CN114480633A - Application of DCAF13 gene in serving as target for inhibiting breast cancer cell proliferation - Google Patents

Application of DCAF13 gene in serving as target for inhibiting breast cancer cell proliferation Download PDF

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CN114480633A
CN114480633A CN202110366588.2A CN202110366588A CN114480633A CN 114480633 A CN114480633 A CN 114480633A CN 202110366588 A CN202110366588 A CN 202110366588A CN 114480633 A CN114480633 A CN 114480633A
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潘巍巍
张昕
单宝迁
王晓敏
徐营
刘生兵
程树群
韩瑶
陈颢飒
郑丽
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Abstract

The invention relates to a novel action substrate of DCAF13 gene and application thereof, belonging to the technical field of molecular medicine. The DCAF13 gene is found to have the application of inhibiting the proliferation of the breast cancer cells by regulating and controlling the PERP protein; the detection result shows that the expression amount of the DCAF13 gene in breast cancer tissues and cells is obviously increased, the survival rate of a patient with DCAF13 high-expression breast cancer is obviously reduced, the phenotype that the DCAF13 protein lacks and inhibits cell proliferation can be saved by inhibiting the PERP protein, which indicates that the DCAF13 protein possibly plays an important role in the occurrence of the breast cancer by influencing the PERP protein, and the DCAF13 deletion can obviously inhibit the tumor proliferation in vivo and in vitro by constructing 4T1, 4T07 and MCF-7 cells knocked out by DCAF 13.

Description

Application of DCAF13 gene in serving as target for inhibiting breast cancer cell proliferation
Technical Field
The invention relates to a new application of DCAF13 gene in inhibiting breast cancer proliferation through a new molecular target PERP, belonging to the technical field of molecular medicine.
Background
The incidence of breast cancer worldwide has been on the rise in recent years. According to the disease onset data published by the national cancer center and the disease prevention and control bureau of the Ministry of health, the method comprises the following steps: the incidence of breast cancer in tumor registration areas in China is the 1 st of female malignant tumors, and the global breast cancer death rate shows a descending trend from the 90 s in the 20 th century; firstly, the breast cancer screening work is carried out, so that the proportion of early cases is increased; secondly, the development of comprehensive treatment of breast cancer improves the curative effect.
With the rapid development of precise medical technology, the concept of cancer treatment has also changed fundamentally from empirical science to evidence-based medicine and from cell attack to targeted therapy. The targeted therapy is that corresponding therapeutic drugs are designed aiming at the defined carcinogenic sites on the cellular molecular level, and after the drugs enter the human body, the drugs are accurately combined with the carcinogenic sites and take effect. The therapeutic action is mainly limited to the death of tumor cells, and the function of normal cells, tissues or organs is not influenced, so that the curative effect is improved, and the toxic and side effects are reduced.
Therefore, it is of great importance to develop new molecular targets with tumor cell inhibitory effects.
Disclosure of Invention
A first object of the present invention is to provide:
the application of a reagent for knocking out the RNA sequence of the gene guide of DCAF13 in preparing a small molecule inhibitor for inhibiting the proliferation of breast cancer cells.
A second object of the present invention is to provide:
use of an agent for knocking out DCAF13 gene in preparation of an agent for promoting DNA damage of tumor cells.
A third object of the present invention is to provide:
the DCAF13 gene knockout agent is used for preparing an agent for inhibiting the tumor cell cycle and promoting cell senescence.
A fourth object of the present invention is to provide:
use of an agent that knocks out the DCAF13 gene in the preparation of a medicament for activating the P63/PERP signaling pathway.
A fourth object of the present invention is to provide:
use of a reagent for knocking out DCAF13 gene in preparation of a medicament for inhibiting CRL4 ubiquitination linkage.
Advantageous effects
According to the invention, through constructing the tumor cell with the DCAF13 gene knocked out, the expression of the DCAF13 gene is found to obviously influence the relevant characteristics of proliferation, cell cycle and aging of the tumor cell; meanwhile, the invention also discovers that the DCAF13 gene influences the related activity of tumor cells through a P63/PERP signal channel.
Drawings
FIG. 1 shows the results of the expression level of DCAF13 in breast cancer tissues and cells;
FIG. 2 is a graph of the results associated with the inhibition of breast cancer cell proliferation by DCAF13 depletion;
FIG. 3 is a graph showing the relative results of DCAF13 deletion promoting apoptosis in breast cancer cells;
FIG. 4 is a graph showing the relative results of inhibition of tumor proliferation in vivo with a DCAF13 deletion;
FIG. 5 is a graph showing the correlation between the absence of DCAF13 affecting multiple signaling pathways in tumor cells;
Detailed Description
Materials and methods
Material (one):
the 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 miniprep kit were purchased from Axygen corporation. Beta-galactosidase cell senescence assay kit, beta-Actin, ERK1/2, P-ERK1/2, P21, P-AKT, P-PI3K, PI3K, PDK, P-HistonH3, P-H2AX and other primary antibodies and various secondary antibodies are purchased from CST company. FLAG, HA antibodies were purchased from Abcam. The cell cycle detection kit and the Annexin V-FITCII double-staining apoptosis detection kit are purchased from CST (continuous control technology) limited company. DMEM and RPIM1640 cell culture medium, PBS, 0.25% pancreatin + 0.2% EDTA, FBS were purchased from Gibco. DH 5. alpha. was stored in this laboratory.
(II) cell culture
Tumor cell lines 4T1,4T07, OVAR-3, CAOV3, ES-2, SKOV3, HO8910 and A2780 were all purchased from ATCC. Tumor cells were cultured in DMEM and 1640 medium supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin (Gibco) solution at 37 deg.C with humidified 5% CO2Culturing in an incubator.
(III) nude mouse and xenograft model
The nude mice were kept under light/dark at 14 hours/10 hours, bright and dark, and were supplied with food and water at any time. All animal experiments were performed according to the NIH guidelines for laboratory animal care and use. 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 paraffin embedded. Sections (5mm thick) and HE staining.
(IV) retroviral infection
DCAF 13-deleted cells were created by CRISPR genome editing techniques. Lentiviral infection produced MCF-7, 4T07 and 4T1 cells stably expressing empty vector DCAF13 knock-out. 293T LentiV packaging cells were transfected with pLentiV2 empty vector or pLentiV2-DCAF13 knock-out construct. 48 hours after transfection, MCF-7, 4T07 and 4T1 cells were cultured in lentiviral medium. 48 hours after infection, cells were screened with 2-4. mu.g/ml puromycin in culture.
(V) Soft agar colony formation experiment
Preparing 0.5% lower layer gel, spreading 1.5ml mixed solution on six-hole plate, standing at room temperature for 15min for coagulation, suspending the cells in 1.5ml 0.35% agar containing 1 × cell culture medium and 10% fetal calf serum, and pouring into lower layer. Adding 2ml of fresh DMEM culture solution after the gel is solidified, and adding 2-3 days of fresh DMEM culture solution every timeAnd (6) changing the liquid. Triplicate cultures were used for each experiment. Place the plate at 37 ℃ 5% CO2A humidifying incubator. And counting colonies after the 14-20 days later by using crystal violet staining.
(VI) Transwell cell migration assay
Migration and invasion potentials of ES-2 cells transfected with control 4T07 and 4T1 WT and DCAF13 knockout cells were evaluated using 24-well tissue culture plate inserts with 8m well filters and biofilm matrices. The cells were counted and 500. mu.l of normal medium was added to the lower chamber of the Transwell plate, and 1X 10 cells were collected4Cells were placed in the upper chamber and 200. mu.l serum-free medium was added. After 24 hours incubation at 37 ℃, the transwell upper surface cells were gently wiped off with a wet cotton swab and the migrated cells attached to the lower surface. Precooled ice methanol is used for fixing for 30min, hematoxylin is used for staining for 30s and then observed, and the cross well is washed by water and dried by air. Positive cells were quantified using Image-Pro Plus 6.0 software.
(VII) detection of apoptosis by PE Annexin V staining
4T07 and 4T1WT and DCAF13 deficient cells in 6 wells, cold 2mL 1 x PBS washing 3 times, with 1mL 1 x buffer heavy suspension cells, 1 x 105The individual cells were stained with PE-Annexin V and operated according to the kit instructions, and were detected after 15min at room temperature in the dark.
(VIII) mouse and xenograft model
Mice were housed under standard conditions with 14 hours/10 hours light/night and were provided with food and water at any time. All animal experiments were performed according to the NIH guidelines for laboratory animal care and use. To assess cancer cell proliferation in vivo, we deleted 4T07 and 4T1WT and DCAF13 cells (5X 10)5) Subcutaneously transplanted to the dorsal side of 8-week-old female nude mice, 6-8 mice per group. Three weeks later, primary tumor masses were collected from nude mice, fixed in 4% paraformaldehyde, and embedded in paraffin.
(nine) immunohistochemistry
Primary tumor masses were excised at 5 μm with a Leica RM2235 microtome, stained with hematoxylin and eosin (H)&E) In that respect Immunochemical terms, sections were deparaffinized and rehydrated with a xylene and ethanol gradient, then treated at 0.3% H2O2After incubation, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), primary anti-p-H2 AX, KI-67, p-Histon H3, cleaned caspase 3(1:200), at room temperature for 1H, followed by 30 min of reaction with the antibody. Packages and DAB peroxidase substrate packages were performed using ABC staining.
(ten) immunofluorescence
Take 1x105The cells were inoculated in a 24-well plate, washed with PBS and fixed with 4% PFA for 10min at room temperature, the cells were permeabilized with 0.3% Triton X-100 in PBS, blocked with 5% BSA-containing blocking solution for 1H at room temperature, incubated with p-Histon H3, p-H2AX, p-CHK1, HP1, beta-catenin, GOLPH 2-antibody (1:150) for 1H at room temperature, washed with PBST for 3 times and 5 min/time, incubated with secondary antibody at room temperature in the dark for 1H, counterstained with DAPI for 5min, washed with PBST for 3 times and 5 min/time, and mounted.
(eleven) immunoblotting
Total proteins were separated from cell extracts, separated by SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes. After primary antibody detection, the membranes were washed in buffered saline containing 0.05% tween-20 (TBST) and incubated with horseradish peroxidase-linked secondary antibody. Finally, the resulting bands were detected using an enhanced chemiluminescence detection kit. The primary antibody is DCAF13, p-AKT, p-H2AX, cleared caspase 3, p-Histon H3, PERP, Actin, GAPDH (1: 1000).
Extraction of total RNA from cells by (twelve) TRIpure method and quantitative PCR
RNA is extracted by a TRIpure method, the process is described in detail by a reagent, and RNA concentration is detected by using quantitative analysis of nucleic acid. A reverse transcription kit is used. The cDNA was purified in a manner known as 1: and 5, diluting the mixture by DEPC water, and detecting by using a Q-PCR kit. The target gene was amplified using the following primers:
Figure RE-GDA0003187833140000051
(thirteen) statistical methods
Data are expressed as mean ± sd, and comparisons between two groups were analyzed by T-test, with differences of p <0.05 being significant.
Results of the experiment
High expression of DCAF13 in breast cancer tissues and cells
To clarify the role of the DCAF13 protein in breast cancer proliferation, we first examined the expression of DCAF13 protein in human normal breast tissue and breast cancer tissue by immunohistochemical methods. As shown in fig. 1A, DCAF13 was low expressed in normal breast tissue and high expressed in human breast cancer tissue (fig. 1A), TCGA database analysis showed that DCAF13 was low expressed in paracancer tissue and high expressed in cancer tissue (fig. 1B), while we also examined the expression amount of DCAF13 protein in 7 cases of breast Cancer Tissue (CT) and Paracancer Tissue (PT) by immunoblotting, and the results showed that DCAF13 was high expressed in breast cancer tissue and low expressed in paracancer tissue (C of fig. 1), which consistently showed that DCAF13 protein was high expressed in human breast cancer tissue and negatively correlated with patient survival (D of fig. 1). . Next, we examined by immunoblotting that DCAF13 protein was highly expressed in both murine breast cancer cells 4T1,4T07 and human breast cancer cells MDA-MB-231, MCF-7, but was less expressed in normal ovarian surface epithelial cells (FIG. 1E). In addition, the quantitative PCR assay showed that the expression level of Dcaf13 gene was significantly increased in mouse breast cancer cell 4T1 (FIG. 1F). These results indicate that the DCAF13 protein may play an important role in breast cancer development.
Inhibition of breast cancer cell proliferation by DCAF13 depletion
To further clarify the role of DCAF13 in breast cancer cells, we knocked out DCAF13 in mouse and human breast cancer cells using the CRISPR/Cas9 method, and immunoblot detection found partial knock-out of DCAF13 in human breast cancer cells MCF-7, mouse breast cancer cells 4T1 and 4T07 (fig. 2 a). Cell count results showed that partial DCAF13 knock-out significantly inhibited breast cancer cell proliferation (B-2D in FIG. 2), and immunoblot results also showed that p-Histon H3 expression was significantly reduced in DCAF13 knock-out breast cancer cells (A in FIG. 2). The ability of cells to form clones can be tested by soft agar colony formation assay, and the ability of DCAF13 knock-out to inhibit tumor cell colony formation is shown in FIG. 2 as E-G results. In addition, cell migration experiments showed that DCAF13 knockdown significantly inhibited 4T1 and 4T07 cell migration (fig. 2H). Immunofluorescence assay revealed that proliferation-associated proteins p-Histon H3 were under-expressed after DCAF13 knock-out (I in FIG. 2), and DNA damage marker proteins p-H2AX and p-Chk1 (E in FIG. 5). These results indicate that DCAF13 knockdown inhibits cell proliferation, colony formation and migration, promoting cellular DNA damage.
Deletion of DCAF13 promotes apoptosis and senescence in breast cancer cells, affecting the cell cycle
The effect of DCAF13 knockout on breast cancer cell cycle was examined by flow cytometry, and the results indicated that DCAF13 knockout S-phase cells were reduced (fig. 3 a). In addition, cellular senescence assay found that DCAF13 knockout promoted cellular senescence (fig. 3B). In addition, we found that the number of apoptosis of DCAF13 knocked-out breast cancer cells was significantly increased by Annexin V detection method (C of FIG. 3). Cellular senescence assay DCAF13 knock-out was found to promote cellular senescence (C of fig. 3). The p53 and p16/pRB axes are important signaling pathways affecting cellular senescence and cell cycle regulation. Quantitative PCR detection and immunoblotting revealed that DCAF13 knockdown affected P27, P16 and P21 expression (D-3E of fig. 3). These results indicate that loss of DCAF13 promotes apoptosis and senescence by affecting p53 signaling pathway inhibition in breast cancer cells.
Inhibition of tumor proliferation in vivo by DCAF13 depletion
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 measured tumor size periodically. The DCAF13 knockout significantly inhibited tumor cell proliferation (A-B in FIG. 4). Quantitative PCR detection shows that DCAF13 deletion promotes the increase of p63, Perp and p21 expression, and inhibits Mmp13, p53 and Elk-1 gene expression (C in figure 4). Immunohistochemistry results showed that DCAF13 knocked out tumor tissue, the expression level of apoptosis-related protein Cleaved caspase 3 was increased, and the expression level of DNA damage protein p-H2AX was increased (FIG. 4D). The immunoblot detection found that DCAF13 knockout tumor tissue proliferation protein p-Histon H3 was significantly reduced, the expression of apoptosis-related proteins cleared caspase 3 and p-H2AX was increased, and p-AKT was also significantly increased (FIG. 4E). These results indicate that DCAF13 knockdown inhibits tumor cell proliferation in vivo.
Deletion of DCAF13 affecting multiple signaling pathways in tumor cells
To further search for downstream target molecules that DCAF13 knockdown affects breast cancer cell proliferation, we found that DCAF13 knockdown affects signaling pathways such as carcinogenesis, cell proliferation PI3K by RNA-Seq sequencing analysis of transcriptome sequencing (a-C of fig. 5). We detected that the expression level of Slc22a18, Perp, P63 and Dvl genes in DCAF13 knockout cells is obviously increased, while the expression level of Mmp3, ELK-1 and P53 genes is obviously reduced through quantitative PCR (D in figure 5). Meanwhile, the expression level of the DCAF13 knockout cell beta-catenin is obviously reduced through immunofluorescence detection (E in figure 5). We extracted mouse tumor tissue protein and showed by immunoblot detection that the expression level of P-AKT, P-PDK1, P-PI3K, which is a key protein of PI3K signal pathway, was increased (FIG. 5F). These results indicate that the DCAF13 protein can affect multiple signaling pathways and transcriptome of breast cancer cells.
DCAF13 affecting breast cancer cell proliferation by inhibiting the P63/PERP signaling pathway
In order to further search for knocking out downstream target molecules influencing breast cancer cell proliferation by DCAF13, the expression of Slc22a18 and Perp genes is interfered by RNAi, and the quantitative PCR result shows that the expression of Slc22a18 and Perp genes is obviously reduced in DCAF13 knock-out cells, but a cell proliferation experiment shows that Slc22a18 can not knock out a phenotype inhibiting cell proliferation by virtue of cut DCAF13, and the phenotype inhibiting cell proliferation can obviously knock out the phenotype inhibiting cell proliferation by virtue of cut DCAF 13. The CRISPR/Cas9 method is adopted to knock out the PERP protein in the DCAF13 knocked-out breast cancer cells, and the immunoblotting detection result shows that the PERP expression rate is obviously reduced. Cell proliferation experiments showed that the deletion of PERP remedied the phenotype of DCAF13 knockout inhibiting cell proliferation. Clonogenic experiments indicate that PERP knock-out increases the number of cell clonogenic. Co-immunoprecipitation experimental results showed that DCAF13 was associated with PERP protein, but the DCAF13 SOF and WD regions were not binding to PERP.
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Claims (6)

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