Application of Cullin4A and / or DCAF7 in preparation of product for mediating HDAC7 ubiquitination degradation

Abstract

The invention relates to the technical field of medicines, in particular to application of Cullin4A and DCAF7 in preparation of a product for mediating HDAC7 ubiquitination degradation. It is found that knock-down of the Cullin4A gene can obviously up-regulate the HDAC7 content and promote proliferation and transfer of NSCLC cells, and over-expression of the Cullin4A can obviously increase the ubiquitination level of HDAC7 molecules. Therefore, the Cullin4A can mediate the ubiquitination degradation of the HDAC7 and inhibit the malignant development of the NSCLC. It is also found that a substrate recognition molecule DCAF7 of the Cullin4A interacts with HDAC7, after exogenous overexpression of DCAF7, the protein ubiquitination level of HDAC7 is increased, the protein content is reduced, and up-regulation of the DCAF7 gene significantly inhibits proliferation and metastasis of NSCLC cells. The result shows that the DCAF7 is used as a substrate recognition molecule to mediate the ubiquitination degradation of the CRL4A on the HDAC7.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of Cullin4A (CUL4A) and / or DDB1-CUL4 associated factor 7 (DCAF7) in preparing products that mediate ubiquitination degradation of histone deacetylase 7 (HDAC7). Background Art

[0002] Lung cancer is the leading cause of cancer death worldwide. Approximately 85% of lung cancer cases are non-small cell lung carcinoma (NSCLC). More than half of lung cancer patients will develop metastases, and the recurrence rate is high in patients with early-stage lung cancer, resulting in a poor prognosis for lung cancer patients. In addition to surgical treatment, traditional cytotoxic chemotherapy can only modestly improve the survival rate of NSCLC patients, with limited improvement in patient outcomes. Over the past decade, as increasing research has uncovered the profound impact of genetic alterations on tumor development and progression, targeted therapies and immunotherapies have made significant advances, significantly transforming the treatment paradigm and survival outcomes for NSCLC patients. Compared with traditional chemotherapy, patients receiving genotype-targeted therapies can achieve faster and more durable tumor responses, often with less toxicity. With the advancement of research, a growing number of targets have been discovered and approved by the FDA for the treatment of NSCLC patients. However, the treatment of advanced NSCLC remains a clinical challenge, and the exploration of new mechanisms and molecular targets that drive its proliferation and metastasis is of great clinical significance. Summary of the Invention

[0003] To address the above issues, the present invention provides the use of Cullin4A and DCAF7 in the preparation of products that mediate the ubiquitination and degradation of HDAC7. The present invention has discovered a new therapeutic target for NSCLC, namely, Cullin4A can mediate the ubiquitination and degradation of HDAC7 and inhibit the malignant progression of NSCLC. Cullin4A's substrate recognition molecule DCAF7 interacts with HDAC7, and upregulating the DCAF7 gene significantly inhibits the proliferation and metastasis of NSCLC cells. Based on the new therapeutic target discovered by the present invention, products that mediate the ubiquitination and degradation of HDAC7 can be prepared, thereby inhibiting the proliferation and metastasis of non-small cell lung cancer.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides the use of Cullin4A protein and / or DCAF7 protein in preparing a product for degrading HDAC7.

[0006] Preferably, the HDAC7 degradation product is a product that mediates HDAC7 ubiquitination degradation.

[0007] The present invention provides use of Cullin4A protein and / or DCAF7 protein in preparing a product for treating non-small cell lung cancer.

[0008] Preferably, the product for treating non-small cell lung cancer is a product that inhibits the proliferation and / or metastasis of non-small cell lung cancer.

[0009] Preferably, the product is a medicine.

[0010] The present invention provides use of a reagent for increasing the expression of Cullin4A protein and / or DCAF7 protein in preparing a product for degrading HDAC7.

[0011] Preferably, the HDAC7 degradation product is a product that mediates HDAC7 ubiquitination degradation.

[0012] The present invention provides use of a reagent for increasing the expression of Cullin4A protein and / or DCAF7 protein in preparing a product for treating non-small cell lung cancer.

[0013] Preferably, the product for treating non-small cell lung cancer is a product that inhibits the proliferation and / or metastasis of non-small cell lung cancer.

[0014] Preferably, the product is a medicine.

[0015] Beneficial effects:

[0016] The present invention provides the use of Cullin4A protein and / or DCAF7 protein in the preparation of products that degrade HDAC7. The present invention has discovered a new mechanism and molecular targets (Cullin4A and DCAF7) for the proliferation and metastasis of NSCLC. Through experiments, the present invention found that knocking down the Cullin4A (CUL4A) gene can significantly upregulate HDAC7 levels and promote the proliferation and metastasis of NSCLC cells. Overexpression of the CUL4A gene can significantly increase the ubiquitination level of the HDAC7 molecule. As the expression level of CUL4A increases, the ubiquitination level of HDAC7 also increases. Therefore, CUL4A can mediate the ubiquitination degradation of HDAC7 and inhibit the malignant progression of NSCLC. The present invention also discovered that DCAF7, a specific substrate recognition molecule for CUL4A, interacts with HDAC7. Exogenous overexpression of the DCAF7 gene increased HDAC7 protein ubiquitination levels and decreased protein content. Upregulating DCAF7 significantly inhibited NSCLC cell proliferation and metastasis. Truncating and point-mutating HDAC7 revealed that DCAF7 promoted ubiquitination of the K173 and K179 sites of the HDAC7 molecule. This suggests that DCAF7, as a substrate recognition molecule, mediates the ubiquitination and degradation of HDAC7 by CRL4A (Cullin-RING ubiquitin E3 ligase 4A). Low DCAF7 expression in NSCLC tissues hinders this ubiquitination and degradation process, thereby maintaining high HDAC7 expression levels and promoting the malignant progression of NSCLC. Based on the new therapeutic targets discovered by the present invention, products that mediate HDAC7 ubiquitination and degradation can be prepared, thereby inhibiting the proliferation and metastasis of non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0018] Figure 1 To detect the degradation pathway results of HDAC7 in NSCLC cell lines;

[0019] Figure 2 Results of double-transfection Co-IP binding validation of CULs and HDAC7 in 293T cells (A) and semi-endogenous CULs and HDAC7 binding validation in Calu-1 HDAC7OE cells (B);

[0020] Figure 3 Effect of overexpression of CUL1 / 4A in 293T cells on HDAC7 expression level (A), expression level of CUL1 / 4A in 8 NSCLC cell lines and HBE cell lines (B), and expression level of HDAC7 after lentiviral infection of A549 cells to interfere with CUL1 / 4A expression (C);

[0021] Figure 4 Figure 2 shows the HDAC7 ubiquitination level (A), HDAC7 protein degradation rate (B), and HDAC7 protein expression abundance (C) after plasmid transfection into 293T cells; shScr represents the control group A549 cells, i.e., A549 cells transfected with the ShCUL4A negative control plasmid; shCullin 4A represents the A549 ShCUL4A cell line;

[0022] Figure 5 Figure 3: Results of different experiments on the A549 ShCUL4A cell line. A: EdU assay and statistical results; B: clone formation assay and statistical results; C: CCK8 assay and statistical results; D: Transwell assay and statistical results; E: scratch assay and statistical results. ** represents P < 0.01, *** represents P < 0.001. shCON represents the control group, i.e., A549 cells transfected with the ShCUL4A negative control plasmid; shCUL4A represents the A549 ShCUL4A cell line.

[0023] Figures 6-8 is the experimental result of Example 7; wherein, Figure 6 Middle A: protein interaction network analysis results; Figure 6 Middle BE: Co-IP experiments verified the protein interaction between DCAF7 and HDAC7; Figure 6 Middle F: GST pull-down experiment results; Figure 6 Middle G: simulated molecular docking experiment results; Figure 6 Middle H: Experimental results showing increased HDAC7 ubiquitination levels after overexpression of DCAF7; Figure 7 : Immunohistochemical staining results for DCAF7 expression in tissues of 257 NSCLC patients. The scale bars in the local magnification images are 20 μm, and the scale bars in the other figures are 200 μm. Figure 8 Middle A: Differential expression of DCAF7 in cancer and adjacent tissues; Figure 8 Middle B: Analysis of the TCGA database on the correlation between DCAF7 expression levels and prognosis in 1411 NSCLC patients;

[0024] Figure 9The experimental results of Example 8 are shown in Figures AB and C. Detection of HDAC7 protein ubiquitination levels by Western blot. C. Detection of DCAF7 protein levels in NSCLC cell lines and HBE cells by Western blot. D. Detection of changes in HDAC7 protein levels after exogenous overexpression of DCAF7 by Western blot. E. Detection of changes in HDAC7 protein levels after exogenous knockdown of DCAF7 by Western blot. F. Changes in HDAC7 protein degradation rate after DCAF7 overexpression and statistical graph.

[0025] Figure 10 The experimental results of Example 9; A: photographic and statistical results of EdU experiment; B: photographic and statistical results of colony formation experiment; C: statistical results of CCK8 experiment; D: photographic and statistical results of scratch experiment; E: photographic and statistical results of Transwell experiment; ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001;

[0026] Figure 11 The experimental results of Example 10; wherein, A: gross specimen of subcutaneous tumor of nude mice; B: curve of change of subcutaneous tumor volume; C: curve of change of body weight of nude mice; D: comparison of subcutaneous tumor weight of nude mice; ** represents P < 0.01;

[0027] Figure 12 The experimental results of Example 11 are shown in Figure 11; A: DACF7 truncation plasmid and HDAC7 plasmid were double-transfected in 293T cells, and Co-IP was used to verify the effects of different truncation schemes on molecular binding; B: HDAC7 truncation plasmid and DCAF7 plasmid were double-transfected in 293T cells, and Co-IP was used to verify the effects of different truncation schemes on molecular binding;

[0028] Figure 13 The experimental results of Example 12 are as follows; Figure 13 Middle AB: DCAF7 promotes the degradation effect of HDAC7 truncations; Figure 13 Middle CD: DACF7 degradation-promoting effect and binding of HDAC7 molecules retaining amino acids 100-285; Figure 13 Middle EJ: Verification of the degradation-promoting and ubiquitination effects of DCAF7 on mutant HDAC7;

[0029] Figure 14 :The cBioPortal database found that HDAC7 K179R mutation exists in NSCLC;

[0030] Figure 15 Schematic diagram of the degradation mechanism of HDAC7 molecules. DETAILED DESCRIPTION

[0031] The present invention provides the use of Cullin4A protein and / or DCAF7 protein in preparing a product for degrading HDAC7.

[0032] Technical Terminology: The Cullin4A protein described in this article is a key scaffolding molecule for the ubiquitin E3 ligase family within the ubiquitin-proteasome system. DDB1-CUL4-associated factor 7 (DCAF7) is a protein belonging to the DDB1-CUL4-associated factor (DCAF) family and acts as a substrate receptor for E3 ubiquitin ligases. RBX, Cullin4A, DDB1, and DCAF7 together constitute the E3 ubiquitin ligase complex CRL4A.

[0033] This invention first explains the degradation mechanism of HDAC7 ( Figure 15), providing technical support for targeted drug development. The present invention first systematically screened Cullin family members and, combined with mass spectrometry and Co-IP, discovered that HDAC7 interacts with Cullin4A (CUL4A), suggesting that Cullin-RING ubiquitin E3 ligase 4A (CRL4A) may be involved in regulating HDAC7 ubiquitination. Subsequently, experiments revealed that knocking down the CUL4A gene significantly upregulated HDAC7 levels and promoted NSCLC cell proliferation and metastasis, while overexpressing CUL4A significantly increased HDAC7 ubiquitination. This ubiquitination also increased with increasing CUL4A expression. Therefore, the CUL4A-containing complex CRL4A mediates HDAC7 ubiquitination degradation and inhibits NSCLC malignant progression. The specificity of CRL4A components in mediating ubiquitination degradation of different substrates depends on the specific substrate recognition functions of different DDB1-CUL4 associated factors (DCAFs). By screening the DCAFs family of molecules, the present invention discovered that DCAF7, a specific substrate recognition molecule for CUL4A, interacts with HDAC7. Immunohistochemical staining and survival prognosis analysis of clinical patient specimens revealed that DCAF7 expression was significantly lower in NSCLC than in adjacent tissues, suggesting that low DCAF7 expression may be closely associated with poor patient prognosis. Subsequent experiments revealed that exogenous overexpression of the DCAF7 gene increased HDAC7 ubiquitination and decreased protein content. Upregulating the DCAF7 gene significantly inhibited NSCLC cell proliferation and metastasis. Truncations and point mutations of HDAC7 revealed that DCAF7 promoted ubiquitination of HDAC7 at K173 and K179. The present invention discovered that DCAF7, as a substrate recognition molecule, mediates the ubiquitination and degradation of HDAC7 by the CRL4A complex. Low DCAF7 expression in NSCLC tissues inhibits this ubiquitination and degradation process, thereby maintaining high HDAC7 expression and impacting NSCLC malignant progression. Based on the new therapeutic targets discovered in the present invention, products that mediate HDAC7 ubiquitination and degradation can be prepared by utilizing Cullin4A protein and / or DCAF7 protein, or reagents that increase the expression of Cullin4A protein and / or DCAF7 protein (such as lentiviral expression vectors), thereby inhibiting the proliferation and metastasis of non-small cell lung cancer.

[0034] Since there are currently no specific inhibitors or PROTAC molecules targeting HDAC7, and CRBN, one of the specific substrate recognition molecules of CUL4A, is one of the most commonly used E3 ubiquitin ligase recruitment molecules for designing PROTAC molecules, combined with the new molecular mechanism discovered by the present invention that HDAC7 can be degraded by CRL4A, and based on the technical support provided by the present invention, PROTAC drugs that can target and degrade HDAC7 using DCAF7 as the E3 ubiquitin ligase recruitment molecule are designed. This will provide new treatment options for patients who are currently resistant to targeted drugs and can improve the treatment effect of patients who do not respond well to immunotherapy.

[0035] As an embodiment, the HDAC7 degradation product is a product that mediates HDAC7 ubiquitination degradation.

[0036] Based on the above advantages, the present invention provides the use of Cullin4A protein and / or DCAF7 protein in the preparation of a product for treating non-small cell lung cancer. In one embodiment, the product for treating non-small cell lung cancer inhibits the proliferation and / or metastasis of non-small cell lung cancer. In one embodiment, the product for treating non-small cell lung cancer mediates the ubiquitination and degradation of HDAC7, thereby inhibiting the proliferation and / or metastasis of non-small cell lung cancer. In one embodiment, the product is a pharmaceutical.

[0037] Based on the above advantages, the present invention provides the use of a reagent for increasing the expression of Cullin4A protein and / or DCAF7 protein in the preparation of a product for degrading HDAC7.

[0038] As an embodiment, the HDAC7 degradation product is a product that mediates HDAC7 ubiquitination degradation.

[0039] Based on the above advantages, the present invention provides the use of an agent that increases the expression of Cullin4A protein and / or DCAF7 protein in the preparation of a product for treating non-small cell lung cancer. In one embodiment, the product for treating non-small cell lung cancer inhibits the proliferation and / or metastasis of non-small cell lung cancer. In one embodiment, the product for treating non-small cell lung cancer mediates the ubiquitination and degradation of HDAC7, thereby inhibiting the proliferation and / or metastasis of non-small cell lung cancer. In one embodiment, the product is a pharmaceutical.

[0040] To further illustrate the present invention, the application of Cullin4A and / or DCAF7 provided by the present invention in preparing products that mediate ubiquitination and degradation of HDAC7 is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Preparation Example

[0042] The antibody preparation ratios and product numbers used in the present invention are: CUL1 (1:1000, sc-17775, SANTA), CUL4A (1:2000, ab92554, Abcam), CUL4B (1:1000, 12916-1-AP, Proteintech), HDAC7 (1:2000, #33418, CST), FLAG-TAG (1:1000, #14793, CST), HA-TAG (1:2000, #3724, CST), HIS-TAG (1:1000, #12698, CST), MYC-TAG (1:1000, ab32, Abcam), and β-actin (1:1000, GB11001, Servicebio).

[0043] Example 1 HDAC7 molecules are degraded through the ubiquitin proteasome pathway

[0044] Different types of proteasome inhibitors, MG132 (10 μmol / L) and MLN4924 (Pevonedistat, 10 μmol / L), and lysosomal inhibitors, chloroquine (CQ, 20 μmol / L) and BafA1 (Bafilomycin A1, 25 nmol / L), were added to NSCLC lung cancer cell lines A549 and H1299 over time (0, 1, 3, 6, and 12 hours). Western blot analysis was performed to detect changes in HDAC7 expression levels in cells. Figure 1 .

[0045] The results showed that when the proteasome inhibitor MG132 was added to the cells, the expression level of HDAC7 molecules in the cells increased significantly within 1 to 3 hours after drug addition, and the protein stability increased; however, when the lysosomal degradation inhibitor CQ was added, no inhibition of HDAC7 degradation was observed ( Figure 1 (A). We further replaced the proteasome inhibitor MLN4924, which inhibits the activation of the E3 ubiquitin ligase CRL, with the lysosomal inhibitor BafA1 to directly compare their effects on HDAC7 expression levels. It can be seen that after the CRL-mediated ubiquitination degradation process was inhibited, the degradation of HDAC7 molecules was more significantly inhibited, especially in H1299 cells. In particular, at 1 hour and 3 hours after drug addition, the HDAC7 expression level was significantly higher than that of the BafA1 group at the same time ( Figure 1 Middle B). This indicates that HDAC7 molecules are degraded in vivo via the proteasome pathway, particularly through the ubiquitination pathway mediated by the E3 ubiquitin ligase CRL.

[0046] Example 2 Lentiviral vector system used in the present invention

[0047] 1. CUL1 or CUL4A interferes with the lentiviral packaging process as follows:

[0048] This lentiviral vector system consists of a vector that stably expresses shRNA, the psPAX2 (gag / pol / rev / tat elements) vector, and the pMD2.G (VSVG element) vector. shRNAs were designed and synthesized targeting either the human CUL1 (NM_003592) or CUL4A (NM_003589) genes. The shRNA primer sequences are as follows:

[0049] shCUL1-1: 5'-GATTTGATGGATGAGAGTGTA-3' (SEQ ID NO. 1);

[0050] shCUL1-2: 5'-GCCAGCATGATCTCCAAGTTA-3' (SEQ ID NO. 2);

[0051] shCUL4A-1: 5'-GTGTGGAGAAACAGCTATTAG-3' (SEQ ID NO. 3);

[0052] shCUL4A-2: 5'-GGACAAGAAGATGTTACTAAA-3' (SEQ ID NO. 4);

[0053] The shRNA primers were cloned into the pLKO.1 vector (pLKO.1puro) between the AgeⅠ and EcoRI restriction sites to obtain the coding vector (shCUL1 or shCUL4A).

[0054] The encoding vector was then mixed with psPAX2 and pMD2.G at a mass ratio of 6:4:2 and transfected into 293T cells using EZ Trans (Heyuan Liji (Shanghai) Biotechnology Co., Ltd.). Viral supernatants were collected 24 and 48 hours after transfection and filtered through a 0.45 μm polyvinylidene fluoride membrane. The filtrates were collected into sterile 50 ml centrifuge tubes and stored at -80°C for short-term use.

[0055] 2. The DCAF7 interference (shDCAF7) lentiviral vector system consists of an interference vector containing shRNA and green fluorescent protein (GFP) and packaging plasmids psPAX2 and pMD2.G. The interference vector was constructed as follows: shRNA was designed and synthesized using the human DCAF7 gene (NM_005828) as the target gene. The target sequence is as follows:

[0056] RNAi1: 5'-GCCAGGTTAAACAACCATCGA-3' (SEQ ID NO.5);

[0057] RNAi2: 5'-CACAGCACCATCATTTACGAA-3' (SEQ ID NO. 6);

[0058] RNAi3: 5'-GCCCATGACAAAGAGGTCTAT-3' (SEQ ID NO.7);

[0059] The shRNA primer sequences corresponding to the three target sequences are as follows:

[0060] DCAF7-RNAi1-a (SEQ ID NO.8): 5'-ccgggccaggttaaacaaccatcgactcgagtcgatggttgtttaacctggctttttg-3';

[0061] DCAF7-RNAi1-b (SEQ ID NO.9): 5'-aattcaaaaagccaggttaaacaaccatcgactcgagtcgatggttgtttaacctggc-3';

[0062] DCAF7-RNAi2-a (SEQ ID NO. 10): 5'-ccggcacagcaccatcatttacgaactcgagttcgtaaatgatggtgctgtgtttttg-3';

[0063] DCAF7-RNAi2-b (SEQ ID NO. 11): 5'-aattcaaaaacacagcaccatcatttacgaactcgagttcgtaaatgatggtgctgtg-3';

[0064] DCAF7-RNAi3-a (SEQ ID NO. 12): 5'-ccgggcccatgacaaagaggtctatctcgagatagacctctttgtcatgggctttttg-3';

[0065] DCAF7-RNAi3-b (SEQ ID NO. 13): 5'-aattcaaaaagcccatgacaaagaggtctatctcgagatagacctctttgtcatgggc-3';

[0066] The backbone vector of the interference vector is GV493, and the three shRNAs are respectively inserted between the AgeⅠ and EcoRI restriction sites of the GV493 vector to obtain three interference vectors; the lentiviral packaging process is the same as step 1.

[0067] 3. The DCAF7 overexpression (DCAF7 OE) lentiviral vector was constructed by Shanghai GeneCare Gene Medical Technology Co., Ltd. (GeneCare Gene for short). It consists of a vector expressing the DCAF7 gene, a psPAX2 vector, and a pMD2.G vector. The lentiviral packaging process is the same as step 1, except that the encoding vector is replaced with a vector expressing the DCAF7 gene. The backbone vector of the DCAF7 gene-expressing vector is CV572, and the DCAF7 gene is inserted between the AgeI and NheI restriction sites of CV572. The amplification primers for the DCAF7 gene are as follows:

[0068] DCAF7-F (SEQ ID NO. 14): 5'-gaggatccccgggtaccggtcgccaccatgtccctgcacggcaaacg-3';

[0069] DCAF7-R (SEQ ID NO. 15): 5'-cacacattccacaggctagcctaagcatagtctgggacatcataag-3'.

[0070] Example 3 CUL1 / 4A binds to HDAC7 in lung cancer cell lines

[0071] 1. After transiently transfecting Myc-CULs (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., referred to as Sigma) and Flag-HDAC7 plasmid (purchased from GeneCare Gene) into 293T cells, a co-immunoprecipitation (Co-IP) experiment was performed, including the following steps:

[0072] 1) Prepare IP buffer: Tris-HCl 50 mmol, NaCl 150 mmol, EDTA 1 mmol, Triton X-100 1 ml, and dilute to 100 ml with deionized water (adjust pH to 7.4 after dilution).

[0073] 2) Total cell protein extraction; 3) Protein concentration determination by BCA; 4) Co-IP experimental steps:

[0074] ① After BCA, separate 80μl as the Input group and the rest as the IP group; ② Add 25μl anti-FlagM2 affinity gel beads to the IP group (note that the tip of the pipette should be cut off when aspirating the beads to avoid clogging), and incubate on a shaker at 4℃ for 4-8h; Add 20μl 5× Loading buffer to the Input group, shake and mix, boil the sample for 10min, and then cool the sample to a -80℃ refrigerator to obtain the Input group sample; ③ Remove the EP tube, centrifuge and discard the supernatant, add 1ml IP buffer, gently turn it upside down, centrifuge again, repeat 4 times, and after the last centrifugation, use a spotting pipette tip to aspirate the IP buffer; ④ Add 40μl IP buffer and 10μl 5× Loading buffer to each tube. Add buffer (note that after the last aspiration of the supernatant, IP buffer should be added quickly to prevent the beads from drying out), vortex to mix, and boil the sample for 10 minutes. Then, centrifuge and aspirate the supernatant, which is the Co-IP group sample, and store it in a -80℃ refrigerator until use. Note: When setting up the IgG group for endogenous IP, replace the affinity gel with empty beads and rabbit IgG antibody;

[0075] 5) Western blot detection of target protein. Figure 2 Middle A. Co-IP results showed that CUL1, CUL4A, and CUL4B molecules can bind to HDAC7 molecules.

[0076] 2. Co-IP experiments were performed on Calu-1 cells overexpressing Flag-HDAC7. The results are shown in Figure 2 Middle B. The results show that CUL1 or CUL4A binds to HDAC7 molecules.

[0077] These results indicate that CUL1 or CUL4A may be important scaffold proteins involved in the ubiquitination and degradation of HDAC7 in lung cancer.

[0078] Example 4 CUL4A is an E3 ubiquitin ligase scaffold molecule that mediates HDAC7 ubiquitination and degradation

[0079] 1. 293T cells were cultured in 10 cm diameter culture dishes and transiently transfected with different concentrations (0, 4, and 6 μg / dish) of CUL1 or CUL4A plasmids (purchased from Sigma) into 293T cells. Western blot was used to detect the effect of artificial overexpression of CUL1 and CUL4A on HDAC7 expression levels in 293T cells. The results are shown in Table 1. Figure 3 Middle A. The results showed that after artificial overexpression of CUL4A, the expression level of HDAC7 molecules was significantly reduced; however, after overexpression of CUL1, the expression level of HDAC7 did not change significantly.

[0080] 2. Western blot was used to detect the expression levels of CUL1 and CUL4A in 8 NSCLC cell lines (SK-LU-1, H838, PC-9, H1299, H460, Calu-1, H1975, A549) and HBE cell lines. The results are shown in Figure 3 Middle B.

[0081] 3. Based on the expression levels of CUL1 and CUL4A in lung cancer cell lines, A549 cells with high expression levels and wide application were selected to construct stable cell lines with CUL1 and CUL4A interference. The method is as follows:

[0082] 1) Cultivate A549 cells to be infected; 2) Lentiviral transfection steps are as follows:

[0083] After the cells adhere and expand, select two wells with uniform density and the best cell status for transfection, and the other wells serve as controls. Aspirate the culture medium in the wells, rinse twice with PBS, add 1 mL of DMEM high-glucose medium and 2 mL of the virus solution prepared in step 1 of Example 2, and transfect the control group with the corresponding negative control virus solution. After mixing, continue to culture for 12 hours, observe the cell status during this period, and if no obvious cell death is observed, continue to switch to the same culture system and continue transfection for 12 hours, then switch to normal culture medium for culture.

[0084] 3) Puromycin selection of stably transfected cell lines: When the transfected cells to be screened grow to 80% density in normal culture medium, replace the medium with 4 μg / mL puromycin for selection. Untransfected cells are also added to this medium as a control. After 48 hours, the cells in the control group die, and the culture medium of the lentiviral-infected group is replaced with normal culture medium.

[0085] 4) Identification of Stable Cell Lines: Whether the transfected cells acquire puromycin resistance can be used to preliminarily determine whether the transfection is successful. Further, a small amount of transfected cell protein can be extracted and the expression level of the target protein can be detected by Western blot to determine the extent of viral interference.

[0086] See the results Figure 3 Middle C. The results showed that the expression levels of CUL1 and CUL4A were significantly disrupted, but the decreased expression of CUL1 did not significantly affect the expression of HDAC7. However, after the disruption of CUL4A expression, the expression level of HDAC7 molecules was significantly increased. This indicates that CUL4A is a key scaffold molecule that mediates the ubiquitination and degradation of HDAC7 molecules in NSCLC.

[0087] Example 5 CUL4A regulates HDAC7 ubiquitination and degradation

[0088] Five large dishes of 293T cells were plated according to the following protocols: blank control group, HA-HDAC7, HA-HDAC7+His-Ub, HA-HDAC7+His-Ub+Myc-CUL4A, and HA-HDAC7+His-Ub+Myc-CUL4A. After transient transfection of the corresponding plasmids in 293T cells, the Ni-NTAHis-Tag ubiquitination level assay was used to determine whether CUL4A could promote the ubiquitination level of HDAC7. The different plasmids were purchased from the following manufacturers: HA-HDAC7 was purchased from GeneCare, His-Ub was purchased from Hunan Fenghui Biotechnology Co., Ltd. (referred to as Fenghui Bio), and Myc-CUL4A was purchased from Sigma.

[0089] The experimental steps are as follows:

[0090] 1) Prepare Buffer A: Guanidine-HCl 6 mol, Imidazole 10 mmol, NaH2PO4 100 mmol, Na2HPO4 100 mmol, and dilute to 250 ml with deionized water (adjust pH to 8.0 after dilution).

[0091] Prepare TI Buffer: Tris-HCl 20 mmol, Imidazole 20 mmol, dilute to 200 ml with deionized water (adjust pH to 6.8 after dilution);

[0092] 2) Cell preparation:

[0093] ① Select HEK-293T cells in good condition and seed them into large dishes (10 cm in diameter) in advance. When the cell density reaches 70%-80%, transiently transfect His-Ub, HA-HDAC7, and Myc-CUL4A according to the grouping; ② Prepare the transfection system as follows: Solution A: 500 μl serum-free and double-antibody-free medium + 4 μg / plasmid; Solution B: 500 μl serum-free and double-antibody-free medium + 48 μl EZ Trans (Liji Biotechnology, Shanghai); Add Solution B to Solution A; Incubate at room temperature for 10-15 minutes to form EZ Trans-DNA complexes, and replace the medium for the cells to be transfected with new culture medium; ③ Add the above EZ Evenly drip the Trans-DNA transfection complex into the culture dish containing cells, gently shake to evenly disperse the complex into the culture medium, and return to the incubator for culture; ④ After 6-8 hours, replace the new culture medium again and continue to culture the cells for 24 hours; ⑤ 3 hours before protein extraction and ubiquitination level determination, add MG132 (10μmol / L) to the cells to inhibit ubiquitination degradation of the target protein.

[0094] 3) Extraction of protein to be tested:

[0095] ① Discard the culture medium, wash the large dish twice with pre-cooled PBS and then discard; ② Add 10mL PBS to each dish and gently pipette the 293T cells down, collect them into a 15mL sterile centrifuge tube, and pipette evenly; ③ Divide the collected cells into two parts, input: aspirate 1mL cell suspension into a 1.5mL EP tube, incubate at 4000rpm for 5min at room temperature, discard the supernatant, add 80μL RIPA (with protease phosphatase inhibitors), lyse on ice for 10min, centrifuge at 13000rpm for 10min at 4℃, and measure BCA. Protein concentration; IP: Incubate the remaining 9 mL of cell suspension at 4000 rpm at room temperature for 5 min, discard the supernatant, resuspend the cells with 1 mL of Buffer A, and transfer to a 1.5 mL EP tube (the cells become viscous after adding Buffer A); ④ Ultrasonicate the cells (35% power, 10 s, 1 s interval each time) until the cell suspension is clear and no longer viscous when blown by the pipette tip; ⑤ Centrifuge the lysed cell solution at 13000 rpm at room temperature for 5 min. During this period, prepare Ni-NTA beads according to the number of samples and label the EP tubes. ⑥ Vortex mix the beads, add 50 μL beads to each tube of sample (be careful to cut the pipette tip), add 1 mL of Buffer A to each tube, gently invert up and down 10 times to mix, incubate at 13,000 rpm at room temperature for 2 minutes, aspirate the supernatant, repeat the above steps and wash again; ⑦ Aspirate the supernatant after centrifugation in step 5 and add it to the washed Ni-NTA beads. Be sure to leave at least 50 μL of liquid when aspirating the supernatant to prevent the cell pellet from being aspirated; ⑧ Place the mixture on a rotor and rotate at room temperature for 3 hours; ⑨ Take the mixture and incubate at 13,000 rpm at room temperature for 1 minute, aspirate the supernatant, and be sure to leave 100 μL to prevent the beads from being aspirated; ⑩ Add 1 mL of Buffer A, gently invert up and down 10 times, incubate at 13,000 rpm at room temperature for 2 minutes, and aspirate the supernatant to wash away the unbound proteins; Prepare Buffer A / TI Buffer (1:3 volume), add 1 mL to each sample, continue to gently invert, centrifuge and discard the supernatant; Repeat step ⑩ for each sample with 1 mL of TIBuffer; Use a spotting pipette tip to completely aspirate the supernatant, add 40μL IP buffer + 10μL 5× Loading buffer to the beads (note that the liquid addition process should be rapid to prevent the beads from drying out), vortex the beads to mix them, centrifuge them briefly, and boil them in boiling water for 8 minutes. Then use a spotting pipette tip to aspirate the supernatant. The result is the purified protein solution bound to His-Ub. The loading amount (20 μg) was calculated based on the BCA results of the Input group.

[0096] 4) Western blot detection of target protein. Figure 4Middle A. The results showed that CUL4A could significantly increase the ubiquitination level of HDAC7 molecules. As the expression level of CUL4A increased, the ubiquitination level of HDAC7 also increased.

[0097] Cycloheximide (CHX, 30 μmol / L) was added to the A549 ShCUL4A cell line constructed in Example 4 to inhibit cell protein synthesis. Western blot was used to detect the degradation cycle of HDAC7 protein in the control group A549 cells (transfected with the negative control plasmid of ShCUL4A) compared with the ShCUL4A A549 cells after CHX treatment. The results are shown in Figure 4. Figure 4 Middle B: Image J quantitatively detected the abundance of HDAC7 protein expression at different times, and the graph shows the changes in protein expression abundance over time. The expression level of β-actin in the two groups of samples was used as a reference to normalize the protein abundance of HDAC7. The results are shown in Figure 4 Middle C. It can be clearly seen that the decrease in CUL4A expression level significantly prolonged the half-life of HDAC7 in A549 cells.

[0098] These results indicate that HDAC7 can be degraded through the CUL4A-mediated ubiquitin-proteasome pathway.

[0099] Example 6 Inhibiting CUL4A-mediated HDAC7 degradation and promoting A549 cell proliferation and migration

[0100] The A549 ShCUL4A cell line constructed in Example 4 was subjected to cell scratch test, Transwell cell migration test, CCK8 cell proliferation assay, EdU cell proliferation assay and clone formation assay. The results are shown in Figure 5 The results showed that knockdown of CUL4A enhanced the proliferation and metastasis ability of NSCLC cells.

[0101] Example 7 DCAF7 directly binds to HDAC7, DCAF7 is lowly expressed in NSCLC and is associated with poor prognosis

[0102] Protein interaction network analysis and Co-IP experiments revealed that HDAC7 interacted with DCAF7, and overexpression of DCAF7 could reduce the expression level of HDAC7 ( Figure 6 AE), GST pull-down experiments and simulated molecular docking experiments all showed that DCAF7 could directly bind to HDAC7 ( Figure 6 FG), HDAC7 ubiquitination level increased after overexpression of DCAF7 ( Figure 6The expression levels of DCAF7 in NSCLC tissue and adjacent non-tumor tissue of 257 NSCLC patients were detected by immunohistochemistry ( Figure 7 ), and found that the expression of DCAF7 in cancer tissues was significantly lower than that in paired adjacent tissues ( Figure 8 Middle A), TCGA database analysis of 1411 NSCLC patients with DCAF7 expression levels and prognosis, the results showed that NSCLC patients with low DCAF7 expression had a poor prognosis ( Figure 8 Middle B).

[0103] Example 8 DCAF7 mediates K48-type polyubiquitination of HDAC7 and regulates HDAC7 expression levels in NSCLC cell lines

[0104] Wild-type (WT) ubiquitin, K6, K11, K27, K29, K33, K48, and K63 ubiquitin (only one lysine site for ubiquitin attachment) plasmids (all purchased from Fenghui Bio) were sequentially added to 293T cells, and the Ni-NTAHis-Tag ubiquitination level assay was used to determine which type of ubiquitination DCAF7 promotes on HDAC7. The results are shown in Figure 9 In Figures A and B, DCAF7 promotes K48-type polyubiquitination of HDAC7, leading to its degradation.

[0105] Western blot was used to detect the DCAF7 protein content in NSCLC cell lines and HBE cells. Figure 9 Middle C. The results showed that the expression level of DCAF7 in most NSCLC cell lines was lower than that in normal lung epithelial cells (HBE).

[0106] The changes in HDAC7 expression levels were detected after lentiviral infection of A549 and Calu-1 cells overexpressing DCAF7. Figure 9 In middle D, it can be seen that the expression level of HDAC7 was significantly decreased after overexpression of DCAF7 in A549 and Calu-1 cells.

[0107] The changes in HDAC7 expression levels were detected after lentivirus infection of H460 and H1299 cells to interfere with DCAF7 expression. The results are shown in Figure 9 Middle E, after interfering with DCAF7 protein expression in H460 and H1299 cells, the expression level of HDAC7 increased, and shDCAF7-2 (i.e., shDCAF7 with RNAi2 as the target sequence in Example 2) had the best interference effect.

[0108] Cycloheximide (CHX, 30 μmol / L) was added to the Calu-1 overexpressing DCAF7 cell line to inhibit cell protein synthesis. Western blot was used to detect the degradation cycle of HDAC7 protein in the control (CON) Calu-1 cells and DCAF7 OE Calu-1 cells after CHX treatment. The results are shown in Figure 9 In middle F, it can be clearly seen that the upregulation of DCAF7 expression level significantly shortened the half-life of HDAC7 in Calu-1 cells.

[0109] Example 9: DCAF7 inhibits NSCLC cell proliferation and metastasis

[0110] The DCAF7 knockdown cell lines and DCAF7 overexpression cell lines were subjected to CCK8 assay, Transwell cell migration assay and wound healing assay. Figure 10 The results showed that knockdown of DCAF7 enhanced the proliferation and metastasis of NSCLC cells, while overexpression of DCAF7 decreased the proliferation and metastasis of NSCLC cells.

[0111] Example 10 DCAF7 inhibits NSCLC cell proliferation

[0112] The Calu-1 DCAF7 OE cell line constructed in the previous experiment was inoculated subcutaneously into mice to establish a subcutaneous tumor model. The tumor volume and mouse weight were measured every 3 days. The time of sacrifice was determined according to the tumor growth rate (28 days or 35 days. According to ethical requirements, the tumor radius should not exceed 1.5 cm). The results are shown in Figure 11 The results showed that the proliferation ability of NSCLC cells was significantly inhibited after overexpression of DCAF7.

[0113] Example 11 DCAF7 binds to HDAC7 via two WD40 sequences at positions 164-254

[0114] DCAF7 and HDAC7 truncation molecules were designed based on their molecular domains, and Beijing Qingke Biotechnology Co., Ltd. (Qingke Biotechnology) was commissioned to synthesize mutant plasmids. The complete DCAF7 was designated as WT 1-342 (SEQ ID NO. 16), and the DCAF7 truncation molecules were 55-342, 105-342, 155-342, 210-342, 254-342, 1-107, 1-164, and 1-210, respectively. The numbering of the DCAF7 truncation molecules is the first and last amino acid position numbering in the amino acid sequence of the complete DCAF7. The amino acid sequences of 55-342, 105-342, 155-342, 210-342, 254-342, 1-107, 1-164, and 1-210 are shown in SEQ ID NO. 17 to SEQ ID NO. 24, respectively. The complete HDAC7 was designated as WT 1-952 (SEQ ID NO. NO.25), the truncations of the HDAC7 molecule are 100-952, 285-952, 500-952 and 1-500 respectively; the numbering of the truncations of the HDAC7 molecule is the first and last amino acid position numbering in the amino acid sequence of the complete HDAC7, the amino acid sequences of 100-952, 285-952, 500-952 and 1-500 are shown in SEQ ID NO.26 to SEQ ID NO.29, respectively, and the same applies to the other numbers.

[0115] Intact DCAF7 (WT 1-342):

[0116] MSLHGKRKEIYKYEAPWTVYAMNWSVRPDKRFRLALGSFVEEYNNKVQLVGLDEESSEFICRNTFDHPYPTTKLMWIPDTKGVYPDLLATSGDYLRVWRVGETETRLECLLNNNKNSDFCAPLTSFDWNEVDPYLLGTSSIDTTCTIWGLETGQVLGRVNLVSGHVKTQLIAH DKEVYDIAFSRAGGGRDMFASVGADGSVRMFDLRHLEHSTIIYEDPQHHPLLRLCWNKQDPNYLATMAMDGMEVVILDVRVPCTPVARLNNHRACVNGIAWAPHSSCHICTAADDHQALIWDIQQMPRAIEDPILAYTAEGEINNVQWASTQPDWIAICYNNCLEILRV(SEQ ID NO.16);

[0117] Intact HDAC7 (WT 1-952):

[0118] MDLRVGQRPPVEPPPEPTLLALQRPQRLHHHLFLAGLQQQRSVEPMRLSMDTPMPELQVGPQEQELRQLLHKDKSKRSAVASSVVKQKLAEVILKKQQAALERTVHPNSPGIPYRTLEPLETEGATRSMLSSFLPPVPSLPSDPPEHFPLRKTVSEPNLKLRYKPKKSLERRKNPLLRKESAPPSLRRRPAETLGDSSPSSSSTPASGCSSPNDSEHGPNPILGSEALLGQRLRLQETSVAPFALPTVSLLPAITLGLPAPARADSDRRTHPTLGPRGPILGSPHTPLFLPHGLEPEAGGTLPSRLQPILLLDPSGSHAPLLTVPGLGPLPFHFAQSLMTTERLSGSGLHWPLSRTRSEPLPPSATAPPPPGPMQPRLEQLKTHVQVIKRSAKPSEKPRLRQIPSAEDLETDGGGPGQVVDDGLEHRELGHGQPEARGPAPLQQHPQVLLWEQQRLAGRLPRGSTGDTVLLPLAQGGHRPLSRAQSSPAAPASLSAPEPASQARVLSSSETPARTLPFTTGLIYDSVMLKHQCSCGDNSRHPEHAGRIQSIWSRLQERGLRSQCECLRGRKASLEELQSVHSERHVLLYGTNPLSRLKLDNGKLAGLLAQRMFVMLPCGGVGVDTDTIWNELHSSNAARWAAGSVTDLAFKVASRELKNGFAVVRPPGHHADHSTAMGFCFFNSVAIACRQLQQQSKASKILIVDWDVHHGNGTQQTFYQDPSVLYISLHRHDDGNFFPGSGAVDEVGAGSGEGFNVNVAWAGGLDPPMGDPEYLAAFRIVVMPIAREFSPDLVLVSAGFDAAEGHPAPLGGYHVSAKCFGYMTQQLMNLAGGAVVLALEGGHDLTAICDASEACVAALLGNRVDPLSEEGWKQKPNLNAIRSLEAVIRVHSKYWGCMQRLASCPDSWVPRVPGADKEEVEAVTALASLSVGILAEDRPSEQLVEEEEPMNL(SEQ ID NO.25)。

[0119] DACF7 truncation plasmid and HDAC7 plasmid were double-transfected into 293T cells, and Co-IP was used to verify the effect of different truncation schemes on molecular binding. The results are shown in Figure 12 Middle A: HDAC7 truncation plasmid and DCAF7 plasmid were double-transfected into 293T cells, and Co-IP was used to verify the effect of different truncation schemes on molecular binding. The results are shown in Figure 13 Middle B. The results showed that DCAF7 binds to HDAC7 through two WD40 sequences at positions 164-254; however, there is more than one region on the HDAC7 molecule that can bind to DCAF7.

[0120] Example 12 DCAF7 promotes ubiquitination of HDAC7 at K173 and K179 sites

[0121] The degradation effect of DCAF7 on different HDAC7 truncations showed that the range of DCAF7-promoted HDAC7 ubiquitination degradation was between amino acids 100-285 ( Figure 13 AD); Ubiquitination modification omics detection of HDAC7 ubiquitination modification sites in this region and site mutation (plasmid purchased from Qingke Bio) followed by degradation and ubiquitination level detection ( Figure 13 The results showed that DCAF7 could no longer promote the degradation and ubiquitination of HDAC7 after mutations at K173 and K179 of HDAC7; the cBioPortal database found that K179R mutations were present in HDAC7 in NSCLC ( Figure 14 ), which suggests that the high expression of HDAC7 in NSCLC is related to its inability to be ubiquitinated and degraded after point mutation.

[0122] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of Cullin4A protein and / or DCAF7 protein in the preparation of products for degrading HDAC7.

2. The use according to claim 1, characterized in that The HDAC7 degradation product is a product that mediates HDAC7 ubiquitination degradation.

3. Use of Cullin4A protein and / or DCAF7 protein in the preparation of products for the treatment of non-small cell lung cancer.

4. The use according to claim 3, characterized in that The product for treating non-small cell lung cancer is a product for inhibiting the proliferation and / or metastasis of non-small cell lung cancer.

5. The use according to any one of claims 1 to 4, characterized in that: The product described is a drug.

6. Use of reagents for increasing the expression of Cullin4A protein and / or DCAF7 protein in the preparation of products for degrading HDAC7.

7. The use according to claim 6, characterized in that The HDAC7 degradation product is a product that mediates HDAC7 ubiquitination degradation.

8. Use of a reagent for increasing the expression of Cullin4A protein and / or DCAF7 protein in the preparation of a product for treating non-small cell lung cancer.

9. The use according to claim 8, characterized in that The product for treating non-small cell lung cancer is a product for inhibiting the proliferation and / or metastasis of non-small cell lung cancer.

10. The use according to any one of claims 6 to 10, characterized in that: The product described is a drug.

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