CN109321656B - Use of protein DEPDC1 as marker for diagnosing triple-negative breast cancer - Google Patents
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Abstract
The invention provides application of a protein DEPDC1 as a marker for diagnosing triple negative breast cancer. The invention also provides application of the miR-26b in preparation of a medicine for treating triple negative breast cancer. The invention also provides application of the miR-26b in preparation of a drug for inhibiting DEPDC1mRNA expression. The invention finds that DEPDC1 expression is obviously higher than that of matched paracarcinoma tissues in triple-negative breast cancer tissues. Overexpression of DEPDC1 in triple negative breast cancer cells can promote cell growth and tumor formation by up-regulating FOXM1 expression. In contrast, knockdown of DEPDC1 showed the opposite effect. Moreover, in triple negative breast cancer, miR-26b as a tumor suppressor can directly inhibit DEPDC1 expression and weaken the promoting effect of DEPDC1 on cell growth and clonogenic formation. The invention provides a new important mechanism for the generation, development and regulation of triple negative breast cancer.
Description
Technical Field
The invention belongs to the field of biological medicine, and relates to a protein, in particular to application of a protein DEPDC1 as a diagnostic marker.
Background
Breast Cancer (BC) is one of the most prevalent cancers and can be classified into four different types based on gene expression profiles: luminel a subtype, luminel B subtype, HER2 overexpressed, and basal-like breast cancer (BLBC). Triple Negative Breast Cancer (TNBC) is characterized by a lack of ER, PR and HER2 expression and belongs to basal-like breast cancer. TNBC is highly invasive, has a large tumor volume and tumor burden, and is more likely to metastasize than other subtypes. At present, the diagnosis rate of new TNBC is between 9% and 16%, while the incidence rate of young women is significantly increased. However, TNBC is not sensitive to targeted drugs currently used in clinical practice, and its primary therapeutic approach is still chemotherapy. Therefore, the search for specific carcinogenic factors is a problem to be solved urgently in order to realize targeted therapy and improve the clinical prognosis effect of triple negative breast cancer.
DEPDC1, protein 1 containing DEP (Dishevelled and EGL-10, Pleckstrin) structural domain, is a highly conserved protein in multiple species of caenorhabditis elegans and mammals (see https:// www.ncbi.nlm.nih.gov/protein/NP-001107592.1 for specific information and amino acid sequence). Although numerous reports indicate that DEP domain-containing proteins (promiscuous and EGL-10, Pleckstrin) can regulate a variety of cellular functions, including a number of signaling proteins, the pathophysiological role of DEPDC1 has not been well studied. Since Kanehira et al first discovered a novel gene DEPDC1 for bladder cancer and reported its important role in bladder cancer cell growth, the important function of DEPDC1 and its possible regulation will be further elucidated. Mi et al found DEPDC1 to be a novel cell cycle-associated gene that regulates mitosis. They also found that DEPDC1 is associated with cell growth and apoptosis in other tumors such as liver cancer, colorectal cancer and glioblastoma, and may be a novel diagnostic marker or prognostic predictor. However, DEPDC1 was poorly known for its function in breast cancer.
The triple negative breast cancer is characterized in that estrogen receptor, progestogen receptor and HER2 genes are all negative, the malignancy degree of the tumor is higher than that of other subtypes, and no target medicine sensitive to the tumor exists at present. Clinically, the prognosis of triple negative breast cancer is worse than that of other types of breast cancer, and no effective treatment strategy exists so far. Therefore, it is necessary to find out important regulatory factors involved in the development of triple negative breast cancer.
Disclosure of Invention
The invention aims to provide application of protein DEPDC1 as a diagnostic marker, and aims to solve the technical problem of poor effect on diagnosing and treating triple negative breast cancer in the prior art by using the protein DEPDC1 as the diagnostic marker.
The invention provides application of a protein DEPDC1 as a marker for diagnosing triple negative breast cancer.
The invention also provides application of the protein DEPDC1 as a marker in preparation of a reagent for diagnosing triple negative breast cancer.
The invention also provides application of the miR-26b in preparation of a drug for inhibiting DEPDC1mRNA expression.
The invention also provides application of the miR-26b in preparation of a medicine for treating triple negative breast cancer.
Specifically, the miR-26b gene sequence is shown in SEQ ID NO. 1.
Specifically, the amino acid sequence of the protein DEPDC1 is shown in SEQ ID NO. 2.
The invention detects the expression conditions of DEPDC1 and miR-26b by Western blotting and real-time quantitative PCR. The invention also applies luciferase reporter gene detection to determine the interaction between miR-26b and DEPDC1, and determines the effects of DEPDC1 and miR-26b on cell proliferation and tumor formation in both in vivo and in vitro aspects.
The invention finds that DEPDC1 expression is obviously higher than that of matched paracarcinoma tissues in triple-negative breast cancer tissues. Overexpression of DEPDC1 in triple negative breast cancer cells can promote cell growth and tumor formation by up-regulating FOXM1 expression. In contrast, knockdown of DEPDC1 showed the opposite effect. Moreover, miR-26b in triple negative breast cancer as a tumor suppressor can directly inhibit DEPDC1 expression and weaken the promoting effect of DEPDC1 on cell growth and clonogenic formation.
The research result of the invention shows that: DEPDC1 is normally up-regulated in triple negative breast cancer, it promotes cell growth and colony formation and is an oncogenic factor for TNBC. The study by the present invention shows that miR-26b negatively regulates DEPDC1 in TNBC. DEPDC1 has a promoting effect on cell proliferation mediated by FOXM 1. These results provide new therapeutic targets for improving the therapeutic efficacy of TNBC.
Compared with the prior art, the invention has the advantages of positive and obvious technical effect. The result of the invention shows that the DEPDC1 negatively regulated by the miR-26b can promote cell proliferation and tumor formation by up-regulating the expression of FOXM1, and a new important mechanism is pointed out for the generation, development and regulation of triple negative breast cancer.
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FIG. 1: DEPDC1 was upregulated as a carcinogenic factor in triple negative breast cancer. A and B show expression of DEPDC1 in 10 pairs of triple negative breast cancer tissue and paired non-tumor tissue, respectively. C and D show DEPDC1 expression in 33 pairs of triple negative breast cancer tissues and paired non-tumor tissues, respectively. E and F show the correlation of DEPDC1 and cell growth markers (PCNA and Ki 67) in triple negative breast cancer tissue, respectively. G and H and I show the correlation between DEPDC1 and cell cycle associated genes (CCNA 2, CCNB1 and CCNE 2) in triple negative breast cancer tissue, respectively.
FIG. 2: a cell line overexpressing DEPDC1 was constructed in MDA-MB-436 cells. A mRNA expression level of DEPDC1 in a series of triple negative breast cancer cell lines. B and C show the validation of the efficiency of DEPDC1 overexpression by mRNA expression level (panel B) and protein expression level (panel C), respectively, in MDA-MB-436 cells.
FIG. 3: DEPDC1 overexpression promoted triple negative breast cancer cell proliferation and tumor growth. A shows that overexpression of DEPDC1 in MDA-MB-436 cells results in increased cell viability. B shows that the ability of BrdU intercalation into synthetic DNA was also enhanced by over-expression of DEPDC 1. C shows that DEPDC1 overexpression upregulated PCNA protein expression compared to control. D shows that overexpression of DEPDC1 inhibited the proportion of cells entering G0/G1 phase, increasing the number of cells entering S phase. E shows that DEPDC1 overexpression promotes MDA-MB-436 cell clonogenic formation. A scale: 100 microns. F shows representative tumors isolated from nude mice in predetermined groups. G and H show that overexpression of DEPDC1 increased the volume (panel G) and weight (panel H), respectively, of the transplanted tumors.
FIG. 4: the knockout efficiency of DEPDC1 was verified in MDA-MB-231 cells using real-time quantitative PCR and immunoblotting methods. A and B show the detection of mRNA and protein expression levels after knockout of DEPDC1 using shRNA technology in MDA-MB-231 cells, respectively.
FIG. 5: knockout of DEPDC1 in triple negative breast cancer cells inhibited cell proliferation and clonogenic formation. A shows that knockout DEPDC1 significantly inhibited cell growth. B shows that knocking out DEPDC1 inhibits BrdU intercalation into synthetic DNA in MDA-MB-231 cells. C shows down-regulation of PCNA expression following knockdown of DEPDC 1. D shows that knockout DEPDC1 inhibited cell cycle progression, leaving more cells in G0/G1 phase. E shows that clonogenic capacity in MDA-MB-231 cells is diminished after knocking out DEPDC 1. A scale: 100 microns. F shows representative tumors isolated from nude mice in designated groups. G and H show a decrease in both volume (panel G) and weight (panel H), respectively, of subcutaneous neoplasia following knockdown of DEPDC 1.
FIG. 6: DEPDC1 was directly down-regulated by miR-26b in triple negative breast cancer cells. A shows a schematic diagram of a candidate DEPDC1 function regulator. B shows miR-26B expression in triple negative breast cancer tissues and paired paracarcinoma tissues. C shows the correlation of miR-26b and DEPDC1 expression in the same triple negative breast cancer tissue. D shows the potential binding site of the 3' non-transcribed region miR-26b of DEPDC 1. E shows that luciferase reporter assay shows that miR-26b significantly reduces luciferase activity of wild-type 3' non-transcribed region plasmids, whereas luciferase activity of mutant plasmids was unchanged. F and G show that miR-26b inhibits the mRNA (panel H) and protein (panel I) expression levels of DEPDC1 in MDA-MB-231 cells, respectively. H and I show increased expression levels of DEPDC1 following treatment with anti-miR-26b in MDA-MB-436 cells, respectively.
FIG. 7: cell proliferation and clonal formation inhibited by miR-26b in triple negative breast cancer cells can be attenuated by DEPDC1 overexpression. A shows that inhibition of miR-26b in MDA-MB-436 cells results in an increase in cell viability. B shows that miR-26B inhibition promotes BrdU inhalation. C shows that the inhibition of miR-26b can increase PCNA protein expression. D: anti-miR-26b can promote the clone formation of MDA-MB-436 cells. A scale: 100 microns. E shows that cell growth inhibition by miR-26b can be attenuated after the addition of DEPDC1 again. F and G show that inhibition of miR-26b in BrdU inhalation (panel F) and PCNA protein expression (panel G), respectively, can be antagonized by overexpressing DEPDC 1. H shows that miR-26b treatment in MDA-MB-231 cells can inhibit colony formation, and the effect can be reversed by DEPDC1 overexpression. A scale: 100 microns.
FIG. 8: DEPDC1 promoted cell growth and proliferation by up-regulating FOXM1 expression in triple negative breast cancer. A and B show a clear upregulation of FOXM1 expression in triple negative breast cancer tissue compared to paired non-tumor tissue, respectively. C shows that there is a correlation in expression between FOXM1 and DEPDC1 in the same triple negative breast cancer tissue. D shows that overexpression of DEPDC1 in triple negative breast cancer cells can promote FOXM1 expression. E shows that protein levels of FOXM1 were inhibited after knockout of DEPDC 1. F and G show that both mRNA and protein expression levels of FOXM1 were used to validate the knockout efficiency of siFOXM1, respectively. H shows that increased cell viability by overexpression of DEPDC1 was attenuated by siFOXM 1. I shows that DEPDC1 promoted the antagonism of BrdU intercalation of synthetic DNA by siFOXM 1. J shows that the promotion of PCNA expression by over-expression of DEPDC1 is attenuated by siFOXM 1.
Detailed Description
Materials and methods
1. Cell culture and clinical samples
MDA-MB-231, MDA-MB-436, MDA-MB-468, MDA-MB-157, MDA-MB-435 and BT549 cells (human TNBC cell line) were obtained from American Tissue Culture Collection (Manassas USA). All cell lines were in 5% CO2And (3) culturing in Dulbecco's modified Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) at 37 ℃.
2、CCK-8
Cells were seeded in 96-well plates at 2X 10 per well3Cells/well, 5% CO at 37 ℃2And (4) incubating under the condition. Cell viability assays were performed at various time points using Cell Counting Kit-8 (CCK-8; Dojindo laboratories, Inc.) according to the manufacturer's protocol. The absorbance at 450nm was then measured.
3. BrdU embedding experiments
Cells were seeded in 96-well microplates at a density of 5000 cells/well and allowed to attach overnight. Cell proliferation was assessed by incorporating 5 '-bromo-2' -deoxyuridine (BrdU) into newly synthesized DNA using a cell proliferation ELISA kit (roche). The optical density at 595 nm was measured using a microplate reader (baits HT) and the proliferation rate was expressed as a percentage of the control group.
4. Western blotting
Cells were washed three times with cold PBS, harvested using cell lysis buffer (RIPA) and cell spoons, and quantified by BCA method. Equal masses of protein extract (50. mu.g) were electrophoresed on polyacrylamide gel for 80mV for 90 min and then transferred to nitrocellulose membrane at 100mV for 60 min at room temperature. Blocking in 5% skim milk, and then incubating with primary antibody overnight. This was followed by 5 washes with PBS-T over 30 minutes and incubation with anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology) for 1 hour. PBS-T was washed 5 times within 30 minutes again and the immune complexes were detected using Enhanced Chemiluminescence (ECL).
5. RNA extraction and real-time fluorescent quantitative PCR
Extracting total RNA by TRIzol (Invitrogen) according to a preset program; extracting micro RNA (miRNA) (14) by using an Ambion miRNA RNA extraction kit. Reverse transcription of miRNA and qRT-PCR Taqman miRNA reverse transcription kits (Applied Bio-systems, Calsbad, Calif.) and Taqman premix (Japanese Shiga, Takara) were used. Specific reverse transcription primers for miR-26b and snRNA U6 (internal reference) and qRT-PCR Taqman probe were purchased from Applied Biosystems. For mRNA analysis, total RNA was reverse transcribed using the Tarara Prime-Script RT kit and samples were amplified using SYBR Green Real-time PCR Master Mix (Applied Bio-systems). Beta-actin serves as an internal control for mRNA levels.
6. Cell cycle analysis
Cells were collected (1X 10)6) And resuspended in PBS after trypsinization. After fixation in 70% cold ethanol at 20 ℃ for at least 1.5 hours, staining was performed with PI staining system (40. mu.g/ml PI, 100. mu.g/ml RNaseA and 0.1% Triton X-100) for 1 hour at 37 ℃ followed by analysis of cell cycle distribution using flow cytometry. Flow Analysis was performed by Epics Altra flow cytometer and the results were analyzed by EXPO32 Multicomp and EXPO32 v1.2 Analysis software (Beckman Coulter).
7. Clone formation experiments
Cell growth ability was examined by soft agar colony formation assay. 20% FBS DMEM medium was added to 24-well plates plated with 0.8% agarose (lower layer) and 0.4% agarose (upper layer), and 3X 10 cells were inoculated per well3And (4) suspending the cells. The colonies formed were observed after 21 days and counted under a 40-fold magnification, only the clearly visible colonies (diameter) being counted>50µm）。
8. Luciferase reporter gene detection
Seeding HEK-293T cells in 24-well plates at approximately 1X 105Per well. Cells were co-transfected with 200 ng of reporter vector, 5 ng pRL-CMV (Promega, USA) and 5 nM miR-26b analogue or control (Ambion, USA) using Lipofectamine 2000 (Invitrogen). Luciferase activity was measured after 24 hours using the dual luciferase reporter system (Promega). The luciferase activity of the reporter gene should be normalized to the internal reference Renilla luciferase activity in all samples.
9. Nude mouse transplantation tumor model
Female BALB/c nude mice (Stanford linac center, Shanghai, China) six to eight weeks old were used to establish a breast cancer transplantable tumor model. All experimental animal related procedures were approved by the university of shanghai transportation laboratory animal care and use committee (IACUC). Mice were randomly grouped (n = 6), 5 x 10 suspended in 100 μ Ι _ PBS, respectively6The DEPDC1 overexpression and vector MDA-MB-436 cells or shDEPDC1 and shControl MDA-MB-231 cells were injected subcutaneously into the abdomen of mice, and the mice were sacrificed 30 days later to measure the mass and volume of the tumor.All animal studies were conducted in the Mingji Hospital under the animal Care guidelines to minimize animal pain.
10. Statistical analysis
Statistical analysis was performed by Windows GraphPad Prism v5.0 (GraphPad software ltd, san diego, ca, usa). All data were passed through at least three independent experiments and are expressed as mean ± standard deviation of the mean (s.e.m.). Statistical analysis using the t-test for two groups of samples or the Dunnett test for one-way analysis of variance for multiple groups of samples, p < 0.05 was considered statistically significant.
Example 1 DEPDC1 upregulated in triple negative breast cancer
In order to determine the effect of DEPDC1 in the triple negative breast cancer, the invention analyzes the data of two sets of chips (GSE 76250 and GSE 81838) on Gene Expression Omnibus (the website is http:// www.ncbi.nlm.nih.gov/geo), and determines the Expression conditions of DEPDC1 in the triple negative breast cancer and paired paracarcinoma tissues. As shown in fig. 1A and 1B, DEPDC1 expression was significantly increased in tissues of triple negative breast cancer compared to paired paraneoplastic tissues. Similar results may be obtained in other databases.
In 33 pairs of triple negative breast cancer and paraneoplastic tissues, the present inventors found that DEPDC1 expression was higher in most triple negative breast cancer tissues than in paraneoplastic tissues (see fig. 1C and 1D). Furthermore, the expression of PCNA (nuclear proliferation antigen) and Ki67 (a marker of proliferation, representing an important indicator of tumor cell growth) was positively correlated with the expression of DEPDC1 in 165 cases of triple negative breast cancer tissues (see fig. 1E and 1F). And the expression level of DEPDC1 and the expression levels of other cell cycle related genes (CCNA 2, CCNB1 and CCNE 2) were also significantly correlated in triple negative breast cancer tissues (see fig. 1G-1I). These results suggest that DEPDC1 appears to be a pro-cancer factor that regulates the progression of triple negative breast cancer.
Example 2 overexpression of DEPDC1 in triple negative breast cancer cells can promote cell proliferation and colony formation.
The present invention then examined the mRNA expression of DEPDC1 in a panel of three negative breast cancer cell lines. In these cells, the expression of endogenous DEPDC1 was significantly higher in MDA-MB-231 than in other cell lines, while the expression of DEPDC1 was minimal in MDA-MB-436 cells (see FIG. 2A). Therefore, a DEPDC1 stable overexpression cell line was constructed in MDA-MB-436 cells.
The real-time quantitative PCR and western blot methods detected the mRNA and protein expression levels of DEPDC1, respectively, to confirm transfection efficiency (fig. 2B and 2C). As shown in FIG. 3A, the results of the CCK-8 assay showed a significant increase in cell viability following overexpression of DEPDC 1. BrdU intercalation assay and PCNA expression were used to examine cell proliferative capacity.
The present inventors have found that overexpression of DEPDC1 increases the amount of BrdU incorporated into new DNA and induces expression of PCNA (FIGS. 3B and 3C). Furthermore, the present invention also investigated the cell cycle distribution by flow cytometry and showed that overexpression of DEPDC1 decreased the number of G0/G1 phase cells from 70.21 to 49.82, while DEPDC1 overexpression increased the proportion of S phase cells from 20.44% to 36.55% (fig. 3D).
In vitro experiments found that overexpression of DEPDC1 in MDA-MB-436 cells promoted colony formation (FIG. 3E). In addition, the invention also constructs a nude mouse subcutaneous tumor formation model, and applies in vivo experiments to detect the effect of DEPDC1 in tumor growth (figure 3F).
The results of the present invention indicate that DEPDC1 overexpression can significantly increase the tumorigenicity of MDA-MB-436 cells, with both tumor volume (FIG. 3G) and weight (FIG. 3H) increased compared to the control group. Taken together, these results suggest that DEPDC1 overexpression may promote cell growth and tumor formation by accelerating the cell cycle progression of triple negative breast cancer cells.
Example 3 knockout of DEPDC1 in triple negative breast cancer can attenuate cell proliferation
To determine the physiological function of DEPDC1 in triple negative breast cancer, the present invention also knockdown DEPDC expression in MDA-MB-231 cells. The efficiency of the knockdown was also determined by real-time quantitative PCR and western blot (fig. 4A and 4B). As shown in FIG. 5A, knockdown of DEPDC1 inhibited the growth of MDA-MB-231 cells. Furthermore, both BrdU inhalation and PCNA protein expression levels were attenuated by knockout DEPDC1 (fig. 5B and 5C). The results of cell cycle distribution showed that knocking down DEPDC1 expression increased the proportion of tumor cells that were growth arrested at G0/G1 (from 56.31% to 78.52%) with a concomitant decrease in S phase cells from 32.41% to 16.31% (fig. 5D).
As shown in FIG. 5E, the knockout of DEPDC1 clone formation was inhibited in MDA-MB-231 cells (FIG. 5E). Furthermore, the subcutaneous tumorigenesis results in nude mice showed that the knockdown of DEPDC1 significantly inhibited the tumor-origin of MDA-MB-231 cells (FIG. 5F). Consistently, tumor volume and weight were also significantly reduced after knockdown of DEPDC1 (fig. 5G and 5H). These results suggest that knockout of DEPDC1 inhibits cell proliferation and tumorigenic capacity in triple negative breast cancer cells.
Example 4 DEPDC1 expression was negatively regulated by microRNA-26b in triple-negative breast cancer
MicroRNAs are negative regulators of their target genes and act by binding to the 3' UTR region of the target gene. In order to determine the upstream regulatory factor of DEPDC1 in triple negative breast cancer, targetScan (http:// www.targetscan.org /) and Miranda (http:// www.microrna.org) were used in the present invention to predict the microRNAs that could potentially bind to DEPDC 1. In combination with the expression of the microRNAs in the triple-negative breast cancer tissues (tissues selected from the database GSE 76250), the invention finds that the expression of the microRNA26b is obviously reduced, and the expression of the microRNA is negatively related to the expression of DEPDC1 in the same triple-negative breast cancer tissues, thereby indicating that miR-26b is likely to be a functional regulatory factor of DEPDC1 (FIGS. 6A-6C).
The invention further detects whether the DEPDC1 can be directly regulated by the miR-26b, as shown in FIG. 6D, the potential binding site of the miR-26b exists in the 3 ' UTR region of the DEPDC1, and the Luciferase reporter gene result shows that the miR-26b can obviously inhibit the Luciferase activity of the plasmid carrying the wild-type DEPDC 13 ' UTR, while the activity of the mutant plasmid is not obviously changed, which indicates that the miR-26b can be directly bound in the predicted 3 ' UTR region of the DEPDC1 (FIG. 6E). Moreover, miR-26b can obviously inhibit the expression of DEPDC1mRNA and protein, and an inhibitor (anti-miR-26 b) of miR-26b can cause the expression of DEPDC1 in breast cancer cells to be increased (FIG. 6F-6I). These results indicate that miR-26b can inhibit its expression as a negative regulator of DEPDC1 in triple negative breast cancer.
Example 5 miR-26b inhibits triple-negative breast cancer cell proliferation by targeting DEPDC1
The invention further researches the effect of miR-26b in the proliferation of triple negative breast cancer cells. As shown in FIG. 7A, inhibition of miR-26b in MDA-MB-436 cells can result in an increase in cell viability (FIG. 7A). And BrdU intercalation into synthetic DNA and protein expression of PCNA increased significantly after miR-26B inhibition (fig. 7B and 7C). Moreover, the clonogenic capacity of anti-miR-26 b-treated MDA-MB-436 cells was significantly enhanced (FIG. 7D). Conversely, miR-26b was able to inhibit MDA-MB-231 cell viability, but this inhibition was abrogated after administration of DEPDC1 (FIG. 7E). Moreover, miR-26b was able to inhibit BrdU intercalation, attenuate PCNA protein expression, and inhibit cell clonogenic whereas miR-26 b's inhibition of cell proliferation and clonogenic was significantly reversed after restoration of DEPDC1 expression (fig. 7F-7H). These results demonstrate that miR-26b is able to inhibit the proliferation and progression of triple negative breast cancer cells by reducing expression of DEPDC 1.
Example 6 DEPDC1 promotes the expression of FOXM1 in triple negative breast cancer cells
Previous results indicate that there is an interaction between DEPDC1 and FOXM1, and FOXM1 regulates cell cycle progression as an important mediating molecule. To elucidate the potential mechanism by which DEPDC1 promotes cell proliferation and cell cycle progression, the present invention then examined whether there is a link between DEPDC1 and FOXM1 in triple negative breast cancer cells. As shown in fig. 8A and 8B, FOXM1 expression was significantly increased in triple negative breast cancer tissues relative to normal paraneoplastic tissues. And expression of FOXM1 was positively correlated with expression of DEPDC1 in the same triple negative breast cancer tissue (fig. 8C). Also overexpression of DEPDC1 in triple negative breast cancer cells enhanced the expression of FOXM1, while knockdown of DEPDC1 inhibited its expression (fig. 8D and 8E). The above results demonstrate that DEPDC1 is able to positively regulate expression of FOXM1 in triple negative breast cancer.
Example 7 enhancement of cell proliferation by DEPDC1 was significantly attenuated following knockdown of FOXM1
To investigate the role of FOXM1 in DEPDC1 in promoting cell proliferation, the present invention applied siRNA of FOXM1 to inhibit its expression, and Real-time PCR and Western blot were used to determine the efficiency of knockdown (fig. 8F and 8G). The present inventors found that the increase in cell viability caused by overexpression of DEPDC1 was significantly attenuated after interfering with FOXM1 (fig. 8H). And knock-down FOXM1 was able to counteract cell proliferation promoted by over-expression of DEPDC1 (fig. 8I and 8J). These results indicate that DEPDC1 promotion of cell proliferation is mediated at least in part by FOXM 1.
Conclusion
In conclusion, the present study shows that DEPDC1 up-regulated in triple negative breast cancer tissues can increase expression of FOXM1 to promote growth and proliferation of tumor cells, and DEPDC1 can be negatively regulated by miR-26 b. These results confirm the role of DEPDC1 in regulating the growth of triple negative breast cancer cells and elucidate the relevant molecular mechanisms, which in turn may provide new potential therapeutic targets for future molecular therapies of triple negative breast cancer.
Sequence listing
<110> Shanghai university of traffic medical college affiliated renji hospital
Application of <120> protein DEPDC1 as marker for diagnosing triple-negative breast cancer
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Claims (3)
1. Use of the protein DEPDC1 as a marker for the diagnosis of triple negative breast cancer.
2. Use of the protein DEPDC1 as a marker in the preparation of a reagent for diagnosing triple negative breast cancer.
The application of miR-26b in preparing a medicine for treating triple negative breast cancer is that miR-26b inhibits the proliferation of triple negative breast cancer cells through targeting DEPDC 1.
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| CN111269910B (en) * | 2019-12-12 | 2024-02-02 | 中国人民解放军陆军军医大学第二附属医院 | Application of human DEPDC1 gene and related products |
| CN111562362B (en) * | 2020-04-02 | 2022-05-20 | 臻悦生物科技江苏有限公司 | Marker for predicting three-negative breast cancer immune combined anti-angiogenesis treatment effect and application and kit thereof |
| CN112680476A (en) * | 2020-12-23 | 2021-04-20 | 南京景瑞康分子医药科技有限公司 | KLHL22-DEPDC5 protein interaction screening system |
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| WO2007095186A2 (en) * | 2006-02-14 | 2007-08-23 | Dana-Farber Cancer Institute, Inc. | Compositions, kits, and methods for identification, assessment, prevention, and therapy of cancer |
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