Application of DEPTOR as biomarker in pancreatic cancer diagnosis

Abstract

The invention provides application of DEPTOR as a biomarker in preparation of a glutamine metabolism level evaluation reagent or a pancreatic cancer early diagnosis reagent. The invention further provides a kit capable of quantitatively or semi-quantitatively detecting the protein, and the kit contains an antibody capable of being specifically combined with DEPTOR. According to the application disclosed by the invention, the glutamine metabolism level of a subject individual can be more effectively evaluated by detecting the expression level of DEPTOR, so that the pancreatic cancer is subjected to early diagnosis. The application of the invention is helpful for early diagnosis and early treatment of pancreatic cancer. The kit disclosed by the invention can be suitable for evaluating the metabolism level of glutamine in individuals or diagnosing the early stage, the middle stage and the late stage of pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to the application of DEPTOR with metabolic reprogramming function as a biomarker in the diagnosis of pancreatic cancer, and belongs to the field of tumor medicine. Background Art

[0002] Pancreatic cancer is a highly aggressive malignant solid tumor with a 5-year survival rate of only 10%. It is expected that by 2040, pancreatic cancer will surpass colorectal cancer and become the leading cause of cancer-related deaths after lung cancer. There are many factors driving the high mortality rate of pancreatic cancer. The most important reason is that the early symptoms of the disease are very hidden and there is a lack of biomarkers for early tumor diagnosis. In addition, due to the special anatomical location of the pancreas, routine clinic screening is impossible, resulting in 90% of pancreatic cancer patients reaching the progressive stage of the disease at the time of initial diagnosis, and more than half of the patients have systemic metastasis. Therefore, in-depth research on the molecular mechanisms of the occurrence and development of pancreatic cancer, finding important tumor markers, effective therapeutic targets, and solutions to alleviate chemotherapy resistance are crucial to achieve early diagnosis, early treatment, delayed recurrence, and improved prognosis of pancreatic cancer.

[0003] At present, the methods of tumor diagnosis are divided into five levels: ① Clinical diagnosis: a speculative diagnosis based on clinical symptoms, signs and imaging examinations, with reference to the law of disease development; ② Surgical diagnosis: a judgment made only on the tumor seen by the naked eye through surgery or various endoscopic examinations, without pathological confirmation; ③ Physical and chemical diagnosis: clinically consistent with cancer manifestations, and supported by positive results of physical and chemical examinations, such as X-ray, B-ultrasound, CT and MRI nuclear magnetic resonance examinations, or carcinoembryonic antigen, alpha-fetoprotein determination, etc.; ④ Cytopathological diagnosis: a diagnosis made based on various exfoliated cells and puncture cell examinations; ⑤ Histopathological diagnosis: a diagnosis made after pathological analysis of tissue obtained after coarse needle puncture. Among these five levels, the reliability of the diagnosis increases in sequence, with the fifth level being the most ideal. However, the above methods cannot achieve early detection and diagnosis of tumors. More often than not, after the tumor develops to a certain extent, the patient may even develop serious symptoms and signs. At this time, the best treatment period is often missed, making the later treatment extremely passive. In addition, pathological diagnosis with better diagnostic effect requires puncture to obtain cells or tissue slices, which has great damage and negative effects on the patient's mental and physical health. It is crucial to find more effective biomarkers to achieve early diagnosis of tumors.

[0004] Metabolic reprogramming is one of the characteristic signs of tumor development. It not only appears in the early stages of tumor development, but also continues to evolve during the development of the tumor. Unlike normal cells, cancer cells are highly dependent on glutamine metabolism. "Glutamine addiction" provides tumor cells with a large amount of energy support required to maintain proliferation and survival, and to cope with stress conditions such as radiotherapy, chemotherapy, hypoxia or starvation. Abnormal tumor metabolism is closely related to the occurrence and development of tumors, and can serve as an important marker for the early diagnosis of tumors. Therefore, quantifying the level of abnormal tumor metabolism in a specific way is of great clinical significance for the early diagnosis and treatment of pancreatic cancer. In addition, a comprehensive and systematic understanding of the mechanism of tumor metabolism can better target tumor metabolic biomarkers, break through the current bottleneck in our research on tumor diagnosis, and realize the improvement of early diagnosis of pancreatic cancer from the perspective of metabolic abnormalities.

[0005] In view of the above background, in order to more accurately grasp the abnormalities of tumor metabolism and achieve early diagnosis and treatment of pancreatic disease, it is necessary to find key markers that are closely related to the occurrence and development of abnormal pancreatic tumor metabolism and use them for the early diagnosis and treatment of pancreatic cancer. Summary of the invention

[0006] The technical problem or primary purpose to be solved by the present invention is to use a new tumor marker to evaluate the individualized glutamine metabolism level of a patient, and based on this, to perform early diagnosis of pancreatic cancer in the subject.

[0007] Another object of the present invention is to provide a product for assessing a patient's individualized glutamine metabolism level.

[0008] Another object of the present invention is to provide a product for early diagnosis of pancreatic cancer.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention uses DEPTOR as a biomarker to prepare a reagent for evaluating the individualized glutamine metabolism level of a patient.

[0011] Furthermore, the present invention also uses DEPTOR as a biomarker for preparing an early diagnostic reagent for pancreatic cancer.

[0012] In the solution of the present invention, DEPTOR (DEP-domain containing mTOR-interacting protein) is a protein with metabolic reprogramming function, and its NCBI sequence number is: NP_001269941.1.

[0013] In the scheme of the present invention, by using an immunoassay reagent for DEPTOR to detect the expression level of DEPTOR in a sample from a subject, the individualized glutamine metabolism level of the subject can be effectively evaluated; further, based on the evaluation result of the individualized glutamine metabolism level, early diagnosis of pancreatic cancer is achieved. The sample from the subject is any one of the subject's venous peripheral blood, paracancerous tissue or tumor tissue.

[0014] In a preferred embodiment of the present invention, the reagent for evaluating the individualized glutamine metabolism level of a patient is an immunoassay reagent based on the Elisa or Western Blot method.

[0015] In a second aspect, the present invention further provides an antibody, wherein the antibody is an antibody capable of specifically binding to the DEPTOR.

[0016] In a third aspect, the present invention further provides a kit for quantitative or semi-quantitative detection of proteins, comprising the antibody described in the second aspect of the present invention.

[0017] In a preferred embodiment of the present invention, the kit capable of quantitatively or semi-quantitatively detecting proteins is a kit capable of detecting proteins by an enzyme-linked immunosorbent assay (ELISA) method or a Western Blot method; the kit contains a first antibody and a second antibody; the first antibody is the antibody described in the second aspect of the present invention, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

[0018] Fourthly, the present invention also provides the use of an antibody that specifically binds to the DEPTOR in preparing a kit for evaluating the individualized glutamine metabolism level of a patient or a kit for early diagnosis of pancreatic cancer.

[0019] In a further preferred embodiment of the present invention, the kit further comprises one or more substances selected from the following group: a container, instructions for use, a positive control substance, a negative control substance, a buffer, an adjuvant or a solvent.

[0020] Among them, the instruction manual records how to use the test kit for testing, how to use the test results to evaluate the patient's individualized glutamine metabolism level, and how to judge the development of pancreatic cancer based on the patient's individualized glutamine metabolism level evaluation results.

[0021] The present invention is committed to solving the technical challenges in the field of early diagnosis of pancreatic cancer, and provides a new specific biomarker DEPTOR, which is used to evaluate the individualized glutamine metabolism level of patients and the early diagnosis of pancreatic cancer. By detecting the expression level of the biomarker, the evaluation of the individualized glutamine metabolism level of patients and the early diagnosis of pancreatic cancer can be achieved. This biomarker provides a powerful tool for the early diagnosis of pancreatic cancer from the perspective of abnormal tumor metabolism, and can more effectively perform early preliminary evaluation and prediction of the risk of pancreatic cancer in subjects. The application of this technology will help pancreatic cancer patients to obtain early diagnosis and early treatment. The detection kit prepared based on the biomarker of the present invention can be applied to the evaluation of individualized glutamine metabolism levels and the early diagnosis of pancreatic cancer.

[0022] The DEPTOR described in the present invention is highly correlated with abnormal metabolism of pancreatic cancer tumor cells. Specifically, we found through experimental studies that the mTOR natural inhibitory molecule DEPTOR is related to the activation of glutamine catabolism. Silencing DEPTOR can promote the degradation of the glutamine dehydrogenase (GDH) activity inhibitory molecule SIRT4. In terms of molecular mechanism, DEPTOR directly binds to SIRT4 through its PDZ domain, and this binding inhibits the degradation of SIRT4 through the ubiquitination pathway. Therefore, silencing DEPTOR destroys the stability of SIRT4 and inhibits the expression of SIRT4, thereby promoting GDH enzyme activity and activating the key metabolic node of glutamate to α-ketoglutarate conversion in the process of glutamine catabolism. In clinical samples, we found that the expression level of DEPTOR was significantly negatively correlated with the level of glutamine. These findings reveal the key mechanism by which pancreatic cancer cells inhibit SIRT4 expression by silencing DEPTOR to promote glutamine metabolism, and emphasize the potential of DEPTOR protein in targeted cancer diagnosis and treatment.

[0023] The above findings were obtained through the following research:

[0024] 1 Materials and Methods

[0025] 1.1 Materials used:

[0026] (1) Cell types: PANC1 cell line and SW1990 cell line were obtained from ATCC

[0027] (2) Patient samples: Sample tissues were obtained from the Second Affiliated Hospital of Zhejiang University. IHC staining was performed on human pancreatic cancer tissue specimens and adjacent tissues to compare protein expression between tumor and normal tissue samples. Tissue section staining was quantitatively scored based on the percentage of positive cells and staining intensity.

[0028] (3) The siRNA sequences used for gene knockdown are as follows:

[0029] siDEPTOR:5'-GCCATGACAATCGGAAATCTA-3'

[0030] 1.2 Test methods

[0031] (1) Protein level detection: First, extract the protein from the cell, then use the corresponding antibody to react with the protein and determine the expression and action of the specific target protein through substrate color development. The protein level detection system used in the present invention includes: immunoprecipitation and immunoblotting. The antibody used is DEPTOR (CST, #11816).

[0032] Glutamine metabolism detection: Intracellular and cell supernatant glutamine content, intracellular glutamate content, intracellular α-KG level, glutaminase activity, glutamine dehydrogenase activity and glutamine transaminase activity were all detected using commercial detection kits to ensure that the cells were in good condition before the test, and the operation was performed strictly in accordance with the instructions of each kit.

[0033] 2 Results and analysis

[0034] 2.1DEPTOR protein level is significantly downregulated in human pancreatic cancer tissues and is associated with poor prognosis

[0035] The results of IHC staining of clinical cases showed that the protein expression level of DEPTOR in tumor tissue was significantly lower than that in adjacent tissues (results shown in Figure 1 As shown in the figure, the left side shows the DEPTOR expression results of the tissue chips of the tumor and adjacent tissues of pancreatic cancer patients after surgery at different magnifications after immunohistochemical staining with DEPTOR specific antibodies, and the right side shows the corresponding statistical results). Survival analysis also shows that the overall survival rate of pancreatic cancer patients with low DEPTOR expression is significantly lower than that of patients with high DEPTOR expression (the results are shown in Figure 2 As shown, the blue curve below represents the data of pancreatic cancer patients with low DEPTOR expression, and the red curve above represents the data of patients with high DEPTOR expression). In order to explore the changes in DEPTOR protein levels during the progression of pancreatic cancer, we further analyzed the expression of DEPTOR in different pathological stages. The results showed that with the increase of the patient's pathological stage, the protein expression level of DEPTOR gradually decreased, indicating that the expression level of DEPTOR was significantly inhibited during the progression of pancreatic cancer (results are shown in Figure 3As shown in the figure, the left side shows the DEPTOR expression results at different magnifications after DEPTOR-specific antibody immunohistochemical staining of cancer and adjacent tissue chips of pancreatic cancer patients with different pathological grades (G1, G2 and G3) after surgery, and the right side shows the corresponding statistical results). Univariate and multivariate analysis showed that the expression level of DEPTOR can be independent of other clinical indicators and serve as an independent clinical prognostic factor for pancreatic cancer. This also shows that the expression level of DEPTOR has a good predictive effect on the prognosis of pancreatic cancer patients (the results of univariate and multivariate COX analysis of DEPTOR expression level and multiple clinical factors are shown in the figure). Figure 4 shown).

[0036] 2.2 Inhibition of DEPTOR can promote the proliferation and survival of pancreatic cancer cells by activating the mTOR signaling pathway

[0037] The expression of DEPTOR was knocked down using small interfering RNA (siRNA) of DEPTOR in two human pancreatic cancer cell lines, PANC1 and SW1990. The survival and proliferation abilities of cells after silencing DEPTOR were tested by CCK-8 assay and clone formation assay, respectively. The results showed that compared with the control group, PANC1 and SW1990 cells showed a significant enhancement in cell proliferation ability after knocking down DEPTOR (experimental results are shown in Figure 2). Figure 5 As shown, the blue line of siCtrl represents the control group, and the red line of siDEPTOR represents the DEPTOR knockdown group). At the same time, the results of the clone formation experiment also confirmed that inhibiting DEPTOR can significantly promote the survival ability of cells (the experimental results are shown in Figure 6 As shown, the left side shows the observation results of the cell cloning experiment, and the right side shows the corresponding statistical results; in each figure, siCtrl represents the control group, and siDEPTOR represents the DEPTOR knockdown group).

[0038] 2.3DEPTOR promotes glutamine catabolism in pancreatic cancer by affecting glutamine dehydrogenase (GDH) activity

[0039] GSVA functional enrichment analysis was performed on the pancreatic cancer cohort, and the results showed that changes in DEPTOR expression were closely related to changes in multiple tumor metabolism-related signaling pathways (the results of the whole genome GSVA enrichment analysis based on the pancreatic cancer cohort are shown in Figure 2). Figure 7 Metabolomics analysis found that with the inhibition of DEPTOR expression, the contents of many compounds also changed, including a significant decrease in glutamine and glutamate (metabolic analysis results are shown in Figure 8 shown).

[0040] 2.4 Inhibition of DEPTOR promotes glutamine catabolism in pancreatic cancer by activating GDH enzyme activity

[0041] After silencing DEPTOR, pancreatic cancer cells consumed significantly more glutamine in the culture supernatant than the control group without silencing DEPTOR, and the intracellular glutamine and glutamate levels were significantly decreased compared with the control group without silencing DEPTOR, while the intracellular α-KG level did not change significantly (experimental results are shown in Fig. 9 As shown in the figure, "siControl" represents the control group without DEPTOR silencing, and "siDEPTOR" represents the corresponding pancreatic cancer cell group with DEPTOR silencing), and the enzyme activity of GDH in cells increased significantly after DEPTOR silencing (the experimental results are shown in Fig.10 As shown, the left column graph is the result of PANC1 cells, and the right column graph is the experimental result of SW1990 cells; in each column graph, "siControl" represents the control group without silencing DEPTOR, and "siDEPTOR" represents the corresponding pancreatic cancer cell group with silencing DEPTOR)

[0042] 2.5 Inhibition of DEPTOR promotes the enzymatic activity of GDH by degrading SIRT4

[0043] By analyzing the protein components bound by DEPTOR through mass spectrometry, we detected several known DEPTOR-binding proteins, such as mTOR, RICTOR, etc. We also focused on a new interacting protein SIRT4 (analysis results are shown in Fig.11 As shown in Figure 2 ). Previous studies have shown that SIRT4 is a core regulatory factor affecting GDH enzyme activity. Subsequently, WB experiments verified that after silencing DEPTOR in pancreatic cancer cell lines, the protein level of SIRT4 was significantly downregulated compared with the control group without silencing DEPTOR (WB experimental results are shown in Figure 2 ). Fig.12 As shown in the figure, siControl and Vector represent the control group, and "siDEPTOR" represents the corresponding pancreatic cancer cell group with DEPTOR silenced).

[0044] 2.6DEPTOR expression level is significantly negatively correlated with glutamine level

[0045] The Pearson correlation analysis showed that the expression level of DEPTOR in tumor tissues of pancreatic cancer patients was significantly negatively correlated with the glutamine level (Pearson correlation analysis results are shown in Figure 2). Fig.13 shown).

[0046] Through the above experiments, we found that in patients with low DEPTOR expression levels, their classical mTOR signaling pathway function was significantly activated, which significantly promoted the occurrence and development of tumors, while their non-classical glutamine metabolism function was significantly activated.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] 1. Based on the mechanism of glutamine metabolism in pancreatic cancer, the present invention has found a tumor marker for glutamine metabolism. By detecting the expression level of the tumor marker, early diagnosis of tumors can be achieved, and the glutamine metabolism level of the patient can be individually evaluated.

[0049] 2. The test can be completed by collecting samples from venous peripheral blood, which greatly reduces the psychological burden and physical damage of the subjects. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It reflects the immunohistochemical staining and statistical results of the cancer and adjacent tissue chips of pancreatic cancer patients after surgery using DEPTOR's specific antibodies.

[0051] Figure 2 It reflects the correlation between DEPTOR expression level and overall survival of pancreatic cancer patients.

[0052] Figure 3 It reflects the differences in DEPTOR protein expression in patients with pancreatic cancer of different pathological grades.

[0053] Figure 4 The results of univariate and multivariate COX analysis of DEPTOR expression levels and various clinical factors are reflected.

[0054] Figure 5 The results of the CCK8 cell proliferation experiment were shown 48 hours after pancreatic cancer cells were transfected with DEPTOR small interfering RNA.

[0055] Figure 6 This shows the results of a cell clone formation experiment 48 hours after pancreatic cancer cells were transfected with DEPTOR small interfering RNA.

[0056] Figure 7 It reflects the results of genome-wide GSVA enrichment analysis based on pancreatic cancer cohort.

[0057] Figure 8 The metabolomics analysis results of wild-type and DEPTOR-stable knockdown PANC1 are shown, and the most significantly different compounds are displayed in the form of a difference heat map.

[0058] Fig. 9 It reflects the intracellular glutamine content, glutamate content, glutamine consumption level and α-kg content after silencing DEPTOR.

[0059] Fig.10 It reflects the activity of intracellular glutamine dehydrogenase after silencing DEPTOR.

[0060] Fig.11 It reflects the introduction of a Flag-tagged DEPTOR plasmid into cells, and the use of immunoprecipitation and mass spectrometry analysis to reveal the protein components bound to DEPTOR.

[0061] Fig.12 It reflects the changes in SIRT4 expression levels after knocking down DEPTOR.

[0062] Fig.13 It reflects the results of the Pearson correlation analysis between DEPTOR expression levels and glutamine levels in clinical samples. DETAILED DESCRIPTION

[0063] In order to make the purpose and technical solution of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings. The experimental methods described in the following experiments are all conventional methods unless otherwise specified; if no specific technology or conditions are specified in the experiment, the technology or conditions described in the literature in the field or the product instructions are used; the reagents and materials described can be obtained from commercial channels unless otherwise specified.

[0064] The present invention provides the use of DEPTOR as a biomarker in the evaluation of glutamine metabolism level and the early diagnosis of pancreatic cancer.

[0065] The present invention also provides the use of DEPTOR in preparing a reagent for evaluating glutamine metabolism level or a reagent for early diagnosis of pancreatic cancer.

[0066] The reagent includes a reagent for detecting the expression level of DEPTOR in a sample. The sample is venous peripheral blood, paracancerous tissue or tumor tissue.

[0067] The reagents include a first antibody and a second antibody. The first antibody is an antibody that can specifically bind to the DEPTOR. The second antibody is an antibody that is homologous to the first antibody and is labeled with horseradish peroxidase.

[0068] The present invention also provides an amine metabolism level assessment kit or a pancreatic cancer early diagnosis kit based on Elisa or Western Blot methods, wherein the kit contains a reagent for detecting DEPTOR, and each reagent in the kit includes a first antibody and a second antibody, wherein the first antibody can specifically bind to an antibody of DEPTOR, and the second antibody is an antibody homologous to the first antibody and labeled with horseradish peroxidase. The kit may also include a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, and a solvent. The instructions for use describe how to use the kit for detection, and how to use the detection results to judge tumor development and select a treatment plan. The components of the kit may be packaged in the form of an aqueous medium or in a lyophilized form.

[0069] The method for evaluating the glutamine metabolism level of a patient or diagnosing pancreatic cancer at an early stage using the kit of the present invention comprises the following steps:

[0070] 1) detecting the expression level of the biomarker in the sample;

[0071] When venous peripheral blood is used as a sample, the sample processing method includes: after collecting venous peripheral blood, it needs to be placed on ice for 15 minutes, and then centrifuged at 3500rpm for 15 minutes to obtain serum or plasma, which is then tested or stored at -80°C. Samples that cannot be tested immediately need to be stored at -80°C and thawed on ice during testing.

[0072] Using Elisa or Western Blot to detect the expression level of DEPTOR in the treated samples;

[0073] 2) Evaluating the glutamine metabolism level of the subject or making an early diagnosis of pancreatic cancer based on the DEPTOR expression level in the sample detected in 1).

[0074] The Western Blot method mentioned in the evaluation or diagnosis method of the present invention, i.e., protein immunoblotting experiment, is a technique commonly used to separate and identify proteins in research. It utilizes SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in a specified sample, and then transfers the separated proteins to a nitrocellulose or PVDF membrane, and then incubates the membrane with a specific antibody to the target protein. During the membrane washing process, unbound antibodies are washed away, leaving only antibodies bound to the target protein, and finally the bound antibodies are detected by developing film or fluorescence scanning. Since the antibody is only bound to the target protein, generally only a clear band can be seen, and the thickness of the band corresponds to the amount of protein. By analyzing the position and intensity of a specific reaction, the expression information of the target protein in a given cell or tissue homogenate can be obtained. Due to the high resolution of gel electrophoresis and the strong specificity and high sensitivity of immunity, Western blot analysis can detect target proteins as low as 1ng. This method is widely used in molecular biology, biochemistry, immunogenetics and other molecular biology fields.

[0075] Based on the description of the above specific implementation methods, the present invention specifically lists the following embodiments to further illustrate the technical solution and technical effects of the present invention in detail.

[0076] Example 1.

[0077] An ELISA detection kit comprises a pre-coated microplate, a standard substance, an enzyme-labeled secondary antibody, a substrate solution, a stop solution, a washing buffer solution, a sample diluent, a control sample, an instruction manual and a plate sealing film; a capture antibody (an antibody that can specifically bind to DEPTOR) is pre-fixed in the wells of the microplate; and the enzyme-labeled secondary antibody is an antibody that is homologous to the capture antibody and is labeled with horseradish peroxidase.

[0078] Example 2.

[0079] A WB detection kit containing the following reagents and items:

[0080] Primary antibody: DEPTOR;

[0081] Secondary antibody (antibody homologous to DEPTOR and labeled with horseradish peroxidase);

[0082] Substrate solution;

[0083] Blocking solution: milk powder blocking solution;

[0084] Wash buffer: TBS / T;

[0085] Transfer buffer;

[0086] PVDF membrane;

[0087] Electroporation fluid;

[0088] Membrane staining solution;

[0089] Sample buffer;

[0090] Standard protein or marker protein: used to estimate the molecular weight of the target protein;

[0091] Instructions for use.

[0092] Example 3.

[0093] A method for evaluating glutamine metabolism level or early diagnosis of pancreatic cancer in a subject, comprising the following steps:

[0094] 1. The WB detection kit described in Example 2 was used to detect the DEPTOR expression level in the venous peripheral serum of the subjects.

[0095] The specific detection steps are as follows:

[0096] 1. Sample Preparation

[0097] 1) Sample collection: Collect venous peripheral blood and use the Bio-Tech Mitochondrial Extraction Kit to extract mitochondria from plasma.

[0098] 2) Preparation of lysis solution: 100 μl lysis solution + 0.1 μl phosphatase inhibitor mixture + 1 μl protease inhibitor mixture were added to the extracted mitochondria.

[0099] 3) Centrifuge at 4°C, 12,000 g for 15 min and carefully collect the supernatant into another 1.5 ml tube.

[0100] 4) Take 2.5 μl of sample and dilute it with 22.5 μl of triple-distilled water for measuring protein concentration using the BCA method.

[0101] 5) Add 5× Loading Buffer (2.5 ml Buffer / 10 ml protein) to the remaining samples and cook at 95°C for 10 minutes.

[0102] Oscillate once during the process and directly load the sample onto the gel for running or aliquot and store at -80℃ for long term.

[0103] 2. SDS-PAGE polyacrylamide gel electrophoresis

[0104] 1) Prepare separation gel (6 ml / gel).

[0105] 2) Carefully inject the separation gel, leaving about 2 cm of space for the concentrated gel, cover the top layer with deionized water, and let it stand for about

[0106] 30min.

[0107] 3) Prepare stacking gel (2 ml / gel).

[0108] 4) Inject the concentrated gel into the upper end of the separation gel, taking care to avoid bubbles.

[0109] 5) Insert the comb and wait for the concentrated gel to solidify (there should be a clear boundary between the gel and the comb, and the solidification time of the separation gel should be greater than 2 hours), wash the wells with double distilled water to remove the gel fragments, and then absorb the water with filter paper.

[0110] 6) Place the gel into the electrophoresis tank and add 1× electrophoresis buffer to both the upper and lower tanks (do not reuse more than 3 times).

[0111] 7) Sample loading: Take 5 μl prestained marker from the marker well and add appropriate amount of 1× sample loading buffer to make the total

[0112] The volume is the same as that of the sample well. The sample loading volume is generally 15-25μl. First, use a heating block at 95℃ for 5-10 minutes, shake once during the period, then quickly centrifuge and then load the sample and run the gel; add an equal volume of 1× loading buffer to the well without loading sample.

[0113] 8) Electrophoresis: Start with a constant voltage of 60-80V. After running through the concentrated gel, increase the current to 100-120V. The electrophoresis time is determined according to the size of the target protein and the position of the marker. Generally, the target protein runs to two-thirds of the separation gel.

[0114] 3. Membrane Transfer

[0115] 1) Cut the gel according to the marker and the position of the target band (make sure to mark the gel cutting angle), and immerse the eluted gel in transfer buffer for 15 minutes.

[0116] 2) After the PVDF membrane is marked, immerse it in methanol for 1 minute, and then immerse it in transfer buffer together with 4 pieces of 3mm filter paper and sponge for 15 minutes.

[0117] 3) Prepare the transfer equipment: follow the following order: fiber pad - filter paper - PVDF membrane - gel - filter paper - fiber pad; Note: each item must be aligned to ensure there are no bubbles.

[0118] 4) Transfer: The transfer time is determined according to 1). Connect one side of the PVDF membrane to the positive electrode (red) and one side of the gel to the negative electrode (black).

[0119] 4. Blocking and Antibody Incubation:

[0120] 1) After transfer, wash the membrane with 10 mL of 1× TBS at room temperature for 10 min;

[0121] 2) Incubate 5 mL of milk powder blocking solution at room temperature for 2 hours or gently shake at 4°C overnight. Since milk powder is difficult to dissolve, it should be prepared at least 1 hour in advance;

[0122] 3) Wash the membrane three times with 10 mL of TBS / T, 5 min each time;

[0123] 4) Add 5 mL of primary antibody dilution buffer (antibodies are diluted according to the instructions), shake gently at room temperature for 2 hours or at 4°C overnight, recover the primary antibody, add sodium azide (which can inhibit bacterial growth) at 5 μL / mL of primary antibody solution, store at 4°C (uncommon antibodies can be stored at -20°C for a long time), and can be reused;

[0124] 5) Wash the membrane three times with 10 mL of TBS / T, 5 min each time;

[0125] 6) Add secondary antibody (usually 1:2000 dilution) and shake gently at room temperature for 1 hour;

[0126] 7) Wash the membrane three times with 10 mL of TBS / T, 5 min each time.

[0127] 5. Development:

[0128] According to the instructions of the ultra-high sensitivity enhanced chemiluminescence (ECL) developer, solution A and solution B were prepared in a ratio of 1:1. The prepared developer was dropped onto the PVDF membrane incubated with the antibody, and after a certain period of light-proof reaction, it was exposed and developed in an exposure machine.

[0129] 2. Evaluate the patient's glutamine metabolism level based on the protein expression results detected in step 1.

[0130] Diagnostic analysis of test results:

[0131] If a protein band of the following size appears on the NC membrane: DEPTOR (48KD), and is compared with the standard protein sample, grayscale quantification is performed using ImageJ software. If the subject's protein grayscale is lower than the standard, it indicates that the subject's glutamine metabolism level is relatively active, the subject has a higher risk of tumor development, and close imaging testing is required.

[0132] In summary, the present invention can well assess the individualized risk of pancreatic cancer in the population by detecting the DEPTOR level in the body and incorporating it into existing large cohort studies.

[0133] In addition, the present invention will provide new ideas and effective solutions for the development of anti-tumor drugs targeting glutamine metabolic reprogramming and the early diagnosis of tumors.

[0134] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Application of DEPTOR as a biomarker in assessing individualized glutamine metabolism levels.

2. Application of DEPTOR as a biomarker in the preparation of personalized glutamine metabolism level assessment reagents or early diagnosis reagents for pancreatic cancer.

3. The use according to claim 2, characterized in that: The individualized glutamine metabolism level assessment reagent or pancreatic cancer early diagnosis reagent is a reagent used to detect the expression level of DEPTOR in a sample from a subject.

4. The use according to claim 3, characterized in that: The detection of the expression level of DEPTOR in the sample from the subject is achieved by Elisa or Western Blot method.

5. The use according to any one of claims 3 or 4, characterized in that: The detecting of the expression level of DEPTOR in the sample from the subject is detecting the expression level of DEPTOR in the venous peripheral blood, paracancerous tissue and / or tumor tissue from the subject.

6. A kit for quantitative or semi-quantitative detection of proteins, comprising an antibody that can specifically bind to DEPTOR.

7. The kit according to claim 6, characterized in that: The kit for quantitative or semi-quantitative detection of proteins is a kit for detecting proteins by an enzyme-linked immunosorbent assay (Elisa) method or a Western Blot method; the kit contains a first antibody and a second antibody; the first antibody is an antibody that can specifically bind to DEPTOR, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

8. The kit according to any one of claims 6 to 7, characterized in that: The antibody that can specifically bind to DEPTOR is a monoclonal antibody of DEPTOR.

9. Use of antibodies that can specifically bind to DEPTOR in the preparation of a personalized glutamine metabolism level assessment kit or a pancreatic cancer diagnosis kit.

10. Use of the kit according to claims 6-8 in assessing individualized glutamine metabolism levels.

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