Application of PGM2L1 in preparation of product for predicting prognosis condition of triple negative breast cancer

Abstract

The invention discloses application of PGM2L1 in preparation of a product for predicting the prognosis condition of triple negative breast cancer, and belongs to the technical field of biological medicine and molecular biology. It is proved for the first time that PGM2L1 expression is increased along with increase of the malignant degree of the triple negative breast cancer and is in negative correlation with the survival rate. After interference of PGM2L1 expression, proliferation of triple-negative breast cancer cells is inhibited, transfer of the triple-negative breast cancer cells is inhibited, and the PGM2L1 can be used as an effective medicine for preventing and / or treating triple-negative breast cancer. The invention provides a more favorable means for diagnosis and prognosis evaluation analysis of the triple-negative breast cancer, which is of great significance for research and treatment of the triple-negative breast cancer, and also lays a test foundation and provides a new view for development of efficient medicines related to treatment of the triple-negative breast cancer, thereby having good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to the application of PGM2L1 in preparing a product for predicting the prognosis of triple-negative breast cancer. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Breast cancer is the most common malignant tumor in women worldwide and the second leading cause of cancer-related death, which seriously threatens women's life and health. Breast cancer shows high heterogeneity in morphology and molecular genetics. According to its molecular classification, breast cancer can be divided into luminal A type, luminal B type, HER2 overexpression type, basal type and normal breast type. Among them, the molecular phenotype of triple-negative breast cancer (TNBC) is characterized by negative expression of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (Her2). It has clinical characteristics such as rapid progression, strong invasiveness, strong metastasis ability and poor prognosis. It is a molecular subtype with a higher degree of malignancy in breast cancer. Since TNBC lacks the above receptors, it cannot be given effective endocrine or anti-HER2 targeted therapy, which brings great challenges to the treatment of TNBC. Therefore, revealing the molecular mechanism of progression and metastasis of triple-negative breast cancer will help to discover new molecular therapeutic targets, which is of great clinical significance for improving the efficacy and prognosis of patients.

[0004] Metabolic reprogramming can control changes in tumor energy and biosynthetic pathways. Glucose metabolic reprogramming is the most representative metabolic characterization in tumors. Tumor cells have a unique energy metabolism mode. Even in aerobic conditions, they do not use mitochondrial oxidative phosphorylation to generate energy, but show active aerobic glycolysis, which is called the Warburg effect (aerobic glycolysis). Compared with other breast cancer subtypes, TNBC shows a high metabolic reprogramming effect. TNBC tumor cells provide themselves with substances and energy through glucose metabolic reorganization to meet their rapid growth needs. Various metabolic enzymes and signaling molecules involved in the glucose metabolism process play an important role in the occurrence and development of tumors, and are considered to be important targets for the diagnosis and treatment of TNBC.

[0005] The main function of the phosphoglucomutase (PGM) family is to promote the conversion between glucose-1-phosphate (G-1-P) and glucose-6-phosphate (G-6-P). In the context of glucose metabolism, G-1-P is involved in glycogen synthesis, while G-6-P is related to glycolysis. Therefore, members of the PGM family play a key role in regulating the metabolic balance between glycogen synthesis and glycolysis. Existing studies have shown that abnormal expression of PGM is closely related to a variety of tumors, cardiovascular diseases, etc. However, there are still few studies on the role of PGM in triple-negative breast cancer. Therefore, it is necessary to explore more effective PGM as a marker and target for clinical diagnosis and treatment of triple-negative breast cancer. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide the use of PGM2L1 in the preparation of a product for predicting the prognosis of triple-negative breast cancer. The present invention first discovered that PGM2L1 is abnormally highly expressed in triple-negative breast cancer and is closely related to the prognosis of triple-negative breast cancer patients. Further studies have shown that interfering with PGM2L1 expression can inhibit the proliferation and metastasis of triple-negative breast cancer. Therefore, PGM2L1 can be used as a prognostic indicator and potential therapeutic target for triple-negative breast cancer, thereby completing the present invention.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] The first aspect of the present invention provides the use of a substance for detecting PGM2L1 in the preparation of any one or more of the following drugs or preparations;

[0009] (1) Drugs or preparations for diagnosis or auxiliary diagnosis of triple-negative breast cancer;

[0010] (2) Drugs or preparations for prognosis assessment or auxiliary prognosis assessment of triple-negative breast cancer.

[0011] The present invention found that PGM2L1, a member of the phosphoglucomutase family, plays an important role in the malignant progression of triple-negative breast cancer and is involved in metabolic reprogramming that promotes glycolysis. Therefore, exploring the specific functions and mechanisms of PGM2L1 in tumor progression and metastasis is expected to become an important breakthrough point for improving the diagnosis and treatment of breast cancer. The present invention found through research that the expression of PGM2L1 in triple-negative breast cancer is significantly higher than that in normal tissues, suggesting that PGM2L1 plays a role in the progression of triple-negative breast cancer. At the same time, the prognosis of patients with high expression of PGM2L1 is significantly worse than that of patients with low expression of PGM2L1. Therefore, PGM2L1 can be used as a new prognostic marker for triple-negative breast cancer, providing a basis for patient survival assessment.

[0012] A second aspect of the present invention provides a system for triple-negative breast cancer prognosis assessment or auxiliary prognosis assessment, the system comprising:

[0013] An analysis unit, the analysis unit comprising: a detection substance for determining the expression level of PGM2L1 in a test sample of a subject;

[0014] An evaluation unit, comprising: performing prognosis evaluation on a plurality of subjects according to the PGM2L1 expression level determined by the analysis unit.

[0015] The third aspect of the present invention provides the use of PGM2L1 as a target in the treatment of triple-negative breast cancer and / or screening of triple-negative breast cancer drugs.

[0016] A fourth aspect of the present invention provides use of a substance that inhibits the expression of PGM2L1 in the preparation of a product for treating triple-negative breast cancer;

[0017] Wherein, the substance that inhibits the expression of PGM2L1 includes RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, shRNA, and gene knockout substances targeting PGM2L1.

[0018] The functions of the product for treating triple-negative breast cancer are any one or more of the following:

[0019] (1) Inhibit G-6-P content in triple-negative breast cancer cells;

[0020] (2) Inhibit glycolysis in triple-negative breast cancer cells;

[0021] (3) Inhibit the proliferation of triple-negative breast cancer cells;

[0022] (4) Inhibit the malignant invasion of triple-negative breast cancer cells;

[0023] (5) Inhibit the growth of triple-negative breast cancer;

[0024] (6) Inhibit the metastasis of triple-negative breast cancer.

[0025] The fifth aspect of the present invention provides a drug for treating triple-negative breast cancer, wherein the active ingredient is a substance for inhibiting the expression level of PGM2L1, and the substance includes RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, shRNA, and gene knockout substances targeting PGM2L1.

[0026] The functions of the drug are any one or more of the following:

[0027] (1) Inhibit G-6-P content in triple-negative breast cancer cells;

[0028] (2) Inhibit glycolysis in triple-negative breast cancer cells;

[0029] (3) Inhibit the proliferation of triple-negative breast cancer cells;

[0030] (4) Inhibit the malignant invasion of triple-negative breast cancer cells;

[0031] (5) Inhibit the growth of triple-negative breast cancer;

[0032] (6) Inhibit the metastasis of triple-negative breast cancer.

[0033] The beneficial effects of the present invention are:

[0034] The technical solution of the present invention proves for the first time that PGM2L1 expression increases with the increase of triple-negative breast cancer malignancy and is negatively correlated with the survival rate. Interference with PGM2L1 expression inhibits the proliferation of triple-negative breast cancer cells and inhibits their metastasis, and can be used as an effective drug for the prevention and / or treatment of triple-negative breast cancer.

[0035] The technical solution of the present invention provides a more advantageous means for the diagnosis and prognosis evaluation and analysis of triple-negative breast cancer, which is of great significance for the research and treatment of triple-negative breast cancer. It also lays an experimental foundation and provides a new perspective for the development of highly effective drugs for the treatment of triple-negative breast cancer, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0037] Figure 1 The figure is an analysis of the correlation between PGM2L1 and the prognosis of patients with triple-negative breast cancer; A is the ROC curve of PGM2L1 in clinical samples, B is the ROC curve of PGM2L1 in breast cancer samples in the TCGA database, C is the expression level of PGM2L1 in TNBC tumor tissues in the TCGA database, D is the expression level of PGM2L1 in metastatic TNBC tissues in the TCGA database, E is the immunohistochemical staining of breast cancer tissues and adjacent normal tissues of TNBC patients, F is the Kaplan–Meier analysis of the overall survival rate of breast cancer patients with high and low expression of PGM2L1, and G is the overall survival rate of breast cancer patients with high and low expression of PGM2L1 in triple-negative breast cancer samples;

[0038] Figure 2The figure is related to the level of glycolysis promoted by PGM2L1 in breast cancer cells; A is the glucose consumption, lactate secretion and ATP production in triple-negative breast cancer cells after knocking down PGM2L1; B is the G-6-P content in triple-negative breast cancer cells after knocking down PGM2L1; C is the glucose consumption, lactate secretion and ATP production in triple-negative breast cancer cells after overexpressing PGM2L1; B is the G-6-P content in triple-negative breast cancer cells after overexpressing PGM2L1;

[0039] Figure 3 Figure 1 is a graph showing the ability of PGM2L1 to promote the proliferation and metastasis of breast cancer cells; A is the proliferation of triple-negative breast cancer cells after knocking down PGM2L1; B is the migration and invasion of triple-negative breast cancer cells after knocking down PGM2L1; C is the expression levels of FN1, N-cad, and E-cad after knocking down PGM2L1; D is the proliferation of triple-negative breast cancer cells after overexpressing PGM2L1; E is the migration and invasion of triple-negative breast cancer cells after overexpressing PGM2L1; F is the expression levels of FN1, N-cad, and E-cad after overexpressing PGM2L1;

[0040] Figure 4 These are the in vivo verification related graphs of PGM2L1 promoting breast cancer proliferation and metastasis; among them, A is the actual picture of triple-negative breast cancer tumor in nude mouse subcutaneous tumor formation experiment; B is the change of triple-negative breast cancer tumor volume in nude mouse subcutaneous tumor formation experiment; C is the weight of triple-negative breast cancer tumor in nude mouse subcutaneous tumor formation experiment; D is the actual picture of lung in lung metastasis experiment; E is the statistical graph of lung metastasis number in lung metastasis experiment; F is the HE staining result in lung metastasis experiment. DETAILED DESCRIPTION

[0041] According to the present invention, the concept of "treatment" refers to any measure applicable to the treatment of triple-negative breast cancer related diseases, or preventive treatment of such diseases or symptoms, or avoiding the recurrence of such diseases, such as recurrence after the end of the treatment period or treatment of symptoms of the disease that has already occurred, or pre-interventional prevention, inhibition or reduction of the occurrence of such diseases or symptoms.

[0042] The inventors previously screened genes specifically expressed in triple-negative breast cancer using gene sequencing technology, among which PGM2L1 was a candidate gene with great variability and no reports in triple-negative breast cancer, suggesting that it may be used as a new molecular biomarker for diagnosing triple-negative breast cancer.

[0043] In view of this, in a first typical embodiment of the present invention, there is provided use of a substance for detecting PGM2L1 in the preparation of any one or more of the following drugs or preparations;

[0044] (1) Drugs or preparations for diagnosis or auxiliary diagnosis of triple-negative breast cancer;

[0045] (2) Drugs or preparations for prognosis assessment or auxiliary prognosis assessment of triple-negative breast cancer.

[0046] The present invention has found through research that the expression of PGM2L1, a member of the phosphoglucomutase family, in triple-negative breast cancer is significantly higher than that in normal tissues, suggesting that PGM2L1, a member of the phosphoglucomutase family, plays a role in the progression of triple-negative breast cancer. At the same time, the prognosis of patients with high expression of PGM2L1, a member of the phosphoglucomutase family, is significantly worse than that of patients with low expression of PGM2L1. Therefore, PGM2L1 can be used as a new prognostic marker for triple-negative breast cancer, providing a basis for evaluating the patient's survival period.

[0047] In some examples of this embodiment, the triple-negative breast cancer prognostic assessment or auxiliary prognostic assessment includes the prediction of the overall survival of patients with triple-negative breast cancer.

[0048] In some examples of this embodiment, the substance for detecting PGM2L1 includes substances used for detecting the expression level of PGM2L1 by RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing.

[0049] In some examples of this embodiment, the dosage form of the drug or preparation includes, but is not limited to, one or more of an injection, a tablet, a capsule, an oral liquid form, or a granule.

[0050] In some examples of this embodiment, the administration of the drug or preparation includes, but is not limited to, one or more of sublingual administration, oral administration, intramuscular injection, or intravenous injection.

[0051] In some examples of this embodiment, the drug or formulation further comprises a pharmaceutically acceptable excipient;

[0052] Preferably, the auxiliary materials include, but are not limited to, one or more of: a chelating agent, a protective agent, a surfactant, an excipient or a stabilizer.

[0053] In a second typical embodiment of the present invention, a system for triple-negative breast cancer prognosis assessment or auxiliary prognosis assessment is provided, the system comprising:

[0054] An analysis unit, the analysis unit comprising: a detection substance for determining the expression level of PGM2L1 in a test sample of a subject;

[0055] An evaluation unit, comprising: performing prognosis evaluation on a plurality of subjects according to the PGM2L1 expression level determined by the analysis unit.

[0056] In some examples of this embodiment, the prognostic assessment or auxiliary prognostic assessment includes assessing the overall survival of the subject;

[0057] When the subject's PGM2L1 expression level is higher than the threshold, it is considered high expression, indicating that the subject's overall survival is shorter;

[0058] When the subject's PGM2L1 expression level is lower than the threshold, it is considered low expression, indicating that the subject has a longer overall survival;

[0059] The threshold is the optimal cutoff value between the PGM2L1 expression level and the overall survival of patients with triple-negative breast cancer.

[0060] In some examples of this embodiment, the sample to be tested may be breast tissue of a subject.

[0061] In a third typical embodiment of the present invention, there is provided the use of PGM2L1 as a target in the treatment of triple-negative breast cancer and / or screening of triple-negative breast cancer drugs.

[0062] In some examples of this embodiment, the triple-negative breast cancer drug is a drug for preventing and / or treating triple-negative breast cancer.

[0063] In a fourth typical embodiment of the present invention, a method for screening triple-negative breast cancer drugs is provided, comprising:

[0064] (1) treating a system expressing and / or containing the PGM2L1 with a candidate substance; and setting up a parallel control without treatment with the candidate substance;

[0065] (2) After completing step (1), detecting the expression level of PGM2L1 in the system; if the expression level of PGM2L1 in the system treated with the candidate substance is significantly reduced compared with the parallel control, the candidate substance can be used as a candidate triple-negative breast cancer drug.

[0066] In some examples of this embodiment, the system can be a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

[0067] In some examples of this embodiment, the cells in the cell system may be triple-negative breast cancer cells.

[0068] In some examples of this embodiment, the tissue in the tissue system can be triple-negative breast cancer breast tissue.

[0069] In some examples of this embodiment, the organ in the organ system can be a mammary gland.

[0070] In some examples of this embodiment, the animal in the animal system can be a mammal, such as rats, mice, guinea pigs, rabbits, monkeys, humans, etc.

[0071] In a fifth typical embodiment of the present invention, there is provided use of a substance that inhibits the expression of PGM2L1 in the preparation of a product for treating triple-negative breast cancer;

[0072] Wherein, the substance that inhibits the expression of PGM2L1 includes RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, shRNA, and gene knockout substances targeting PGM2L1.

[0073] In some examples of this embodiment, the siRNA includes siRNA-1 and siRNA-2, the nucleic acid sequence of siRNA-1 is: sense GAACAGAACCAAAGAUAAAT T (SEQ ID NO.1), antisense UUUAUCUUUGGUUCUGUUCTT (SEQ ID NO.2); the nucleic acid sequence of siRNA-2 is: sense GGAGAAUGGUUGUUGGAAA TT (SEQ ID NO.3), antisense UUUCCAACAACCAUUCUCCTT (SEQ ID NO.4).

[0074] In some examples of this embodiment, the shRNA includes shRNA1 and shRNA2, the nucleic acid sequence of shRNA1 is: GGGACTGCAGGACTTCGTTCT (SEQ ID NO.5), and the nucleic acid sequence of shRNA2 is: GCAAGGAAGACAATGGAT ACA (SEQ ID NO.6).

[0075] In some examples of this embodiment, the functions of the product for treating triple-negative breast cancer are any one or more of the following;

[0076] (1) Inhibit G-6-P content in triple-negative breast cancer cells;

[0077] (2) Inhibit glycolysis in triple-negative breast cancer cells;

[0078] (3) Inhibit the proliferation of triple-negative breast cancer cells;

[0079] (4) Inhibit the malignant invasion of triple-negative breast cancer cells;

[0080] (5) Inhibit the growth of triple-negative breast cancer;

[0081] (6) Inhibit the metastasis of triple-negative breast cancer.

[0082] In some examples of this embodiment, the product may be a medicine.

[0083] In a sixth typical embodiment of the present invention, a drug for treating triple-negative breast cancer is provided, wherein the active ingredient is a substance for inhibiting the expression level of PGM2L1, and the substance includes RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, shRNA, and gene knockout substances targeting PGM2L1.

[0084] In some examples of this embodiment, the siRNA includes siRNA-1 and siRNA-2, the nucleic acid sequence of siRNA-1 is: sense GAACAGAACCAAAGAUAAAT T (SEQ ID NO.1), antisense UUUAUCUUUGGUUCUGUUCTT (SEQ ID NO.2); the nucleic acid sequence of siRNA-2 is: sense GGAGAAUGGUUGUUGGAAA TT (SEQ ID NO.3), antisense UUUCCAACAACCAUUCUCCTT (SEQ ID NO.4).

[0085] In some examples of this embodiment, the shRNA includes shRNA1 and shRNA2, the nucleic acid sequence of shRNA1 is: GGGACTGCAGGACTTCGTTCT (SEQ ID NO.5), and the nucleic acid sequence of shRNA2 is: GCAAGGAAGACAATGGAT ACA (SEQ ID NO.6).

[0086] In some examples of this embodiment, the function of the drug is any one or more of the following;

[0087] (1) Inhibit G-6-P content in triple-negative breast cancer cells;

[0088] (2) Inhibit glycolysis in triple-negative breast cancer cells;

[0089] (3) Inhibit the proliferation of triple-negative breast cancer cells;

[0090] (4) Inhibit the malignant invasion of triple-negative breast cancer cells;

[0091] (5) Inhibit the growth of triple-negative breast cancer;

[0092] (6) Inhibit the metastasis of triple-negative breast cancer.

[0093] In some examples of this embodiment, the drug further comprises at least one inactive drug ingredient. The inactive drug ingredient may be a carrier, excipient, diluent, etc. commonly used in pharmacy. Moreover, according to the usual method, the drug may be prepared into a dosage form in the form of an oral agent, an external agent, a suppository, or a sterile injection solution such as a powder, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc.

[0094] Among them, the non-drug active ingredients such as carriers, excipients and diluents commonly used in medicine are well known in the art, and ordinary technicians in the field can determine that they meet clinical standards.

[0095] In some examples of this embodiment, the carrier, excipient and diluent include but are not limited to: lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, etc.

[0096] In some examples of this embodiment, the drug can be administered to the body in a known manner. For example, it can be delivered to the tissue of interest by intravenous systemic delivery or local injection. It can be optionally administered via intravenous, percutaneous, intranasal, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered in the present invention can vary depending on a variety of factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, routes of administration, modes of administration, etc.

[0097] In some examples of this embodiment, the drug administration subject can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.

[0098] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0099] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0100] Example

[0101] 1. Materials and Methods

[0102] 1. Test materials

[0103] 1.1 Genetic information

[0104] Amino acid sequence of PGM2L1:

[0105] MAENTEGDLNSNLLHAPYHTGDPQLDTAIGQWLRWDKNPKTKEQIENLLRNGMNKELRDRLCCRMTFGTAGLRSAMGAGFCYINDLTVIQSTQGMYKYLERCFSDFKQRGFVVGYDTRGQVTSSSCSSQRLAKLTAAVLLAKDVPVYLFSRYVPTP FVPYAVQKLKAVAGVMITASHNRKEDNGYKVYWETGAQITSPHDKEILKCIEECVEPWNGSWNDNLVDTSPLKRDPLQDICRRYMEDLKKICFYRELNSKTTLKFVHTSFHGVGHDYVQLAFKVFGFKPPIPVPEQKDPDPDFSTVKCPNPEEGES VLELSLRLAEKENARVVLATDPDADRLAAAELQENGCWKVFTGNELAALFGWWMFDCWKKNKSRNADVKNVYMLATTVSSKILKAIALKEGFHFEETLPGFKWIGSRIIDLLENGKEVLFAFEESIGFLCGTSVLDKDGVSAAVVVAEMASYLET MNITLKQQLVKVYEKYGYHISKTSYFLCYEPPTIKSIFERLRNFDSPKEYPKFCGTFAILHVRDVTTGYDSSQPNKKSVLPVSKNSQMITFTFQNGCVATLRTSGTEPKIKYYAEMCASPDQSDTALLEEELKKLIDALIENFLQPSKNGLIWRSV

[0106] 1.2 Cell information:

[0107] Cell Name Cell Chinese name Source species MDA-MB-231 Triple negative breast cancer cells people MDA-MB-468 Triple negative breast cancer cells people

[0108] 2. Test methods

[0109] 2.1 Cell culture

[0110] The triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were cultured in DMEM containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin at 37° C. in a humidified incubator containing 5% CO 2 .

[0111] 2.2 Cell proliferation (MTT assay, Transwell assay, western blot experiment)

[0112] MTT assay

[0113] The cells were plated at 5×10 3 Inoculate 100 μL of culture medium / well into a 96-well plate. After 24 hours, add 20 μL MTT solution to each well and incubate at 37°C for 4-6 hours. Aspirate the supernatant from each well and add 100 μL DMSO to each well. Shake to dissolve the crystals. Detect the absorbance at a wavelength of 570 nm in an ELISA reader for statistical analysis.

[0114] Transwell assay

[0115] Cell migration assay: Digest and count the cells, using 7×10 4 DA-MB-231 cells or 9×10 4 MDA-MB-468 cells / chamber were plated in a transwell chamber placed in a 24-well plate. 200 μL of serum-free medium was added to the upper chamber, and 700 μL of complete medium containing 20% ​​serum was added to the lower chamber. After incubation at 37°C for 24 hours, the chamber was placed in 4% paraformaldehyde, fixed at room temperature for 15 minutes, and then stained with crystal violet for 10 minutes. The crystal violet inside the chamber was washed in water, and the cells inside the chamber were carefully wiped with a cotton swab. After drying, they were observed and photographed under a microscope, counted, and statistically analyzed.

[0116] Cell invasion assay: Dilute the matrix gel and serum-free medium at a ratio of 1:6, evenly spread 70 μL per well on the bottom of the chamber, and place at 37°C for 2 hours to allow the matrix gel to solidify. The rest of the steps are the same as the "cell migration assay".

[0117] Western blot assay

[0118] Cellular proteins were extracted using protein lysis buffer, and protein concentration was measured using the BCA method. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to protein concentration. The protein blot was transferred to a PVDF membrane, after which the bands were blocked with 5% skim milk for 1 hour and then incubated with diluted primary antibody solution at 4°C overnight. After washing, the blot was incubated with HRP-conjugated secondary antibody solution at room temperature for at least 1 hour. After washing, the blot was developed in a chemiluminometer using ECL colorimetric solution.

[0119] 2.3 Nude mouse subcutaneous tumor formation test

[0120] Balb / c-nude female mice aged 4 to 6 weeks were purchased and randomly divided into groups, with 3 mice in each group. According to the assumption that 1×10 7Cells were expanded and stabilized. Cells were collected after digestion, and the cells were resuspended after diluting the matrix gel with PBS at a ratio of 1:6. The cell suspension was injected subcutaneously into the back of nude mice. When a stable tumor was observed under the skin of nude mice, the length and width of the tumor were measured every 5 days with a vernier caliper, and the volume was calculated as 1 / 2 length × width. 2 The nude mice were killed 4 weeks later, and the tumors were removed, photographed, and weighed.

[0121] 2. Results

[0122] 1. Expression and prognostic analysis

[0123] The expression of PGM2L1 in triple-negative breast cancer was analyzed using data from 1073 breast cancer samples and 112 normal tissues in the TCGA database. The analysis found that the expression of PGM2L1 in breast cancer tissues was significantly higher than that in normal tissues (p = 0.0003), and it had a good diagnostic value for breast cancer patients (AUC = 0.6856, p < 0.001) ( Figure 1 B).

[0124] The expression of PGM2L1 in TNBC tumor tissues was significantly higher than that in non-TNBC tissues (p = 0.0076) ( Figure 1 C), and the PGM2L1 content in metastatic TNBC tissues was significantly higher than that in the non-metastatic group (p<0.0001) ( Figure 1 D). This suggests that PGM2L1 plays a role in the progression of triple-negative breast cancer.

[0125] Subsequently, we performed immunohistochemical staining on breast cancer tissues and adjacent normal tissues of TNBC patients in Shandong Qilu Hospital. The results showed that the expression level of PGM2L1 in TNBC breast cancer tissues was significantly higher than that in adjacent normal tissues ( Figure 1 E).

[0126] The Kaplan–Meier analysis of the correlation between the expression level of PGM2L1 and the prognosis of breast cancer patients showed that the overall survival rate (OS) of breast cancer patients with high expression of PGM2L1 was significantly lower than that of the low expression group (HR=1.72, p=0.012) ( Figure 1 F). It was also found that breast cancer patients with high expression of PGM2L1 in triple-negative breast cancer samples had worse OS (HR=3.32, p=0.0156) ( Figure 1 G) demonstrated that high expression of PGM2L1 was significantly associated with poor prognosis in triple-negative breast cancer.

[0127] 2. PGM2L1 promotes glucose metabolism reprogramming in triple-negative breast cancer cells

[0128] After knocking down PGM2L1 in triple-negative breast cancer cells, the levels of glycolysis-related metabolites were detected, and it was found that the glucose consumption, lactate secretion, and ATP production of breast cancer cells were significantly reduced ( Figure 2 A). In addition, the study showed that the PGM family is one of the key molecules that regulate the conversion between G-1-P and G-6-P. Therefore, the G-6-P content was detected and it was found that the G-6-P content in breast cancer cells was significantly inhibited after knocking down PGM2L1 ( Figure 2 B). Overexpression of PGM2L1 significantly increased glucose consumption, lactate secretion, ATP production, and G-6-P content ( Figure 2 CD). The above results prove that PGM2L1 can promote glycolysis by increasing the intracellular G-6-P content.

[0129] 3. PGM2L1 promotes the proliferation and metastasis of triple-negative breast cancer cells

[0130] Tumor cells use enhanced glycolysis to provide more energy for their biological functions, thereby affecting the malignant progression of tumors. Therefore, the effect of PGM2L1 on the proliferation and metastasis of tumor cells was detected. Through the MTT test, it was observed that the proliferation ability of breast cancer cells was weakened after knocking down PGM2L1 ( Figure 3 A). Transwell assay demonstrated that knockdown of PGM2L1 weakened the migration and invasion abilities of breast cancer cells ( Figure 3 B). Epithelial-mesenchymal transition (EMT) is an important biological process for tumor cells to acquire migration and invasion capabilities. Western blot experiments were used to detect EMT-related marker proteins, and it was found that after knocking down PGM2L1, the expression level of epithelial cell marker E-cadherin (E-cad) increased, while the expression of mesenchymal cell markers N-cadherin (N-cad) and fibronectin (FN1) was inhibited ( Figure 3 C). Overexpression of PGM2L1 enhances the proliferation, invasion and metastasis of tumor cells ( Figure 3 DE), tumor cell to mesenchymal cell transformation ( Figure 3 F). In summary, PGM2L1 can promote the proliferation and metastasis of triple-negative breast cancer cells and participate in regulating the EMT process.

[0131] 4. In vivo verification of the ability of PGM2L1 to promote breast cancer proliferation and metastasis

[0132] In order to verify the effect of PGM2L1 on tumor malignant progression in vivo, a series of in vivo experiments were conducted. Through the subcutaneous tumor formation test in nude mice, it was found that the tumor size of breast cancer cells with stable knockdown of PGM2L1 was significantly reduced, and the tumor formation speed was slowed down ( Figure 4AC); The results of lung metastasis assay and HE staining showed that knocking down PGM2L1 led to a significant decrease in the number of lung metastatic nodules ( Figure 4 DF), demonstrating that PGM2L1 can effectively promote the proliferation and metastasis of breast cancer cells in vivo.

[0133] 5. Correlation between PGM2L1 and prognosis of patients with triple-negative breast cancer

[0134] To verify the feasibility of PGM2L1 as a clinical marker, its clinical prognostic relevance was analyzed. 313 breast cancer samples and 73 normal tissues were selected from the biological sample bank of Qilu Hospital of Shandong University. The mRNA expression level of PGM2L1 in clinical tissue samples was detected by qRT-PCR. The results showed that the expression of PGM2L1 in breast cancer tissue was significantly higher than that in adjacent normal tissue (p<0.001). The ROC curve was drawn and it was found that PGM2L1 had a good diagnostic value for breast cancer patients (AUC=0.8317, p<0.001)( Figure 1 A).

[0135] Subsequently, immunohistochemical staining was performed on breast cancer tissues and adjacent normal tissues of TNBC patients in Shandong Qilu Hospital. The results showed that the expression level of PGM2L1 in TNBC breast cancer tissues was significantly higher than that in adjacent normal tissues ( Figure 1 E). The Kaplan–Meier analysis showed that the correlation between the expression level of PGM2L1 and the prognosis of breast cancer patients showed that the overall survival rate (OS) of breast cancer patients with high expression of PGM2L1 was significantly lower than that of the low expression group (HR=1.72, p=0.012) ( Figure 1 F). It was also found that breast cancer patients with high expression of PGM2L1 in triple-negative breast cancer samples had worse OS (HR=3.32, p=0.0156) ( Figure 1 G) demonstrated that high expression of PGM2L1 was significantly associated with poor prognosis in triple-negative breast cancer.

[0136] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a substance for detecting PGM2L1 in the preparation of any one or more of the following drugs or preparations; (1) Drugs or preparations for diagnosis or auxiliary diagnosis of triple-negative breast cancer; (2) Drugs or preparations for prognosis assessment or auxiliary prognosis assessment of triple-negative breast cancer.

2. The use according to claim 1, characterized in that In the application (2), the prognostic assessment or auxiliary prognostic assessment of triple-negative breast cancer includes the prediction of the overall survival of patients with triple-negative breast cancer.

3. The use according to claim 1, characterized in that The substances for detecting PGM2L1 include substances used for detecting the expression level of PGM2L1 by RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing.

4. The use according to claim 1, characterized in that The dosage form of the drug or preparation includes: one or more of injection, tablet, capsule, oral liquid or granule; Preferably, the administration of the drug or preparation includes one or more of sublingual administration, oral administration, intramuscular injection or intravenous injection.

5. The use according to claim 1, characterized in that The medicine or preparation further comprises a pharmaceutically acceptable excipient; Preferably, the auxiliary material includes one or more of a chelating agent, a protective agent, a surfactant, an excipient or a stabilizer.

6. A system for triple-negative breast cancer prognosis assessment or auxiliary prognosis assessment, characterized in that: The system comprises: An analysis unit, the analysis unit comprising: a detection substance for determining the expression level of PGM2L1 in a test sample of a subject; An evaluation unit, the evaluation unit comprising: performing a prognosis evaluation on a plurality of subjects according to the PGM2L1 expression level determined by the analysis unit; The prognostic assessment or auxiliary prognostic assessment includes assessing the overall survival of the subject; When the subject's PGM2L1 expression level is higher than the threshold, it is considered high expression, indicating that the subject's overall survival is shorter; When the subject's PGM2L1 expression level is lower than the threshold, it is considered low expression, indicating that the subject has a longer overall survival; The threshold is the optimal cutoff value between the PGM2L1 expression level and the overall survival of patients with triple-negative breast cancer.

7. Application of PGM2L1 as a target in the treatment of triple-negative breast cancer and / or screening of triple-negative breast cancer drugs.

8. Use of substances that inhibit the expression of PGM2L1 in the preparation of products for treating triple-negative breast cancer; in, The substances that inhibit the expression of PGM2L1 include RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, shRNA, and gene knockout substances targeting PGM2L1; Preferably, the siRNA includes siRNA-1 and siRNA-2; the nucleic acid sequence of the siRNA-1 is shown as SEQ ID NO.1-2, and the nucleic acid sequence of the siRNA-2 is shown as SEQ ID NO.3-4; Preferably, the shRNA includes shRNA1 and shRNA1, wherein the nucleic acid sequence of shRNA1 is shown as SEQ ID NO.5, and the nucleic acid sequence of shRNA2 is shown as SEQ ID NO.6; Preferably, the functions of the product for treating triple-negative breast cancer are any one or more of the following: (1) Inhibit G-6-P content in triple-negative breast cancer cells; (2) Inhibit glycolysis in triple-negative breast cancer cells; (3) Inhibit the proliferation of triple-negative breast cancer cells; (4) Inhibit the malignant invasion of triple-negative breast cancer cells; (5) Inhibit the growth of triple-negative breast cancer; (6) Inhibit metastasis of triple-negative breast cancer; Preferably, the product is a medicine.

9. A drug for treating triple-negative breast cancer, characterized in that: The active ingredients are substances for inhibiting the expression level of PGM2L1, including RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, shRNA, and gene knockout substances targeting PGM2L1; Preferably, the siRNA includes siRNA-1 and siRNA-2; the nucleic acid sequence of the siRNA-1 is shown as SEQ ID NO.1-2, and the nucleic acid sequence of the siRNA-2 is shown as SEQ ID NO.3-4; Preferably, the shRNA includes shRNA1 and shRNA1, the nucleic acid sequence of shRNA1 is shown as SEQ ID NO.5, and the nucleic acid sequence of shRNA2 is shown as SEQ ID NO.

6.

10. The drug according to claim 9, characterized in that The functions of the drug are any one or more of the following: (1) Inhibit G-6-P content in triple-negative breast cancer cells; (2) Inhibit glycolysis in triple-negative breast cancer cells; (3) Inhibit the proliferation of triple-negative breast cancer cells; (4) Inhibit the malignant invasion of triple-negative breast cancer cells; (5) Inhibit the growth of triple-negative breast cancer; (6) Inhibit the metastasis of triple-negative breast cancer.

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