Target tRF-18-YR66EFD2 for treating multiple myeloma and application of target tRF-18-YR66EFD2

Abstract

The invention discloses a multiple myeloma treatment target point tRF-18-YR66EFD2 and an application of the multiple myeloma treatment target point tRF-18-YR66EFD2. Based on the result of early-stage next-generation sequencing, further research finds that tRF-18-YR66EFD2 molecules highly expressed in recurrent refractory MM (R / RMM) are degraded in a specific targeting manner, so that effective killing of MM cells is caused, and the research provides a new theoretical basis for treatment of MM.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor molecular biology, and specifically relates to a multiple myeloma therapeutic target tRF-18-YR66EFD2 and an application thereof. Background Art

[0002] Multiple myeloma (MM) is the second most common hematological malignancy caused by abnormal proliferation of clonal plasma cells in the bone marrow and secretion of monoclonal globulin. With the advancement of medical technology, the introduction and continuous updating of molecular, genetic and cytological techniques, preclinical studies have greatly promoted the understanding of the pathogenesis of MM in the past two decades. Functionally, it is known that there are genomic changes accumulated in tumor cells, as well as MM-specific defects (i.e., the high dependence of tumor cells 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 the research breakthroughs at the pathogenesis level, the clinical treatment strategy for MM has made great progress. Before the 1960s, the treatment of MM was aimed at relieving symptoms rather than controlling the disease. Since the first application of alkylating agents and corticosteroids in the 1960s, the treatment strategy of MM has gradually evolved. By the end of the 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 cornerstones 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 (panobinostat), alkylating agents (melphalan, cyclophosphamide, and bendamustine), corticosteroids (dexamethasone and prednisone), and monoclonal antibodies (rantoumab and elotuzumab). The new treatment model has greatly improved the quality of life and survival time of MM patients. According to statistics, the overall survival time of MM patients has been extended by 6-10 years compared with the past.

[0004] However, MM is still an incurable disease. As time goes by, patients will inevitably face the dilemma of relapse and drug resistance. Therefore, new and more effective therapeutic targets need to be broken through. tsRNA (tRNA derived small RNA) is a small RNA fragment derived from tRNA, which is essentially a small non-coding RNA. Therefore, the corresponding mimics or antisense molecules can antagonize its function and provide the possibility of being used as a small molecule drug, which also makes some tsRNA have great potential as a therapeutic target for hematological malignancies. In MM, a small number of studies have shown the significance of bone marrow tsRNA in predicting the risk of progression of smoldering MM to MM and in the prognostic stratification of MM patients, but the therapeutic value of tsRNA in MM is still unclear. Summary of the invention

[0005] The primary purpose of the present invention is to provide a bone marrow tsRNA molecule tRF-18-YR66EFD2, the sequence of which is: TTCCCGGCCAACGCACCA. The molecule has great potential in the diagnosis and treatment of multiple myeloma.

[0006] The second object of the present invention is to provide the use of an agent for inhibiting the expression of tRF-18-YR66EFD2 in the preparation of a therapeutic preparation for multiple myeloma.

[0007] The agent for inhibiting the expression of tRF-18-YR66EFD2 includes an inhibitor of tRF-18-YR66EFD2.

[0008] The sequence of inhibitor is: TGGTGCGTTGGCCGGGA.

[0009] The third object of the present invention is to provide a reagent for detecting the expression of tRF-18-YR66EFD2 for use in preparing a diagnostic or prognostic preparation for multiple myeloma.

[0010] Specifically, it is 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 the previous second-generation sequencing results, the present invention further discovered that the tRF-18-YR66EFD2 molecule highly expressed in relapsed and refractory MM (R / RMM) was specifically targeted for degradation, resulting in effective killing of MM cells. The present invention provides a new theoretical basis for the treatment of MM. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0017] Figure 2 : Regulation of tRF-18-YR66EFD2 expression by siRNA (i.e. inhibitor) in MM cells;

[0018] Figure 3 : Inhibition of the effect of tRF-18-YR66EFD2 on the proliferation of ARP1 cells;

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

[0020] Figure 5 : Inhibit the effect of tRF-18-YR66EFD2 on apoptosis of ARP1 cells;

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

[0022] Figure 7 :Effect of tRF-18-YR66EFD2 on VEGF protein expression in MM cells. DETAILED DESCRIPTION

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

[0024] method

[0025] 1. Clinical samples

[0026] A total of 52 R / RMM (relapsed / refractory multiple myeloma, R / RMM) and 48 NDMM patients from the Third Xiangya Hospital of Central South University from March 2016 to March 2020 were selected in the present invention. The diagnosis of multiple myeloma referred to the diagnostic criteria for symptomatic multiple myeloma defined by the National Comprehensive Cancer Network (NCCN) of the United States. The relevant clinical information of the subjects was collected, and bone marrow specimens were collected for tsRNA extraction.

[0027] 2. Cell Culture

[0028] 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 subcultured in an incubator at 37°C and 5% CO2 using RPMI 1640 medium containing 10% FBS and 1% double antibody, with the medium changed every other day. The culture density was about 0.2-0.5×10 6 / mL, and cell counts were performed using trypan blue staining.

[0029] 3. RNA Extraction

[0030] According to the kit procedure, TRIzol (Invitrogen, USA) was used to extract RNA from bone marrow specimens. The specific procedure was as follows: (1) the specimen was placed on ice, and Trizol was added to lyse the specimen for 5-10 minutes. The liquid was aspirated and placed in an EP tube after being gently blown with a pipette tip. 1 / 5 volume of chloroform was added, the liquid was mixed by up and down, and then allowed to stand at 4°C for 10-15 minutes.

[0031] (2) Centrifuge at 4°C for 15 minutes. After centrifugation, the tube is separated into three layers. Gently remove the EP tube from the centrifuge, aspirate the supernatant, avoiding the lower precipitate, and place the liquid in a new EP tube.

[0032] (3) Add isopropanol at a rate of 0.5 ml isopropanol / ml Trizol, let stand for 10 min, then centrifuge at 12,000 rpm at 4°C for 10 min, discard the supernatant, add 75% ethanol at a rate of 1 ml 75% ethanol / ml Trizol, centrifuge at 4°C for 5 min, and discard the supernatant;

[0033] (4) Place the EP tube in the centrifuge again and centrifuge for a short time, discard the residual liquid on the tube wall. Then place the EP tube in a clean bench to dry for 5-10 minutes. Add 50ul of DEPC-treated water and shake to fully dissolve the precipitate;

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

[0035] 4. RNA interference

[0036] (1) Preparation of transfection complex

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

[0038] (2) Cell transfection

[0039] When the transfection complex is at rest, take 1-2×10 6 Resuspend the cells to 4 mL in a 60 mm culture dish; add 1 mL of transfection complex to the culture dish, the final system is 5 mL, then gently shake the culture dish to mix the complex, culture the cells at 37°C, replace with complete culture medium after 4-6 hours, and use 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 5min, aspirate the culture medium, then add 4℃ pre-cooled PBS to wash the samples, centrifuge at 300g for 5min, and discard the supernatant; 6 Lysis buffer was added at a ratio of cells / 100 μL lysis buffer, and the cells were lysed on ice for 30 min after shaking, with shaking every 10 min. After lysis, the proteins were collected by cold centrifugation at 26000 g for 15 min at 4 °C, and the protein concentration was determined by 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, place in a 95℃ metal bath for 5 minutes, cool to room temperature, and prepare for electrophoresis;

[0045] (3) Protein electrophoresis and transfer

[0046] Place the prepared SDS-PAGE gel in the electrophoresis tank, add a sufficient amount of 1× electrophoresis solution, add the sample, and then start electrophoresis at 80V for 30min. After bromophenol blue runs into the separation gel, adjust the voltage to 120V, and stop the electrophoresis when bromophenol blue just runs out, and then proceed to the next step of membrane transfer; prepare the transfer solution in advance, pre-cool it at -20℃, pour it into the plate, peel the gel from the electrophoresis device, then cover the separation gel on the filter paper, cover the membrane on the gel, cover the filter paper, and finally cover the sponge pad, and transfer the mold at 100mA for 90min. After the transfer is completed, seal it;

[0047] (4) Antibody incubation

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

[0049] (5) Development

[0050] Add developer solution onto the membrane, put it into LAS-4000 instrument for development and take pictures.

[0051] 6. Real-time fluorescence quantitative PCR

[0052] (1) Reaction system configuration

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

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

[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 fluorescence quantitative PCR

[0061] Add the mixture to the corresponding wells of the 384-PCR plate, affix the sealing film, centrifuge briefly to mix, then place the PCR plate in the Realtime PCR instrument and set the PCR amplification conditions: 95℃, 10min, 1 cycle; 95℃, 10s, 60℃, 60s, 40 cycles. -ΔΔCt The expression level was calculated.

[0062] 7. Flow Cytometry

[0063] (1) Cell resuspension: After cell processing and counting, resuspend the cells in PBS containing 2% BSA to a cell concentration of 1×10 7 / mL;

[0064] (2) Control setting: 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, and 5-20uL of isotype antibody and target antibody were added to the isotype control tube and the sample tube to be tested (refer to the instructions for specific usage), mixed thoroughly, and incubated at 4°C for 30 min; an appropriate amount of cell washing solution was added, centrifuged at 1000rpm for 5 min, the supernatant was discarded, and the cells were washed twice and resuspended in 100μL of cell washing solution; an appropriate amount of fluorescently labeled secondary antibody was added to the isotype control tube and the test tube, mixed thoroughly, and incubated at 4°C in the dark for 30 min;

[0066] (4) Detection on the machine: Add an appropriate amount of cell washing solution, centrifuge at 1000 rpm for 5 min, discard the supernatant, and repeat the washing twice; resuspend the cells with 100-200 μL of cell washing solution and then load on the machine.

[0067] 8. CCK-8 detection of cell proliferation

[0068] (1) Take logarithmic phase cells, count them, resuspend them in fresh complete medium to an appropriate density, and add 100 μL of cell suspension to each well of a 96-well plate (the general number of cells is about 1,000-10,000, which is determined according to the corresponding cell size, proliferation rate, intervention conditions, etc.);

[0069] (2) Provide the preset interference measures for the experiment and culture the cells for the required time; after the intervention, perform the test by adding 10 μL of CCK-8 solution to each well, taking care to avoid bubbles that may affect the OD value, and gently shake the well before incubating for 1-4 hours; (3) Turn on the microplate reader, select a wavelength of 450 nm, measure the absorbance of each well, and then calculate the cell proliferation rate.

[0070] 9. Statistical Analysis

[0071] SPSS23.0 software was used for statistical analysis, and the qPCR values ​​were taken as mean ± standard deviation. The KS test was used to determine whether the data conformed to normal distribution. For data that conformed to normal distribution, a two-tailed unpaired t-test was used for statistical analysis, otherwise a nonparametric test (Mann-Whitney test) was selected, and a P value < 0.05 was considered statistically significant. Data visualization was achieved using GraphPad 8.4.3 and Adobe Illustrater software.

[0072] result

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

[0074] Based on the previous second-generation sequencing results, we further identified the tsRNA molecules that were significantly upregulated in R / RMM. qPCR detection of tRF-18-YR66EFD2 (base sequence 5'-TTCCCGGCCAACGCACCA-3') expression showed that tRF-18-YR66EFD2 was significantly upregulated in R / RMM ( Figure 1 A), ROC curve analysis showed that tRF-18-YR66EFD2 had a certain test efficacy for distinguishing R / RMM from NDMM (sensitivity 0.673, specificity 0.79, AUC 0.798, CI 0.702-0.877).

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

[0076] To further explore the potential function of tRF-18-YR66EFD2, we first regulated the level of tRF-18-YR66EFD2 in MM cells by RNA interference technology, and verified the transfection efficiency by qPCR. The results showed that the level of tRF-18-YR66EFD2 in the mimics group was upregulated ( Figure 2 A), the level of tRF-18-YR66EFD2 decreased in the inhibitor group ( Figure 2 B), indicating successful transfection.

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

[0078] We further tested the effect of inhibiting tRF-18-YR66EFD2 on MM cell proliferation by CCK-8 assay. APR1 cells were transfected with tRF-18-YR66EFD2 inhibitor and blank control, and plated in 96-well plates. Cell proliferation was detected by CCK-8 after 24h, 48h, and 72h. The results showed that inhibiting tRF-18-YR66EFD2 led to a significant decrease in 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 in 96-well plates, and detected cell proliferation with CCK-8. The results showed that upregulation of tRF-18-YR66EFD2 led to a significant increase in MM cell proliferation ( Figure 4 ).

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

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

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

[0084] Similarly, we overexpressed tRF-18-YR66EFD2 in MM.1S cells by mimics transfection, and flow cytometry analysis was performed by Annexin V / PI double staining at 72 hours. The results showed that overexpression of tRF-18-YR66EFD2 led to a decrease in both early and late apoptosis of MM cells ( Figure 6 ).

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

[0086] Finally, we used western blot to detect the effect of tRF-18-YR66EFD2 levels on VEGF expression in MM 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 caused decreased VEGF expression in ARP1 cells ( Figure 7 We chose to inhibit tRF-18-YR66EFD2 in ARP1 cells where tRF-18-YR66EFD2 was relatively highly expressed, and to overexpress it in MM.1S cells where tRF-18-YR66EFD2 was relatively low, in order to observe the difference in VEGF expression. It was difficult to observe obvious differences in results when both overexpression and inhibition were performed in the same cell line.

[0087] result

[0088] MM is a complex disease. A new generation of biomarkers based on cytogenetics, epigenetics, focal lesions, clonal status, and gene expression profiles are beginning to provide clinicians with more information about the clinical behavior of the disease, providing a basis for the continuous improvement of risk stratification models and therapies. Biomarker-driven personalized treatments can maximize benefits while reducing toxicity. Such potential biomarkers need to have stable expression and repeatable measurement characteristics.

[0089] The function of tRF-18-YR66EFD2 in MM proliferation suggests the possibility of being a target for RNA interference-mediated gene therapy, which has many advantages over traditional treatment. First, the mechanism of RNA interference therapy is clear, with high consistency, effectiveness and specificity; second, small RNA binds to the lead compound quickly and has high selectivity; third, RNA efficacy has the possibility of targeting any gene, including some genes that have been found to encode protein products that cannot synthesize drugs; fourth, the synthesis of this small RNA for interference does not involve complex protein purification and folding, and is relatively simple and efficient. In short, the research of the present invention provides a potential new therapeutic target for the treatment of MM, with good clinical application prospects.

Claims

1. A tsRNA molecule tRF-18-YR66EFD2, the sequence of which is: TTCCCGGCCAACGCACCA.

2. Use of the agent for inhibiting the expression of tRF-18-YR66EFD2 according to claim 1 in the preparation of a therapeutic preparation for multiple myeloma.

3. The use according to claim 2, characterized in that: The agent for inhibiting the expression of tRF-18-YR66EFD2 includes an inhibitor of tRF-18-YR66EFD2.

4. The use according to claim 3, characterized in that: The sequence of inhibitor is: TGGTGCGTTGGCCGGGA.

5. Use of the reagent for detecting the expression of tRF-18-YR66EFD2 according to claim 1 in the preparation of a diagnostic or prognostic preparation for multiple myeloma.

6. The use according to claim 5, characterized in that: Specifically, it is the diagnosis or prognosis of relapsed refractory multiple myeloma R / RMM.

7. The use according to claim 5 or 6, characterized in that: Reagents for detecting tRF-18-YR66EFD2 expression include PCR detection reagents.

8. The use according to claim 7, characterized in that: The primer sequences in the PCR detection reagent are as follows: F:5'-GCTAAGGAAGTCCTGTGCT-3', R:5'-GTCCAGTTTTTTTTTTTTTTAAAACTGA-3'.

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