Fusion RNA transcript and application thereof as multiple myeloma biomarker

Abstract

The invention discloses a biomarker fusion RNA transcript CADPSE-RNF148 related to occurrence and development of multiple myeloma and plasma cell leukemia and a corresponding protein, and early screening, diagnosis, curative effect and prognosis evaluation of multiple myeloma are realized by detecting whether a patient expresses the fusion RNA transcript CADPSE-RNF148 and detecting the expression quantity of the fusion RNA transcript CADPSE-RNF148. The invention also discloses a multiple myeloma detection kit. The number of plasma cells in bone marrow and peripheral blood can be calculated by detecting the expression quantity of the fusion transcript CADPSE-RNF148 of the peripheral blood, which has great medical application value for reducing the medical cost, shortening the diagnosis time, relieving the pain of a patient and easily obtaining a sample clinically.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology and tumor marker medical technology, and in particular to a fusion RNA transcript CADPS2-RNF148 based on peripheral blood detection and a biomarker for multiple myeloma diagnosis and prognosis evaluation, as well as a medicine and application thereof. Background Art

[0002] Multiple myeloma (MM) is a malignant disease characterized by abnormal proliferation of clonal plasma cells. In many countries, it is the second most common malignant tumor of the blood system (Wang SF, Xu L, Feng JN, et al. Prevalence and Incidence of Multiple Myeloma in Urban Area in China: A National Population-Based Analysis. Frontiers in Oncology 2020; 9). Epidemiology suggests that the prevalence of the disease is 2 to 3 / 100,000, slightly higher in men than in women, and mostly in the elderly. The clinical staging standards for multiple myeloma include the traditional Durie-Salmon (DS) staging system, the international staging system ISS staging system, and the revised international staging system R-ISS staging system. Although the clinical items of the staging are different, they are all divided into stages I, II, and III according to the severity. In the DS staging system, it is divided into subtypes A and B according to whether the renal function is normal or not.

[0003] In terms of treatment, although new drugs and new methods including chimeric antigen receptor T cell (CAR-T) immunotherapy continue to emerge, which have significantly improved the overall efficacy and progression-free survival of the disease, the overall prognosis is still poor. Most patients will experience tumor recurrence, and the remission rate is not good after re-treatment. The mortality rate accounts for 20% of hematological malignancies, and the 5-year survival rate is about 40%.

[0004] From the initial monoclonal globulinemia to the progression of smoldering multiple myeloma to symptomatic multiple myeloma, the development of multiple genetic abnormalities has gone through two major stages. First, chromosomal abnormalities such as hyperdiploidy, hypodiploidy, polyploidy, and chromosomal translocations are typical features of the early stages of the disease. Then, as the disease progresses, more genetic abnormalities appear, including changes in genes such as NRAS, KRAS, and MYC. Due to the in-depth study of multiple myeloma and the search for better treatments, the overall management goals now focus more on cytogenetics and molecular biological characteristics. Therefore, studying the genetic abnormalities of multiple myeloma is of great significance in guiding clinical diagnosis and treatment.

[0005] In terms of biological examination of chromosomes and fluorescence in situ hybridization (FISH), chromosomes and FISH are widely used. Chromosomes and FISH detect bone marrow chromosome 17p13 deletion, and / or t(4;14) and / or t(14;16) abnormalities, which often indicate high risk. FISH, especially FISH after purification with CD138 (positive expression in most myeloma cells) magnetic beads, iFISH examination, can further increase the positive rate of the test. This test has been used in the newly revised international prognostic staging system (R-ISS staging system) in 2015.

[0006] Fusion is a somatic mutation that causes cancer and is associated with up to 20% of tumors (Mitelman F, Johansson B, Mertens F. The impact of translocations and gene fusions on cancer causation. Nature Reviews Cancer 2007; 7: 233-45). Translocation, copy number changes and inversions can all lead to fusion phenomena, uncontrolled gene expression and the generation of new functional molecules. Fusion phenomena may define some tumor types, such as BCR-ABL1 in chronic myeloid leukemia. The ectopic t(9;22) leads to BCR-ABL1 fusion and produces a fusion protein. The ABL1 kinase domain in this fusion protein is continuously activated, signaling cells to continue dividing instead of apoptosis. Imatinib, as a BCR-ABL1 fusion protein inhibitor, became the first FDA-approved drug specifically targeting fusion proteins in 2001 (Mertens F, Johansson B, Fioretos T, Mitelman F. The emerging complexity of gene fusions in cancer. Nature Reviews Cancer 2015; 15: 371-81).

[0007] RNA fusion transcripts are composed of exons from two or more genes and have RNA sequences that have the potential to encode new proteins and change cell phenotypes. With the growing demand for early diagnosis and prognosis evaluation of tumors, as well as targeted therapy and personalized medication in the clinic, specific fusion RNA is becoming a new research direction. At present, in multiple myeloma, there is no detection of the expression level of fusion RNA transcripts in peripheral blood as a marker for screening and diagnosis of plasma cells in bone marrow and peripheral blood. Therefore, the development of fusion RNA transcripts as a marker for diagnosis and prognosis evaluation of multiple myeloma and a target for drug treatment has very important significance and clinical value. Summary of the invention

[0008] Based on this, the purpose of the present invention is to provide a nucleic acid molecule and / or protein molecule based on peripheral blood detection, and its fusion RNA transcript CADPS2-RNF148, etc. are used in drugs as markers for diagnosis and prognosis evaluation of multiple myeloma and targets for drug treatment.

[0009] The first aspect of the present invention is to provide a nucleic acid molecule and / or protein molecule selected from at least one of the following:

[0010] A. Fusion RNA transcript CADPS2-RNF148, the sequence of which is shown in SEQ ID NO: 1;

[0011] B. The open reading frame of the fusion RNA transcript CADPS2-RNF148, the sequence of which is shown in SEQ ID NO: 6;

[0012] C. The encoded protein of the fusion RNA transcript CADPS2-RNF148, whose sequence is shown in SEQ ID NO:7.

[0013] A specific or enriched CADPS2-RNF148 fusion RNA transcript exists in multiple myeloma cells and / or plasma cell leukemia, and its specific fusion site, upstream and downstream nucleotide sequences and predicted protein spatial structure are shown in SEQ ID NO: 1; the expression and content of the transcript are related to the stage, tumor load, physical status and overall survival time of multiple myeloma, and can be applied to the diagnosis of multiple myeloma cells and / or plasma cell leukemia, tumor load or stage detection, and prognosis evaluation.

[0014] The second aspect of the present invention is to provide the use of the above-mentioned nucleic acid molecules and / or protein molecules including the fusion RNA transcript CADPS2-RNF148 as biomarkers in the preparation of products for diagnosis and prognosis evaluation of multiple myeloma and / or plasma cell leukemia.

[0015] Or use of a reagent for detecting nucleic acid molecules and / or protein molecules including the fusion RNA transcript CADPS2-RNF148 in the preparation of products for diagnosis and prognosis evaluation of multiple myeloma and / or plasma cell leukemia.

[0016] The product may be a kit well known in the art, such as a new product, or a device, such as a sequencing device.

[0017] The third aspect of the present invention is to provide a multiple myeloma and / or plasma cell leukemia detection kit, which includes a reagent for detecting the expression amount of the above-mentioned fusion RNA transcript CADPS2-RNF148 in a sample.

[0018] In some of these embodiments, the sample is peripheral blood or human multiple myeloma cell lines RPMI-8226, MM.1R and MM.1S.

[0019] In some of the embodiments, the reagents include detection primers for detecting CADPS2-RNF148 fusion RNA transcripts and fusion sites, and the sequences of the detection primers include but are not limited to those shown in SEQ ID NO:2 and SEQ ID NO:3.

[0020] In some embodiments, the reagent further comprises an internal reference primer, and the sequence of the internal reference primer includes but is not limited to SEQ ID NO:4 and SEQ ID NO:5.

[0021] The fourth aspect of the present invention is to provide a primer capable of detecting the above-mentioned fusion RNA transcript CADPS2-RNF148, and the sequence of the detection primer is shown in SEQ ID NO:2 and SEQ ID NO:3.

[0022] The fifth aspect of the present invention is to provide a pharmaceutical composition, the active ingredients of which include an inhibitor of the expression of the above-mentioned fusion RNA transcript CADPS2-RNF148, or an inhibitor of the protein encoded by the above-mentioned fusion RNA transcript CADPS2-RNF148.

[0023] The inhibitor is that those skilled in the art can administer corresponding shRNA or siRNA inhibitors to the fusion RNA transcript CADPS2-RNF148 according to the prior art to reduce the expression of the fusion RNA transcript CADPS2-RNF148.

[0024] The sixth aspect of the present invention is to provide the use of the fusion RNA transcript CADPS2-RNF148 as a molecular target in the preparation of medicines for treating multiple myeloma and / or plasma cell leukemia.

[0025] In some of these embodiments, the multiple myeloma and / or plasma cell leukemia is positive for expression of the fusion RNA transcript CADPS2-RNF148 and is stage III multiple myeloma.

[0026] Or the multiple myeloma is positive for the expression of the fusion RNA transcript CADPS2-RNF148, accompanied by renal impairment (serum creatinine ≥176.8 μmol / L), high tumor burden including serum β2-MG level ≥10 mg / L (serum β2-MG ≥5.5 mg / L can be judged as stage III) and serum LDH>240 U / L, and / or patients with positive expression of the fusion RNA transcript CADPS2-RNF148 also have a lower physical status (ECOG score ≥3). Patients with positive expression of the fusion RNA transcript CADPS2-RNF148 had a significantly shorter overall survival time (the 3-year survival rate of MM patients who did not express this transcript was 94.44%, while the 3-year survival rate of patients who expressed this transcript was 61.25%, P = 0.0199). This trend was particularly significant in people diagnosed with multiple myeloma before the age of 60 (the 3-year survival rate of MM patients diagnosed before the age of 60 was 100% for those who did not express this transcript, while the 3-year survival rate of MM patients who expressed this transcript was only 40%, P = 0.0017).

[0027] In some of the embodiments, the multiple myeloma is positive for the expression of the fusion RNA transcript CADPS2-RNF148, and the expression of CADPS2-RNF148 is linearly positively correlated with the number of plasma cells in peripheral blood and bone marrow. The expression of the fusion RNA transcript CADPS2-RNF148 of the present invention can also directly reflect the plasma cell level in the patient's peripheral blood and bone marrow (the expression of CADPS2-RNF148 is linearly positively correlated with the number of plasma cells in peripheral blood and bone marrow) (the higher the number of plasma cells in peripheral blood and bone marrow, the higher the malignancy of multiple myeloma).

[0028] The present invention has found in the research that the fusion RNA transcript CADPS2-RNF148 is expressed in 13.8% (15 / 109 cases) of total peripheral blood lymphocytes (PBMCs) of multiple myeloma patients, not expressed in normal human peripheral blood PBMCs, and also specifically highly expressed in human multiple myeloma cell lines RPMI-8226, MM.1R and MM.1S. In particular, it is found that the expression of the CADPS2-RNF148 fusion transcript and the expression amount are closely related to the multiple myeloma stage and tumor load (serum β2-MG and LDH levels), physical fitness score (ECOG score), plasma cell levels in peripheral blood and bone marrow, and overall survival. The fusion RNA transcript CADPS2-RNF148 is used as a marker for the diagnosis and prognosis evaluation of multiple myeloma. The fusion RNA transcript provides a molecular target for the treatment of multiple myeloma and can be used in the preparation of anti-multiple myeloma drugs.

[0029] The fusion RNA transcript CADPS2-RNF148 of the present invention has the first (462 bp) and second exons (114 bp) of CADPS2 (RefSeq NM_017954) fused at its 5' end, and the 433-1255 fusion (648 bp) downstream of the ATG of RNF148 (RefSeq NM_198085) fused at its 3' end.

[0030] The advantages of the present invention include the following:

[0031] 1. The present invention has discovered for the first time the biomarker fusion RNA transcript CADPS2-RNF148 associated with the occurrence and development of multiple myeloma and plasma cell leukemia. By detecting whether the patient's peripheral blood expresses the fusion RNA transcript CADPS2-RNF148 and the expression level, early screening, diagnosis and prognosis assessment of multiple myeloma can be achieved.

[0032] 2. The present invention provides a molecular target for treating multiple myeloma and / or plasma cell leukemia, which has specificity in treating the disease by targeting molecular markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The expression rates of CADPS2-RNF148 in peripheral blood PBMCs of MM patients and healthy donors were 15 / 109 (13.8%) and 0 / 101 (0), respectively. The vertical axis represents percentage; ****, P < 0.0001.

[0034] Figure 2 Schematic diagram of the mechanism of production of RNA fusion transcript CADPS2-RNF148.

[0035] Figure 3 A is to further verify the expression of the encoded fusion protein CADPS2-RNF148 in tumor cells. We attached Flag tags to the N-terminus and C-terminus of the coding sequence, respectively, and constructed a lentiviral vector. Anti-Flag antibodies were used to detect the expression of the fusion protein in the Nalm6 cell line; B is the predicted protein structure analysis of human CADPS2-RNF148 using XtalPred-RF; C is the three-dimensional spatial structure diagram of the fusion protein CADPS2-RNF148 predicted by Alphafold2.

[0036] Figure 4 This is an agarose gel electrophoresis diagram of qPCR amplification products of three multiple myeloma cell lines RPMI-8226, MM.1R, MM.1S and PBMCs isolated from peripheral blood samples of 15 MM patients with positive expression.

[0037] Figure 5Figure 2 is an agarose gel electrophoresis diagram of the qPCR amplification products of the RNA fusion transcript CADPS2-RNF148 in PBMCs isolated from peripheral blood samples of 12 MM cases (MPP-1 to MPP-4).

[0038] Figure 6 This is a schematic diagram of the nucleotide sequence sequencing results of the RNA fusion transcript CADPS2-RNF148 fusion site.

[0039] Figure 7 This is a schematic diagram of the analysis results of the number of peripheral blood and bone marrow plasma cells screened from the bone marrow puncture reports of 15 multiple myeloma patients with positive CADPS2-RNF148 expression.

[0040] Figure 8 This is a schematic diagram of the survival analysis results of 109 MM cases using Kaplan-Meier analysis. DETAILED DESCRIPTION

[0041] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0042] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0043] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0044] We performed Z-test analysis on RNA-Seq data from 675 cases of multiple myeloma (dbGap: phs000749) and 452 healthy people in GTEx (dbGap: phs000424), and found that the expression of CADPS2-RNF148 fusion transcripts in multiple myeloma (357 / 675, 52.89%) and healthy people (0 / 452, 0) was significant (Z = 31.3; P < 0.00001). Subsequently, we collected peripheral blood from 109 multiple myeloma cases and 101 healthy people for verification, isolated total lymphocytes (PBMCs), extracted total RNA, detected the expression of the transcript by qRT-PCR, and used sangerdideoxy sequencing. Combined with the analysis of clinical data, we found that the expression of CADPS2-RNF148 fusion transcript was closely related to the multiple myeloma stage and tumor burden (serum β2-MG and LDH levels), physical fitness score (ECOG score) and overall survival (OS), and the expression of the fusion transcript was linearly positively correlated with the number of plasma cells in the bone marrow and peripheral blood.

[0045] The present invention will be further described below in conjunction with the accompanying drawings. It should be noted that this embodiment is based on the technical solution and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to this embodiment.

[0046] Example 1. Application of RNA fusion transcript CADPS2-RNF148 as a risk marker for diagnosis and prognosis assessment of multiple myeloma.

[0047] 1. Research Objects and Methods

[0048] 1. Research subjects

[0049] From September 2018 to March 2023, peripheral blood specimens of 109 patients with multiple myeloma (all patients gave informed consent) admitted to the Department of Hematology of Shenzhen People's Hospital and the First Affiliated Hospital of Jinan University (Guangzhou Overseas Chinese Hospital) were collected as research subjects, including 58 males and 51 females, with a median age of 64 years and an age range of 35 to 87 years. They were followed up until death, loss of follow-up or May 2023. The diagnostic criteria for multiple myeloma refer to the guidelines of the National Comprehensive Cancer Network (NCCN) of the United States, and the staging is carried out according to the Durie-Salmon (DS) staging system, the International Staging System (ISS) and the Revised International Staging System (R-ISS). The treatment and efficacy evaluation of patients with multiple myeloma refer to the above guidelines. Overall survival (OS) is defined as the time from the start of the first treatment to death due to any cause, and death is an event. Normal control peripheral blood specimens were obtained from 101 adult healthy volunteers. The samples used in the research scheme of the present invention were approved by the Ethics Committee of Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. All healthy volunteers and patients signed informed consent.

[0050] 2. Isolation and extraction of peripheral blood total lymphocytes (PBMCs) and RT-qPCR

[0051] PBMCs were separated from EDTA anticoagulated whole blood by gradient centrifugation using lymphocyte separation solution (Ficol), and total RNA of PBMCs was extracted by Trizol method; 4 ml of anticoagulated whole blood was diluted with 4 ml of PBS at room temperature, 4 ml of Ficol was added to another 15 ml centrifuge tube, 8 ml of diluted whole blood was slowly added to the upper layer of Ficol, centrifuged at 800g for 30 minutes, the middle layer (buffy coat layer) of the centrifuged blood sample was transferred to a 1.5 ml enzyme-free EP tube, washed with PBS 3 times, centrifuged at 800g, the supernatant was discarded, the cell precipitate was PBMCs, 1 ml of Trizol was added and mixed thoroughly by blowing, and then allowed to stand at room temperature for 5 minutes. 200 μl of chloroform was added to each tube, shaken vigorously for 15-20 seconds, allowed to stand at room temperature for 5 minutes, and then centrifuged at 12000rpm in a 4°C centrifuge for 15 minutes. Pipette the supernatant into a new 1.5ml RNase-free EP tube, add 1μl glycogen (Glycogen) and 500μl isopropanol, let stand at room temperature for 10 minutes, and centrifuge at 12000rpm for 15 minutes in a 4℃ centrifuge. Discard the supernatant, add 1ml 75% ethanol to each tube, wash the precipitate upside down, centrifuge at 12000rpm for 5 minutes, and discard the supernatant. Repeat this step again. Discard the supernatant. Use a 10μl pipette tip to remove all the supernatant as much as possible, and leave it open at room temperature for about 5 minutes. Add 30-50μl RNase-Free ddH2O water to fully dissolve the precipitate, place on ice for 2 minutes and measure the concentration.

[0052] RT-PCR used ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan.). The experimental process is as follows: Thaw the reagents required for the experiment at room temperature, and place them on ice with the template RNA for later use. Before use, all reagents were mixed, and then centrifuged briefly to collect all the liquid on the tube wall. 1μg of total RNA and 2μL Oligo dT were supplemented with RNase-Free ddH2O to 10μL, 65℃, 5 minutes, ice bath for 5 minutes, and 10μL of the mixture was mixed according to the reverse transcription reaction system in Table 1, vortexed and mixed, and then centrifuged instantly. Then put it into the PCR instrument and run the program: 42℃, 60 minutes; 99℃, 5 minutes; 16℃ forever. After the reaction is completed, cDNA can be obtained. After diluting 20 times with RNase-Free ddH2O, it can be used for subsequent experiments. cDNA is stored in a -20℃ refrigerator.

[0053] Table 1 Reverse transcription reaction system

[0054]

[0055] The qRT-PCR experiment used Vazyme's ChamQ SYBR qPCR Master Mix. The experimental process is as follows: the reagents required for the experiment were thawed at room temperature and placed on ice for later use. Before use, all reagents should be vortexed and mixed, and then centrifuged briefly to collect all the liquid on the tube wall.

[0056] Prepare the mixture on ice according to the qRT-PCR reaction system in Table 2, vortex and mix well, and then centrifuge instantly. Transfer the prepared mixture to a 96-well plate dedicated to qRT-PCR and seal it with an optical sealing film. Centrifuge the 96-well plate for 30 seconds and then put it into the real-time fluorescence quantitative PCR instrument. Run the program: pre-denaturation: 95℃, 2 minutes and 20 seconds; 5℃ denaturation for 10 seconds, 61℃ annealing for 30 seconds, 70℃ extension for 1 second, run 40 cycles; melting curve: 70℃-94℃, 5 seconds.

[0057] Table 2 qRT-PCR reaction system

[0058]

[0059] Agarose gel electrophoresis, the experimental process is as follows: prepare agarose gel, use 4% agarose gel. When preparing, first weigh 4g of agarose dry powder, pour it into a conical flask, then add 120ml of 1×TAE solution, put it in a microwave to heat and melt, mix well, pour it into the gel tank, cool and solidify for use;

[0060] Take 5 μl of the qPCR product in a total volume of 10 μl and add 1 μl of 6× DNA loading buffer. Mix well and add to the sample well of agarose gel. Run the gel and set the program voltage to 110 V for 30 minutes.

[0061] T clone sequencing: TOYOBO's Convenient TA Cloning System for PCR Products Target Clone was used TM / Target Clone TM -Plus-T cloning kit; Take 3μl qPCR product and prepare the connection solution according to the composition in Table 3, fully suspend and centrifuge, and react at 4℃ overnight. Thaw the Top10 competent cells on ice, add 10μl of the above connection solution, mix gently and ice bath for 30 minutes; heat shock at 42℃ for 90 seconds, immediately cool in ice bath for 2 minutes, add 900μl of LB medium without ampicillin, and shake and culture at 37℃ for 1 hour; centrifuge at 3000rpm for 5 minutes, discard the supernatant, resuspend with 100μl LB medium containing 1 / 1000 ampicillin, apply on LB / AP / IPTG / X-gal plate, and culture at 37℃ overnight. Select the white recombinant colonies by blue-white judgment, place them in 500μl LB medium containing 1 / 1000 ampicillin, shake and culture at 37℃ for 5-6 hours, and send the bacterial solution for sequencing. Sequencing was performed using Sanger dideoxy sequencing.

[0062] Table 3 Preparation of ligation solution

[0063]

[0064] Cell recovery: Place the cryotube containing 1mL of cell suspension in a 37℃ water bath to thaw quickly, transfer to a centrifuge tube containing 5mL of culture medium (RPMI-1640+10% FBS+1% P / S) aseptically, mix thoroughly, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 4-6mL of culture medium to resuspend the cells, transfer to a T25 culture flask, culture in suspension, culture overnight at 37℃, 5% CO2, and observe the cell status after 24 hours. Record the cell name, generation, date, and operator.

[0065] Cell passaging: When the cell density reaches 80-90%, it can be passaged. Collect cells into centrifuge tubes, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 1-2mL culture medium and blow evenly, distribute to new culture bottles at a ratio of 1:2-1:3, add appropriate amount of culture medium, culture in suspension, culture at 37℃, 5% CO2 overnight, and observe the cell status after 24 hours. Record the cell name, generation, date and operator.

[0066] Cell freezing: collect cells into centrifuge tubes, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add freezing solution (90% FBS + 10% DMSO) according to the number of frozen cells, mix well and add to cell freezing tubes, and mark the cell name, generation, freezing date and operator on the tube. Place the frozen cells in a programmed cooling box that has returned to room temperature, and put them in a -80℃ refrigerator in time. Transfer them to a liquid nitrogen tank the next day and register them.

[0067] The Ct values ​​of 18S and CADPS2-RNF148 were obtained by amplification curves of the sample internal reference genes 18S and CADPS2-RNF148 (primer sequences are shown in Table 4), and the melting curve peaks were referred to. Then, according to the formula: ΔCt = CADPS2-RNF148 Ct value - 18S Ct value, the final value was 2 -ΔCt =The expression level of CADPS2-RNF148 in the samples is presented.

[0068] Table 4. CADPS2-RNF148 and 18S primer sequences

[0069] Primers Sequence (5'-3') CADPS2-RNF148 upstream primer 5'-CGGTTCCAGGCCTTCCTC-3'(SEQ ID NO.2) CADPS2-RNF148 downstream primers 5'-GTCACATAGACTCCTTTCTG-3'(SEQ ID NO.3) 18S upstream primer 5'-TAGTCGCCGTGCCTACCA-3'(SEQ ID NO.4) 18S downstream primer 5'-TGCTGCCTTCCTTGGATGTG-3'(SEQ ID NO.5)

[0070] The multiple myeloma RNA sample was amplified using the above CADPS2-RNF148 primers to obtain a 167 bp cDNA product, which contained a specific fusion site (as marked in the figure below, AGTCGC The 3' end of the CADPS2 sequence and the fusion point of RNF148), the nucleotide sequences of the upstream (CADPS2) and downstream (RNF148) were cloned and sequenced as follows:

[0071] CGGTTCCAGGCCTTCCTCAATGGGGAAACCCAAATTGTAGCTGACGAAGCATTTTGCAACGCAGTTCGGAGTTATTATGAGGGGACGGAAAATAT AGTCGC GGTGATGATAAGCAACCTGAAAGGCATGGAAATTTTGCACTCGATTCAGAAAGGAGTCTATGTGAC(SEQ ID NO.1)

[0072] 3. Statistical analysis

[0073] SPSS27.0 and Graphpad Prism 7 were used for statistical analysis. The differences between the two groups of data were compared by chi-square test for categorical variables and t-test for continuous variables. P < 0.05 was considered statistically significant. Linear regression was used to calculate the correlation between CADPS2-RNF148 expression (CADPS2-RNF148 aEX) and peripheral blood and bone marrow plasma cell counts, and the regression equation was predicted. Survival analysis was performed using Kaplan-Meier analysis and log-rank test. Cox proportional hazard regression model was used for multivariate analysis. The variables with P > 0.1 were gradually eliminated by backward regression method, and P < 0.05 was considered statistically significant.

[0074] 2. Research Results

[0075] 1. CADPS2-RNF148 is specifically expressed in the peripheral blood of multiple myeloma patients

[0076] CADPS2-RNF148 was expressed in 15 peripheral blood PBMCs of 109 multiple myeloma patients (15 / 109; 13.76%; median expression level 5.34E-08; range 1.67E-08-1.75E-06), and was not expressed in 101 healthy donor controls (****P<0.0001)( Figure 1 ). The expression levels of the fusion transcript CADPS2-RNF148 in the three multiple myeloma cell lines RPMI-8226, MM.1R, and MM.1S used as positive controls were 1.77E-06, 7.71E-07, and 9.48E-07, respectively.

[0077] The fusion RNA transcript CADPS2-RNF148 is shown in the schematic diagram Figure 2 As shown, unlike most genes, RNF133 and RNF148 genes are located in intron 1 and intron 2 of the CADPS2 gene, respectively. RNF133 and RNF148 genes are located in the CADPS2 genomic sequence; Figure 2 As shown in B, CADPS2 is transcribed into CADPS2 pre-mRNA, which contains the RNF144 pre-mRNA sequence, with triangle lines indicating introns and dotted lines indicating omitted exons and introns; Figure 2 As shown in C, the first two exons of the CADPS2 gene and the last two exons of the RNF144 gene are cis-spliced ​​to form the CADPS2-RNF148 fusion RNA transcript. The red and black rectangles are the 5' and 3' ends, respectively.

[0078] The fusion RNA transcript CADPS2-RNF148 fused the first (462 bp) and second exons (114 bp) of CADPS2 (RefSeq NM_017954) at the 5' end, and fused 433-1255 fusions (648 bp, the underlined portion in SEQ ID NO.6, and the underlined portion of the amino acid sequence encoded by SEQ ID NO.7) downstream of the ATG of RNF148 (RefSeq NM_198085) at the 3' end. The total ORF length is 936 bases, encoding 311 amino acids.

[0079] The total ORF length is 936 bases and the sequence is as follows:

[0080] ATGCTGGACCCGTCTTCCAGCGAAGAGGAGTCGGACGAGGGGCTGGAAGAGGAAAGCCGCGATGTGCTGGTGGCAGCCGGCAGCTCGCAGCGAGCTCCTCCAGCCCCGACTCGGGAAGGGCGGCGGGACGCGCCGGGGCGCGGCGGCGCCAGATCTGTGAGCCCGAGCCCCTCTGTGCTCAGCGAGGGGCGAGACGAGCCCCAGCGGCAGCTGGACGATGAGCA GGAGCGGAGGATCCGCCTGCAGCTCTACGTCTTCGTCGTGAGGTGCATCGCGTACCCCTTCAACGCCAAGCAGCCCACCGACATGGCCCGGAGGCAGCAGAAGCTTAACAAACAACAGTTGCAGTTACTGAAAGAACGGTTCCAGGCCTTCCTCAATGGGGAAACCCAAATTGTAGCTGACGAAGCATTTTGCAACGCAGTTCGGAGTTATTATGAGGGGACGGAAAATATAGTCGC GGTGATGATAAGCAACCTGAAAGGCATGGAAATTTTGCACTCGATTCAGAA AGGAGTCTATGTGACAGTCATCATTGAAGTGGGGAGAATGCACATGCAGTGGGTGAGCCATTACATCATGTATCTA TTTACCTTCCTGGCTGCCACAATTGCCTACTTTTACTTAGATTGCGTCTGGAGACTTACACCTAGAGTGCCCAATT CTTTCACCAGGAGGCGAAGTCAAATAAAGACAGATGTGAAGAAAGCTATTGACCAGCTTCAACTGCGAGTTCTCAA AGAAGGGGATGAGGAATTAGACCTAAATGAAGACAACTGTGTTGTTTGCTTTGACACATACAAACCCCAAGATGTA GTACGCATTTTAACTTGCAAACATTTTTTCCATAAGGCATGCATTGACCCTGGCTTTTAGCCCATAGGACATGTC CCATGTGCAAGTGTGACATCCTGAAAACTTAA (SEQ ID NO.6)

[0081] The encoded amino acid sequence is: MLDPSSSEEESDEGLEEESRDVLVAAGSSQRAPPAPTREGRRDAPGRAGGGGAARSVSPSPSVLSEGRDEPQRQLDDEQERRIRLQLYVFVVRCIAYPFNAKQPTDMARRQQKLNKQQLQLLKERFQAFLNGETQIVADEAFCNAVRSY YEGTENIVAVMISNLKGMEILHSIQKGVYVTVIIEVGRMHMQWVSHYIMYLF TFLAATIAYFYLDCVWRLTPRVPNSFTRRRSQIKTDVKKAIDQLQLRVLKEGDEELDLNEDNCVVCFDTYKPQDVV RILTCKHFFHKACIDPWLLAHRTCPMCKCDILKT (SEQ ID NO.7).

[0082] To further verify the expression of the encoded fusion protein in tumor cells, we attached Flag tags to the N-terminus and C-terminus of the coding sequence and constructed a lentiviral vector. Then, we used anti-Flag antibody to detect the expression of the fusion protein in the Nalm6 cell line. Figure 3 A

[0083] In order to obtain information about the possible role of CADPS2-RNF148 fusion protein in humans, the XtalPred-RF program was used to perform predicted protein structure analysis on human CADPS2-RNF148, such as Figure 3 As shown in B: The CADPS2-RNF148 fusion protein is composed of approximately 311 amino acids, with a molecular weight and isoelectric point of 35.58KD and 6.61, respectively. Black represents loops in the protein structure, red represents α-helical structures, blue represents β-folded structures, yellow represents transmembrane regions, red represents the N-terminus of the fusion protein, and purple represents the N-terminus. Its three-dimensional structure is determined by Alphafold2, as shown in Figure 3 As shown in C.

[0084] Agarose gel bands of PCR products of 15 cases with positive expression of CADPS2-RNF148 are as follows Figure 4 As shown, primer sequences primer qF and primer qR of the fusion RNA transcript CADPS2-RNF148 were used to perform qPCR amplification on three multiple myeloma cell lines RPMI-8226, MM.1R, MM.1S and PBMCs isolated from peripheral blood samples of MM patients. The PCR products of the samples expressing the transcript were subjected to agarose gel electrophoresis. The results showed that the fusion RNA transcript was present in the three cell lines and peripheral blood PBMCs of 15 patients out of 109 MM cases.

[0085] The nucleotide sequencing results of the CADPS2-RNF148 fusion site are as follows Figure 6 As shown, a total of 167 bp.

[0086] 2. CADPS2-RNF148 expression is linearly correlated with the number of plasma cells in peripheral blood and bone marrow of patients with multiple myeloma

[0087] To further study the relationship between CADPS2-RNF148 level and clinical indicators of multiple myeloma, the bone marrow puncture reports of 15 multiple myeloma patients with positive CADPS2-RNF148 expression were analyzed.

[0088] Regression analysis showed that in 15 cases of MM with positive expression of fusion RNA transcript CADPS2-RNF148, the expression of this fusion transcript was significantly correlated with the percentage of bone marrow plasma cells bm-PLAC% (r=0.81, P=2.71e-04) (e.g. Figure 7 A) and peripheral plasma cell pb-PLAC% (r = 0.96, P = 2.25e-08) (as shown in Figure 7 B) showed a linear positive correlation. The prediction equations were: bm-PLAC% = 4.595e + 007 * CADPS2-RNF148 expression + 14.55 and pb-PLAC% = 5.406e + 007 * CADPS2-RNF148 expression - 2.603.

[0089] 3. Relationship between CADPS2-RNF148 expression and general clinical features of multiple myeloma

[0090] According to whether CADPS2-RNF148 was expressed (see Research Results 1), 109 patients with multiple myeloma were divided into CADPS2-RNF148 positive expression group (CADPS2-RNF148+) and CADPS2-RNF148 negative expression group (CADPS2-RNF148-).

[0091] The results showed that patients in the CADPS2-RNF148 positive expression group were more likely to have stage III (P=0.009) in ISS (P=0.024) and R-ISS at the time of initial diagnosis. At the same time, patients in the CADPS2-RNF148 positive expression group were more likely to have renal impairment, namely DS stage B (serum creatinine ≥176.8μmol / L, P=0.002), high tumor burden, specifically manifested as high serum β2 microglobulin (β2-microglobulin, β2-MG ≥10mg / L, P<0.0001****) and high LDH (Lactate dehydrogenase>240U / L, P<0.001***). In addition, patients in the CADPS2-RNF148 positive expression group had a higher ECOG score (P=0.0003***) (Table 5).

[0092] Since a low age at diagnosis is an independent risk factor affecting the prognosis of multiple myeloma, we conducted a statistical analysis of people aged less than 60 years at diagnosis and found that in this population, patients in the CADPS2-RNF148 positive expression group were more likely to have high LDH (LDH>240U / L, P=0.011*) and higher ECOG scores (P<0.0001****) (Table 5).

[0093] Table 5. Relationship between CADPS2-RNF148 expression and general clinical characteristics of multiple myeloma

[0094]

[0095]

[0096]

[0097]

[0098] 4. Relationship between CADPS2-RNF148 expression level and prognosis of multiple myeloma patients

[0099] Survival analysis of 109 patients was performed using the Kaplan-Meier method, and the 3-year overall survival (OS) rates of patients in the CADPS2-RNF148 positive expression group and the negative expression group were 61.25% and 94.44%, respectively. The overall survival time of patients in the CADPS2-RNF148 positive expression group was shorter than that in the negative expression group (median 1.5 vs. 1.58 years; P = 0.02*; Figure 8 (A) (Table 6).

[0100] Similarly, we conducted a statistical analysis on the population aged ≤60 years at diagnosis and found that in this population, the 3-year OS of patients in the CADPS2-RNF148 positive expression group and the CADPS2-RNF148 negative expression group were 100% and 40%, respectively. The overall survival time of patients in the CADPS2-RNF148 positive expression group was shorter than that in the negative expression group (median 1.5 vs. 1.58 years; P = 0.002**; Figure 8 (B) (Table 6), indicating that the positive expression of CADPS2-RNF148 has a more obvious trend of shorter overall survival in people diagnosed with MM before 60 years of age.

[0101] Table 6. Analysis of CADPS2-RNF148 expression and overall survival (OS) in patients with multiple myeloma

[0102]

[0103] Example 2

[0104] We collected 12 multiple myeloma patients for further verification. CADPS2-RNF148 was expressed in peripheral blood PBMCs in 2 of the 12 multiple myeloma patients (diagnosed age range 54-80 years, median 70 years, 8 males, 4 females) (MPP-1, female, diagnosed at 70 years old and MPP-4, male, diagnosed at 77 years old) (2 / 12; 16.67%; median expression 2.996E-07; MPP-1 expression 5.32e-08~5.46e-07), while the other 10 cases were not detected to express the fusion transcript. Since the experimental process is the same as that of Example 1, it will not be repeated. Similarly, combined with clinical data, the chi-square test Fisher's exact test analysis results showed that the two patients with positive CADPS2-RNF148 expression (2 / 2, 100%) were both ISS and R-ISS stage III, and the patients with negative expression were not ISS and R-ISS. Stage III (0 / 10, 0) (ISS stage I 4 cases, stage II 6 cases; R-ISS stage I 3 cases, stage II 7 cases) (P=0.0152*); 2 patients with positive expression had serum creatinine levels greater than or equal to 176.8μmol / L, i.e. DS stage B type (2 / 2, 100%), while only 1 of the 10 negative patients (CADPS2-RNF148 negative expression) had serum creatinine level ≥176.8μmol / L (DS stage B type) (1 / 10, 10%) (P=0.046*); 2 patients with positive expression had serum β2-MG levels greater than or equal to 10mg / L (2 / 2, 100%, MPP-1 was 17.68mg / L, MPP-4 was 10.93mg / L, median was 14.14mg / L), while 1 Only one of the 0 negative patients had a serum β2-MG level ≥ 10 mg / L (1 / 10, 10%, median 3.10 mg / L), and the serum LDH of the 2 positive patients was greater than 240 U / L (2 / 2, 100%, MPP-1 was 306 U / L, MPP-4 was 931 U / L, median 618.5 U / L), while only one of the 10 negative patients had a serum LDH > 240 U / L (1 / 10, 10%, median 170.5 U / L) (P values ​​were 0.046*); the ECOG score of the 2 positive patients was 3 points (2 / 2, 100%), while the ECOG score of the negative patients was < 3 points (ECOG score of 1 point in 4 cases and ECOG score of 2 points in 6 cases) (P = 0.0152*). Due to the small number of cases and short follow-up time, no overall survival analysis was performed.

[0105] In addition, the expression level of the fusion transcript CADPS2-RNF148 in peripheral blood PBMCs of MPP-1 was 5.46e-07, and the bone marrow puncture results showed that the proportion of bone marrow (bm-PLAC%) and peripheral plasma cell number (pb-PLAC%) were 71% and 49%, respectively; the expression level of the fusion transcript of MPP-4 was 5.32e-08, and the bone marrow puncture results showed that the proportion of bone marrow (bm-PLAC%) and peripheral plasma cell number (pb-PLAC%) were 12% and 0, respectively, which were consistent with the calculation formula in Example 1.

[0106] The positive expression of 15 fusion RNA transcripts CADPS2-RNF148 in Example 1 was detected and regression analysis was performed. The expression of 2 transcripts in Example 2 and the number of bone marrow and peripheral plasma cells in the bone marrow puncture report were substituted into the formula for verification. The above results show that by detecting the expression of the fusion transcript CADPS2-RNF148 in peripheral blood, the number of plasma cells in the bone marrow and peripheral blood can be calculated by the formula. The increase in visible plasma cells in the bone marrow cavity is an important basis for diagnosing multiple myeloma. The average reference value of bone marrow plasma cells is 1.3%, usually <2%. Foreign countries believe that >2-4% of plasma cells in the bone marrow are the diagnostic criteria for plasma cell increase, and >3% is used as the diagnostic criteria for plasma cell increase in China. If the number of plasma cells in the bone marrow exceeds 10-20%, the diagnosis of multiple myeloma can be confirmed. At present, the diagnosis and efficacy evaluation of multiple myeloma need to rely on invasive bone marrow puncture for bone marrow biopsy to detect the number of plasma cells, and because the cells in the bone marrow cavity of patients with multiple myeloma are often unevenly infiltrated, a puncture may be negative, and multiple sites or multiple punctures are required to find characteristic changes, and the results of each puncture may be very different; in addition, plasma cells do not appear in peripheral blood under normal circumstances. The number of plasma cells in peripheral blood ≥ 20% is defined as plasma cell leukemia, which is also a malignant disease. Plasma cell leukemia can appear alone or develop from multiple myeloma, and is often a manifestation of terminal multiple myeloma. Therefore, monitoring the number of plasma cells in peripheral blood is particularly important for diagnosing plasma cell leukemia and evaluating the progression of multiple myeloma. If the expression amount of the fusion transcript CADPS2-RNF148 detected in peripheral blood can be used to infer the number of plasma cells in the bone marrow and peripheral blood, it has a very large medical application value in terms of reducing medical costs, shortening diagnosis time, alleviating patient pain, and the ease of clinical sample acquisition.

[0107] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A nucleic acid molecule and / or a protein molecule selected from at least one of the following: A. Fusion RNA transcript CADPS2-RNF148, the sequence of which is shown in SEQ ID NO: 1; B. The open reading frame of the fusion RNA transcript CADPS2-RNF148, the sequence of which is shown in SEQ ID NO: 6; C. The protein encoded by the fusion RNA transcript CADPS2-RNF148, whose sequence is shown in SEQ ID NO:

7.

2. Use of the nucleic acid molecule and / or protein molecule according to claim 1 as a biomarker in the preparation of a product for diagnosis and prognosis evaluation of multiple myeloma and / or plasma cell leukemia.

3. Use of a reagent for detecting the nucleic acid molecule and / or protein molecule according to claim 1 in the preparation of a product for diagnosis and prognosis evaluation of multiple myeloma and / or plasma cell leukemia.

4. A detection kit for multiple myeloma and / or plasma cell leukemia, characterized in that: It comprises a reagent for detecting the expression amount of the nucleic acid molecule and / or protein molecule as claimed in claim 1 in a sample.

5. The detection kit according to claim 4, characterized in that The samples are peripheral blood or human multiple myeloma cell lines RPMI-8226, MM.1R and MM.1S.

6. The detection kit according to claim 4, characterized in that The reagents include detection primers for detecting CADPS2-RNF148 fusion RNA transcripts and fusion sites, and the sequences of the detection primers include but are not limited to those shown in SEQ ID NO:2 and SEQ ID NO:

3.

7. The detection kit according to claim 6, characterized in that The reagents also include internal reference primers, and the sequences of the internal reference primers include but are not limited to those shown in SEQ ID NO:4 and SEQ ID NO:

5.

8. A primer for detecting the fusion RNA transcript CADPS2-RNF148 described in claim 1, wherein the sequence of the detection primer includes but is not limited to SEQ ID NO: 2 and SEQ ID NO:

3.

9. A pharmaceutical composition, characterized in that The active ingredient targets an inhibitor of the expression of the fusion RNA transcript CADPS2-RNF148 described in claim 1, or an inhibitor of the protein encoded by the fusion RNA transcript CADPS2-RNF148 described in claim 1.

10. Use of the nucleic acid molecule and / or protein molecule of claim 1 as a molecular target in the preparation of a drug for treating multiple myeloma and / or plasma cell leukemia.

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