Application of UFL1-T426p protein in the preparation of products for detecting, preventing or treating cancer

Abstract

The present invention discloses the application of UFL1‑T426p protein in the preparation of products for detecting, preventing or treating cancer, and belongs to the field of biomedical technology. The present invention proves for the first time at the cellular level and in a mouse model that phosphorylation of UFL1 is necessary for the growth and metastasis of breast cancer. The protein level of UFL1‑T426p protein in breast cancer tissue is significantly higher than that in adjacent normal breast tissue, and its high expression reduces the survival rate of breast cancer patients. Therefore, UFL1‑T426p protein can be used as a new diagnostic marker for the occurrence and metastasis of breast cancer, improving the accuracy of breast cancer diagnosis and prediction of breast cancer metastasis, making breast cancer diagnosis faster and more accurate. UFL1‑T426p protein also provides a new molecular target for breast cancer treatment, laying a theoretical foundation for the development of new drugs for the treatment of breast cancer. The above-mentioned application can be extended to non-small cell lung cancer, pancreatic cancer and liver cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of UFL1-T426p protein in the preparation of products for detecting, preventing or treating cancer. Background Art

[0002] Currently, the most commonly used cancer treatments include surgical resection, radiotherapy, chemotherapy, or a combination of surgical resection with postoperative radiotherapy and chemotherapy. However, the chemotherapeutic drugs on the market are usually not specific and can cause indelible damage to normal body cells while killing tumor cells.

[0003] Breast cancer is one of the most common malignant tumors in women. The molecular mechanism of breast cancer development has not been clearly studied, and breast cancer is prone to drug resistance and recurrence. At present, X-rays and ultrasound are commonly used methods for screening breast cancer, but they are difficult to detect early lesions and need to be further confirmed in combination with cytology or pathology results. However, existing breast cancer markers generally have the disadvantages of low sensitivity and poor specificity. This problem also exists in the detection, prevention and treatment of non-small cell lung cancer, pancreatic cancer and liver cancer. Therefore, there is an urgent need to develop new diagnostic markers and therapeutic targets.

[0004] Human UFL1 protein is UFL1 UFL1 is encoded by the gene (Genebank NM_015323.5) and is the E3 ligase in the UFMylation modification system. UFL1 covalently links UFM1 to substrate proteins with the assistance of UBA1 (E1), UFC1 (E2) and UFBP1, which promotes the UFMylation modification of substrates, thereby regulating processes such as tumor cell metastasis, DNA damage repair, endoplasmic reticulum homeostasis and immune response. Studies have shown that UFL1 promotes the occurrence and development of tumors such as breast cancer and is an important molecular target for tumor therapy. However, as the only E3 ligase in the UFMylation system, the molecular mechanism of UFL1 itself being regulated has rarely been reported. There are currently no reports on the role of UFL1 post-translational modification in breast cancer and other tumors.

[0005] ATK is a serine / threonine protein kinase. As a key molecule in the PI3K / Akt signaling pathway, it can activate or inhibit a series of downstream substrates through phosphorylation to participate in the regulation of cell proliferation, differentiation, apoptosis and metabolism. ATK has three subtypes, AKT1, AKT2 and AKT3. There is a high degree of homology between the amino acid sequences and domains of the three subtypes, so they have many of the same substrates, regulatory factors and functions. However, there are also different or even opposite functions among the three subtypes of AKT. Whether AKT promotes the phosphorylation of UFL1 and whether it regulates the occurrence and development of tumors through UFL1 has not been reported. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the present invention proposes the use of UFL1 protein (UFL1-T426p protein) in which the threonine at position 426 of UFL1 is phosphorylated in the preparation of products for detecting, preventing or treating cancer. In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides an application of a UFL1-T426p protein in preparing a product for detecting, preventing or treating cancer. The UFL1-T426p protein is a UFL1 protein in which the threonine at position 426 is phosphorylated.

[0008] Preferably, the UFL1-T426p protein is used as a biomarker to prepare a product for detecting cancer occurrence and metastasis.

[0009] Furthermore, the products include products for detecting UFL1-T426p protein at the protein level and products for quantitatively detecting UFL1-T426p protein.

[0010] Preferably, the product is selected from one or more of a reagent, a kit, a test paper and a chip.

[0011] Preferably, the product comprises a substance specific to the UFL1-T426p protein, and the substance specific to the UFL1-T426p protein is selected from one or more of the following a1), a2) and a3):

[0012] a1) Antibodies that specifically recognize and bind to UFL1-T426p protein;

[0013] a2) a ligand protein or peptide that specifically recognizes and binds to the UFL1-T426p protein;

[0014] a3) Non-protein compounds that specifically recognize and bind to UFL1-T426p protein.

[0015] Preferably, the UFL1-T426p protein is used as a therapeutic target for preparing a drug for preventing or treating cancer. Based on this, the present invention also provides the use of an agent using the UFL1-T426p protein as a therapeutic target in preparing a drug for preventing or treating cancer.

[0016] Preferably, the drug is selected from:

[0017] b1) an oligonucleic acid or an oligonucleotide expression vector that reduces the level of UFL1-T426p protein, wherein the expression vector comprises one or more of a eukaryotic cell plasmid expression vector and a eukaryotic cell virus expression vector;

[0018] b2) Proteins or peptides that inhibit the activity of UFL1-T426p protein;

[0019] b3) Small molecule compounds that inhibit the function of UFL1-T426p protein;

[0020] b4) one or more PROTACs (protein degradation targeting chimeras) and molecular glues that target degradation of UFL1-T426p protein and their derivatives;

[0021] b5) Proteins or peptides that inhibit the interaction between UFL1 and protein kinase AKT and reduce the level of UFL1-T426p protein;

[0022] b6) Small molecule compounds that inhibit the interaction between UFL1 and protein kinase AKT and reduce the level of UFL1-T426p protein.

[0023] Among them, the PROTAC refers to one or more ubiquitin-proteasome targeting chimeras that target the degradation of UFL1-T426p protein; the derivative technology of the PROTAC refers to one or more autophagy-lysosome targeting chimeras that target the degradation of UFL1-T426p protein, such as AUTOTAC (Autophagy-Targeting Chimera), ATTEC (Autophagosome-Tethering Compound), and LYTAC (Lysosome-Targeting Chimaeras); the molecular glue refers to one or more small molecules that enhance the interaction between UFL1-T426p protein and E3 ubiquitin ligase and thereby promote the degradation of UFL1-T426p protein through the proteasome pathway.

[0024] Preferably, the protein kinase AKT includes AKT1, AKT2 and AKT3.

[0025] Preferably, the UFL1-T426p protein is used as a therapeutic target for screening drugs for preventing or treating cancer. Based on this, the present invention also provides the use of a reagent using the UFL1-T426p protein as a therapeutic target in the preparation and screening of products for preventing or treating cancer.

[0026] Preferably, the cancer includes breast cancer, non-small cell lung cancer, pancreatic cancer and liver cancer.

[0027] The present invention has demonstrated for the first time at the cellular level and in mouse models that phosphorylation of threonine 426 of human UFL1 (UFL1-T426p) is necessary for UFL1 to promote breast cancer growth and metastasis. Experiments have found that the level of UFL1-T426p protein in breast cancer tissue is significantly higher than that in adjacent normal breast tissue, and its high expression reduces the survival rate of breast cancer patients. In addition, the level of UFL1-T426p is also significantly upregulated in other cancer tissues such as non-small cell lung cancer, pancreatic cancer, and liver cancer. Based on the above research and experimental results, the present invention provides the use of UFL1-T426p protein in the preparation of products for detecting, preventing or treating cancer.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The UFL1-T426p protein provided by the present invention can be used as a new diagnostic marker for the occurrence and metastasis of cancers such as breast cancer, thereby improving the accuracy of diagnosing and predicting the metastasis of cancers such as breast cancer, and making the diagnosis of cancers such as breast cancer faster and more accurate.

[0030] 2. The UFL1-T426p protein provided by the present invention also provides a new molecular target for the treatment of cancers such as breast cancer, and lays a theoretical foundation for the development of new drugs for the treatment of cancers such as breast cancer.

[0031] 3. The present invention also proves that protein kinase AKT catalyzes UFL1 to produce UFL1 protein (UFL1-T426p protein) with phosphorylation at threonine 426, which provides a new research and development idea for the preparation of drugs for preventing or treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] 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.

[0033] Figure 1The results of Example 1 confirming the interaction between UFL1 and AKT, wherein A shows that in HEK293T cells, UFL1 interacts with AKT1, AKT2 and AKT3, and the interaction between AKT1 and UFL1 is the strongest; B shows that UFL1 can immunoprecipitate AKT1 in MDA-MB-231 breast cancer cells; C shows that AKT1 can immunoprecipitate UFL1 in MDA-MB-231 breast cancer cells.

[0034] Figure 2 These are the result graphs confirming that AKT1 promotes the phosphorylation level of UFL1 in Example 2, wherein A is the phosphorylation signal of UFL1 detected using a specific AKT1 phosphorylation substrate antibody, and the phosphorylation signal was significantly reduced after knocking down AKT1; B is the highly conserved motif that is recognized and phosphorylated by AKT1 in the amino acid sequence of UFL1 of humans, goats, cattle, and monkeys; C is the T426A mutation in UFL1 that makes the specific AKT1 phosphorylation substrate antibody unable to detect the phosphorylation signal of UFL1.

[0035] Figure 3 The specificity of the UFL1-T426p antibody in recognizing phosphorylated UFL1 in Example 3 is detected, wherein A is a p-UFL1(T426) signal diagram detected in MDA-MB-231 cells under insulin stimulation, and the phosphorylation signal disappears after knocking down AKT1; B is that the p-UFL1(T426) antibody can detect the phosphorylation signal of wild-type (WT) UFL1 in MDA-MB-231 breast cancer cells, but cannot detect the phosphorylation signal of T426A mutant UFL1; C is that the p-UFL1(T426) antibody can detect the phosphorylation signal of wild-type (WT) UFL1 in MDA-MB-468 breast cancer cells, but cannot detect the phosphorylation signal of T426A mutant UFL1.

[0036] Figure 4 The results of Example 4 confirming that the protein level of UFL1-T426p is upregulated in breast cancer (BRCA) tissues and significantly reduces the survival rate of breast cancer patients at the tissue level, wherein A is a graph of the protein level of UFL1-T426p in tumor tissues (T) and adjacent normal tissues (N) such as breast cancer (BRCA), non-small cell lung cancer (NCSLC), pancreatic cancer (PADD) and liver cancer (HCC) detected by Western Blot; B is a graph of the protein level of UFL1-T426p in breast cancer tissues detected by immunohistochemistry; C is a graph showing the effect of the protein level of UFL1-T426p in breast cancer tissues on the overall survival rate of breast cancer patients.

[0037] Figure 5The data graphs of UFL1-T426p protein promoting the growth and metastasis of breast cancer in Example 5, wherein A is an invasion picture of shSC cells, shUFL1#1 cells, shUFL1#1+UFL1-WT cells and shUFL1#1+UFL1-T426A cells; B is a picture of tumors formed by injecting shSC cells, shUFL1#1 cells, shUFL1#1+UFL1-WT cells and shUFL1#1+UFL1-T426A cells into the fat pad of female NOD-SCID mice; C is a picture of tumors formed by injecting shSC cells, shUFL1#1 cells, shUFL1#1+UFL1-WT cells and shUFL1#1+UFL1-T426A cells into the fat pad of female NOD-SCID mice. A is a volume comparison of tumors formed by injecting shSC cells, shUFL1#1 cells, shUFL1#1+UFL1-WT cells, and shUFL1#1+UFL1-T426A cells into the fat pad of female NOD-SCID mice to form tumors. B is a volume comparison of tumors formed by injecting shSC cells, shUFL1#1 cells, shUFL1#1+UFL1-WT cells, and shUFL1#1+UFL1-T426A cells into the fat pad of female NOD-SCID mice to form tumors. DETAILED DESCRIPTION

[0038] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0039] Human UFL1 protein is UFL1 The gene is encoded by the gene (Genebank NM_015323.5), and the sequence is shown in SEQ ID NO.1.

[0040] A first typical embodiment of the present invention provides: use of UFL1-T426p protein as a biomarker in the preparation of a product for detecting the occurrence and metastasis of breast cancer.

[0041] In one or more examples of this embodiment, the products include products for detecting UFL1-T426p at the protein level and products for quantitatively detecting UFL1-T426p protein.

[0042] In one or more examples of this embodiment, the product includes one or more of a reagent, a kit, a test paper, and a chip.

[0043] In one or more examples of this embodiment, the product contains a substance specific to the UFL1-T426p protein, and the substance specific to the UFL1-T426p protein includes one or more of the following a1), a2) and a3):

[0044] a1) Antibodies that specifically recognize and bind to UFL1-T426p protein;

[0045] a2) a ligand protein or peptide that specifically recognizes and binds to the UFL1-T426p protein;

[0046] a3) Non-protein compounds that specifically recognize and bind to UFL1-T426p protein.

[0047] In one or more examples of this embodiment, the substance specific to UFL1-T426p protein is suitable for one or more of immunoblotting, immunocoprecipitation, immunofluorescence, radioimmunoassay, enzyme-linked immunosorbent assay, enzyme-linked immunospot, colloidal gold immunoassay, immunochemiluminescence, fluorescent immunochromatography, immunohistochemistry, mass spectrometry, high performance liquid chromatography, capillary gel electrophoresis, flow cytometry, and proteomics.

[0048] In one or more examples of this embodiment, the test sample of the product includes one or more of tumor tissues, body fluids, and cells isolated from breast cancer patients.

[0049] In one or more examples of this embodiment, the antibody comprises an antibody that specifically recognizes UFL1-T426p protein produced in mice, rats, guinea pigs, chickens, rabbits, pigs, monkeys, or any other organism using any UFL1 short peptide or protein containing T426p modification.

[0050] A second typical embodiment of the present invention provides: use of UFL1-T426p protein as a therapeutic target in the preparation of a drug for preventing or treating the occurrence and metastasis of breast cancer.

[0051] In one or more examples of this embodiment, the drug includes an oligonucleic acid or oligonucleotide expression vector that reduces the level of UFL1-T426p protein, a protein or peptide that inhibits the activity of UFL1-T426p protein, a small molecule compound that inhibits the function of UFL1-T426p protein, one or more of PROTAC and molecular glue that target the degradation of UFL1-T426p protein and their derivative technologies, a protein or peptide that inhibits the interaction between UFL1 and protein kinase AKT to reduce the level of UFL1-T426p protein, and a small molecule compound that inhibits the interaction between UFL1 and protein kinase AKT to reduce the level of UFL1-T426p protein.

[0052] In one or more examples of this embodiment, the expression vector includes a eukaryotic expression vector, and the eukaryotic expression vector includes one or more of a plasmid expression vector and a viral expression vector.

[0053] In one or more examples of this embodiment, the PROTAC refers to one or more protein degradation targeting chimeras that target the degradation of UFL1-T426p protein. PROTAC is a hybrid bifunctional small molecule compound containing two different ligands, one is an E3 ubiquitin ligase ligand, and the other is a ligand that binds to the target protein in the cell. The two ligands are connected by a linker to form a "target protein ligand-linker-E3 ligand" polymer. ROTAC forcibly shortens the distance between the target protein and the E3 ubiquitin ligase in the cell, and uses the "ubiquitin-proteasome" pathway to specifically degrade the target protein.

[0054] In one or more examples of this embodiment, the derivative technology of PROTAC refers to one or more autophagy-lysosome targeting chimeras that target the degradation of UFL1-T426p protein, such as AUTOTAC (Autophagy-Targeting Chimera), ATTEC (Autophagosome-Tethering Compound), and LYTAC (Lysosome-Targeting Chimaeras).

[0055] In one or more examples of this embodiment, the molecular glue refers to one or more small molecules that enhance the interaction between the UFL1-T426p protein and the E3 ubiquitin ligase and thus promote the degradation of the UFL1-T426p protein through the proteasome pathway. The molecular glue is usually a single small molecule that promotes the interaction between the target protein or the E3 ubiquitin ligase by changing the conformation of the target protein or the E3 ubiquitin ligase without requiring a specific connecting chain or a high-affinity ligand for the targeting protein.

[0056] A third typical embodiment of the present invention provides: use of UFL1-T426p protein as a therapeutic target in screening drugs for preventing and inhibiting the occurrence and metastasis of breast cancer.

[0057] A fourth typical embodiment of the present invention provides: applying the above embodiment to non-small cell lung cancer, pancreatic cancer, liver cancer, etc.

[0058] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0059] Example 1: UFL1 interacts with AKT

[0060] Co-immunoprecipitation (Co-IP), sodium dodecyl sulfate polyacrylamide (SDS-PAGE) electrophoresis and Western Blot experiments

[0061] Use cell lysis buffer to lyse MDA-MB-231 breast cancer cells or HEK293T cells transiently transfected with expression plasmids for 24 hours, centrifuge and collect the supernatant, and determine the protein concentration. Take the supernatant and add IgG or specific antibodies, then add immunoprecipitation magnetic beads and incubate overnight. Wash the immunoprecipitation magnetic beads three times with cell lysis buffer, retain the precipitate, add loading buffer and boil at 100 ° C for 8 minutes. The above samples were subjected to SDS-PAGE electrophoresis, transfer, blocking, antibody incubation and gel imaging system development, and finally the result of the interaction between UFL1 and AKT was obtained. Figure 1 As shown in A, UFL1 interacts with AKT1, AKT2, and AKT3 in HEK293T cells, and the UFL1-AKT1 interaction is the strongest; Figure 1 As shown in B and C, UFL1 and AKT1 interact in MDA-MB-231 breast cancer cells.

[0062] Example 2: AKT1 promotes UFL1 phosphorylation

[0063] 2.1. Construction of cell lines

[0064] shSC cells: The shSC plasmid was packaged into viruses, and then breast cancer cells were infected with the viruses and selected by puromycin to obtain shSC breast cancer cells that could not knock down AKT1.

[0065] shAKT1 cells: The shAKT1 plasmid with knockdown of AKT1 was packaged into virus, and then breast cancer cells were infected with the virus and selected by puromycin to obtain shAKT1 breast cancer cells with knockdown of AKT1.

[0066] Flag-UFL1-WT cells: The Flag-UFL1-WT plasmid was packaged into virus, and then breast cancer cells were infected with the virus and selected by puromycin to obtain breast cancer cells that stably expressed Flag-UFL1-WT.

[0067] Flag-UFL1-T426A cells: The Flag-UFL1-T426A plasmid was packaged into virus, and then breast cancer cells were infected with the virus and selected by puromycin to obtain breast cancer cells that stably expressed Flag-UFL1-T426A.

[0068] Puromycin is an antibiotic widely used in biological laboratories, mainly for screening cells that efficiently express exogenous genes.

[0069] 2.2 Co-IP, SDS-PAGE and Western Blot Experiments

[0070] Lyse MDA-MB-231 breast cancer cells with cell lysis buffer, centrifuge and collect the supernatant, and determine the protein concentration. Take the supernatant and add IgG or special antibody, then add immunoprecipitation magnetic beads and incubate overnight. Wash the immunoprecipitation magnetic beads 3 times with cell lysis buffer, retain the precipitate, add loading buffer and boil at 100 ° C for 8 minutes. Perform SDS-PAGE electrophoresis, transfer, block, antibody incubation and gel imaging system development on the above samples, and finally detect the protein level of UFL1-T426p. Figure 2 As shown in A, the AKT1 phosphorylation substrate antibody (p-AKT1-sub) signal shows that AKT1 promotes the phosphorylation level of UFL1; Figure 2 As shown in B, the amino acid sequence of UFL1 contains an evolutionarily conserved motif that may be phosphorylated by AKT1, namely RRRKAT(426), and the threonine (T) at position 426 is a potential phosphorylation site; Figure 2 As shown in C, wild-type (WT) UFL1 can be phosphorylated by AKT1, while T426 mutant UFL1 (T426A) cannot be phosphorylated by AKT1.

[0071] Example 3, Preparation and Application of UFL1-T426p Antibody

[0072] 3.1. Antibody Preparation

[0073] A short peptide of UFL1 containing phosphorylated T426 (i.e., C-ERRRKA[pT]EGSGS-N) was synthesized by a biological company as an antigen. The short peptide solution was mixed with an immune adjuvant and emulsified, and then injected into the rabbit subcutaneously for immunization. After 5 immunizations, the serum was collected and the titer of the antibody that specifically recognizes the UFL1-T426p protein (referred to as p-UFL1(T426) antibody) was detected by enzyme-linked immunosorbent (ELISA) experiment. The highest titer serum was combined with protein A / G immunoprecipitation magnetic beads for 2 hours, and the magnetic beads were washed with TBS buffer. The magnetic beads were rotated and incubated in an acidic elution solution (0.1M Glycine-HCl, pH3.0) for 5 minutes, and then a neutralizing solution (0.5M Tris-HCl, pH7.4, 1.5M NaCl) was added. Finally, the purified UFL1-T426p antibody was obtained and stored in a refrigerator at -80 ℃ after aliquoting.

[0074] 3.2. Construction of cell lines

[0075] Flag-UFL1-WT+Myc-AKT1 cells: Flag-UFL1-WT cells were infected with a virus overexpressing Myc-AKT1, and then screened with G418 to obtain Flag-UFL1-WT+Myc-AKT1 breast cancer cells.

[0076] Flag-UFL1-T426A+Myc-AKT1 cells: Flag-UFL1-T426A cells were infected with a virus overexpressing Myc-AKT1, and then selected with G418 to obtain Flag-UFL1-WT+Myc-AKT1 breast cancer cells.

[0077] G418 (Geneticin) is an aminoglycoside antibiotic and the most commonly used resistance screening reagent for stable transfection in molecular genetic experiments.

[0078] 3.3 Co-IP, SDS-PAGE and Western Blot Experiments

[0079] The specificity and titer of UFL1-T426p antibody were further tested by SDS-PAGE and Western Blot experiments. Figure 3 As shown in Figure A, p-UFL1(T426) signal can be detected in MDA-MB-231 cells under insulin stimulation, and this signal disappears when AKT1 is knocked down, indicating that the p-UFL1(T426) antibody does specifically recognize the UFL1 protein phosphorylated by AKT1. Figure 3 As shown in B and C, the p-UFL1(T426) antibody only recognizes wild-type (WT) UFL1 phosphorylated by AKT1 in MDA-MB-231 and MDA-MB-468 breast cancer cells, but not mutant (T426A) UFL1 phosphorylated by AKT1. Therefore, UFL1-T426p does exist in breast cancer cells, and the p-UFL1(T426) antibody specifically recognizes UFL1 protein (UFL1-T426p) phosphorylated by AKT1 at Thr426.

[0080] It should be pointed out that any UFL1 short peptide containing T426p, regardless of its length, has the potential to prepare p-UFL1 (T426) antibodies.

[0081] Example 4: UFL1-T426p protein levels increase in tumor cells and breast cancer tissues and reduce the survival rate of breast cancer patients

[0082] 4.1. SDS-PAGE and Western Blot Experiments

[0083] The chopped tumor tissue and adjacent normal tissue were immersed in cell lysis buffer and the cells were broken by ultrasonic disruption. Centrifuge and collect the supernatant to determine the protein concentration. Loading buffer was added to the cell lysis buffer and boiled at 100 °C for 8 minutes. The above samples were subjected to SDS-PAGE electrophoresis, membrane transfer, blocking, antibody incubation and gel imaging system development to finally obtain the results of UFL1-T426p protein levels in tumor tissue and adjacent normal tissue. Figure 4 As shown in A, the level of UFL1-T426p in breast cancer, non-small cell lung cancer, pancreatic cancer and liver cancer tissues was significantly higher than that in adjacent normal tissues.

[0084] 4.2 Immunohistochemistry

[0085] The breast cancer tissue was fixed in formalin, then embedded in paraffin and sliced ​​with a thickness of 10 μm. The slices were transferred to glass slides for dewaxing, hydration and antigen retrieval. Protein blocking, endogenous enzyme blocking, homemade UFL1-T426p antibody (primary antibody) incubation, HRP-labeled secondary antibody incubation, color development and hematoxylin counterstaining were performed. Then, the coverslip was sealed with nail polish. Finally, the above-stained samples were scanned and imaged using a high-resolution scanner. Figure 4 As shown in B, the content of UFL1-T426p protein in breast cancer tissues is significantly higher than that in adjacent normal tissues.

[0086] 4.3 Kaplan-Meier analysis

[0087] According to the UFL1-T426p immunohistochemical signal intensity, breast cancer patients were divided into two groups. The 80 cases with stronger signal intensity were defined as the high expression group, and the 75 cases with weaker signal intensity were classified as the low expression group. Then, the survival time of the patients was input into GraphPadPrism 8.0.1 to obtain the overall survival curve. Figure 4 As shown in C, high levels of UFL1-T426p reduce the survival rate of breast cancer patients.

[0088] The above experiments show that the UFL1-T426p protein level is significantly upregulated in breast cancer tissues and reduces the survival rate of breast cancer patients. In addition, the UFL1-T426p protein level is also significantly increased in non-small cell lung cancer, pancreatic cancer and liver cancer tissues.

[0089] Example 5: UFL1 protein promotes breast cancer growth and metastasis by its phosphorylation modification

[0090] 5.1. Construction of cell lines

[0091] shSC cells: The shSC plasmid was packaged into viruses, and then breast cancer cells were infected with the viruses and screened with G418 to obtain shSC breast cancer cells that could not knock down UFL1.

[0092] shUFL1#1 cells: The shUFL1#1 plasmid with UFL1 knockdown was packaged into virus, and then breast cancer cells were infected with the virus and screened with G418 to obtain shUFL1#1 breast cancer cells with UFL1 knockdown.

[0093] shUFL1#1+UFL1-WT cells: shUFL1#1 breast cancer cells were infected with a virus overexpressing wild-type UFL1 (UFL1-WT), and then screened with puromycin to obtain shUFL1#+UFL1-WT breast cancer cells.

[0094] shUFL1#1+UFL1-T426A cells: shUFL1#1 breast cancer cells were infected with a virus overexpressing mutant UFL1 (UFL1-T426A, threonine [T] at position 426 mutated to alanine [A]), and then screened with puromycin to obtain shUFL1#+UFL1-T426A breast cancer cells.

[0095] 5.2 Cell invasion assay

[0096] 5×10 4 shSC breast cancer cells, shUFL1#1 breast cancer cells, shUFL1#1+UFL1-WT breast cancer cells and shUFL1#1+UFL1-T426A breast cancer cells were suspended in serum-free DMEM medium and then plated on the upper chamber of the transwell pre-laid with matrix gel, and DMEM medium containing 10% fetal bovine serum was added to the lower chamber of the transwell. After 17 hours of culture in an incubator at 37°C with 5% CO2, the cells were fixed with formaldehyde, stained with crystal violet, washed with PBS and photographed. Figure 5 As shown in A, knockdown of UFL1 protein significantly inhibited the invasive ability of cells. Overexpression of UFL1-WT in shUFL1#1 breast cancer cells could compensate for the decreased invasive ability of cells caused by knockdown of UFL1, while overexpression of UFL1-T426A could not compensate for the phenotype of shUFL1#1 cells, indicating that UFL1 promotes the invasion of breast cancer cells by relying on its phosphorylation modification.

[0097] 5.3. Fat pad tumor-bearing mouse experiments

[0098] 1×10 6shSC breast cancer cells, shUFL1#1 breast cancer cells, shUFL1#1+UFL1-WT breast cancer cells, and shUFL1#1+UFL1-T426A breast cancer cells were suspended in a mixture of 50 μL serum-free DMEM medium and 50 μL matrix gel. The cells were injected into the second mammary gland tissue on the left side of female NOD-SCID mice. Starting from the 24th day, the tumor volume was measured every two days. On the 57th day, the mice were euthanized and the lungs were perfused. The tumors were dissected out and photographed, and the lungs were fixed, paraffin-embedded, sectioned, H&E stained, and photographed. Figure 5 As shown in B, C, and D, knockdown of UFL1 significantly inhibited breast tumor growth and lung metastasis, and this inhibitory effect could be effectively complemented by overexpressed UFL1-WT, but not by overexpressed UFL1-T426A. The above experiments indicate that UFL1 protein promotes breast cancer growth and metastasis by its phosphorylation modification.

[0099] 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 UFL1-T426p protein in the preparation of a product for detecting, preventing or treating cancer, characterized in that: The UFL1-T426p protein is a UFL1 protein in which the threonine at position 426 is phosphorylated.

2. Use of the UFL1-T426p protein according to claim 1 in preparing a product for detecting, preventing or treating cancer, characterized in that: The UFL1-T426p protein is used as a biomarker to prepare products for detecting cancer occurrence and metastasis.

3. Use of the UFL1-T426p protein according to claim 1 in preparing a product for detecting, preventing or treating cancer, characterized in that: The products include products for detecting UFL1-T426p protein at the protein level and products for quantitatively detecting UFL1-T426p protein.

4. Use of the UFL1-T426p protein according to claim 1 in preparing a product for detecting, preventing or treating cancer, characterized in that: The product is selected from one or more of a reagent, a test kit, a test paper and a chip.

5. Use of the UFL1-T426p protein according to claim 1 in preparing a product for detecting, preventing or treating cancer, characterized in that: The product comprises a substance specific to the UFL1-T426p protein, wherein the substance specific to the UFL1-T426p protein is selected from one or more of the following a1), a2) and a3): a1) Antibodies that specifically recognize and bind to UFL1-T426p protein; a2) a ligand protein or peptide that specifically recognizes and binds to the UFL1-T426p protein; a3) Non-protein compounds that specifically recognize and bind to UFL1-T426p protein.

6. Use of the UFL1-T426p protein according to claim 1 in preparing a product for detecting, preventing or treating cancer, characterized in that: The UFL1-T426p protein is used as a therapeutic target for preparing drugs for preventing or treating cancer.

7. Use of the UFL1-T426p protein according to claim 6 in preparing a product for detecting, preventing or treating cancer, characterized in that: The drug is selected from: b1) an oligonucleic acid or an oligonucleotide expression vector that reduces the level of UFL1-T426p protein, wherein the expression vector comprises one or more of a eukaryotic cell plasmid expression vector and a eukaryotic cell virus expression vector; b2) Proteins or peptides that inhibit the activity of UFL1-T426p protein; b3) Small molecule compounds that inhibit the function of UFL1-T426p protein; b4) one or more of protein degradation targeting chimeras and molecular glues for targeted degradation of UFL1-T426p protein and their derivatives; b5) Proteins or peptides that inhibit the interaction between UFL1 and protein kinase AKT and reduce the level of UFL1-T426p protein; b6) Small molecule compounds that inhibit the interaction between UFL1 and protein kinase AKT and reduce the level of UFL1-T426p protein.

8. Use of the UFL1-T426p protein according to claim 7 in preparing a product for detecting, preventing or treating cancer, characterized in that: The protein kinase AKT includes AKT1, AKT2 and AKT3.

9. Use of the UFL1-T426p protein according to claim 1 in preparing a product for detecting, preventing or treating cancer, characterized in that: The UFL1-T426p protein is used as a therapeutic target for screening drugs for preventing or treating cancer.

10. Use of the UFL1-T426p protein according to any one of claims 1 to 9 in preparing a product for detecting, preventing or treating cancer, characterized in that: The cancers include breast cancer, non-small cell lung cancer, pancreatic cancer and liver cancer.

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