A treatment target tRF-18-YR66EFD2 for multiple myeloma and its applications

Abstract

The present invention discloses a multiple myeloma treatment target tRF-18-YR66EFD2 and its application. Based on the previous next-generation sequencing results, the present invention further studies and discovers that specifically targeting and degrading the highly expressed tRF-18-YR66EFD2 molecule in relapsed and refractory MM (R / RMM) leads to effective killing of MM cells, and this research provides a new theoretical basis for the treatment of MM.

Description

Technical Field

[0001] This invention belongs to the field of tumor molecular biology technology, specifically relating to a therapeutic target for multiple myeloma, tRF-18-YR66EFD2, and its application. Background Technology

[0002] Multiple myeloma (MM) is the second most common hematologic malignancy caused by the abnormal proliferation of clonal plasma cells in the bone marrow and their secretion of monoclonal globulins. With advancements in medical technology and the introduction and continuous updating of molecular, genetic, and cellular techniques, preclinical research has greatly advanced our understanding of the pathogenesis of MM over the past two decades. Functionally, we now know of the accumulation of genomic alterations within tumor cells, MM-specific defects (i.e., tumor cells' high dependence on the ubiquitin-proteasome system), and MM-induced changes in the bone marrow microenvironment. These abnormalities can induce tumor cell proliferation, survival, migration, drug resistance, osteolytic destruction, angiogenesis, and immunosuppression.

[0003] Based on breakthroughs in pathogenesis research, clinical treatment strategies for multiple myeloma (MM) have made significant progress. Prior to the 1960s, MM treatment aimed to alleviate symptoms rather than control the disease. Since the initial use of alkylating agents and corticosteroids in the 1960s, MM treatment strategies have gradually evolved. By the late 20th century, high-dose melphalan combined with autologous stem cell transplantation further improved patient survival. In the past decade, proteasome inhibitors and immunomodulatory drugs have become the cornerstone of MM treatment. Currently, FDA-approved anti-MM drugs include proteasome inhibitors (bortezomib, carfilzomib, and ixazomib), immunomodulators (thalidomide, lenalidomide, and pomalidomide), histone deacetylase (HDAC) inhibitors (pabistat), alkylating agents (melphalan, cyclophosphamide, and bendamustine), corticosteroids (dexamethasone and prednisone), and monoclonal antibodies (raptumab and erlotuzumab). The new treatment model has greatly improved the quality of life and survival time of MM patients. According to statistics, the total survival time of MM patients has now been extended by 6-10 years compared to the past.

[0004] However, multifocal malignancy (MM) remains an incurable disease, and patients inevitably face relapse and drug resistance over time. Therefore, new and more effective therapeutic targets are needed. tsRNA (tRNA-derived small RNA) is a small RNA fragment derived from tRNA, essentially a small non-coding RNA. Therefore, corresponding mimics or antisense molecules can antagonize its function and offer the possibility of acting as small molecule drugs. This also makes some tsRNAs have great potential as therapeutic targets for hematologic malignancies. In MM, a few studies have shown the significance of bone marrow tsRNA in predicting the risk of progression from smoking MM to MM and in prognostic stratification of MM patients, but the therapeutic value of tsRNA in MM remains unclear. Summary of the Invention

[0005] The primary objective of this invention is to provide a bone marrow tsRNA molecule, tRF-18-YR66EFD2, with the sequence: TTCCCGGCCAACGCACCA. This molecule shows great potential in the diagnosis and treatment of multiple myeloma.

[0006] A second objective of this invention is to provide the use of the reagent that inhibits the expression of tRF-18-YR66EFD2 in the preparation of a therapeutic agent for multiple myeloma.

[0007] Agents that inhibit tRF-18-YR66EFD2 expression include tRF-18-YR66EFD2 inhibitors.

[0008] The sequence of the inhibitor is: TGGTGCGTTGGCCGGGA.

[0009] A third objective of this invention is to provide the use of a reagent for detecting the expression of tRF-18-YR66EFD2 in the preparation of diagnostic or prognostic agents for multiple myeloma.

[0010] Specifically, it is used for the diagnosis or prognosis of relapsed / refractory multiple myeloma (R / RMM).

[0011] Reagents for detecting tRF-18-YR66EFD2 expression include PCR detection reagents.

[0012] The primer sequences in the PCR detection reagent are as follows:

[0013] F:5'-GCTAAGGAAGTCCTGTGCT-3',

[0014] R:5'-GTCCAGTTTTTTTTTTTTTTAAAACTGA-3'.

[0015] Based on previous next-generation sequencing results, this invention further discovered that the tRF-18-YR66EFD2 molecule, which is highly expressed in relapsed / refractory MM (R / RMM), is specifically targeted and degraded, leading to effective killing of MM cells. This invention provides a new theoretical basis for the treatment of MM. Attached Figure Description

[0016] Figure 1 :tRF-18-YR66EFD2 is expressed in R / RMM;

[0017] Figure 2 : In MM cells, tRF-18-YR66EFD2 expression is regulated by siRNA (i.e., inhibitor);

[0018] Figure 3 Effect of tRF-18-YR66EFD2 on ARP1 cell proliferation;

[0019] Figure 4 Effect of tRF-18-YR66EFD2 overexpression on MM.1S cell proliferation;

[0020] Figure 5 The effect of tRF-18-YR66EFD2 on ARP1 cell apoptosis;

[0021] Figure 6 Effect of tRF-18-YR66EFD2 overexpression on apoptosis in MM.1S cells;

[0022] Figure 7 Effects of tRF-18-YR66EFD2 on VEGF protein expression in MM cells. Detailed Implementation

[0023] The following examples are intended to further illustrate the present invention, but not to limit it.

[0024] method

[0025] 1. Clinical Samples

[0026] This invention selected 52 patients with relapsed / refractory multiple myeloma (R / RMM) and 48 patients with non-relapsed multiple myeloma (NDMM) from Xiangya Third Hospital of Central South University between March 2016 and March 2020. The diagnosis of multiple myeloma was based on the diagnostic criteria for symptomatic multiple myeloma defined by the National Comprehensive Cancer Network (NCCN). Relevant clinical information of the subjects was collected, and bone marrow samples were collected for tsRNA extraction.

[0027] 2. Cell Culture

[0028] Both MM.1S and ARP1 myeloma cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai). Both were suspension cells, cultured and passaged in RPMI 1640 medium containing 10% FBS and 1% penicillin antibiotics at 37°C in a 5% CO2 incubator, with medium changed every other day, at a density of approximately 0.2-0.5 × 10⁻⁶ cells / day. 6 / mL, cell count was performed using trypan blue staining.

[0029] 3. RNA extraction

[0030] According to the kit procedure, RNA was extracted from bone marrow specimens using TRIzol (Invitrogen, USA). The specific procedure was as follows: (1) Place the specimen on ice, add Trizol to lyse for 5-10 minutes, gently blow with a pipette tip and then draw the liquid into an EP tube, add 1 / 5 volume of chloroform, mix the liquid up and down, and let stand at 4°C for 10-15 minutes.

[0031] (2) Centrifuge at 4℃ for 15 minutes. After centrifugation, the liquid will separate into three layers. Gently remove the EP tube from the centrifuge, aspirate the supernatant, and avoid aspirating the lower sediment. Place the liquid in a new EP tube.

[0032] (3) Add isopropanol at 0.5 ml isopropanol / ml Trizol, let stand for 10 min, then centrifuge at 12000 rpm at 4℃ for 10 min, discard the supernatant, add 75% ethanol at 1 ml 75% ethanol / ml Trizol, centrifuge at 4℃ for 5 min, and discard the supernatant.

[0033] (4) Place the EP tube back into the centrifuge for a brief centrifugation, discarding any residual liquid on the tube wall. Then place the EP tube in a clean bench to dry for 5-10 minutes. Add 50 μL of DEPC-treated water and shake thoroughly to dissolve the precipitate.

[0034] (5) RNA concentration and activity were determined using a NanoDrop ND-1000 (NanoDrop, USA), and the purity and integrity of RNA were then detected by formaldehyde denaturing agarose gel electrophoresis. The extracted RNA was stored at -80℃.

[0035] 4. RNA interference

[0036] (1) Prepare the transfection complex

[0037] First, place the GP-transfect-Mate transfection reagent at room temperature and shake well. Then, add 500 μL of serum-free culture medium and an appropriate amount of GP-transfect-Mate transfection reagent to a 1.5 mL sterile centrifuge tube and mix well. Let it stand at room temperature for 5 min. Simultaneously, add 500 μL of serum-free culture medium and an appropriate amount of tRF-18-YR66EFD2 mimics (5'-UUCCCGGCCAACGCACCA-3') or inhibitor (5'-TGGTGCGTTGGCCGGGA-3') or blank control to a new 1.5 mL centrifuge tube and mix well. Let it stand at room temperature for 5 min. Mix the two solutions thoroughly and let it stand at room temperature for 15-20 min before starting transfection.

[0038] (2) Cell transfection

[0039] While the transfection complex is standing, take 1-2 × 10 6 Cells were resuspended in a 60 mm culture dish to 4 mL; 1 mL of the transfection complex was added to the culture dish, bringing the final volume to 5 mL. The culture dish was then gently shaken to mix the complex. Cells were cultured at 37°C for 4-6 hours, and then replaced with complete culture medium. Cells were used for subsequent functional experiments after 24 hours.

[0040] 5. Western blot

[0041] (1) Protein collection

[0042] Collect the cell samples to be tested, centrifuge at 300g for 5 min, aspirate the culture medium, then add 4℃ pre-cooled PBS to wash the samples, centrifuge at 300g for 5 min, and discard the supernatant; add 1×10 6 Add lysis buffer at a ratio of 100 μL cells / 100 μL lysis buffer, vortex, and lyse on ice for 30 min, vortexing once every 10 min. After lysis, collect protein by centrifugation at 26000g for 15 min at 4℃, and determine protein concentration using Nanodrop.

[0043] (2) Protein denaturation

[0044] Calculate the volume of protein required for loading, add loading buffer at a ratio of protein:5×SDS-PAGE loading buffer = 4:1, mix well, incubate at 95℃ for 5 minutes, cool to room temperature, and prepare for electrophoresis.

[0045] (3) Protein electrophoresis and transduction

[0046] Place the pre-prepared SDS-PAGE gel into the electrophoresis tank, add sufficient 1× electrophoresis buffer, add the sample, and then start electrophoresis at 80V for 30 minutes. After the bromophenol blue reaches the separating gel, adjust the voltage to 120V and stop electrophoresis when the bromophenol blue just runs out. Then proceed to the next step of membrane transfer. Prepare the transfer buffer in advance, pre-cool it to -20℃, pour it into a tray, peel the gel from the electrophoresis apparatus, then cover the separating gel on the filter paper, cover the membrane on the gel, cover with filter paper, and finally cover with a sponge pad. Transfer at 100mA for 90 minutes. After the transfer is complete, seal the membrane.

[0047] (4) Antibody incubation

[0048] Remove the membrane and place it in blocking buffer. Block on a shaker for 1 hour. After washing with TBST, add primary antibody dilution buffer and incubate overnight at 4°C on a shaker. After washing with TBST, add secondary antibody dilution buffer and incubate on a shaker at room temperature for 2 hours. Then wash with TBST and prepare for development.

[0049] (5) Development

[0050] Add developer to the membrane, place it in the LAS-4000 instrument for development and photographing.

[0051] 6. Real-time quantitative PCR

[0052] (1) Reaction system configuration

[0053] Real-time quantitative PCR reaction system: 5 μL 2×Master Mix, 0.5 μL 10 μM forward primer, 0.5 μL 10 μM reverse primer, 2 μL cDNA, 2 μL water.

[0054] The primer sequence for tRF-18-YR66EFD2 is as follows:

[0055] F:5'-GCTAAGGAAGTCCTGTGCT-3',

[0056] R:5'-GTCCAGTTTTTTTTTTTTTTAAAACTGA-3',

[0057] The U6 primer sequence is:

[0058] F:5'-GCTTCGGCAGCACATATACTAAAAT-3',

[0059] R:5'-CGCTTCACGAATTTGGCGTGTCAT-3'.

[0060] (2) Real-time quantitative PCR

[0061] Add the mixture to the corresponding wells of the 384-PCR plate, seal with Sealing Film, briefly centrifuge to mix, then place the PCR plate in a Realtime PCR instrument and set the PCR amplification conditions: 95℃, 10 min, 1 cycle; 95℃, 10 s, 60℃, 60 s, 40 cycles. -ΔΔCt The method calculates the level of expression.

[0062] 7. Flow cytometry

[0063] (1) Cell resuspending: After cell treatment and counting, the cells were resuspended in PBS containing 2% BSA to achieve a cell concentration of 1×10⁻⁶. 7 / mL;

[0064] (2) Control settings: Set up blank control, isotype control and test sample, and take 100uL of the above resuspended cells in each tube;

[0065] (3) Antibody incubation: No antibody was added to the blank control tube. 5-20 μL of isotype antibody and target antibody were added to the isotype control tube and the test sample tube, respectively (refer to the instructions for specific dosage). The mixture was thoroughly mixed and incubated at 4°C for 30 min. An appropriate amount of cell washing buffer was added, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the mixture was washed twice. The cells were resuspended in 100 μL of cell washing buffer. An appropriate amount of fluorescently labeled secondary antibody was added to the isotype control tube and the test tube, and the mixture was thoroughly mixed and incubated at 4°C in the dark for 30 min.

[0066] (4) Detection on the machine: Add an appropriate amount of cell washing buffer, centrifuge at 1000 rpm for 5 min, discard the supernatant, and wash twice; resuspend the cells in 100-200 μL of cell washing buffer before testing.

[0067] 8. CCK-8 assay for cell proliferation

[0068] (1) Take logarithmic phase cells, count them, resuspend them in fresh complete culture medium to a suitable density, and add 100 μL of cell suspension to each well of a 96-well plate (generally, the number of cells is about 1,000-10,000, depending on the corresponding cell size, proliferation rate, intervention conditions, etc.).

[0069] (2) Provide the experimentally preset interference measures and culture for the required duration; after the intervention is completed, perform the detection by adding 10 μL of CCK-8 solution to each well, taking care to avoid the generation of bubbles that may affect the OD value, gently shake and incubate for 1-4 hours; (3) turn on the microplate reader, select the 450 nm wavelength, measure the absorbance of each well and calculate the cell proliferation rate.

[0070] 9. Statistical Analysis

[0071] SPSS 23.0 software was used for statistical analysis. qPCR values ​​were expressed as mean ± standard deviation. The KS test was used to determine whether the data conformed to a normal distribution. For normally distributed data, a two-tailed unpaired t-test was used for statistical analysis; otherwise, a nonparametric test (Mann-Whitney test) was used. A p-value < 0.05 was considered statistically significant. Data visualization was achieved using GraphPad 8.4.3 and Adobe Illustrator software.

[0072] result

[0073] 1. tRF-18-YR66EFD2 expression was significantly upregulated in R / RMM.

[0074] Based on previous next-generation sequencing results, we further identified tsRNA molecules that were significantly upregulated in the R / RMM. qPCR detection of tRF-18-YR66EFD2 (5'-TTCCCGGCCAACGCACCA-3') expression showed that tRF-18-YR66EFD2 was significantly upregulated in the R / RMM. Figure 1 A) ROC curve analysis showed that tRF-18-YR66EFD2 has certain diagnostic power in distinguishing between R / RMM and NDMM (sensitivity 0.673, specificity 0.79, AUC 0.798, CI 0.702-0.877).

[0075] 2. siRNA regulates the expression level of tRF-18-YR66EFD2

[0076] To further explore the potential function of tRF-18-YR66EFD2, we first regulated tRF-18-YR66EFD2 levels in MM cells using RNA interference technology, and then verified the transfection efficiency by qPCR. The results showed that tRF-18-YR66EFD2 levels were upregulated in the mimics group (…). Figure 2 A), the inhibitor group showed a decrease in tRF-18-YR66EFD2 levels ( Figure 2 B) indicates successful transfection.

[0077] 3. Inhibition of tRF-18-YR66EFD2 leads to decreased proliferation of MM cells.

[0078] We further investigated the effect of tRF-18-YR66EFD2 inhibition on MM cell proliferation using a CCK-8 assay. APR1 cells were transfected with tRF-18-YR66EFD2 inhibitor and a blank control, and seeded in 96-well plates. Cell proliferation was assessed using a CCK-8 assay at 24, 48, and 72 hours. The results showed that tRF-18-YR66EFD2 inhibition significantly reduced MM cell proliferation. Figure 3 ).

[0079] 4. Overexpression of tRF-18-YR66EFD2 leads to increased proliferation of MM cells.

[0080] Similarly, we overexpressed tRF-18-YR66EFD2 in MM.1S cells by transfection with tRF-18-YR66EFD2 mimics, incubated them in 96-well plates, and assessed cell proliferation using CCK-8 assay. The results showed that upregulation of tRF-18-YR66EFD2 significantly increased MM cell proliferation. Figure 4 ).

[0081] 5. Inhibition of tRF-18-YR66EFD2 leads to increased apoptosis in MM cells.

[0082] We also examined the effect of tRF-18-YR66EFD2 on apoptosis in MM cells. First, ARP1 cells were transfected with either tRF-18-YR66EFD2 inhibitor or control. Apoptosis was detected by flow cytometry 72 hours later. The results showed that inhibition of tRF-18-YR66EFD2 increased both early and late apoptosis in MM cells. Figure 5 ).

[0083] 6. Overexpression of tRF-18-YR66EFD2 leads to decreased apoptosis in MM cells.

[0084] Similarly, we transfected MM.1S cells with mimics to overexpress tRF-18-YR66EFD2. Flow cytometry analysis using Annexin V / PI double staining after 72 hours showed that overexpression of tRF-18-YR66EFD2 led to a decrease in both early and late apoptosis in MM cells. Figure 6 ).

[0085] 7. tRF-18-YR66EFD2 promotes VEGF expression in MM cells.

[0086] Finally, we used Western blot to examine the effect of tRF-18-YR66EFD2 levels on VEGF expression in MM.1S cells. The results showed that overexpression of tRF-18-YR66EFD2 led to increased VEGF expression in MM.1S cells, while inhibition of tRF-18-YR66EFD2 resulted in decreased VEGF expression in ARP1 cells. Figure 7 We chose to inhibit VEGF expression in ARP1 cells with relatively high tRF-18-YR66EFD2 expression and overexpress it in MM.1S cells with relatively low tRF-18-YR66EFD2 expression to observe the difference in VEGF expression. It was difficult to observe significant differences when overexpressing and inhibiting VEGF in the same cell line.

[0087] result

[0088] Multivariate disease (MM) is a complex condition. A new generation of biomarkers based on cytogenetics, epigenetics, focal lesions, clonal states, and gene expression profiles is beginning to provide clinicians with more information about the clinical behavior of the disease, laying the foundation for continuously refining risk stratification models and therapies. Biomarker-driven personalized treatments can maximize benefits while minimizing toxicity; biomarkers for this potential application need to possess characteristics of stable expression and reproducible measurement.

[0089] The function of tRF-18-YR66EFD2 in MM proliferation suggests its potential as a target for RNA interference-mediated gene therapy, offering several advantages over traditional treatments. Firstly, RNA interference therapy has a well-defined mechanism with high consistency, efficacy, and specificity. Secondly, small RNAs bind rapidly to lead compounds with high selectivity. Thirdly, RNA therapy has the potential to target any gene, including some genes previously found to encode protein products that cannot be synthesized into drugs. Fourthly, the synthesis of this small RNA for interference does not involve complex protein purification and folding, making it relatively simple and efficient. In conclusion, this research provides a potential novel therapeutic target for MM treatment and has promising clinical application prospects.

Claims

1. Use of a reagent for inhibiting tRF-18-YR66EFD2 expression in preparing a therapeutic preparation for multiple myeloma, wherein the sequence of tRF-18-YR66EFD2 is: TTCCCGGCCAACGCACCA; the reagent for inhibiting tRF-18-YR66EFD2 expression is an inhibitor of tRF-18-YR66EFD2; and the sequence of the inhibitor is: TGGTGCGTTGGCCGGGA.

2. Use of a reagent for detecting tRF-18-YR66EFD2 expression in the preparation of a multiple myeloma diagnostic preparation, wherein the sequence of tRF-18-YR66EFD2 is: TTCCCGGCCAACGCACCA.

3. The use according to claim 2, characterized in that Specifically, it is used to diagnose relapsed or refractory multiple myeloma (R / RMM).

4. The use according to claim 2 or 3, characterized in that Reagents for detecting tRF-18-YR66EFD2 expression include PCR detection reagents.

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