Use of agents that knock down or inhibit pseudokinase trib3 in the manufacture of a medicament for treating multiple myeloma

Abstract

The application discloses application of a reagent for knocking down or inhibiting pseudokinase TRIB3 in preparation of an anti-multiple myeloma drug. Through a series of experiments, it is proved that knocking down TRIB3 has the activity of resisting multiple myeloma. Moreover, TRIB3 can promote bortezomib resistance, and targeting TRIB3 is expected to develop a new type of anti-tumor drug. Through analysis on a MM database, it is found that TRIB3 is highly expressed in MM, and the high expression is related to poor prognosis. Through in-vitro activity experiments on TRIB3, it is found that knocking down TRIB3 has significant anti-multiple myeloma activity. More importantly, it is also found in mice in-vivo that knocking down TRIB3 can significantly inhibit MM proliferation. Diagnosis of diseases caused by abnormal expression of TRIB3 protein, such as MM, is expected to be developed as a new type of anti-tumor targeted drug.

Description

Technical Field

[0001] This invention relates to novel uses of the pseudokinase TRIB3, specifically the application of reagents that knock down or inhibit the pseudokinase TRIB3 in the preparation of drugs for treating multiple myeloma. It belongs to the field of pharmaceutical technology. Background Technology

[0002] Protein kinases are important kinases that play a crucial role in cell signaling. They are considered a potential resource of drug targets, and small-molecule inhibitors targeting protein kinases have been successfully used in cancer treatment. Pseudokinases belong to the protein kinase superfamily and are structurally highly similar to their homokinase members. However, their catalytic domains have lost function during evolution, instead functioning by assembling large complexes or allosterically transmitting signals. The pseudoactive site structures on pseudokinases are diverse, thus small-molecule drugs targeting pseudokinases have higher specificity and a wider range of medicinal chemistry design possibilities.

[0003] The pseudokinase TRIB3 participates in chronic inflammation, metabolism, and malignant diseases through interactions with signaling and functional proteins. TRIB3 plays an important role in the development of various tumors, such as promoting the progression and drug resistance of non-small cell lung cancer through interaction with epidermal growth factor receptor (EGFR); promoting colorectal cancer stem cell characteristics and tumorigenesis through interaction with β-catenin and TCF4; and promoting the development of MYC-associated lymphoma by inhibiting UBE3B-regulated MYC degradation.

[0004] Hematologic malignancies are malignant tumors originating from the hematopoietic system. Among them, multiple myeloma (MM) is the second most common hematologic malignancy after leukemia. Its main characteristic is the clonal proliferation of plasma cells, which accumulate in the bone marrow and produce excessive amounts of monoclonal immunoglobulins. The excessive proliferation and drug resistance of malignant plasma cells play a crucial role in the development and relapse of MM, but the molecular mechanisms are not fully understood. In recent years, proteasome inhibitors, such as bortezomib, have significantly improved the prognosis of MM, but they can lead to treatment resistance in MM patients, posing a new challenge to MM treatment. Therefore, MM remains an incurable tumor. Elucidating the pathogenesis of MM and discovering new diagnostic and treatment strategies has important basic and clinical translational value for the prevention and treatment of MM. TRIB3, as a potential therapeutic target for tumors, has not yet been applied in multiple myeloma and has significant research value. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of reagents that knock down or inhibit pseudokinase TRIB3 in the preparation of anti-multiple myeloma drugs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] 1. Application of reagents for detecting the expression level of pseudokinase TRIB3 in the preparation of diagnostic test strips / kits for multiple myeloma.

[0008] 2. Application of reagents for detecting the expression level of pseudokinase TRIB3 in the preparation of test strips / kits for assessing bortezomib resistance in patients with multiple myeloma.

[0009] 3. Application of reagents that knock down or inhibit pseudokinase TRIB3 in the preparation of anti-multiple myeloma drugs.

[0010] Preferred use of agents that knock down or inhibit the pseudokinase TRIB3 in the preparation of anti-multiple myeloma drugs that reduce bortezomib resistance.

[0011] Preferred applications of agents that knock down or inhibit the pseudokinase TRIB3 in the preparation of drugs to improve the prognosis of multiple myeloma.

[0012] 4. Application of pseudokinase TRIB3 as a drug target in screening anti-multiple myeloma drugs.

[0013] 5. A composition comprising a reagent for detecting the expression level of the pseudokinase TRIB3.

[0014] 6. The use of the aforementioned composition in the preparation of diagnostic test strips / kits for multiple myeloma or in test strips / kits for assessing bortezomib resistance in patients with multiple myeloma.

[0015] 7. A diagnostic test strip / kit for multiple myeloma or a test strip / kit for assessing bortezomib resistance in patients with multiple myeloma, comprising a reagent for detecting the expression level of the pseudokinase TRIB3.

[0016] 8. A pharmaceutical composition comprising an agent that knocks down or inhibits the pseudokinase TRIB3.

[0017] 9. Application of the aforementioned pharmaceutical composition in the preparation of drugs for treating multiple myeloma.

[0018] 10. An anti-multiple myeloma drug comprising an agent that knocks down or inhibits the pseudokinase TRIB3.

[0019] The beneficial effects of this invention are:

[0020] This invention discloses a novel application of the pseudokinase TRIB3 in the clinical diagnosis of multiple myeloma. Through a series of experiments, this invention demonstrates that knocking down TRIB3 has anti-multiple myeloma activity. Furthermore, TRIB3 can promote bortezomib resistance, and targeting TRIB3 holds promise for developing novel anti-tumor drugs.

[0021] This invention, through analysis of the MM database, revealed that TRIB3 is highly expressed in MM, and this high expression is associated with poor prognosis. In vitro activity experiments on TRIB3 showed that knockdown of TRIB3 had significant anti-multiple myelopathy activity. More importantly, in mice, knockdown of TRIB3 also significantly inhibited MM proliferation. This technology has the potential to diagnose diseases caused by abnormal TRIB3 protein expression, such as MM, and may be developed into a novel targeted anti-tumor drug. Attached Figure Description

[0022] Figure 1 TRIB3 is highly expressed in MM patients and leads to poor prognosis. A. Analysis of pseudokinase expression in MM patients in GSE5900 and GSE2658: the top five molecules are the most significantly expressed high-expression pseudokinases in MM, and the bottom five molecules are the most significantly expressed low-expression pseudokinases in MM. B. Analysis of the relationship between pseudokinases TRIB3, JAK3, SCYL2, ROR2, and NRBP1 and patient prognosis in GSE2658. "High-risk factor" indicates low overall survival in MM patients with high expression of this gene; "protective factor" indicates high overall survival in MM patients with high expression of this gene. C. Analysis of TRIB3 in CD138+ cells of normal (n=22) and MM patients (n=559) in GSE5900 and GSE2658. D. Patients in GSE2658 were divided into high-expression and low-expression groups using X-title software, and then Kaplan-Meier survival analysis was performed. E. CD138+ cells from 10 healthy donors (HD) and 18 MM patients were detected by qPCR, and the expression of TRIB3 at the mRNA level was determined by qPCR. F. The expression level of TRIB3 protein was detected by Western Blot (WB) in CD138+ cells from 3 healthy donors (HD) and 4 MM cell lines.

[0023] Figure 2TRIB3 promotes MM cell proliferation at the cellular level. A. Knockdown of TRIB3 in ARP1, MM.1S, RPMI8226, and ANBL6 MM cells, with Western blotting to detect changes in TRIB3 protein levels. B. Knockdown of TRIB3 in ARP1, MM.1S, RPMI8226, and ANBL6 MM cells, with CCK8 assay to detect changes in cell colony-forming ability. C. Knockdown of TRIB3 in ARP1 and MM.1S MM cells, with soft agar colony-forming assay to detect changes in cell colony-forming ability. D. Overexpression of TRIB3 in ARP1, MM.1S, RPMI8226, and ANBL6 MM cells, with Western blotting to detect changes in TRIB3 protein levels. E. Overexpression of TRIB3 in ARP1, MM.1S, RPMI8226, and ANBL6 MM cells, with CCK8 assay to detect changes in cell viability. F. Overexpression of TRIB3 in ARP1 and MM.1S MM cells, with soft agar colony-forming assay to detect changes in cell colony-forming ability.

[0024] Figure 3 Knockdown of TRIB3 inhibits ARP1 cell tumorigenesis. A xenograft model in MM mice was established by intravenous injection of ARP1 cells carrying luciferase into the tail vein of immunodeficient NCG mice. A. After intraperitoneal injection of 100 mg / kg D-luciferin into each mouse, imaging was performed using a small animal imaging system 5 minutes after the luciferin substrate reacted with the luciferase in the mouse. B. Numerical analysis and statistical analysis of the fluorescence of each mouse were performed. C. The time of death for each mouse was recorded, and mouse survival curves were plotted. D. The weight change curve of the mice during the experiment was recorded.

[0025] Figure 4 TRIB3 promotes bortezomib resistance in MM. A. The expression levels of TRIB3, USP10, and SSRP1 proteins were detected by Western blotting in wild-type MM cells (ARP1, MM.1S) and BTZ-resistant cells (ARP1-BR, MM.1S-BR). B. TRIB3 was knocked down in BTZ-resistant cells (ARP1-BR and MM.1S-BR), followed by the addition of BTZ, and cell viability was assessed using CCK8 assay. C. TRIB3 was overexpressed in MM cells (ARP1 and MM.1S), followed by the addition of BTZ, and cell viability was assessed using CCK8 assay. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0027] Example 1:

[0028] Bioinformatics analysis was used to analyze TRIB3 expression in MM patients. MM patients were then divided into two groups based on TRIB3 expression differences: high and low expression, using X-title software. The changes in overall survival rates between the two groups were analyzed. Furthermore, using samples from healthy individuals and MM patients collected from Xiangya Second Hospital, the applicant analyzed TRIB3 expression at both the mRNA and protein levels using real-time quantitative PCR (qPCR) and Western blotting (WB).

[0029] 1. Using information from CD138+ cells in MM patients and normal individuals from GSE2658 and GSE5900, we analyzed the expression of TRIB3 in MM patients.

[0030] (1) Information on CD138+ cells of newly diagnosed MM patients in gene chips from GSE2658 (n=559) was obtained from the NCBI public database, and information on CD138+ cells of normal people in gene chips from GSE5900 (n=22) was obtained. The two information were then integrated.

[0031] (2) The relevant probes of 45 pseudokinases in GSE2658 and GSE5900 were analyzed, and the expression values ​​of the 45 pseudokinase probes were extracted. The expression values ​​of the 5 pseudokinases with the most significant high expression and the most significant low expression were analyzed and plotted as a heatmap.

[0032] (3) Select the five pseudokinases that are most significantly overexpressed, input the MM patient number, pseudokinase expression value, follow-up time and survival status into the X-title software, determine the optimal cut-off value of the pseudokinases, and divide them into two groups: high expression and low expression.

[0033] (4) Input the follow-up time and survival status of 5 patients with high and low pseudokinase expression in Graph Pad, analyze the changes in overall survival of the two groups of MM patients, calculate all P values ​​and plot them as a bar chart.

[0034] 2. Using X-title software, MM patients in the GSE2658 database were divided into two groups based on differences in TRIB3 expression: high TRIB3 expression and low TRIB3 expression. The changes in overall survival rates between the two groups of MM patients were analyzed.

[0035] (1) Input the MM patient number, TRIB3 expression value, follow-up time and survival status into the X-title software to determine the optimal cut-off value of TRIB3 and divide them into two groups: high TRIB3 expression and low TRIB3 expression.

[0036] (2) Enter the follow-up time and survival status of patients with high and low TRIB3 expression in Graph Pad to analyze the changes in overall survival of the two groups of MM patients.

[0037] 3. qPCR was used to detect TRIB3 expression in CD138+ cells of normal individuals and MM patients.

[0038] (1) Collect CD138+ cells from normal individuals and CD138+ cells from MM patients.

[0039] (2) RNA extraction: Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL TRIzol (from ThermoFisher Scientific) to the cell pellet, mix thoroughly, and place on ice for 10 min to allow the cells to completely dissolve in TRIzol.

[0040] (3) Add 300 μL of chloroform to TRIzol, invert to mix, and let stand at room temperature for 15 min. Transfer the colorless and transparent upper aqueous phase to a new 1.5 mL centrifuge tube. Add an equal volume of isopropanol to the upper aqueous phase, invert to mix, and let stand at room temperature for 15 min. Centrifuge at 12000 rpm and 4℃ for 10 min, and discard the supernatant.

[0041] (4) Add 1 mL of 75% ethanol solution to the cell pellet, centrifuge at 8000 rpm and 4℃ for 5 min, and discard the supernatant.

[0042] (5) After filtering, dissolve in DEPC water and measure the RNA concentration.

[0043] (6) Reverse transcription: RNA was reverse transcribed using a reverse transcription kit (Nanjing Novizan) to obtain cDNA.

[0044] (7) qPCR: Prepare a 10 μL qPCR reaction system, including 3 μL cDNA, 5 μL 2×SYBR Green Mix, 1 μL forward primer and 1 μL reverse primer (Nanjing Novizan qPCR kit), and perform qPCR experiments under the following reaction conditions:

[0045] 95℃ for 10 minutes; 95℃ for 20 seconds, 60℃ for 20 seconds, 70℃ for 20 seconds, 40 cycles.

[0046] The qPCR primer sequences are as follows:

[0047] GAPDH:

[0048] Forward primer: 5'-CGCTGAGTACGTCGTGGAGTC-3', as shown in SEQ ID NO.1;

[0049] Reverse primer: 5'-GCTGATGATCTTGAGGCTGTTGTC-3', as shown in SEQ ID NO.2;

[0050] TRIB3

[0051] Forward primer: 5'-TCAAGCTGTGTCGCTTTGTC-3', as shown in SEQ ID NO.3;

[0052] Reverse primer: 5'-TGTCCCACAGGGAATCATCT-3', as shown in SEQ ID NO.4;

[0053] 4. Western blot analysis was performed to detect the difference in TRIB3 expression between normal human CD138+ cells and MM cell lines.

[0054] (1) Collect 2×10 6 A number of normal human CD138+ cells and MM cells (ARP1, MM.1S, RPMI8226, and ANBL6) in the logarithmic growth phase (from Tianjin Institute of Hematology).

[0055] (2) Extraction of total cellular protein: Collect cells and centrifuge at 800 rpm and 4°C for 5 min. Add RIRA lysis buffer containing 2% (v / v) protease inhibitor cocktail and 2% (v / v) phosphatase inhibitor, mix the cells and cell lysis buffer thoroughly, and incubate on ice for 30 min. Sonicate 20 times. Centrifuge at 13800 rpm and 4°C for 15 min, and collect the supernatant.

[0056] (3) BCA method for determining protein concentration

[0057] (4) Western Blot protein separation: The protein sample was boiled and denatured for 5 minutes, separated by 10% SDS-PAGE, transferred to an NC membrane, and then blocked at room temperature for 2 hours with blocking buffer (PBST solution (pH 7.4, 0.01 mol / L) in 5% skim milk). TRIB3 primary antibody (from Abcam) was added and incubated for 12 hours, followed by 3 washes with PBST. Then, the sample was incubated at room temperature for 2 hours with secondary antibody diluted 10,000 times (from Jackson Laboratory), followed by 3 washes with PBST.

[0058] (5) The expression changes of protein TRIB3 were detected by ECL imaging and gel imaging system.

[0059] Experimental results are as follows Figure 1Through a meta-analysis of expression values ​​of 45 pseudokinases in myeloma data, the applicant identified five significantly overexpressed pseudokinases: TRIB3, SCYL2, JAK3, NRBP1, and ROR2, and five significantly underexpressed pseudokinases: ROR1, PTK7, EPHB6, POMK, and KSR2. Figure 1 (A). Further prognostic analysis of five significantly overexpressed pseudokinases—TRIB3, SCYL2, JAK3, NRBP1, and ROR2—revealed that TRIB3 and JAK3 are high-risk factors in MM, meaning patients with high TRIB3 and JAK3 expression have lower overall survival. TRIB3 is a greater risk factor than JAK3, indicating a more severe risk factor; therefore, we hypothesize that TRIB3 plays a more important role in the progression of MM. SCYL2, NRBP1, and ROR2 are all protective factors; patients with high SCYL2, NRBP1, and ROR2 expression have higher overall survival. Figure 1 (Middle B). Compared with normal human CD138+ cells, the expression level of TRIB3 in CD138+ cells of MM patients is higher ( Figure 1 In addition, MM patients with high TRIB3 expression had a lower overall survival rate. Figure 1 (D). In the MM samples and cell lines collected by the applicant, TRIB3 also showed high expression at both the mRNA and protein levels. Figure 1 (E, F)

[0060] Example 2:

[0061] Silencing TRIB3 expression inhibits the growth and colony formation ability of MM cells, while overexpression of TRIB3 promotes the growth and colony formation ability of MM cells.

[0062] After lentiviral knockdown and overexpression of TRIB3, Western blot was used to detect changes in TRIB3 protein levels in MM cells, CCK8 assay was used to detect changes in MM cell survival, and soft agar colony formation assay was used to detect changes in MM cell colony formation ability.

[0063] 1. After adding TRIB3 shRNA interference, Western blot was used to detect changes in the protein level of TRIB3 in ARP1, MM.1S, RPMI8226 and ANBL6 cells.

[0064] (1) Interference with TRIB3 expression: Cells in the logarithmic growth phase were mixed by pipetting in a culture dish, and 0.5 ml of cell suspension was transferred to four wells of a six-well plate, labeled as shNC group (control group) and sh1, sh2 group (knockdown groups). shRNA was added to the knockdown groups, and an auxiliary well was set up. 1.5 ml of RPMI-1640 medium was added to each well containing cells, and the plates were cultured for 24 h. The medium was then changed, and 2 ml of complete medium (RPMI-1640 medium (from Hyclone) + 10% fetal bovine serum (from Vivacell)) was added to each well for further culture.

[0065] (2) Extract total cell protein, using the same method as described above.

[0066] (3) Protein concentration was determined by the BCA method, which is the same as the method described above.

[0067] (4) Western blotting is used to separate proteins, in the same way as described above.

[0068] (5) Develop with ECL, in the same way as above.

[0069] 2. After adding shRNA to reduce TRIB3 expression, the changes in ARP1, MM.1S, RPMI8226 and ANBL6 cell survival were detected by CCK8 assay.

[0070] (1) Interfere with TRIB3 expression, using the same method as described above.

[0071] (2) Resuspend the cells in a 15 mL centrifuge tube and mix them thoroughly. Dilute the cells 10 times in a 1.5 mL centrifuge tube and calculate the cell density using a cell counting chamber.

[0072] (3) Add 1×10 to the 96-well plate 4 Cells were cultured for 2 days, then incubated with 10% CCK8 solution for 3 hours, and the OD value was detected at a wavelength of 450 nm.

[0073] 3. After adding shRNA to reduce TRIB3 expression, the changes in the colony-forming ability of ARP1 and MM.1S cells were detected by soft agar colony formation assay.

[0074] (1) Interfere with TRIB3 expression, using the same method as described above.

[0075] Prepare 3.5% and 1.66% agarose solutions respectively, autoclave, and let stand at room temperature until ready for use.

[0076] (2) Melt the agarose prepared in step 1 using a microwave oven. Place 3.5% and 1.66% agarose in water baths at 42°C and 37°C respectively. At the same time, place RPMI-1640 medium (from Hyclone) containing 20% ​​fetal bovine serum and 1% penicillin / streptomycin in a water bath at 42°C.

[0077] (3) Preparation of the lower gel: Mix 3.5% agarose and the above complete culture medium at a volume ratio of 1:5. Note that when adding agarose to the culture medium, avoid generating air bubbles during mixing. After mixing, immediately add the mixture dropwise to the 12-well plate (drop from the center of the well), 1 ml / well, and let it stand at room temperature for 10 minutes.

[0078] (4) Preparation of the upper gel: Dilute the cells in the culture medium at a certain density, then mix 1.66% agarose and the cell-containing culture medium at a volume ratio of 1:5. Note that the agarose should be added to the culture medium. After mixing thoroughly, add 0.5 mL of the mixture dropwise onto the lower gel, being careful not to generate air bubbles. Let it stand at room temperature for 5 minutes, then transfer the 12-well plate to a cell culture incubator for incubation. 12-well plate: 3000 cells / well.

[0079] (5) Add a drop of fresh culture medium to the well every 2-3 days, take a picture after 2-3 weeks, and calculate the cloning rate.

[0080] 4. Add TRIB3-overexpressing lentivirus and detect changes in TRIB3 protein levels in ARP1, MM.1S, RPMI8226 and ANBL6 cells by Western blotting.

[0081] (1) TRIB3 overexpression: Cells in the logarithmic growth phase in the culture dish were mixed by pipetting, and 0.5 ml of cell suspension was transferred to two wells of a six-well plate, labeled as EV group (control group) and OE TRIB3 group (overexpression group). 1.5 ml of 1640 medium was added to each well of the six-well plate containing cells, and the plates were cultured for 24 h.

[0082] (2) Change the medium and add 2 mL of complete culture medium to each well for incubation.

[0083] (3) Extract total cell protein, using the same method as described above.

[0084] (4) Protein concentration was determined by the BCA method, which is the same as the method described above.

[0085] (5) Western blotting is used to separate proteins, in the same way as described above.

[0086] (6) Develop with ECL, in the same way as above.

[0087] 5. Add TRIB3 overexpressing lentivirus, and use CCK8 assay to detect changes in the survival of ARP1, MM.1S, RPMI8226 and ANBL6 cells.

[0088] (1) Overexpression of TRIB3, the same method as above.

[0089] (2) Resuspend the cells in a 15 mL centrifuge tube and mix them thoroughly. Dilute the cells 10 times in a 1.5 mL centrifuge tube and calculate the cell density using a cell counting chamber.

[0090] (3) Add 1×10 to the 96-well plate 4 Cells were cultured for 2 days, then incubated with 10% CCK8 solution for 3 hours, and the OD value was detected at a wavelength of 450 nm.

[0091] 6. The changes in the colony-forming ability of ARP1 and MM.1S cells were detected by adding TRIB3-overexpressing lentivirus and performing a soft agar colony formation assay.

[0092] (1) Overexpression of TRIB3, using the same method as described above.

[0093] (2) Prepare 3.5% and 1.66% agarose solutions by autoclaving and place them at room temperature for later use.

[0094] (3) Melt the agarose prepared in step 1 using a microwave oven. Place 3.5% and 1.66% agarose in water baths at 42°C and 37°C respectively. At the same time, place RPMI-1640 medium containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin in a water bath at 42°C.

[0095] (4) Preparation of the lower gel: Mix 3.5% agarose and culture medium at a volume ratio of 1:5. Note that when adding agarose to the culture medium, avoid generating air bubbles during mixing. Immediately after mixing, drop the mixture into the 12-well plate (dropping from the center of the well), 1 ml / well, and let it stand at room temperature for 10 minutes.

[0096] (5) Preparation of the upper gel: Dilute the cells in the culture medium at a certain density, then mix 1.66% agarose and the cell-containing culture medium at a volume ratio of 1:5. Note that the agarose should be added to the culture medium. After mixing thoroughly, add 0.5 mL of the mixture dropwise onto the lower gel, being careful not to generate air bubbles. Let it stand at room temperature for 5 minutes, then transfer the 12-well plate to a cell culture incubator for incubation. 12-well plate: 3000 cells / well.

[0097] (6) Add a drop of fresh culture medium to the well every 2-3 days, take a picture after 2-3 weeks, and calculate the cloning rate.

[0098] Experimental results are as follows Figure 2 As shown, A represents the changes in TRIB3 protein levels in ARP1, MM.1S, RPMI8226, and ANBL6 cells detected by Western blotting after TRIB3 shRNA interference. GAPDH was used as an internal control (glyceraldehyde-3-phosphate dehydrogenase). B represents the changes in cell growth in ARP1, MM.1S, RPMI8226, and ANBL6 cells detected by CCK8 assay after TRIB3 shRNA interference. C represents the changes in colony-forming ability of ARP1 and MM.1S cells detected by soft agar colony formation assay after TRIB3 shRNA interference. *p<0.05, **p<0.01, ***p<0.001. Interference with TRIB3 expression significantly decreased TRIB3 protein expression, significantly inhibited MM cell growth, and significantly suppressed colony-forming ability. D represents the changes in TRIB3 protein levels in ARP1, MM.1S, RPMI8226, and ANBL6 cells detected by Western blotting after the addition of TRIB3 overexpression lentivirus. GAPDH, glyceraldehyde-3-phosphate dehydrogenase, was used as an internal control. E represents the changes in the growth of ARP1, MM.1S, RPMI8226, and ANBL6 cells detected by CCK8 assay after the addition of TTRIB3-overexpressing lentivirus. F represents the changes in the colony-forming ability of ARP1 and MM.1S cells detected by soft agar colony formation assay after the addition of TRIB3-overexpressing lentivirus. *p<0.05, **p<0.01, ***p<0.001. Overexpression of TRIB3 significantly increased TRIB3 protein expression, enhanced MM cell proliferation, and improved colony-forming ability.

[0099] Example 3:

[0100] (1) Collect ARP1 cells with luciferase and ARP1 cells with luciferase after TRIB3 knockdown using 1640 medium, and adjust the concentration to 200 μl containing 1×10⁻⁶ cells / mL. 6 Cell sap of ARP1 cells.

[0101] (2) Select 30 6-week-old NCG (full name NOD / ShiLtJGpt-Prkdc) em26Cd52 Il2rg em26Cd22 / Gpt) mice (from Jicui Yaokang), weighing between 18-22g. The cells were mixed in a 1.5mL centrifuge tube, and 100μl of cell solution was drawn into a 1mL syringe and inoculated into the tail vein of NCG mice.

[0102] (3) Thirty NCG mice were randomly divided into three groups of 10 mice each.

[0103] (4) Record the tumor volume and weight of each mouse once a day.

[0104] (5) On days 18 and 27, each mouse was injected intraperitoneally with 100 mg / kg of D-fluorescein (from MCE), and the mice were photographed 5 minutes later using a small animal imaging device.

[0105] (6) Euthanize the mice and plot the survival curve and the mouse weight change curve.

[0106] Experimental results are as follows Figure 3 As shown in the figure, A represents images taken using a small animal imaging system after intraperitoneal injection of 100 mg / kg D-fluorescein into each mouse; B represents the statistical analysis of graph A; C represents the mouse survival curve; and D represents the mouse body weight change curve. Mice with TRIB3 knockdown showed significantly lower fluorescence imaging intensity than the control group, and their survival time was longer. Furthermore, the body weight of mice with TRIB3 knockdown did not change significantly, indicating that TRIB3 knockdown had no significant side effects on mice. These results demonstrate that TRIB3 knockdown significantly inhibits the development of multiple myeloma (MM) both in vivo and in vitro.

[0107] Example 4:

[0108] Differences in TRIB3 protein expression in wild-type MM cell lines ARP1 and MM.1S, and BTZ-resistant cell lines ARP1-BR and MM.1S-BR were detected by Western blotting. After knocking down TRIB3 in ARP1-BR and MM.1S-BR cells and then adding BTZ, changes in cellular sensitivity to BTZ were detected using the CCK8 assay. Furthermore, after overexpressing TRIB3 in ARP1 and MM.1S cells and then adding BTZ, changes in cellular sensitivity to BTZ were detected using the CCK8 assay.

[0109] 1. Western blot analysis was performed to detect the differences in TRIB3 protein expression in wild-type MM cell lines ARP1 and MM.1S and BTZ-resistant cell lines ARP1-BR and MM.1S-BR.

[0110] (1) Collect 2×10 6 ARP1, MM.1S and BTZ resistant cell lines ARP1-BR, MM.1S-BR cells.

[0111] (2) Extract total cell protein, using the same method as described above.

[0112] (3) Protein concentration was determined by the BCA method, which is the same as the method described above.

[0113] (4) Western blotting is used to separate proteins, in the same way as described above.

[0114] (5) Develop with ECL, in the same way as above.

[0115] 2. After knocking down TRIB3 in ARP1-BR and MM.1S-BR cells, BTZ was added, and the changes in cell sensitivity to BTZ were detected by CCK8 assay.

[0116] (1) Add TRIB3 shRNA lentivirus to ARP1-BR and MM.1S-BR.

[0117] (2) Resuspend the cells in a 15 mL centrifuge tube and mix them thoroughly. Dilute the cells 10 times in a 1.5 mL centrifuge tube and calculate the cell density using a cell counting chamber.

[0118] (3) Add 1×10 to the 96-well plate 4 10 cells were treated with BTZ at concentrations of 0, 5, and 10 nM, respectively.

[0119] (4) After culturing for 2 days, add 10% CCK8 and incubate for 3 hours. Detect the OD value at a wavelength of 450 nm.

[0120] 3. After overexpressing TRIB3 in ARP1 and MM.1S cells, BTZ was added, and the changes in cell sensitivity to BTZ were detected by CCK8 assay.

[0121] (1) Overexpression of TRIB3, the same method as above.

[0122] (2) Resuspend the cells in a 15 mL centrifuge tube and mix them thoroughly. Dilute the cells 10 times in a 1.5 mL centrifuge tube and calculate the cell density using a cell counting chamber.

[0123] (3) Add 1×10 to the 96-well plate 4 10 cells were treated with BTZ at concentrations of 0, 5, and 10 nM, respectively.

[0124] (4) After culturing for 2 days, add 10% CCK8 and incubate for 3 hours. Detect the OD value at a wavelength of 450 nm.

[0125] Experimental results are as follows Figure 4 As shown, TRIB3 protein is highly expressed in BTZ-resistant strains ARP1-BR and MM.1S-BR, suggesting that TRIB3 may be associated with BTZ resistance. Figure 4 (A). Furthermore, knockdown of TRIB3 enhanced the sensitivity of ARP1-BR and MM.1S-BR cells to BTZ. Figure 4 In the middle B), while after overexpression of TRIB3, the resistance of ARP1 and MM.1S cells to BTZ was enhanced. Figure 4 (C) This indicates that TRIB3 promotes BTZ resistance, and TRIB3 is expected to become one of the clinical indicators for assessing BTZ resistance in MM patients.

[0126] While the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1. Application of reagents for detecting the expression level of pseudokinase TRIB3 in the preparation of diagnostic test strips / kits for multiple myeloma.

2. Application of reagents for detecting the expression level of pseudokinase TRIB3 in the preparation of test strips / kits for assessing bortezomib resistance in patients with multiple myeloma.

3. The use of the composition in the preparation of diagnostic test strips / kits for multiple myeloma or in the preparation of test strips / kits for assessing bortezomib resistance in patients with multiple myeloma, characterized in that, The composition contains a reagent for detecting the expression level of the pseudokinase TRIB3.

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