompanying drawings. However, it should be understood that the protection scope of the present invention is not limited to the specific embodiments.

[0026] Example 1:

[0027] 1. Experimental Materials

[0028] The CUL7 siRNA sequence (SEQ ID NO. 1) involved in the embodiment of the present invention is: 5'-TGAGATCCTAGCTGAACTG-3', and the negative control (NC) (SEQ ID NO. 2) is: 5'-TTCTCCGAACGTGTCACGT-3' (purchased from GenePharm).

[0029] 2. Experimental methods:

[0030] The GEO database was used to analyze the expression of CUL7 in patients with colitis. The specific process is as follows:

[0031] The GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ) was used to retrieve data on IBD patients (GEO database number GSE9452) and patients with different types of inflammatory bowel diseases, including CD and UC (GEO database number GSE6731), and the expression of CUL7 in colitis patients was analyzed. Figure 1 CUL7 is highly expressed in colon cancer of patients with inflammatory bowel disease (IBD). Figure 1 A).

[0032] At the same time, a stratified study was conducted for inflammatory bowel disease subtypes, and Crohn's disease (CD) and ulcerative colitis (UC) patient cohorts were included for independent analysis. Figure 1 Results B showed that CUL7 expression was significantly increased in both CD and UC patients. The above database analysis results indicate that CUL7 may be a potential inducing molecule for inflammatory bowel disease.

[0033] Example 2: Knockout of CUL7 alleviates LPS-induced colon cell inflammation

[0034] The cell culture and CUL7 knockdown colon cell construction process are as follows:

[0035] (1) HCT116 and DLD1 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 units of penicillin, and 100 mg / mL of streptomycin in an incubator with 5% CO2 at 37°C. All cells were identified using short tandem repeat (STR) sequencing.

[0036] siRNA transfection was performed using Lipofectamine RNAiMAX transfection reagent (Invitrogen). Transfection was performed according to the manufacturer's instructions. Cells were collected and tested 36 hours after transfection. Total cell protein was extracted and Western blotting was used to detect CUL7 knockdown expression. Figure 2As shown, the CUL7 level in colon cancer cells transfected with CUL7 siRNA was significantly reduced compared with that in colon cells transfected with NC, and can be used for subsequent experiments.

[0037] (2) Construction of LPS-induced colon cell inflammation model

[0038] The NC-transfected colon cells and CUL7-knockdown colon cells (i.e., CUL7 siRNA-transfected colon cancer cells) collected in the above steps were treated with 1000 ng / mL LPS for 24 h, and the cells were collected for subsequent mRNA and protein detection.

[0039] (3) Detection of inflammatory factor mRNA levels

[0040] The reverse transcription RNA-real-time fluorescence quantitative PCR method was used for detection. The specific method is as follows:

[0041] 1) Harvest cells and add 500 μL of Trizol. Place the tubes on a shaker at room temperature for 8 minutes to ensure complete cell lysis. Place on ice for 10 minutes. Add 100 μL of chloroform to each tube, shake vigorously to mix, and let stand at room temperature for 5 minutes. Centrifuge at 12,000 rpm for 15 minutes. Transfer the upper aqueous phase to another Eppendorf tube, add an equal volume of isopropanol, mix, and let stand at room temperature for 15 minutes. Centrifuge at 12,000 rpm for 20 minutes at 4°C. Wash with 1 mL of pre-cooled 75% ethanol and invert the tube to float the white precipitate. Centrifuge at 12,000 rpm at 4°C for 10 minutes, discard the supernatant, aspirate with a small pipette, and repeat this step, centrifuging at 12,000 rpm at 4°C for 5 minutes. Discard the supernatant. Once the ethanol has evaporated, dissolve the supernatant in 10 μL of RNase-free water. Assay the concentration using a Nanodrop analyzer before reverse transcription.

[0042] 2) RNA reverse transcription

[0043] Reverse transcription was performed in a 20 μL reaction system containing 1 μg of total RNA, 2 μL of gDNAClean Reaction Mix, and 4 μL of 5X Evo M-ML VART Reaction Mix. The volume was then made up to 20 μL with RNase-free water. The reaction conditions were 37°C for 15 min and 85°C for 5 s. The resulting cDNA was stored at -80°C until needed.

[0044] 3) Real-time fluorescence quantitative PCR

[0045] The experiment was performed using the SYBR Green method. The reaction system contained 1 μL cDNA, 5 μL 2x Green ProTag HS Premix, 0.5 μL each of upstream and downstream primers (10 μM), and 3 μL of sterile water were used. Detection was performed using a two-step protocol: 95°C for 30 seconds, followed by 40 cycles of (95°C for 5 seconds, 60°C for 30 seconds), followed by a melting curve procedure. Gapdh was used as an internal reference. The primer sequences used are as follows:

[0046] CUL7:

[0047] forward(SEQ ID NO.3):5'-GCCAGTCTTGTGCTGACTGT-3'

[0048] reverse(SEQ ID NO.4):5'-GGTCTTCTGCATAGGCTTGC-3'

[0049] IL-1α:

[0050] forward(SEQ ID NO.5):5'-TGTATGTGACTGCCCAAGATGAAG-3'

[0051] reverse(SEQ ID NO.6):5'-AGAGGAGGTTGGTCTCACTACC-3'

[0052] IL-1β:

[0053] forward(SEQ ID NO.7):5'-ATGATGGCTTATTACAGTGGCAA-3'

[0054] reverse(SEQ ID NO.8):5'-GTCGGAGATTCGTAGCTGGA-3'

[0055] TNFα:

[0056] forward(SEQ ID NO.9):5'-CTCTTCTGCCTGCTGCACTTTG-3'

[0057] reverse(SEQ ID NO.10):5'-ATGGGCTACAGGCTTGTCACTC-3'

[0058] IL-6:

[0059] forward(SEQ ID NO.11):5'-TTTTGTACTCATCTGCACAGC-3'

[0060] reverse(SEQ ID NO.12):5'-GGATTCAATGAGGAGACTTGC-3'

[0061] GAPDH:

[0062] forward(SEQ ID NO.13):5'-ACAACTTTGGTATCGTGGAAGG-3'

[0063] reverse(SEQ ID NO.14):5'-GCCATCACGCCACAGTTTC-3'

[0064] like Figure 2 Statistical results showed that LPS treatment increased mRNA levels of inflammatory factors such as IL-1α, IL-1β, IL-6, and TNF-α in HCT116 and DLD1 colonocytes compared to NC cells. Notably, CUL7 knockdown significantly reduced LPS-induced inflammatory cytokine levels in colonocytes. This suggests that knockdown of CUL7 protein expression by CUL7 siRNA can alleviate / inhibit LPS-induced transcriptional changes in colonocytes.

[0065] Example 3: Knockout of CUL7 improves the expression of key molecular proteins regulating LPS-induced colon cell inflammation

[0066] To test the levels of key molecular proteins regulating inflammation, the specific process is as follows:

[0067] (1) Protein extraction

[0068] Add 200 μL of Western and IP cell lysis buffer (Biyuntian) to each well of a 12-well cell culture plate, scrape the cells with a scraper, and then collect them into an Eppendorf tube. Shake on a rotary shaker at 4°C and 10 rpm for 15 minutes to ensure that the cells are fully lysed. Then centrifuge at 4°C and 12,000 rpm for 15 minutes, and aspirate the supernatant to obtain the total cell protein. Measure the protein concentration using the BCA method, and adjust the protein concentration of all samples to the same level based on the results. Add 5× protein loading buffer (Solybo) to the protein, heat at 100°C for 10 minutes to denature the protein, and then place the sample at -80°C for later use.

[0069] (2) Western blot

[0070] The experiment was performed using a 10% polyacrylamide gel, with a protein loading of 15 μg. After electrophoresis, proteins were transferred from the gel to a NC membrane using wet transfer. The membrane was then blocked and incubated with the primary antibody overnight at 4°C. The membrane was washed and incubated with the secondary antibody for 1 hour at room temperature. The membrane was further washed before chemiluminescence analysis. GAPDH or tubulin was used as an internal control.

[0071] (3) Statistical analysis

[0072] The experimental results are presented as Mean ± SEM. The data were analyzed using the One-way ANOVA method. The statistical significance was *p < 0.05, **p < 0.01, ***p < 0.001.

[0073] Western blotting experiments were used to detect the protein levels of key molecules regulating inflammation. Figure 3 The results showed that compared with the NC group, LPS induction significantly increased the activation of inflammatory signals NF-κB p65 and ERK p42 / p44 in HCT116 and DLD1 colon cells, and CUL7 knockdown significantly reversed this phenomenon, with protein expression levels restored to a level close to that of NC. CUL7 siRNA knockdown of CUL7 protein expression levels could significantly alleviate the expression of key molecular proteins regulating inflammation in intestinal cells induced by LPS.

[0074] In summary, the above experimental results prove that inhibiting CUL7 gene or protein expression can significantly reduce colitis and inhibit key signaling pathways regulating inflammation.

[0075] The above description is only a specific embodiment of the present invention, not all embodiments. Any equivalent transformation of the technical solution of the present invention made by ordinary technicians in this field after reading the specification of the present invention is included in the claims of the present invention.

Claims

1. Use of CUL7 as a target in the preparation of drugs for preventing and / or treating colitis.

2. Application of CUL7 as a target in the preparation of preparations for diagnosing colitis.

3. Use of a CUL7 inhibitor in the preparation of a medicament for preventing and / or treating colitis.

4. The use according to any one of claims 1 to 3, characterized in that The colitis is LPS-induced colitis.

5. The use according to claim 3, characterized in that The CUL7 inhibitor is an agent that can reduce the expression level of CUL7 or inhibit the effect of CUL7.

6. The use according to claim 5, characterized in that The nucleic acid sequence of the siRNAs targeting the CUL7 gene sequence is shown in SEQ ID NO.

1.

7. The use according to claim 3, characterized in that The CUL7 inhibitor is selected from at least one of CUL7 antibodies, modified CUL7, partial peptides of CUL7, siRNAs targeting CUL7 gene sequences, shRNAs targeting CUL7 gene sequences, antisense molecules targeting CUL7 gene sequences, DNA enzymes targeting CUL7 gene sequences, or expression vectors targeting CUL7 gene sequences containing siRNAs, shRNAs, or antisense molecules.

8. The use according to claim 3, characterized in that The CUL7 inhibitor can knock out or weaken the CUL7 gene, or reduce the expression level of CUL7.

9. A pharmaceutical composition for preventing and / or treating colitis, characterized in that: The pharmaceutical composition comprises a CUL7 inhibitor or a pharmaceutically acceptable carrier.

10. The pharmaceutical composition for preventing and / or treating colitis according to claim 9, characterized in that: The pharmaceutically acceptable carrier includes a lentivirus or a vesicle.

Images