Application of CLOCK-based biomarkers in the preparation of tumor diagnostic products

Abstract

The present invention relates to the application of biomarkers based on CLOCK function in the preparation of tumor diagnostic products, and belongs to the field of tumor medicine. The product mainly includes reagents for detecting the expression level of biomarkers, wherein the biomarkers are CLOCK S106 phosphorylation, PRPS1K29 acetylation and PRPS2K29 acetylation. Based on the mechanism of tumor occurrence and development, the present invention finds the above-mentioned biomarkers based on CLOCK function, and early diagnosis of tumors can be achieved by detecting the expression level of biomarkers.

Description

Technical Field

[0001] The present invention relates to the application of a biomarker based on CLOCK function in the preparation of tumor diagnosis products, and belongs to the field of tumor medicine. Background Art

[0002] The key to tumor treatment lies in whether it can be discovered and diagnosed early so that appropriate treatment measures can be taken. At present, tumors can only be completely cured if they are treated early. If the tumor develops to the middle or late stages, the tumor tissue expands or has metastasized, it will be difficult to cure it even if various treatment methods are taken. On the contrary, if the tumor can be discovered in the early or relatively early stages and before metastasis occurs and is given timely and reasonable treatment, a considerable number of patients can achieve satisfactory treatment results, and even achieve a cure.

[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 early 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] At present, there are still many deficiencies in the research on the mechanism of tumor occurrence and development, which seriously hinders the development of new methods for the diagnosis and treatment of tumors. The transcriptional activity of the circadian rhythm motor output cycle gene (CLOCK) controls the circadian rhythm, and damage to the biological clock is related to the development of cancer; phosphoribosyl pyrophosphate synthetase 1 (PRPS1) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2) catalyze the first reaction and rate-limiting reaction of de novo nucleotide synthesis, converting 5-phosphoribose to phosphoribosyl pyrophosphate, which is then used to synthesize purine and pyrimidine nucleotides. The human PRPS1 and PRPS2 genes have three isomers with high sequence consistency. PRPS1 and PRPS2 genes are expressed in multiple tissues, while PRPS3 is specifically expressed in the testis; insulin-like growth factor 1 receptor (IGF1R), its ligand IGF-1 is a polypeptide protein hormone similar to insulin in molecular structure. IGF-1 plays an important role in growth and continues to play an anabolic role in adults. This receptor has a strong growth-promoting effect and plays an important role in the occurrence and development of liver cancer.

[0005] In previous studies, the function of the circadian clock in tumor cells was impaired compared with the corresponding normal tissues, and bioinformatics analysis of 14 different cancer types showed differential expression of many core clock genes and clock-controlled genes. Experimental jet lag and genetic mutations of central circadian clock components BMAL1 and PER2 exacerbated the occurrence of lung tumors, and mice lacking PER1 and PER2, CRY1 and CRY2, or BMAL1 copies all showed increased spontaneous and radiation-induced tumor development. However, in some cases, circadian rhythm genes may also exacerbate cancer progression, and the loss of CLOCK and BMAL1 can hinder the growth of acute myeloid leukemia cells in mice both in vitro and in vivo. In addition, in mice, genetic knockout or knockdown of CLOCK and BMAL1 in patient-derived glioblastoma (GBM) stem cells inhibited cell growth and suppressed tumor growth. These findings indicate that the role of circadian clock genes in different types of cancer is different, and the different effects of core circadian clock defects on tumor formation are not only due to the disruption of their transcriptional function, but also from the disruption of their functions that are independent of gene expression. However, whether CLOCK directly regulates cancer cell proliferation in response to oncogenic signals in a manner independent of its expression and canonical transcriptional function remains unclear. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a biomarker for preparing an early tumor diagnosis product, and by detecting the expression level of the biomarker, an early diagnosis of a subject suffering from a tumor can be performed.

[0007] The present invention provides the use of a biomarker based on CLOCK function in the preparation of a tumor diagnosis product, wherein the biomarker is one or more of CLOCK S106 phosphorylation, PRPS1 K29 acetylation and PRPS2K29 acetylation.

[0008] Among them, CLOCK S106 phosphorylation refers to the phosphorylation at position 106 of the amino acid sequence of CLOCK, PRPS1K29 acetylation refers to the acetylation at position 29 of the amino acid sequence of PRPS1, and PRPS2 K29 acetylation refers to the acetylation at position 29 of the amino acid sequence of PRPS2; the NCBI sequence number of CLOCK is NP_001254772.1, the NCBI sequence number of PRPS1 is NP_002755.1, and the NCBI sequence number of PRPS2 is NP_001034180.1.

[0009] The product includes reagents for detecting the expression level of biomarkers in a sample.

[0010] The reagents include a primary antibody and a secondary antibody.

[0011] The first antibodies include CLOCK S106 phosphorylation antibody CLOCK pS106, PRPS1 K29 acetylation and PRPS2 K29 acetylation antibodies PRPS1 / 2 K29-Acetyl.

[0012] The second antibody is homologous to the first antibody and is labeled with horseradish peroxidase.

[0013] The detection method of the product is Elisa or Western Blot.

[0014] The sample is venous peripheral blood, paracancerous tissue or tumor tissue. 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 for testing or storage at -80℃. Samples that cannot be tested immediately need to be stored at -80℃ and thawed on ice during testing.

[0015] The product is mainly a kit. The kit includes a reagent for detecting the expression level of a biomarker, and one or more substances selected from the following group: a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant or a solvent. The instructions for use record how to use the kit for detection, and how to use the detection results to judge the development of the tumor and select a treatment plan. The components of the kit can be packaged in the form of an aqueous medium or in a lyophilized form.

[0016] The diagnostic product can be applied to the diagnosis of the following tumor diseases in the early, middle or late stages: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, genitourinary system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, biliary system cancer, pheochromocytoma, islet cell carcinoma, Leigh-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. Based on the mechanism of tumor metabolism, the present invention has found new biomarkers for tumor metabolism. Early diagnosis of tumors can be achieved by detecting the expression level of biomarkers.

[0019] 2. The test can be completed by collecting venous peripheral blood, which greatly reduces the psychological burden and physical damage of the subjects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For the immunoprecipitation assay in experiment 1, cells were treated with IGF and then subjected to immunoblot analysis;

[0021] Figure 2 This is a graph showing the results of the luciferase reporter gene assay in Experiment 1;

[0022] Figure 3 For the in vitro phosphorylation assay in Experiment 2, CK2 phosphorylates CLOCK;

[0023] Figure 4 This is the in vitro phosphorylation test of Experiment 2, and the result diagram of the validation test;

[0024] Figure 5 This is the result of the in vitro GST pull-down assay in Experiment 3;

[0025] Figure 6 For experiment 3, immunoprecipitation assay, the effect of CLOCK S106A on the igf-induced dissociation of CLOCK and BMAL1;

[0026] Figure 7 Molecular dynamics (MD) simulation results for Experiment 4, conformational changes of the CLOCK- bmal1 complex induced by CLOCK S106 phosphorylation;

[0027] Figure 8For experiment 4 GST pull-down assay, purified GST- exportin1 was bound to purified WT His-CLOCK in the presence of WT CK2 but not in the absence of inactive His-CK2 K68M;

[0028] Fig. 9 For experiment 4, immunofluorescence assay was performed to determine whether the phosphorylation of CLOCK S106 that inhibits the transcriptional function of CLOCK confers an unknown non-canonical function to CLOCK and showed that CLOCK is mainly localized in the nucleus;

[0029] Fig.10 For the coimmunoprecipitation analysis in experiment 5, IGF1 treatment of Huh7 cells induced the binding of WT CLOCK to PRPS1 / 2, but not the binding of CLOCK S106A or NES mutants to PRPS1 / 2;

[0030] Fig.11 For the in vitro acetylation assay in Experiment 6, purified GST-CLOCK acetylated purified His-PRPS1 / 2;

[0031] Fig.12 For the acetylation test in experiment 6, Huh7 cells were treated with IGF1 for 1 h to induce the acetylation of WT Flag- prps1 and WT Flag-prps2;

[0032] Fig.13 For experiment 6, immunoprecipitation and immunoblot analysis, PRPS1 / 2 K29 acetylation site;

[0033] Fig.14 For experiment 6, immunoprecipitation and immunoblot analysis were performed to analyze the results of recombinant expression of CLOCK (mutant A / B) mutants lacking endogenous CLOCK in Huh7 cells under IGF-1 induction;

[0034] Fig.15 For experiment 6, immunoprecipitation and immunoblot analysis, Huh7 cells recombinantly expressing CLOCK S106A or His-CLOCK NES mutant were subjected to immunoblot analysis under IGF-1 induction;

[0035] Fig.16 For experiment 7, immunoprecipitation and immunoblot analysis, cells were treated with IGF1 in a time-dependent manner, and the PRPS1 / 2 K29 acetylation level was analyzed;

[0036] Fig.17 For experiment 8, immunoprecipitation and immunoblot analysis, PRPS1 / 2 degradation, and analysis of the interaction between HSC70 and Flag-PRPS1 / 2 were performed;

[0037] Fig.18 Immunoprecipitation and immunoblot analysis for Experiment 8, Effect of PRPS1 / 2V30A / v1a mutation on PRPS1 / 2 degradation;

[0038] Fig.19 For test 9, GST pull-down assays showed that both purified WT GST- prps1 / 2 and GST- prps1 / 2 K29R mutant interacted with purified HSC70;

[0039] Fig. 20 For immunoprecipitation and immunoblot analysis in experiment 9, cells overexpressing RPS1 / 2 K29R were treated with IGF1;

[0040] Fig.21 For the experiment 10 luciferase reporter gene assay, IGF1 treatment increased glucose-derived 14C-RNA and 14C-DNA;

[0041] Fig. 22 Experiment 10 Luciferase reporter gene assay, overexpression of PRPS1 / 2 K29R inhibited IGF1- and IGF1Rca increased 14 c-rna 14 c-dna;

[0042] Fig.23 For the experiment 11, luciferase reporter gene assay was performed, and IGF1 treatment of Huh7 cells significantly increased the expression of 13C-labeled PRPP, purine (IMP, AMP, GMP) and pyrimidine (UMP and CMP) intermediates;

[0043] Fig.24 For experiment 11, luciferase reporter gene assay was performed to examine the expression of PRPP, purine (IMP, AMP, GMP), and pyrimidine (UMP and CMP) intermediates after overexpression of PRPS1 / 2 K29R;

[0044] Fig.25 For experiment 12, cell proliferation assay was performed by recombinantly expressing CLOCK S106A and CLOCK NES mutants or knocking in PRPS1 / 2K29R;

[0045] Fig.26 For the tumor size test results of experiment 13 mice, knock-in expression of CLOCK S106A or PRPS1 / 2 K29R inhibited orthotopic and subcutaneous tumor growth;

[0046] Fig. 27 The immunohistochemical results of the mouse tumor samples in Experiment 13;

[0047] Fig.28 The immunohistochemical results of the mouse tumor samples in Experiment 13;

[0048] Fig.29 Results of tumor size detection in mice in Experiment 14, ***P < 0.0001 by two-tailed Student’s t test;

[0049] Fig.30 Immunohistochemistry test of 14 human liver cancer samples (left) and the indicated IHC staining score statistics (right), ***P < 0.0001 by two-tailed Student's t test;

[0050] Fig.31 Survival curves of the expression levels of CLOCK S106 phosphorylation level and PRPS1 / 2K29 acetylation level in experiment 14. DETAILED DESCRIPTION

[0051] 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.

[0052] The tumor diagnostic product of the present invention is a kit, which contains reagents for detecting the expression levels of CLOCK S106 phosphorylation, PRPS1 K29 acetylation and PRPS2 K29 acetylation, each reagent includes a first antibody and a second antibody, the first antibody is a CLOCK S106 phosphorylation antibody CLOCK pS106, a PRPS1 K29 acetylation and PRPS2 K29 acetylation antibody PRPS1 / 2K29-Acetyl, and the second antibody is an antibody homologous to the first antibody and labeled with horseradish peroxidase.

[0053] The kit also includes a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant and a solvent. The instructions for use record how to use the kit for detection, and how to use the detection results to judge the development of the tumor and select a treatment plan. The components of the kit can be packaged in the form of an aqueous medium.

[0054] The kit can be used for the early, middle or late diagnosis of the following tumor diseases: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, genitourinary system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, biliary system cancer, pheochromocytoma, islet cell carcinoma, Leigh-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

[0055] The detection method of the kit is Western Blot.

[0056] Western Blot, or protein immunoblotting, is a technique commonly used to separate and identify proteins in research. It uses SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the various proteins contained in a specified sample, and then transfers the separated proteins to a nitrocellulose or PVDF membrane. Next, the membrane is incubated with specific antibodies against 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 fluorescent scanning. Since the antibody only binds to the target protein, generally only one clear band can be seen, and the thickness of the band corresponds to the amount of protein. By analyzing the location and intensity of specific reactions, 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. The specific detection steps are as follows:

[0057] 1. Prepare the sample

[0058] 1) Sample collection: Collect venous peripheral blood, place it on ice for 15 minutes, and centrifuge it at 4°C, 3500 rpm for 15 minutes to obtain serum for testing.

[0059] 2) Make ice and prepare ice boxes;

[0060] Prepare cell lysis buffer: Determine the required lysis buffer according to the number of cells: 50ul / well of a six-well plate;

[0061] Lysis buffer formula: 100ul cell lysis buffer + 0.1ul phosphatase inhibitor mixture + 1ul protease inhibitor mixture.

[0062] 3) Prepare cells according to experimental requirements: remove the culture medium, add 1×PBS to wash 3 times (remove the serum in the culture medium), add 50ul of lysis buffer containing protease / phosphatase inhibitors to each well (6-well plate). Quickly scrape the cells with a cell scraper and transfer them to a 1.5ml centrifuge tube, place on ice for 20 minutes, shake and mix, and place on ice for another 10 minutes.

[0063] 4) Centrifuge at 12,000 g for 15 min at 4°C and carefully collect the supernatant into another 1.5 ml tube.

[0064] 5) Take 2.5ul of sample and dilute it with 22.5ul of triple-distilled water for measuring protein concentration by BCA method.

[0065] 6) Add 5× Loading Buffer (2.5 ml Buffer / 10 ml protein) to the remaining samples and cook at 95°C for 10 minutes, shaking once during the cooking.

[0066] 7) Load the sample directly onto the gel or aliquot and store at -80°C for long term storage.

[0067] 2. SDS-PAGE polyacrylamide gel electrophoresis

[0068] 1) Prepare separation gel (6 ml / gel).

[0069] 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 30 minutes.

[0070] 3) Prepare stacking gel (2 ml / gel).

[0071] 4) Inject the concentrated gel into the upper end of the separation gel, taking care to avoid bubbles.

[0072] 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.

[0073] 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).

[0074] 7) Sample loading: Take 5ul prestained marker from the marker well and add an appropriate amount of 1× loading buffer to make the total volume the same as that of the sample well. The sample loading volume is generally 15-25μl. First, cook it in a heating block at 95℃ for 5-10 minutes, shake it once, then quickly centrifuge it and load it on the gel; add an equal volume of 1× loading buffer to the well without loading sample.

[0075] 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.

[0076] 3. Membrane transfer

[0077] 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.

[0078] 2) After marking the PVDF membrane, 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.

[0079] 3) Preparation of "Sandwich Cookies":

[0080] In the following order: fiber pad - filter paper - PVDF membrane - gel - filter paper - fiber pad;

[0081] NOTE: Align each item you add to ensure there are no bubbles.

[0082] 4) Transfer: The transfer time is determined according to 1).

[0083] One side of the PVDF membrane is connected to the positive electrode (red), and the other side of the gel is connected to the negative electrode (black).

[0084] 4. Membrane blocking and antibody incubation

[0085] 1) After transfer, wash the membrane with 10 ml of 1× TBS at room temperature for 10 minutes.

[0086] 2) Incubate 5 ml of 5% 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.

[0087] 3) Wash the membrane three times with 10 ml of TBS / T, 5 minutes each time.

[0088] 4) Add 5 ml of primary antibody dilution buffer (antibodies are diluted according to the instructions), gently shake 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 ul / ml primary antibody solution, and store at 4°C (uncommon antibodies can be stored at -20°C for a long time). It can be reused.

[0089] 5) Wash the membrane three times with 10 ml of TBS / T, 5 minutes each time.

[0090] 6) Add secondary antibody (usually 1:2000 dilution) and shake gently at room temperature for 1 hour.

[0091] 7) Wash the membrane three times with 10 ml of TBS / T, 5 minutes each time.

[0092] 5. Development and fixation (or direct fluorescence scanning after incubation with fluorescently labeled secondary antibodies)

[0093] 1) Color development steps: first lay out plastic wrap, then lay out plastic wrap on absorbent paper; pour water, developer (cannot be used if the color becomes darker) and fixer into their respective plates. Mix 2.5ml ECL-A and 2.5ml ECL-B, and protect from light. Take the ECL mixed solution, membrane, etc. into the darkroom. Close the door, lock it, and pull back the cloth. Pour the ECL mixed solution into a small box, slightly dry the membrane with absorbent paper, put it into the ECL mixed solution and shake it at room temperature for 5 minutes (so that the ECL is evenly spread on the membrane), dry it with absorbent paper, put it on the plastic wrap, wrap it face down and put it on the clip, cut the X-film (note that you can only hold the edge of the X-film with your hands) and put it on the membrane, and the corner side is the marker (note: when taking the film, turn the light to the minimum, and do not face the light, but face away from the light), adjust the exposure time according to the brightness of the strip, generally you can expose it for 2 minutes, observe the depth of the strip, and then determine the best exposure time.

[0094] 2) Development and fixing: Take out the X-film and place it in the developer for a certain period of time (depending on the intensity of the target band and the background), wash it with water once and then place it in the fixing solution for more than 5 minutes.

[0095] Finally, the target protein band is obtained on the PVDF membrane. By comparing with the size of the protein marker, the size and position of the target protein can be determined (the size of CLOCK protein is 100KD, and the size of PRPS1 / 2 protein is 35KD), and the expression result of the target protein can be obtained.

[0096] Diagnostic analysis of test results: If the following protein bands of corresponding sizes appear on the PVDF membrane: CLOCK S106 phosphorylation, PRPS1 K29 acetylation, or PRPS2 K29 acetylation, it indicates that the subject may have invasive cancer, be in the progressive stage of invasive cancer, or have a poor prognosis.

[0097] The biomarkers of the present invention are obtained through the following studies:

[0098] 1 Materials and Methods

[0099] 1.1 Materials used

[0100] (1) Cell type:

[0101] Huh7 cells (Human liver cancer cell line Huh7 cells) and Hep3B cells (Human liver cancer cell line Hep3B cells) were from ATCC;

[0102] (2) Athymic nude mice are BALB / c athymic nude mice;

[0103] (3) Patient samples:

[0104] Methods: Surgically resected, formalin-fixed, paraffin-embedded NSCLC tissue samples were retrospectively collected from the biobank of the Zhejiang University Translational Research Institute (Hangzhou, China). Tissue samples from 100 patients with pathologically diagnosed glioma who had not undergone surgical treatment were selected as an independent cohort. Clinical data were obtained by reviewing the patients' medical histories. Pathological staging was assessed by the 8th edition of the American Joint Committee on Cancer / Consortium for International Cancer Control TNM classification system.

[0105] (4) The shRNA sequences used for gene knockdown are as follows:

[0106] HSC70shRNA1: 5'-CCAAGACTTCTTCAATGGAAA-3';

[0107] HSC70 shRNA2: 5'- GCAACTGTTGAAGATGAGAAA-3';

[0108] LAMP2A shRNA1: 5'- GCTCAAGACTGCAGTGCAGAT-3';

[0109] LAMP2A shRNA2: 5'- GCAGTGCAGATGACGACAACT-3'.

[0110] BMAL1 shRNA1: 5'-CTTCTAGGCACATCGTGTTAT -3';

[0111] BMAL1 shRNA2: 5'-GCAGAATGTCATAGGCAAGTT-3'.

[0112] CK2α shRNA1: 5'- GCACAGAAAGCTACGACTAAT-3';

[0113] CK2α shRNA2: 5'- TGGACAAACTGCTGCGATATG-3'.

[0114] 1.2 Test methods

[0115] (1) Detection of phosphorylated protein levels:

[0116] By contacting the sample with an antibody that specifically binds to the phosphorylated polypeptide and determining the amount of bound antibody, for example by detecting or measuring the formation of a complex between the antibody and the polypeptide. The antibody may be labeled (radioactive, fluorescent, etc.) to facilitate detection of the complex.

[0117] The detection systems for the polypeptide levels of the present invention include: autoradiographic immunoassay RIA, immunofluorescence, gel electrophoresis, and Western immunoblotting.

[0118] Antibodies used (added): Antibodies against HA (ab18181) (for IP), c-SRC (ab16885), c-SRC pY418 (ab4816), CK2α pT360 / S362 (ab119410), CLOCK (ab3517) (for IHC), HSC70 (ab154415), IGF1R pY1161 (ab39398), and LAMP2A (ab18528) were purchased from Abcam. Antibodies against CLOCK (#5157) (WB), Exportin1 (#46249), AKT (#4685), AKT pS473 (#4060), acetylated lysine (#9441), HA (#3724) (WB), ERK1 / 2 (#4695), pERK1 / 2 (#4370), c-Jun (#9165), and c-Jun pS73 (#3270) were purchased from CST. Anti-pcna (#610665) antibody was purchased from BD Biosciences. BMAL1 (NBP2-02544) antibody was purchased from Novus Biologicals (Littleton, CO). Normal mouse IgG (sc-2025), normal rabbit IgG (sc-2027), GST (sc-138), CK2α (sc373894) (for WB), PRPS1 / 2 (sc-376440) (for IHC), PRPS1 / 2 (sc-100822) (for IP and WB), and tubulin (sc-8035) antibodies were obtained from SantaCruz Biotechnology. [γ-32P]ATP was from PerkinElmer. We obtained antibodies against p-catenin pS641 (GTX79145), α-catenin (GTX111095), and CLOCK (GTX134464) (for IF) from Genetex (Irvine, CA). CLOCK antibody (A302-617A) (for IP), CK2α antibody (A300-198A) (for WB and IP). CLOCK pS106 and PRPS1 / 2 k29ac-specific antibodies were produced by Signalway Biotechnology.

[0119] (2) Obtaining and analyzing mouse tumor samples:

[0120] Huh7 cells (1 × 10 6 ) were injected intracranially into athymic nude mice (n = 7 / group). Mice were euthanized 28 days after injection and examined for HCC tumor growth.

[0121] (3) Immunohistochemical IHC staining detection of proteins in cells (tissue microarray construction):

[0122] Formalin-fixed, paraffin-embedded tissues were obtained by surgical resection and stained with Mayer's hematoxylin and eosin (H&E; Biogenex Laboratories, San Ramon, CA).

[0123] Tissue microarray (TMA) was performed on tumor samples from cancer cases and normal tissue samples. Cancer tissue (2 mm in diameter, selected by a pathologist) was extracted from each specimen using an automated tissue array instrument (Minicore® 3, Alphelys, Plaisir, France) and fixed in paraffin blocks. After quality control, TMA blocks were cut into sections for immunohistochemical analysis.

[0124] Secondary anti-Rabbit IgG heavy chain (HRP) (ab99702) antibodies were purchased from Abcam (Cambridge, MA).

[0125] (4) Pearson correlation test

[0126] The present invention uses Pearson correlation test to perform correlation between protein expressions in human brain glioma specimens.

[0127] Immunohistochemical analysis was performed according to a previous publication (see Nucleus-Translocated ACSS2 Promotes Gene Transcription for Lysosomal Biogenesis and Autophagy. Molecular cell. 2017;66(5):684-97 e9). After deparaffinization, rehydration, and antigen retrieval, TMA slides were incubated with primary rabbit anti-IGF1R Tyr (Y) 1161 (dilution 1:200), primary rabbit anti-human phospho-CLOCK S106 (dilution 1:200), primary rabbit anti-human PRPS1 / 2 K29 acetylated antibody (dilution 1:500), or nonspecific IgG (as a negative control) at 4°C overnight. Slides were then incubated with anti-rabbit secondary antibody (ready-to-use solution; Cell Signaling Technology; #8114), followed by primary diaminobenzidine (DAB) staining (Cell Signaling Technology) and hematoxylin staining, and mounted in xylene. Tissue slides were quantitatively scored under a microscope based on the percentage of positive cells and staining intensity. The present invention assigns the following ratio scores: 0, 0% of cells are positive; 1, 0% to 1%; 2, 2% to 10%; 3, 11% to 30%; 4, 31% to 70%; and 5, from 71% to 100%. The staining intensity was also rated on a scale of 0 to 3: 0 (negative); 1, weak; 2, moderate; 3, strong. The ratio and intensity scores were then added to obtain a total score (range 0-8) as previously described in the literature. Two pathologists who were blinded to clinical information independently verified the reproducibility of the scoring system.

[0128] (5) Kaplan-Meier plot of overall survival rate of patients

[0129] Data analysis was performed using SPSS version 20.0 software (SPSS Inc., Chicago, IL, USA). The expression levels of biomarkers in tumor and normal tissues were compared using the independent sample t test. One-way analysis of variance (ANOVA, post hoc Bonferroni test) was used for multiple comparisons and the least significant difference test for the correlation between the expression levels of biomarkers and the clinicopathological characteristics of patients. The correlation between the expression levels of biomarkers was analyzed using the Pearson correlation coefficient. Overall survival (OS) was defined as the duration from the date of diagnosis to the date of death or the last follow-up. Survival analysis was performed using K-means cluster analysis to classify the expression levels of relevant markers, the Kaplan-Meier method to plot survival curves, the log-rank test to compare survival rates, and the Cox regression model with a two-way Wald test to calculate the hazard ratio (HR) and 95% confidence intervals (CIs). Censored data were used for patients who were alive at the last follow-up or were lost to follow-up. Variables with a P value of less than 0.05 in univariate analysis were included in the multivariate analysis. P < 0.05 was considered statistically significant. All statistical tests were two-sided.

[0130] 2 Results and analysis

[0131] 2.1 Experiment 1: IGF1 inhibits the formation of the CLOCK-bmal1 complex and suppresses the transcriptional activity of CLOCK.

[0132] (1) IGF1 inhibits the formation of CLOCK-bmal1 complex

[0133] Huh7 and Hep3B human hepatocellular carcinoma (HCC) cells were treated with insulin-like growth factor (IGF1) for 1 h. Figure 1 Co-immunoprecipitation analysis showed that IGF1 reduced CLOCK-BMAL1 heterodimerization.

[0134] (2) Reduced the expression of DBP and NR1D1 in liver cancer cells

[0135] like Figure 2 As shown, the results of luciferase reporter gene assay showed that IGF1 treatment inhibited the transcriptional activity of CLOCK and reduced the expression of CLOCK downstream genes DBP and NR1D1 in HCC cells.

[0136] 2.2 Experiment 2: CK2α phosphorylates CLOCK.

[0137] In vitro phosphorylation assay: Purified GST-CK2 was incubated with purified His-CLOCK for in vitro phosphorylation analysis to determine whether CK2 phosphorylates CLOCK ( Figure 3), this result was verified by mixing purified WT GST-CK2 or inactive GST-CK2 K68M with WT His-CLOCK or His-CLOCK S106A in the presence of [-P]ATP, indicating that only WT GST-CK2 phosphorylated WT His-CLOCK, but not His-CLOCK S106A ( Figure 4 ).

[0138] 2.3 Experiment 3: Mechanism of reduction of the CLOCK-bmal1 complex.

[0139] Purified His-BMAL1 bound to purified WT GST-CLOCK and GST-CLOCK S106A, whereas purified CK2 reduced the binding of His-BMAL1 to WT GST-CLOCK but not to GST-CLOCK S106A ( Figure 5 In addition, CLOCK S106A expressed in Huh7 cells was resistant to IGF-induced dissociation of CLOCK from BMAL1 ( Figure 6 ).

[0140] 2.4 Experiment 4: Phosphorylation of CLOCK S106 leads to cytoplasmic translocation of CLOCK.

[0141] Molecular dynamics (MD) simulations as well as immunofluorescence experiments revealed that mutations in the NES sequence reduced IGF-1-induced immunofluorescence showing CLOCK translocation from the nucleus to the cytoplasm, further supporting that IGF1-induced and CK2α-mediated phosphorylation of CLOCKS106 exposes its NES leading to subsequent CLOCK cytoplasmic translocation.

[0142] 2.5 Experiment 5: CLOCK interacts with PRPS1 / 2 in response to IGF1 stimulation.

[0143] IGF1 treatment of Huh7 cells induced the binding of WT CLOCK to PRPS1 / 2, but not the binding of CLOCK S106A or NES mutants to PRPS1 / 2.

[0144] 2.6 Experiment 6: Nuclear export CLOCK acetylates PRPS1 / 2 K29.

[0145] IGF1 treatment of Huh7 cells for 1 h induced the expression of WT Flag-prps1 ( Fig.11 Left) and WT Flag-prps2 ( Fig.11 , right) acetylation of PRPS1 / 2 K29R knock-in expression inhibits IGF1 treatment ( Fig.13) PRPS1 / 2 K29 acetylation. Recombinant expression of CLOCK mutants (mutants A / B) in Huh7 cells lacking endogenous CLOCK inhibited IGF1-induced K29 ​​acetylation of PRPS1 ( Fig.14 ). Recombinant expression of CLOCK S106A or His-CLOCK NES mutants, but not WT CLOCK, abolished K29 ​​acetylation of PRPS1 ( Fig.15 ).

[0146] 2.7 Experiment 7: IGF1 treatment increases PRPS1 / 2 K29 acetylation levels in a time-dependent manner.

[0147] IGF1 treatment increased PRPS1 / 2 K29 acetylation levels in a time-dependent manner, corresponding to the expression levels of PRPS1 / 2 ( Fig.16 ).

[0148] 2.8 Test 8: PRPS1 / 2 degradation.

[0149] PRPS1 / 2 K29 acetylation inhibits lysosome-dependent PRPS1 / 2 degradation ( Fig.17 ), co-immunoprecipitation analysis showed that HSC70 interacted with Flag-PRPS1 / 2 but not the HSC70 binding motif. Flag-PRPS1 / 2 V30A / v1a mutants with mutations in the binding motif of HSC70 had higher expression levels and longer half-life than the wild type. The PRPS1 / 2V30A / v1a mutation eliminated the effect of the PRPS1 / 2 K29R mutation on PRPS1 / 2 degradation ( Fig.18 ).

[0150] 2.9 Experiment 9: Intrinsic link between PRPS1 / 2 K29 acetylation and CMA-dependent PRPS1 / 2 degradation.

[0151] GST pull-down assay, results ( Fig.19 PRPS1 / 2 K29R expression abolished the association of HSC70 and LAMP-2A with PRPS1 and PRPS2 that was reduced by IGF1 treatment ( Fig. 20 ), indicating that IGF1R activation-induced and intracellular CLOCK-mediated PRPS1 / 2 acetylation inhibits HSC70 binding to PRPS1 / 2 and subsequent CMA / lysosome-dependent PRPS1 / 2 degradation.

[0152] 2.10 Experiment 10: IGF1R activation induces promotion of nucleic acid synthesis in human hepatoma cells.

[0153] In the luciferase reporter gene assay, Huh7 and Hep3B cells were incubated with a small amount of D-[6-14C]glucose (0.01mM), and it was found that IGF1 treatment increased glucose-derived 14C-RNA and 14C-DNA, and overexpression of PRPS1 / 2 K29R inhibited the increase of IGF1- and IGF1R-caused 14C-RNA and 14C-DNA.

[0154] 2.11 Experiment 11. IGF1R activation is mediated by cytoplasmic CLOCK, which promotes de novo nucleotide synthesis through PRPS1 / 2 stabilization.

[0155] IGF1 treatment in Huh7 cells significantly increased the amounts of 13C-labeled PRPP, purine (IMP, AMP, GMP) and pyrimidine (UMP and CMP) intermediates; overexpression of PRPS1 / 2 K29R inhibited the increase in the amounts of PRPP, purine (IMP, AMP, GMP) and pyrimidine (UMP and CMP) intermediates.

[0156] 2.12 Experiment 12: IGF1R activation induces and intracellular CLOCK-mediated PRPS1 / 2 acetylation promotes HCC cell proliferation.

[0157] Recombinant expression of CLOCK S106A and CLOCK NES mutants or knock-in expression of PRPS1 / 2 K29R inhibited cell proliferation.

[0158] 2.13 Experiment 13: CLOCK S106A and CLOCK NES mutants and knock-in expression of PRPS1 / 2 K29R inhibit the occurrence and development of tumors.

[0159] Huh7 cells expressing CLOCK S106A and CLOCK NES mutants and knock-in PRPS1 / 2 K29R were injected orthotopically / subcutaneously into athymic mice, and tumor size was measured 28 days later (e.g. Fig.29 ) Knock-in expression of LOCK S106A or PRPS1 / 2 K29R inhibited orthotopic and subcutaneous tumor growth.

[0160] 2.14 Experiment 14: CLOCK S106 phosphorylation and PRPS1 / 2 K29 acetylation glycosylation are associated with the prognosis of patients with liver cancer.

[0161] Immunohistochemistry was used to compare the levels of IGFR1p1161, CLOCKpS106, PRPS1 / 2, and PRPS1 / 2 K29 acetylation in clinical sample liver cancer tissues and adjacent normal tissues. We analyzed 90 human primary liver cancer and adjacent normal tissues using immunohistochemical staining of K29 acetylation using IGF1R Tyr (Y) 1161 antibody, CLOCK pS106 antibody, prps1 / 2 antibody, and anti-prps1 / 2 antibody. The results showed that the levels of IGF1R pY1161, CLOCKpS106, PRPS1 / 2 K29 acetylation, and PRPS1 / 2 K29 acetylation in the anti-prps1 / 2 antibody were significantly higher than those in the control group compared with adjacent normal tissues.

[0162] In 90 samples of liver cancer patients (73 males and 17 females), the IGF1R Tyr (Y) 1161 phosphorylation level, CLOCK S106 phosphorylation level and PRPS1 / 2 K29 acetylation level were divided into high expression and low expression in 60 samples of the patients, and the overall survival time Kaplan-Meier graph was drawn. The results are as follows Fig.31 As shown, the phosphorylation level of IGF1R Tyr (Y) 1161, the phosphorylation level of CLOCK S106, and the acetylation of K29 of PRPS1 / 2 were negatively correlated with the good prognosis of patients with liver cancer.

[0163] The study revealed that the activation of receptor tyrosine kinases disrupts the effects of CK2-dependent CLOCK S106 phosphorylation on the CLOCK / BMAL1 complex and its transcriptional activity. This phosphorylation leads to exportin1-mediated CLOCK nuclear export, which acetylates PRPS1 / 2 K29 in the cytoplasm and stabilizes the expression of PRPS1 / 2, thereby promoting de novo nucleotide synthesis and hepatocellular carcinoma (HCC) cell proliferation. This invention will provide new ideas and effective solutions for the development of targeted anti-tumor drugs.

Claims

1. Use of a reagent for detecting the expression level of a biomarker in the preparation of a product for predicting the prognosis of liver cancer, wherein the biomarker is one or more of CLOCK S106 phosphorylation, PRPS1 K29 acetylation, and PRPS2 K29 acetylation.

2. The use according to claim 1, characterized in that: The reagents include a primary antibody and a secondary antibody.

3. The use according to claim 2, characterized in that: The primary antibodies included the CLOCK S106 phosphorylation antibody CLOCK pS106, the PRPS1 K29 acetylation and PRPS2 K29 acetylation antibodies PRPS1 / 2 K29-Acetyl.

4. The use according to claim 3, characterized in that: The second antibody is homologous to the first antibody and is labeled with horseradish peroxidase.

5. The use according to claim 1, characterized in that: The detection method of the product is Elisa or Western Blot.

6. The use according to claim 1, characterized in that: The detection samples of the product are venous peripheral blood, paracancerous tissue or tumor tissue.

7. The use according to any one of claims 1 to 6, characterized in that: The products include kits.

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