CN114480633A - Use of DCAF13 gene as a target for inhibiting proliferation of breast cancer cells - Google Patents

Abstract

The invention relates to a new action substrate and application of DCAF13 gene, and belongs to the technical field of molecular medicine. The invention finds that the DCAF13 gene has the purpose of inhibiting the proliferation of breast cancer cells by regulating the PERP protein; the detection results show that the expression of the DCAF13 gene in breast cancer tissues and cells is significantly increased, and the survival rate of breast cancer patients with high DCAF13 expression is significantly reduced, Inhibition of PERP protein can rescue the phenotype of DCAF13 protein deletion inhibiting cell proliferation, which suggests that DCAF13 protein may play an important role in breast cancer by affecting PERP protein. DCAF13 deletion can significantly inhibit tumor proliferation in vitro and in vivo.

Description

Use of DCAF13 gene as a target for inhibiting proliferation of breast cancer cells

technical field

The invention relates to a new use of DCAF13 gene to inhibit the proliferation of breast cancer through a new molecular target PERP, and belongs to the technical field of molecular medicine.

Background technique

The global incidence of breast cancer has been on the rise in recent years. According to the incidence data released by the National Cancer Center and the Bureau of Disease Control and Prevention of the Ministry of Health, the incidence of breast cancer in the national cancer registration area ranks first among female malignant tumors, and the global breast cancer mortality rate has shown a downward trend since the 1990s; The reasons are: first, the development of breast cancer screening has increased the proportion of early cases; second, the development of comprehensive breast cancer treatment has improved the curative effect.

With the rapid development of precision medical technology, the concept of cancer treatment has also undergone a fundamental change, that is, from empirical science to evidence-based medicine, and from cell attack mode to targeted therapy mode. Targeted therapy, that is, at the cellular and molecular level, is aimed at the already defined carcinogenic sites to design corresponding therapeutic drugs. After the drugs enter the human body, they will precisely bind to the carcinogenic sites and act. Its therapeutic effect is mainly limited to the death of tumor cells without affecting the function of normal cells, tissues or organs, thereby improving efficacy and reducing toxic side effects.

Therefore, it is of great significance to develop new molecular targets with inhibitory effects on tumor cells.

SUMMARY OF THE INVENTION

The first object of the present invention provides:

The reagent for knocking out the DCAF13 gene guide RNA sequence is used for preparing a small molecule inhibitor for inhibiting the proliferation of breast cancer cells.

The second object of the present invention provides:

The use of the reagent for knocking out the DCAF1 3 gene in the preparation of a reagent for promoting DNA damage in tumor cells.

The third object of the present invention provides:

The reagent for knocking out the DCAF13 gene is used in the preparation of a reagent for inhibiting tumor cell cycle and promoting cell senescence.

The fourth object of the present invention provides:

The use of the reagent for knocking out the DCAF13 gene in the reagent for preparing the medicine for activating the P63/PERP signaling pathway.

The fourth object of the present invention provides:

Use of a reagent for knocking out DCAF13 gene in a reagent for preparing a drug for inhibiting CRL4 ubiquitination linkage.

beneficial effect

By constructing tumor cells knocking out DCAF13 gene in the present invention, it is found that the expression of DCAF13 gene significantly affects the related characteristics of tumor cell proliferation, cell cycle and aging; at the same time, the present invention also finds that DCAF13 gene affects the P63/PERP signaling pathway to the activity of tumor cells.

Description of drawings

Figure 1 is the correlation result of DCAF13 expression in breast cancer tissues and cells;

Figure 2 is the related results of DCAF13 deletion inhibiting the proliferation of breast cancer cells;

Figure 3 is the related results of DCAF13 deletion promoting breast cancer cell apoptosis;

Figure 4 is the related results of DCAF13 depletion inhibiting tumor proliferation in vivo;

Figure 5 shows the relevant results of DCAF13 deletion affecting various signaling pathways in tumor cells;

Detailed ways

Materials and methods

(1) Materials:

cDNA reverse transcription kit and Q-PCR kit were purchased from TAKARA. DL2000 DNA Marker, PCR product recovery kit, DNA gel recovery kit, and plasmid mini-prep kit were purchased from Axygen. β-Galactosidase Cell Senescence Detection Kit, β-Actin, ERK1/2, p-ERK1/2, P21, p-AKT, AKT, p-PI3K, PI3K, PDK, p-PDK, p-HistonH3, Primary antibodies such as p-H2AX and various secondary antibodies were purchased from CST Company. FLAG and HA antibodies were purchased from Abcam Company. Cell cycle detection kit and Annexin V-FITCPI double-stained apoptosis detection kit were purchased from CST Co., Ltd. DMEM and RPIM1640 cell culture medium, PBS, 0.25% trypsin+0.2% EDTA, FBS were purchased from Gibco. DH5α is preserved by our laboratory.

(2) Cell culture

Tumor cell lines 4T1, 4T07, OVAR-3, CAOV3, ES-2, SKOV3, HO8910 and A2780 were purchased from ATCC. Tumor cells were cultured in DMEM and 1640 supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin (Gibco) solution at 37°C in a humidified 5% CO ₂ incubator.

(3) Nude mice and xenograft models

Nude mice were reared with a light/dark time of 14 hours/10 hours, with light and dark, and were provided with food and water at all times. All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. To assess proliferation, we injected breast cancer cells subcutaneously into 8-week-old female nude mice. After 4 weeks, the primary tumor mass was fixed with 4% PFA and embedded in paraffin. Sections (5 mm thick) were HE stained.

(4) Retrovirus infection

DCAF13-deficient cells were created by CRISPR genome editing technology. Lentiviral infection generated MCF-7, 4T07 and 4T1 cells stably expressing the empty vector DCAF13 knockout. 293T lentiviral packaging cells were transfected with pLentiV2 empty vector or pLentiV2-DCAF13 knockout construct. Forty-eight hours after transfection, MCF-7, 4T07 and 4T1 cells were cultured with lentiviral medium. Forty-eight hours after infection, cells were selected with 2-4 μg/ml puromycin in culture.

(5) Soft agar colony formation experiment

Prepare 0.5% lower layer gel, spread 1.5 ml of the mixture on a six-well plate and let it stand for 15 minutes at room temperature to solidify. Suspend the cells in 1.5 ml of 0.35% agar containing 1× cell culture medium and 10% fetal bovine serum, and pour it into the lower layer. . After the gel has solidified, 2 ml of fresh DMEM medium was added, and the medium was changed every 2-3 days. Triplicate cultures were used for each experiment. Place the plate in a 37 °C 5% _CO humidified incubator. After 14-20 days, the colonies were counted after staining with crystal violet.

(6) Transwell cell migration assay

Migration and invasion potential of ES-2 cells transfected with control 4T07 and 4T1 WT and DCAF13 knockout cells were assessed using 24-well tissue culture plate inserts with 8m-pore filters and biofilm matrix. For cell counting, 500 μl of normal medium was added to the lower chamber of the Transwell culture plate, 1×10 ⁴ cells were taken into the upper chamber, and 200 μl of serum-free culture medium was added. After 24 hours of incubation at 37°C, the cells on the upper surface of the transwell were gently wiped off with a damp cotton swab, and the migrated cells were attached to the lower surface. The samples were fixed in pre-cooled ice methanol for 30 min and observed after hematoxylin staining for 30 s. The cross wells were rinsed with water and air-dried. Positive cells were quantified using Image-ProPlus 6.0 software.

(7) Detection of apoptosis by PE Annexin V staining

4T07 and 4T1WT and DCAF13-deficient cells were cultured in 6 wells, washed 3 times with cold 2mL 1×PBS, resuspended with 1mL 1×buffer, and ¹ ×105 cells were taken and stained with PE-Annexin V according to the kit instructions. , placed at room temperature in the dark for 15 min before detection.

(8) Mice and Xenograft Models

Mice were housed under standard conditions with a light/dark period of 14 h/10 h and were provided with food and water at all times. All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. To assess the proliferation of cancer cells in vivo, we subcutaneously transplanted 4T07 and 4T1 WT and DCAF13-null cells ( ⁵ x 105) into the dorsal flanks of 8-week-old female nude mice, 6-8 mice per group. After three weeks, primary tumor masses were collected from nude mice, fixed in 4% paraformaldehyde, and embedded in paraffin.

(9) Immunohistochemistry

Primary tumor masses were excised at 5 μm with a Leica RM2235 microtome and stained with hematoxylin and eosin (H&E). For immunochemistry, sections were deparaffinized and rehydrated with a gradient of xylene and ethanol, then incubated in 0.3% H2O2, post _- antigen retrieval using ₁₀ mM sodium citrate (pH=6.0), primary antibody p-H2AX, KI-67, p-Histon H3, Cleaved caspase 3 (1:200), 1h at room temperature, followed by reaction with secondary antibody for 30 minutes. Packing and DAB peroxidase substrate packing were performed using ABC staining.

(10) Immunofluorescence

1×10 ⁵ cells were seeded in 24-well plates, washed with PBS, fixed with 4% PFA at room temperature for 10 min, permeabilized with 0.3% TritonX-100 in PBS, blocked with 5% BSA-containing blocking solution for 1 h at room temperature, and mixed with p-Histon H3 , p-H2AX, p-CHK1, HP1, β-catenin, GOLPH2 primary antibody (1:150) was incubated at room temperature for 1 h, washed 3 times with PBST for 5 min/time, incubated with secondary antibody at room temperature for 1 h, counterstained with DAPI for 5 min, washed with PBST 3 times 5min/time, cover with slides.

(11) Western blot

Total proteins were isolated from cell extracts, separated by SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes. Following primary antibody detection, membranes were washed in buffered saline containing 0.05% Tween-20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibodies. Finally, the resulting bands were detected using an enhanced chemiluminescence detection kit. Primary antibodies were DCAF13, p-AKT, AKT, p-H2AX, Cleaved caspased3, p-Histon H3, PERP, Actin, GAPDH (1:1000).

(12) TRIpure method to extract total cellular RNA and quantitative PCR

Extract and extract RNA by TRIpure method. For details, please refer to the instructions of reagents. Use nucleic acid quantitative analysis to detect RNA concentration. Use a reverse transcription kit. The cDNA was diluted 1:5 with DEPC water and detected by Q-PCR kit. Amplify the target gene using the following primers:

(13) Statistical methods

The data were expressed as mean ± standard deviation, and the comparison between the two groups was analyzed by T test, and p<0.05 was considered significant difference.

Experimental results

1.DCAF13 is highly expressed in breast cancer tissues and cells

In order to clarify the role of DCAF13 protein in breast cancer proliferation, we first detected the expression of DCAF13 protein in human normal breast tissue and breast cancer tissue by immunohistochemistry. As shown in Figure 1A, DCAF13 was lowly expressed in normal breast tissue, but highly expressed in human breast cancer tissue (Figure 1A). The TCGA database analysis showed that DCAF13 was lowly expressed in paracancerous tissues and high in cancer tissues (Figure 1B). We also detected the expression of DCAF13 protein in 7 breast cancer tissues (CT) and paracancerous tissues (PT) by western blotting, and the results showed that DCAF13 was highly expressed in breast cancer tissues and lowly expressed in paracancerous tissues (Fig. 1C). ), these results consistently showed that DCAF13 protein was highly expressed in human breast cancer tissues and negatively correlated with patient survival (Figure 1D). . Next, we detected high expression of DCAF13 protein in murine breast cancer cells 4T1, 4T07 and human breast cancer cells MDA-MB-231, MCF-7 by western blotting, but low expression in normal ovarian surface epithelial cells ( Figure 1E). In addition, quantitative PCR detection results showed that the expression of Dcaf13 gene was significantly increased in mouse breast cancer cell 4T1 (FIG. 1F). These results suggest that DCAF13 protein may play an important role in breast cancer development.

2. DCAF13 deletion inhibits breast cancer cell proliferation

In order to further clarify the role of DCAF13 in breast cancer cells, we knocked out DCAF13 in mouse and human breast cancer cells using CRISPR/Cas9 method. DCAF13 was partially knocked out in cancer cells 4T1 and 4T07 (Figure 2A). The results of cell counting showed that partial knockout of DCAF13 significantly inhibited the proliferation of breast cancer cells (B-2D in Figure 2), and the Western blot results also showed that the expression of p-Histon H3 in breast cancer cells with DCAF13 knockout was significantly reduced (A in Figure 2). . The soft agar colony formation assay can detect the ability of cells to form colonies. The E-G results in Figure 2 show that DCAF13 knockout significantly inhibits the ability of tumor cells to form colonies. Furthermore, cell migration experiments showed that DCAF13 knockdown significantly inhibited 4T1 and 4T07 cell migration (Fig. 2H). Immunofluorescence detection showed that after DCAF13 knockout, the proliferation-related protein p-Histon H3 was lowly expressed (I of Figure 2), and the DNA damage marker proteins p-H2AX and p-Chk1 (E of Figure 5). These results suggest that DCAF13 knockdown inhibits cell proliferation, clone formation and migration and promotes cellular DNA damage.

3. DCAF13 deletion promotes apoptosis and senescence in breast cancer cells and affects cell cycle

The effect of DCAF13 knockout on the cell cycle of breast cancer was detected by flow cytometry, and the results showed that DCAF13 knockout reduced cells in S phase (Figure 3A). In addition, the cellular senescence assay found that DCAF13 knockout promoted cellular senescence (Fig. 3B). In addition, we found that the number of apoptotic cells in DCAF13-knockout breast cancer cells was significantly increased by Annexin V assay (Figure 3C). Cell senescence assay found that DCAF13 knockout promoted cellular senescence (Figure 3C). The p53 and p16/pRB axes are important signaling pathways that affect cell senescence and cell cycle regulation. Quantitative PCR detection and immunoblotting revealed that DCAF13 knockdown affected P27, P16 and p21 expression (D-3E in Figure 3). These results suggest that DCAF13 deletion in breast cancer cells promotes apoptosis and senescence by affecting p53 signaling pathway inhibition.

4. DCAF13 deletion inhibits tumor proliferation in vivo

To further verify the effect of DCAF13 deletion on breast cancer cell proliferation in vivo. We injected mice with 4T1 and 4T07 wild-type control cells and DCAF13 knockout cells, respectively, and periodically measured tumor size. DCAF13 knockout significantly inhibited tumor cell proliferation (A-B of Figure 4). Quantitative PCR detection showed that DCAF13 deletion promoted the increased expression of p63, Perp and p21, and inhibited the gene expression of Mmp13, p53 and Elk-1 (Figure 4C). Immunohistochemical results showed that DCAF13 knockout tumor tissue increased the expression of apoptosis-related protein Cleaved caspased 3 and the DNA damage protein p-H2AX (Figure 4D). Western blotting showed that DCAF13 knockout tumor tissue significantly reduced the proliferation protein p-Histon H3, increased the expression of apoptosis-related proteins cleaved caspased 3 and p-H2AX, and significantly increased p-AKT (Figure 4E). These results suggest that DCAF13 knockdown inhibits tumor cell proliferation in vivo.

5. DCAF13 deletion affects multiple signaling pathways in tumor cells

In order to further search for downstream target molecules of DCAF13 knockout affecting breast cancer cell proliferation, we analyzed transcriptome sequencing by RNA-Seq sequencing and found that DCAF13 knockout affected signaling pathways such as cancer occurrence and cell proliferation PI3K (Figure 5A-C). We detected by quantitative PCR that the expression levels of Slc22a18, Perp, P63, and Dvl genes were significantly increased in DCAF13 knockout cells, while the expression levels of Mmp3, ELK-1, and p53 genes were significantly decreased (Figure 5D). At the same time, we found that the expression of β-catenin in DCAF13 knockout cells was significantly reduced by immunofluorescence detection (Figure 5E). We extracted the protein of mouse tumor tissue and detected the increased protein expression of p-AKT, p-PDK1, and p-PI3K, the key proteins of PI3K signaling pathway by western blotting (Fig. 5F). These results suggest that DCAF13 protein can affect multiple signaling pathways and transcriptomes in breast cancer cells.

6. DCAF13 affects breast cancer cell proliferation by inhibiting P63/PERP signaling pathway

In order to further search for the downstream target molecules that DCAF13 knockout affects the proliferation of breast cancer cells, we interfered with the expression of Slc22a18 and Perp genes by RNAi. The quantitative PCR results showed that the expression levels of Slc22a18 and Perp genes were significantly reduced in DCAF13 knockout cells, but cell proliferation experiments showed that Inhibition of Slc22a18 could not rescue the phenotype of DCAF13 knockout inhibited cell proliferation, while inhibition of Perp gene could obviously rescue the phenotype of DCAF13 knockout inhibited cell proliferation. The PERP protein was knocked out in DCAF13 knockout breast cancer cells by CRISPR/Cas9 method, and the results of western blotting showed that the expression rate of PERP was significantly reduced. Cell proliferation experiments showed that PERP deletion rescued the phenotype of DCAF13 knockdown inhibiting cell proliferation. Clonal formation experiments showed that PERP knockout increased the number of cell clones. The results of co-immunoprecipitation experiments showed that DCAF13 and PERP proteins combined with each other, but the SOF and WD regions of DCAF13 were not the regions that combined with PERP.

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Claims (6)

1. Use of a reagent for knocking out DCAF13 gene guideRNA sequence for preparing a small molecule inhibitor for inhibiting breast cancer cell proliferation. 2. Use of a reagent for knocking out DCAF13 gene in the preparation of a reagent for promoting DNA damage in tumor cells. 3. Use of a reagent for knocking out DCAF13 gene in the preparation of a reagent for inhibiting tumor cell cycle and promoting cell senescence. 4. Use of the reagent for knocking out the DCAF13 gene in the reagent for preparing the medicine for activating the P63/PERP signaling pathway. 5. Use of a reagent for knocking out DCAF13 gene in a reagent for preparing a drug for inhibiting CRL4 ubiquitination linkage. 6. The use according to any one of claims 1-5, wherein the tumor cells include but are not limited to: 4T1, 4T07, MCF-7 and the like.

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