CN116773815B - Application of SLC2A6 as treatment target and screening target and breast cancer screening kit - Google Patents
Application of SLC2A6 as treatment target and screening target and breast cancer screening kit Download PDFInfo
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Abstract
The invention provides application of SLC2A6 as a treatment target and a screening target and a breast cancer screening kit, and belongs to the field of bioscience. The invention also provides application of the SLC2A6 protein serving as a target in preparing a medicament for treating breast cancer. The invention discovers that breast cancer and/or breast cancer prognosis are obviously related to the expression level of the SLC2A6 protein of the organism for the first time. The reagent for detecting the SLC2A6 protein expression level is used for preparing the breast cancer and/or breast cancer prognosis screening kit, and can realize effective screening of breast cancer and/or breast cancer prognosis.
Description
Technical Field
The invention belongs to the field of bioscience, and particularly relates to application of SLC2A6 as a treatment target and screening target and a breast cancer screening kit.
Background
Breast cancer is a phenomenon that breast epithelial cells undergo proliferation control under the action of various cancerogenic factors. Early stage of the disease is often manifested by symptoms such as breast lump, nipple discharge, axillary lymphadenectasis, and the like, and late stage of the disease can cause distant metastasis due to cancer cells, and the disease can cause multi-organ lesions, thereby directly threatening the life of patients. In order to be able to treat in time, a timely diagnosis of breast cancer is particularly important. There are many therapeutic drugs for breast treatment at present, but the requirements of the treatment of the present breast cancer patients still cannot be met, and new therapeutic drugs need to be developed.
SLC2A6 is a member of the family of pro-glucoproteins, which encodes GLUT6 protein, uniProt accession No. Q9UGQ3. The expression of the SLC2A6 gene is up-regulated in lung cancer but not widely expressed in normal tissues. SLC2A6 protein is not reported to be related to breast cancer and/or breast cancer prognosis, and is not reported to be used as a breast cancer treatment target.
Disclosure of Invention
The invention aims to provide the application of SLC2A6 protein as a breast cancer detection target and a treatment target.
The invention firstly provides application of a reagent for detecting SLC2A6 protein expression level in preparation of a breast cancer and/or breast cancer prognosis screening kit.
Wherein the reagent for detecting the protein expression level of SLC2A6 comprises a reagent for enzyme-linked immunosorbent assay, a reagent for immunoblotting, a reagent for immunoelectrophoresis, a reagent for tissue immunostaining, a reagent for immunoprecipitation analysis, a reagent for radioimmunoassay, a reagent for complement fixation analysis, a reagent for fluorescence-activated cell sorting, a reagent for mass analysis or a reagent for protein microarray.
Wherein, the reagent for detecting the expression level of SLC2A6 protein is used for detecting SLC2A6 protein in mammary tissue.
The invention also provides a breast cancer and/or breast cancer prognosis screening kit, which comprises a reagent for detecting SLC2A6 protein expression level.
Wherein the reagent for detecting the protein expression level of SLC2A6 comprises a reagent for enzyme-linked immunosorbent assay, a reagent for immunoblotting, a reagent for immunoelectrophoresis, a reagent for tissue immunostaining, a reagent for immunoprecipitation analysis, a reagent for radioimmunoassay, a reagent for complement fixation analysis, a reagent for fluorescence-activated cell sorting, a reagent for mass analysis or a reagent for protein microarray.
The invention firstly provides application of an agent for improving SLC2A6 protein expression level in preparation of a medicine for treating breast cancer.
The invention firstly provides application of an agent for improving the expression level of SLC2A6 protein in preparing medicaments for inhibiting breast cancer metastasis.
Wherein the agent for improving the expression level of SLC2A6 protein is an agent for over-expressing SLC2A 6. Preferably, the agent is a recombinant vector comprising the sequence shown in SEQ ID NO. 1. Further preferably, the recombinant vector is a recombinant pcDNA3.1 plasmid.
The invention has the beneficial effects that abnormal down regulation of SLC2A6 in breast cancer is found, and the down regulation of SLC2A6 is a prognosis predictor of breast cancer patients; the risk of developing breast cancer can thus be screened by detecting the SLC2A6 expression level in the sample to be tested, and a preliminary prediction of whether the prognosis of breast cancer is good. The key point of the invention is that the correlation between SLC2A6 and breast cancer and prognosis of breast cancer are found, so that any method and reagent capable of detecting SLC2A6 expression level can be used for screening breast cancer and prognosis thereof.
The invention also discovers that the SLC2A6 overexpression can inhibit the proliferation of breast cancer cells and can also inhibit the migration of the breast cancer cells; therefore, the reagent for improving the expression level of SLC2A6 protein can be used for preparing medicines for treating breast cancer or inhibiting breast cancer metastasis, and provides a new choice for clinical breast cancer treatment.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a graph of the verification of SLC2A6 protein levels in different samples. (A) Protein level WB panels of SLC2A6 in breast cancer and normal breast tissue; (B) Protein level WB panels of SLC2A6 in mammary epithelial and breast cancer cell lines; (C) Immunohistochemical staining detects a protein expression pattern of SLC2A6 of normal breast tissue in the tissue chip; (D) Immunohistochemical staining detects a protein expression pattern of breast cancer tissue SLC2A6 in the tissue chip; (E) Immunohistochemical staining detects a protein expression scoring graph of SLC2A6 in the tissue chip; (F) And detecting an influence graph, namely a survival graph, of the protein expression level of SLC2A6 in the tissue chip on the prognosis of the patient by immunohistochemical staining.
FIG. 2 is a graph showing the effect of SLC2A6 over-expression on proliferation of breast cancer cells. (A) Protein level WB panels of SLC2A6 after transfection of SLC2A6 over-expression (Flag-SLC 2 A6) plasmid into breast cancer cell lines; (B) Detecting a growth condition diagram of MDA-MB-231 breast cancer cells after SLC2A6 is over-expressed by a cell proliferation experiment; (C) Detecting the growth condition diagram of the MCF-7 breast cancer cells after the overexpression of SLC2A6 by a cell proliferation experiment; (D) Clone formation experiments detect the clone formation ability map of MDA-MB-231 and MCF-7 breast cancer cells after SLC2A6 is over-expressed; (E) A statistical analysis chart of colony formation number of MDA-MB-231 breast cancer cells after the clone formation experiment detects SLC2A6 over expression; (F) A statistical analysis chart of colony formation number of MCF-7 breast cancer cells after the clone formation experiment detects the overexpression of SLC2A 6; (G) Migration experiments detect MDA-MB-231 and MCF-7 breast cancer cell migration ability after SLC2A6 is over-expressed; (H) A statistical analysis chart for detecting the mobility of the MDA-MB-231 breast cancer cells after the SLC2A6 is over-expressed by a migration experiment; (I) A statistical analysis chart for detecting the mobility of the MCF-7 breast cancer cells after the SLC2A6 is over-expressed by a migration experiment; (J) Observing a condition diagram of the change of the substructure in the breast cancer cells of MDA-MB-231 after the overexpression of SLC2A6 by using an electron microscope; (K) And (3) observing a graph of the substructure change in the breast cancer cells of the MCF-7 after the overexpression of SLC2A6 by using an electron microscope. * P <0.001, P <0.01, P <0.05.
FIG. 3 is a graph showing the effect of SLC2A6 over-expression on the biological behavior of breast cancer cells. (A) Flow-through detection of apoptosis level map after transfection of MDA-MB-231 breast cancer cells with SLC2A6 overexpression (Flag-SLC 2A 6) plasmid; (B) Flow-through detection of apoptosis level map after transfection of MCF-7 breast cancer cells with SLC2A6 overexpression (Flag-SLC 2A 6) plasmid; (C) A mitochondrial membrane potential change diagram of cells after transfection of MDA-MB-231 breast cancer cells with SLC2A6 overexpression (Flag-SLC 2A 6) plasmid; (D) A mitochondrial membrane potential change diagram of the cell after transfection of MCF-7 breast cancer cells by SLC2A6 over-expression (Flag-SLC 2A 6) plasmid; (E) Apoptosis rate statistical analysis chart of SLC2A6 over-expression (Flag-SLC 2A 6) plasmid transfected MDA-MB-231 breast cancer cells; (F) Apoptosis rate statistical analysis chart of MCF-7 breast cancer cells transfected by SLC2A6 over-expression (Flag-SLC 2A 6) plasmid; (G) Statistical analysis chart of cell mitochondrial membrane potential change after transfection of MDA-MB-231 breast cancer cells with SLC2A6 over-expression (Flag-SLC 2A 6) plasmid; (H) Statistical analysis chart of cell mitochondrial membrane potential change after transfection of MCF-7 breast cancer cells with SLC2A6 over-expression (Flag-SLC 2A 6) plasmid; (I) Flow detection of Reactive Oxygen Species (ROS) change level patterns of cells after transfection of MDA-MB-231 breast cancer cells with SLC2A6 over-expression (Flag-SLC 2A 6) plasmids; (J) Statistical analysis chart of cell active oxygen content after transfection of MDA-MB-231 breast cancer cells with SLC2A6 over-expression (Flag-SLC 2A 6) plasmid; (K) Flow-through detection of Reactive Oxygen Species (ROS) change level patterns of cells after transfection of MCF-7 breast cancer cells with SLC2A6 over-expression (Flag-SLC 2A 6) plasmids; (L) statistical analysis of cellular reactive oxygen content of SLC2A6 over-expression (Flag-SLC 2A 6) plasmid transfected MCF-7 breast cancer cells. * P <0.001, P <0.01, P <0.05.
Detailed Description
The following examples statistical analysis uses unpaired or paired Student's sttests for group-to-group comparisons, one-factor analysis of variance or two-factor analysis of variance for multiple comparisons. Statistical significance was assessed using GraphPadPrism8 software. P <0.05 is considered statistically significant. All values are expressed as mean ± standard deviation. Prior to statistical analysis, no variance estimation was performed for each set of data.
Example 1 relationship of SLC2A6 protein to prognosis of Breast cancer and/or Breast cancer
1. Experimental method
Relation of SLC2A6 protein expression to breast cancer
1. Cell detection
1) Cell lines and cell cultures (Cell lines and cell culture)
Human breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF-7), human normal mammary epithelial cell line (MCF-10A), human renal epithelial cell line (293T) were purchased from the Withansai Life technologies Co., ltd (Procell, wuhan, china). Breast cancer cell lines were cultured in MEM (procall, wuhan, china), human normal mammary epithelial cells were cultured in DMEM/F-12 (procall, wuhan, china), and human renal epithelial cell lines were cultured in high-sugar DMEM (HyClone, USA). All basal media contained 10% fetal bovine serum (Gibco, thermoFisherScience, USA), penicillin (100U/mL) and streptomycin (100 μg/mL) and were incubated in an incubator at 37℃with 5% carbon dioxide.
2) Immunoblotting experiment (Western blot analysis)
Collecting breast cancer and normal cells and tissues, using a lysis buffer containing phosphatase and protease inhibitor to lyse the samples, and subjecting the cell lysate to ultrasonic disruption and then to lysis on ice for 30min. Then, the supernatant was collected by centrifugation at 12,000rpm for 15 minutes at 4℃and the protein concentration was determined by using a protein quantitative BCA kit (Beyotime, 0012S), and then the protein sample (20. Mu.g/lane) was separated on SDS-PAGE gel and transferred to a 0.45mmNC membrane (Millipore, germeny). The membrane was blocked with 5% skim milk at 37 ℃ for 1h and incubated overnight with primary antibody in a refrigerator at 4 ℃. Membranes were washed 3 times (5 min/time) with 1xTBST, incubated with horseradish peroxidase-labeled secondary antibody for 1h at 37 ℃, then membranes were washed 3 times (5 min/time) with 1xTBST and captured using a chemiluminescent gel imaging system (SHST, hangzhou, china). The antibodies used were as follows: glut6 antibody (SantaCruzB-3, sc-373973), beta-actin antibody (Proteintech, 66009-1-IG, 1:10000), HRP-secondary antibody (CST, #7076/# 7074). Quantitative analysis was performed using beta-actin.
2. Tissue detection
By analyzing the TCGA dataset, SLC2A6 was found to be significantly down-regulated in breast cancer specimens and the prognosis for breast cancer patients was poor compared to normal breast tissue. To confirm the expression of SLC2A6 in breast cancer, we examined the expression of SLC2A6 in breast cancer tissue and normal breast tissue collected from the people's hospitals in the Sichuan province, and also examined the expression level of SLC2A6 in tissue microarrays of breast cancer tissue samples (n=176) and non-tumor breast tissue samples (n=10).
Tissue chip analysis (Tissue microarray analysis) detection: tissue chips were purchased from core superorganisms (Shanghai, china) (HBreD 050Bc01, HBreD136Su 02), and immunohistochemical assays were performed using SLC2A6 antibodies (#ma 5-24979, workingdiluan 1:1500, invitrogen), and we evaluated the protein levels of SLC2A6 in 186 breast tissues according to the IHC procedure. Immunohistochemical results were scored by two independent pathologists, with the following criteria: 1. staining intensity score: no staining was 0min, light yellow was 1 min, dark yellow was 2 min, brown was 3 min; 2. the positive rate scores of stained cells were: 1 score (positive cell percentage 1-25%), 2 scores (26-50%), 3 scores (51-75%), 4 scores (76-100%). The final score is calculated as the sum of the two scores.
(II) relationship between SLC2A6 protein expression and prognosis of Breast cancer
1. Follow-up time: 5.6-10 years. (surgery time 2004.8-2008.12, follow-up time 2014.7).
2. Case selection: the non-clinical stage cases and the non-visit cases were removed, with 132 remaining cases.
3. The analysis method comprises the following steps: the analysis adopts a Kaplan-meier method, and Log Rank statistical test is adopted to analyze the lifetime analysis scoring.
Lifetime analysis criteria: SLC2A6 cytoplasmic staining intensity score less than or equal to 2.5 is a low expression group, and staining intensity score > 2.5 is a high expression group; SLC2A6 cell nuclear staining intensity score 0 is divided into a low expression group, and staining intensity score > 0 is a high expression group; immunohistochemical results were scored by two independent pathologists, with the following criteria: 1. staining intensity score: the non-staining was 0min, the pale yellow was 1 min, the dark yellow was 2 min, and the brown was 3 min.
2. Experimental results
The result shows that:
1. compared to the expression of SLC2A6 in breast cancer cells and normal breast cells, SLC2A6 expression was significantly down-regulated in breast cancer (fig. 1B). Expression levels of SLC2A6 in breast cancer tissue samples compared to non-tumor breast tissue samples SLC2A6 were down-regulated in breast cancer tissue compared to non-tumor breast tissue (fig. 1A, fig. 1C-E and table 1).
2. We analyzed the prognostic role of SLC2A6 in breast cancer. We found that low expression levels of SLC2A6 correlated with poor prognosis of breast cancer (fig. 1F and table 2). Down-regulation of SLC2A6 may be a prognostic predictor in breast cancer patients.
Note that:
in the survival analysis, the estimated average survival time is a biased statistic due to the existence of deletion, and the invention calculates the average survival time by using the following method:
E(T) = S(t)dt
experimental results show that SLC2A6 is abnormally down-regulated in breast cancer, the down-regulation of SLC2A6 is a prognosis predictor of breast cancer patients, the prognosis of breast cancer patients with high SLC2A6 expression is good, and the prognosis of breast cancer patients with low SLC2A6 expression is poor. The SLC2A6 protein can be used for clinical breast cancer and/or auxiliary diagnosis of breast cancer prognosis, and can be used for evaluating the treatment effect of breast cancer medicaments.
Example 2 influence of SLC2A6 protein on breast cancer cells
In view of the aberrant expression of SLC2A6 in breast cancer tissue, the biological role of SLC2A6 in breast cancer cells was further explored. We designed SLC2A6 over-expression (Flag-SLC 2A 6) plasmid and performed SLC2A6 protein over-expression in breast cancer cell lines MDA-MB-231 and MCF-7. Cell growth was detected by colony formation and cell proliferation experiments, and cell migration was detected by scratch experiments.
1. Experimental method
1. Cell lines and cell cultures (Cell lines and cell culture)
The breast cancer cell lines MDA-MB-231 and MCF-7 were derived and cultured in the same manner as in example 1.
2. Method for over-expression of SLC2A6 protein
The transfection efficiency was examined by Western blotting using pcDNA3.1-SLC2A6 and pcDNA3.1-vector plasmids, purchased from Sangon (Shanghai, china), transferred into breast cancer cell lines MDA-MB-231, MCF-7 according to the experimental instructions of Lipofectamine3000 (Invitrogen, USA) transfection kit.
The gene fragment sequence of the transferred SLC2A6 (SEQ ID NO: 1):
atgcaggagccgctgctgggagccgagggcccggactacgacaccttccccgagaagccgcccccgtcgccaggggacagggcgcgggtcgggaccctgcagaacaaaagggtgttcctggccaccttcgccgcagtgctcggcaatttcagctttgggtatgccctggtctacacatcccctgtcatcccagccctggagcgctccttggatcctgacctgcatctgaccaaatcccaggcatcctggtttgggtccgtgttcaccctgggagcagcggccggaggcctgagtgccatgatcctcaacgacctcctgggccggaagctgagcatcatgttctcagctgtgccgtcggcggccggctatgcgctcatggcgggtgcgcacggcctctggatgctgctgctcggaaggacgctgacgggcttcgccggggggctcacagctgcctgcatcccggtgtacgtgtctgagattgctcccccaggcgttcgtggggctctgggggccacaccccagctcatggcagtgttcggatccctgtccctctacgcccttggcctcctgctgccgtggcgctggctggctgtggccggggaggcgcctgtgctcatcatgatcctgctgctcagcttcatgcccaactcgccgcgcttcctgctctctcggggcagggacgaagaggccctgcgggcgctggcctggctgcgtgggacggacgtcgatgtccactgggagttcgagcagatccaggacaacgtccggagacagagcagccgagtatcgtgggctgaggcacgggccccacacgtgtgccggcccatcaccgtggccttgctgatgcgcctcctgcagcagctgacgggcatcacgcccatcctggtctacctgcagtccatcttcgacagcaccgctgtcctgctgccccccaaggacgacgcagccatcgttggggccgtgcggctcctgtccgtgctgatcgccgccctcaccatggacctcgcaggccgcaaggtgctgctcttcgtctcagcggccatcatgtttgctgccaacctgactctggggctgtacatccactttggccccaggcctctgagccccaacagcactgcgggcctggaaagcgagtcctggggggacttggcgcagcccctggcagcacccgctggctacctcaccctggtgcccctgctggccaccatgctcttcatcatgggctacgccgtgggctggggtcccatcacctggctgctcatgtctgaggtcctgcccctgcgtgcccgtggcgtggcctcagggctctgcgtgctggccagctggctcaccgccttcgtcctcaccaagtccttcctgccagtggtgagcaccttcggcctccaggtgcctttcttcttcttcgcggccatctgcttggtgagcctggtgttcacaggctgctgtgtgcccgagaccaagggacggtccctggagcagatcgagtccttcttccgcacggggagaaggtccttcttgcgctag
the preparation method of pcDNA3.1-SLC2A6 recombinant plasmid comprises the following steps: the pcDNA3.1 plasmid was inserted into the SLC2A6 gene fragment (cleavage site BamHI+XhoI) to obtain a positive plasmid.
3. Detection of cell growth and cell migration Capacity
1) Cell proliferation assay
Cell proliferation was measured using the xcelligent system (roccheapplied science and dabio sciences). xcelligent Cell Index (CI) measurements were performed according to the instructions of the supplier. The BCs were resuspended in culture medium and then adjusted to 200,000 cells/ml. After 100. Mu.L of the cell suspension was inoculated into wells of E-pLate96, it was monitored with the xCELLigence system every 10 minutes for 6 days.
2) Colony formation assay (Colony formation assay)
The proliferation of tumor cells was examined using colony formation experiments. Breast cancer cells were seeded in 6-well plates (500 cells/well) and medium containing 10% fetal bovine serum (Gibco, thermoFisherScience, USA), penicillin (100U/mL) and streptomycin (100 μg/mL) were incubated in an incubator at 37 ℃ with 5% carbon dioxide. After 14 days of incubation, cells were fixed with 4% paraformaldehyde for 15min, stained with 1% crystal violet for 15min, and washed 3 times with PBS. The colony numbers were counted. Each set of experiments was repeated three times.
3) Cell migration assay (Cell migration assay)
Migration capacity of breast cancer cells was measured with an 8 μm Transwell chamber, cells were treated with pancreatin and washed twice with Phosphate Buffered Saline (PBS). Diluting cells to 5≡10 4 mu.L of breast cancer cells were inoculated into the upper cavity of a Transwell chamber, MEM medium containing 20% FBS was placed in the lower cavity, and the cells were placed at 37℃and 5% CO 2 The cells were incubated in an incubator for 24h, fixed in 4% paraformaldehyde for 20min, and then stained with 0.1% crystal violet (#c8470, solarbio). The migrated cells were evaluated under a microscope with 3 fields per well. Each set of experiments was repeated three times.
4) Apoptosis detection
According to the instructions of the annexin V-FITC/PI apoptosis detection kit (Keygen Biotech, china). Cells were digested with pancreatin without EDTA. Then, the cell suspension sample was centrifuged at 800rpm for 3min, the supernatant was aspirated off, washed twice with PBS, and centrifuged at 800rpm for 3min, the supernatant was aspirated off to obtain a cell pellet. After resuspension of cells with 500 μl of propidium buffer, 5 μl of LFITC-labeled annexin v was added and gently blown to mix, followed by 5 μl of Propidium Iodide (PI) to mix; the cells were incubated at room temperature for 10min in the absence of light and apoptosis was detected by flow cytometry (BeckmanCoulter).
5) Cell cycle detection
According to the instructions of the cell cycle test kit (Keygen Biotech, china). Cells were digested with pancreatin, the cell suspension samples were centrifuged at 800rpm for 3min, the supernatant was aspirated off, washed twice with an appropriate amount of PBS, centrifuged at 800rpm for 3min, and the supernatant was aspirated off to obtain a cell pellet. Cells were resuspended in 70% pre-chilled ethanol and fixed overnight. The next day, cells were washed twice with PBS. RNaseA: PI is as follows: 9 volumes of the mixed solution were prepared into a staining working solution (used at present), and the cells were resuspended in 500. Mu.LPI/RNaseA staining working solution and were subjected to detection analysis by a flow cytometer (BeckmanCoulter) at room temperature for 30min in the absence of light.
6) Reactive Oxygen Species (ROS) detection
According to the instructions of the active oxygen detection kit (Beyotime, china). Cell samples were collected and DCFH-DA probes were diluted 1:1000 in serum-free medium, with 1mL of DCFH-DA dilution added to each sample. The sample is placed in a cell incubator at 37 ℃ for incubation for 20min, and the mixture is inverted and uniformly mixed every 5min, so that the probe and the cells are fully contacted. Centrifugation at 800rpm for 3min, pipetting the supernatant, washing the cells 3 times with serum-free cell culture medium, centrifugation at 800rpm for 3min, pipetting the supernatant, resuspension with PBS, detection analysis by flow cytometry (BeckmanCoulter).
7) Mitochondrial Membrane Potential (MMP) detection
According to the instructions of the mitochondrial membrane potential detection kit (Beyotime, china). Cell samples were collected and JC-1 probes were diluted 1:200 with JC-1 staining buffer, and 1mL JC-1 staining working solution was added to each sample. The sample is placed in a cell incubator at 37 ℃ for incubation for 30min, and the mixture is inverted and uniformly mixed every 5min, so that the probe and the cells are fully contacted. Centrifugation at 800rpm for 3min, pipetting the supernatant, washing the cells 2 times with JC-1 staining buffer, centrifugation at 800rpm for 3min, pipetting the supernatant, re-suspending with JC-1 staining buffer, and flow cytometry (BeckmanCoulter) detection analysis.
8) Transmission electron microscope
Cell samples were collected, the samples were fixed with 3% glutaraldehyde, 1% osmium tetroxide, stepwise dehydrated with acetone, epon812 embedded, semi-thin sections were optically positioned with toluidine blue dye, ultra-thin sections with diamond blades, re-stained with uranium acetate and lead citrate, and observed with a transmission electron microscope (JEM-1400 FLASH).
2. Experimental results
1. As shown in FIG. 2A, the method obtains MDA-MB-231 and MCF-7 breast cancer cells which over express SLC2A6 protein.
2. Overexpression of SLC2A6 protein can obviously inhibit proliferation of MDA-MB-231 and MCF-7 breast cancer cells (figure 2B-C), inhibit monoclonal formation of MDA-MB-231 and MCF-7 breast cancer cells (figure 2D-F), and inhibit migration of MDA-MB-231 and MCF-7 breast cancer cells (figure 2G-I).
3. Specific mechanisms of SLC2A6 inhibition of cell proliferation: the result of transmission electron microscope observation on MDA-MB-231 and MCF-7 breast cancer cells which over express SLC2A6 plasmid shows that mitochondria of the over-expression SLC2A6 cells are obviously enlarged and the density is reduced. Increased cell density was accompanied by an early apoptotic phenotype (FIG. 2J-K). In view of the result of the electron microscope, the flow cytometry is further adopted to detect the influence on apoptosis, reactive oxygen species ROS and mitochondrial membrane potential after over-expressing SLC2A6 plasmid. The results suggested that overexpression of SLC2A6 promoted apoptosis in MDA-MB-231, MCF-7 breast cancer cells (FIGS. 3A-B, 3E-F), decreased mitochondrial membrane potential (FIGS. 3C-D, 3G-H), and up-regulated the levels of ROS in MDA-MB-231, MCF-7 breast cancer cells (FIGS. 3I-L).
The result shows that SLC2A6 can inhibit proliferation of breast cancer cells, promote apoptosis of the breast cancer cells, inhibit migration of the breast cancer cells, and the agent for improving expression level of SLC2A6 can treat breast cancer or inhibit metastasis of the breast cancer. The experimental results further prove that SLC2A6 is closely related to breast cancer and prognosis thereof, and the SLC2A6 and the prognosis thereof have obvious correlation.
In summary, the kit of the invention can screen the risk degree of breast cancer and/or prognosis of breast cancer of the population to be detected by detecting the level of SLC2A6 protein in breast tissue: if the SLC2A6 protein level is low, the risk of having a prognosis for breast cancer and/or breast cancer is high, and if the SLC2A6 protein level is high, the risk of having a prognosis for breast cancer and/or breast cancer is low; the agent for improving the expression level of SLC2A6 can treat breast cancer or inhibit breast cancer metastasis.
Claims (4)
1. Use of an agent that increases the expression level of SLC2A6 protein in the manufacture of a medicament for inhibiting breast cancer metastasis.
2. Use according to claim 1, characterized in that: the agent for improving the expression level of SLC2A6 protein is an agent for over-expressing SLC2A 6.
3. Use according to claim 2, characterized in that: the reagent is a recombinant vector containing a sequence shown in SEQ ID NO. 1.
4. Use according to claim 3, characterized in that: the recombinant vector is recombinant pcDNA3.1 plasmid.
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