Application of exosome circular RNA in peripheral blood as multiple myeloma bone disease biomarker

Abstract

The invention discloses an application of exosome circular RNA (Ribonucleic Acid) in peripheral blood as a biomarker of multiple myeloma bone diseases. The exosome circular RNA in the peripheral blood is selected from one or more of nucleotide sequences as shown in SEQ ID No.1 to SEQ ID No.20. The invention also discloses an application of the exosome circular RNA in the peripheral blood as the biomarker of the multiple myeloma bone diseases. According to the invention, high-throughput sequencing is combined with a single cell sequencing technology, circular RNA playing a key role in MBD is analyzed, and experiments prove that the expression level of the circular RNA from SEQ ID No.1 to SEQ ID No.20 can be used as a marker of MBD; compared with other genetic information, the exosome circular RNA has the characteristics of high stability, high abundance, high specificity, high conservative property and the like, and an ideal biomarker even a therapeutic target is provided.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to the application of exosome circular RNA in peripheral blood as a biomarker for multiple myeloma bone disease. Background Art

[0002] Multiple myeloma (MM) is a malignant disease of plasma cells originating from the bone marrow, characterized by clonal heterogeneity and bone marrow dependence, and is still incurable. Multiple myeloma bone disease (MM bonediseases, MBD) is a serious complication of MM. Approximately 80% of MM patients have osteolytic lesions at the time of initial diagnosis, and are often accompanied by bone-related events, which seriously affect the quality of life and survival of MM patients. At present, the diagnosis of MM and MBD mainly relies on bone marrow examination and imaging examination. Due to invasiveness and radiation, it is difficult to obtain continuous samples. In contrast, non-invasive liquid biopsy has the advantages of convenience, non-invasiveness, and repeatability, and has gradually become the focus of precise diagnosis and treatment of tumors. Compared with other analytes (such as circulating tumor cells, circulating tumor DNA, etc.), exosomes have higher content in peripheral blood and carry richer information. They are secreted by multiple cell types and can reflect a variety of information such as the tumor microenvironment. Increasing evidence has revealed that the pathogenesis of MBD is closely related to the dysregulation of the bone marrow microenvironment. The interaction between MM cells and bone marrow mesenchymal stem cells, osteoclasts, osteoblasts, osteocytes, and immune cells leads to an imbalance in bone metabolism and bone remodeling, resulting in the occurrence of MBD.

[0003] At present, the effects of exosome-mediated cell communication on MBD are mostly focused on exosomes secreted by MM cells. For example, miR-92a-2-5p and miR-373-3p inhibit the expression of IRF8 and RUNX2 in monocytes or mesenchymal stem cells through exosomes secreted by myeloma cells, induce osteoclast activation, and inhibit osteoblast differentiation. lncRNARUNX2-AS1 in myeloma cells is mediated by exosomes and inhibits its osteogenic differentiation ability by downregulating RUNX2 in mesenchymal stem cells. There is still a lack of relevant research on the effects of exosomes secreted by other cells in the bone marrow microenvironment on MBD. Summary of the invention

[0004] In order to solve the deficiencies of the prior art, the present invention proposes the use of exosome circular RNA in peripheral blood as a biomarker for multiple myeloma bone disease. The technical solution provided by the present invention is as follows:

[0005] The present invention provides the use of exosome circular RNA in peripheral blood as a biomarker for multiple myeloma bone disease, wherein the exosome circular RNA in peripheral blood is selected from one or more of the following:

[0006] The nucleotide sequence is as shown in SEQ ID No. 1, chr2: 2744264-2744443+;

[0007] The nucleotide sequence is as shown in SEQ ID No. 2: chr2: 2744228-2744407+;

[0008] The nucleotide sequence is shown in SEQ ID No. 3 as chr7:75847812-75847976+;

[0009] The nucleotide sequence is shown in SEQ ID No. 4 as chr19:42853602-42853758-;

[0010] The nucleotide sequence is shown in SEQ ID No.5 as chr7:33923121-33923277-;

[0011] The nucleotide sequence is shown in SEQ ID No.6 as chr1:181379313-181379498-;

[0012] The nucleotide sequence is shown in SEQ ID No.7 as chr11:85831034-85831205-;

[0013] The nucleotide sequence is shown in SEQ ID No.8 as chr12:69748686-69748849-;

[0014] The nucleotide sequence is shown in SEQ ID No.9 as chr3:3830938-3831112+;

[0015] The nucleotide sequence is shown in SEQ ID No. 10 as chr7:30728074-30728244+;

[0016] The nucleotide sequence is shown in SEQ ID No. 11 as chr7:151946961-151948051-;

[0017] The nucleotide sequence is shown in SEQ ID No. 12 as chr11: 108046973-108047817-;

[0018] The nucleotide sequence is shown in SEQ ID No. 13: chr5: 138699448-138700432+;

[0019] The nucleotide sequence is shown in SEQ ID No. 14 as chr6:18236683-18258636-;

[0020] The nucleotide sequence is shown in SEQ ID No. 15 as chr8:71126138-71128999-;

[0021] The nucleotide sequence is shown in SEQ ID No. 16 as chr9:79996892-80022523+;

[0022] The nucleotide sequence is shown in SEQ ID No. 17 as chr2:89100616-89104394+;

[0023] The nucleotide sequence is shown in SEQ ID No. 18 as chr17:30267305-30315516+;

[0024] The nucleotide sequence is shown in SEQ ID No. 19: chrX:76907604-76912143-;

[0025] The nucleotide sequence is shown in SEQ ID No.20 as chr19:23541232-23545527-.

[0026] The present invention also provides an application of a substance for detecting exosome circular RNA markers in peripheral blood, including one or more of the following applications:

[0027] a. Use in the preparation of products for diagnosing multiple myeloma bone disease;

[0028] b. Application in the preparation of a product for assessing the prognosis of bone disease in multiple myeloma;

[0029] The exosomal circular RNA marker is one or more nucleotide sequences as shown in SEQ ID No.1 to SEQ ID No.20.

[0030] The product can be a product for diagnosing multiple myeloma bone disease by detecting the expression levels of SEQ ID No. 1 to SEQ ID No. 20 through qRT-PCR, real-time quantitative PCR, or a high-throughput sequencing platform.

[0031] The detection is for an in vitro sample, and the in vitro sample is a peripheral blood sample.

[0032] In the application, the exosomal circular RNA markers shown in the sequence table as SEQ ID No. 1 to SEQ ID No. 10 are significantly upregulated in the peripheral blood samples of multiple myeloma bone disease.

[0033] The exosomal circular RNA markers shown in the sequence table as SEQ ID No. 11 to SEQ ID No. 20 were significantly downregulated in the peripheral blood samples of multiple myeloma bone disease.

[0034] The product can be used as a reagent for detecting the expression level of a sequence list such as SEQ ID No. 1 to SEQ ID No. 20, or atopy recognition, or detecting the content of SEQ ID No. 1 to SEQ ID No. 20.

[0035] The product may be a primer for detecting the expression amount of SEQ ID No. 1 to SEQ ID No. 20, or a primer that specifically recognizes SEQ ID No. 1 to SEQ ID No. 20; or a reagent set or a kit containing the primer.

[0036] The present invention provides the use of exosome circular RNA in peripheral blood as a target for treating multiple myeloma bone disease.

[0037] The inventors studied the whole transcriptome sequencing of peripheral blood exosomes from MM patients and healthy volunteers, screened differentially expressed circular RNAs, analyzed circular RNAs that play a key role in MBD, and integrated single cell RNA sequencing (scRNA-seq) technology to resolve the source cells of key exosomal circular RNAs, and clarified the cellular interactions in the bone marrow microenvironment at the single-cell level, revealing the important role of the bone marrow microenvironment in MBD, providing new insights into the pathogenesis, diagnosis, treatment and monitoring of MBD.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The present invention analyzes circular RNAs that play a key role in MBD by high-throughput sequencing combined with single-cell sequencing technology. Experiments show that SEQ ID No.1 to SEQ ID No.10 are significantly upregulated in peripheral blood exosomes of MM patients, and SEQ ID No.1 to SEQ ID No.10 are significantly downregulated in peripheral blood exosomes of MM patients, indicating that the expression levels of these circular RNAs can be used as markers for MBD;

[0040] 2. The present invention utilizes the characteristics of exosomal circular RNA, which has high stability, high abundance, high specificity, and high conservation compared to other genetic information, and provides an ideal biomarker or even a therapeutic target;

[0041] 3. Through exosomal circular RNA tracing, the present invention speculates that MBD-related differential circular RNA mainly originates from T cells / natural killer cells, B cells and monocytes / macrophages in the bone marrow microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0043] Figure 1 Cluster analysis of differentially expressed circular RNAs.

[0044] Figure 2 GO and KEGG enrichment analysis of circular RNA significantly upregulated in MM; A is the top 10 biological processes in GO enrichment analysis; B is the top 10 signaling pathways in KEGG enrichment analysis.

[0045] Figure 3 It is the UMAP map of the bone marrow microenvironment of MM patients; Figure A is the UMAP visualization form of 7 cell subsets; Figure B is the distribution of characteristic gene expression of each cell subset; Figure C is the volcano map display of significantly differentially expressed genes in each cell subset, and the top 3 significantly upregulated and downregulated differentially expressed genes.

[0046] Figure 4 The expression distribution of MBD-related differentially expressed circular RNA parental genes in each cell subpopulation.

[0047] Figure 5 is the cell-cell interaction strength network; A is the communication strength between different cell subsets; B is the communication strength between monocytes / macrophages and each cell subset.

[0048] Figure 6 It is an analysis of the cell interactions between monocytes / macrophages and various cell subsets; Figure A is a bubble diagram of the interaction between monocytes / macrophages as receptor cells and other cells mediated by ligand-receptor pairs; Figure B is a network diagram of the relationship between the MIF pathway and intercellular communication. DETAILED DESCRIPTION

[0049] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0050] Example 1: Research on exosomal circular RNA in peripheral blood as a biomarker for bone disease in multiple myeloma

[0051] 1 Objects and Methods

[0052] 1.1 Design of peripheral blood clinical sample experiment. Use R software (version 4.2.2) edgeR package for data normalization and differentially expressed circular RNA screening, and calculate differential expression fold based on FPKM value.

[0053] 1.2 Time and Place The experiment was completed in October-November 2020 at the Experimental Center of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine and Shanghai Yunxu Biotechnology Co., Ltd.

[0054] 1.3 Subjects Peripheral blood samples were collected from 6 MM patients and 5 healthy controls. All cases were diagnosed by experienced clinicians and met the diagnostic criteria for active MM established by the International Myeloma Working Group. Peripheral blood was collected in vacuum blood collection tubes for blood coagulation, centrifuged at 1500g for 10 minutes, serum was collected, and then centrifuged at 3000g for 15 minutes to remove cells and cell debris. The supernatant was transferred to a sterile container, and exosomes were isolated using the ExoQuick Exosome Precipitation Kit (SystemBiosciences, USA). 63μl of ExoQuick solution was added to every 250μl of serum, mixed and refrigerated for 30 minutes, then centrifuged at 1500g for 30 minutes, and the supernatant was aspirated. Centrifuged at 1500g for 5 minutes, the remaining liquid was carefully aspirated, and the exosomes were resuspended in PBS and stored at -80℃ until use. This study was approved by the Ethics Committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (Ethics Number: (2020) Ethics Review Number (010)-KY), and written informed consent was obtained from all participants.

[0055] 1.4 Methods

[0056] 1.4.1 High-throughput sequencing All samples were sent to Shanghai Yunxu Biotechnology Co., Ltd. for whole transcriptome sequencing. Ribosomal RNA was removed using the Ribo-Zero rRNA removal kit (Illumina, USA). RNA libraries were prepared using the TruSeqStranded Total RNA Sample Preparation Kit (Illumina, USA). The quantity and quality of the RNA library were analyzed using the BioAnalyzer2100 system (Agilent, USA). The RNA library was then reverse transcribed into cDNA, captured by the illumina flow cell, amplified in situ, and finally sequenced using the HiSeq 4000 sequencing system (Illumina, USA).

[0057] 1.4.2GO and KEGG enrichment analysis

[0058] The differentially expressed circular RNAs between the two groups of samples were screened with a difference fold ≥ 2.0 and P ≤ 0.05 as the standard. The GO database (http: / / www.geneontology.org) and KEGG database (http: / / www.genome.jp / kegg) were used to perform GO and KEGG enrichment analysis on the parental genes of differential circular RNAs (P ≤ 0.05 was considered significant difference), and the R package ggolot2 was used to draw a schematic diagram.

[0059] 1.4.3 ScRNA-seq data acquisition and preprocessing

[0060] The GSE271107 scRNA-seq dataset file was downloaded from the GEO database (http: / / www.ncbi.nlm.nih.gov / geo / ), including 5 healthy volunteers, 4 patients with smoldering myeloma, 6 patients with monoclonal gammopathy of undetermined significance, and 4 patients with MM. Four patients with MM were selected for analysis in this study. The MM samples were converted into Seurat objects using the "CreateSeuratObject" function of the Seurat package. The cells were quality controlled and filtered based on the feature value >200 and <3000, count value <40000, and mitochondrial gene content <20%, and double cells were removed according to the predicted value of 4%.

[0061] 1.4.4 ScRNA-seq data analysis

[0062] The standardized data were integrated using the “FindIntegrationAnchors” function of the Seurat package. 3000 highly variable genes were selected for principal component analysis using “SelectIntegrationFeatures”, and the batch effect was removed using the Harmony method. UMAP clustered 7 cell subpopulations with a resolution of 0.1. The SingleR package was used to perform preliminary annotation of cell types, combining the automatic annotation results with previously published studies, and manually correcting the cell populations.

[0063] 1.4.5 Cell communication analysis

[0064] The R package CellChat was used to infer and visualize intercellular communication in scRNA-seq data and analyze the interactions between ligands and receptors.

[0065] 2 Results

[0066] 2.1 Screening of differentially expressed circular RNAs

[0067] The absolute value of the logarithm of the difference (log 2|fold change(FC)|)>1 and P value<0.05 were used as the threshold, and 2052 differentially expressed circular RNAs were screened and identified. The cluster analysis diagram shows the expression profile of differentially expressed genes in each sample. Figure 1 Compared with healthy volunteers, MM patients had 1265 upregulated circRNAs and 787 downregulated circRNAs. Ten upregulated and downregulated circRNAs with the most significant differential expression were obtained. The relevant information of the top ten upregulated and downregulated circRNAs in peripheral blood exosomes of MM patients compared with the healthy control group is shown in Table 1.

[0068] Table 1 The top 10 most significantly upregulated and downregulated circular RNAs in peripheral blood exosomes of MM patients

[0069]

[0070] 2.2GO analysis and KEGG analysis

[0071] We performed GO and KEGG enrichment analysis on circular RNAs with significantly upregulated expression levels in MM patients ( Figure 2 The results showed that the parental genes of differentially expressed circular RNAs were mainly enriched in pathways in nervous system development, glutamate receptor signaling pathway, cell signal transduction, cancer, PI3K-Akt signaling pathway, Hippo signaling pathway, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy, circadian rhythm synchronization, and hypertrophic cardiomyopathy pathway.

[0072] 2.3 Single-cell dimensionality reduction and clustering analysis

[0073] In order to infer the source cells of differential circular RNA associated with MBD, we selected the bone marrow scRNA-seq data of 4 MM patients in the GSE271107 dataset. Through quality control and UMAP dimension reduction of scRNA-seq data, the transcriptome data of 21,868 cells were obtained. The cells were automatically annotated and manually corrected according to the expression of genes, including T cells / natural killer cells (7,351), monocytes / macrophages (4,077), erythroid progenitor cells (2,849), erythrocytes (2,585), myeloma cells (2,508), B cells (2,380), dendritic cells (118), see Figure 3 A and B. By performing differential analysis on the 7 annotated cell subsets and drawing a volcano plot, we can see the top 3 differentially expressed genes that are significantly upregulated and downregulated in different cell subsets ( Figure 3 C).

[0074] 2.4 Tracing the origin of exosomal circular RNA

[0075] The parent genes of MBD-related differential circular RNA (differential genes involved in the PI3K-Akt pathway and the Hippo pathway) were clustered using the scRNA-seq feature map ( Figure 4 ) visualization, we found that GNB1, JAK1, THEM4, TCF7L2, and GSK3B were distributed in specific cell subsets. GNB1 and JAK1 were mainly distributed in T cells / natural killer cells, B cells, and monocytes / macrophages, GSK3B was mainly distributed in T cells / natural killer cells and monocytes / macrophages, TCF7L2 was mainly distributed in monocytes / macrophages, and THEM4 was mainly distributed in T cells / natural killer cells. Therefore, we speculated that MBD-related differential circular RNAs mainly originated from T cells / natural killer cells, B cells, and monocytes / macrophages in the bone marrow microenvironment.

[0076] 2.5 Interactions between monocytes / macrophages and other cells

[0077] Monocytes / macrophages are the precursor cells of osteoclasts, and their differentiation into osteoclasts plays an important role in the pathological process of MBD. In order to explore the interaction between monocytes / macrophages and other cell types in the bone marrow microenvironment at the single cell level, we used CellChat to detect ligand-receptor pairs and molecular interactions between different cells. The cell-to-cell interaction intensity network is shown in Figure 5 In A and B, monocytes / macrophages interact with erythroid progenitor cells, erythrocytes, myeloma cells, B cells, and dendritic cells, and their interactions with dendritic cells and B cells are stronger.

[0078] 2.6 Ligand-receptor interaction analysis

[0079] CellChat was further used to explore the signaling pathways in which monocytes / macrophages act as receptors ( Figure 6 A), the MIF pathway in which it is mainly involved plays an important role in the differentiation of monocytes / macrophages into osteoclasts. MIF acting on monocytes / macrophages mainly comes from T / natural killer cells, dendritic cells, tumor cells, B cells and monocytes / macrophages ( Figure 6 (middle B).

[0080] In this study, whole transcriptome sequencing was used to obtain the expression profile of peripheral blood exosome circular RNA in MM patients and healthy volunteers, and GO and KEGG analysis were used to predict the possible mechanism of differential circular RNA involved in the pathological process of MBD. The results of scRNA-seq were combined to speculate the bone marrow microenvironment source cells of key circular RNAs, and it was proposed that monocytes / macrophages are osteoclast precursor cells, and the MIF pathway ligand-receptor interaction between them and tumor cells and other immune cells is the key to the occurrence of MBD.

[0081] Exosomes are important mediators of intercellular communication. In MM, exosomes secreted by tumor cells and other cells in the tumor microenvironment participate in MM angiogenesis, immunosuppression, induced drug resistance, and the occurrence of related complications. Exosomes are enriched in body fluids, and the exosomes released by tumor cells and the tumor microenvironment contain a large amount of specific genetic information, which can reflect changes in the physiological and pathological states of their parent cells. These characteristics make circulating exosomes likely to become new biomarkers for tumor liquid biopsy. The inventors identified 1265 circular RNAs that were significantly upregulated in MM peripheral blood exosomes through high-throughput sequencing technology. Bioinformatics analysis showed that the main pathways involved in the proliferation, adhesion, migration and homing of myeloma cells (PI3K-Akt signaling pathway, Hippo signaling pathway), and MM-related complications, such as: MBD (PI3K-Akt signaling pathway, Hippo signaling pathway), cardiac events (arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy, circadian rhythm synchronization, hypertrophic cardiomyopathy pathway) and peripheral neuropathy (nervous system development pathway) play an important role. The present invention further explored the source cells of MBD-related circular RNAs. Based on the scRNA-seq results, we found that the parental genes of MBD-related circular RNAs were enriched in immune cells such as monocytes / macrophages, T / natural killer cells, and B cells. Monocytes / macrophages are osteoclast precursor cells. Under the action of signaling molecules such as M-CSF and RANKL, downstream signaling pathways such as PI3K-Akt, MAPK, and NF-κB are activated, promoting the fusion and differentiation of osteoclast precursors. The Hippo pathway is also involved in the regulation of osteoclast precursor proliferation, osteoclast differentiation, apoptosis, and survival. In addition, related studies have shown that immune cells participate in the regulation of bone metabolism and osteoclast function by secreting a variety of cytokines. For example, Th17 cells secrete IL17 to promote osteoclastogenesis, TGF-β and IL-10 secreted by regulatory T cells inhibit osteoclast differentiation, and B cells can secrete a variety of cytokines, including IL-1, IL-6, IL-7, IL-10, IL-35, IL-37, TNF-α, and TGF-β, which promote or inhibit osteoclastogenesis in different environments. The PI3K-Akt pathway can positively regulate the differentiation of Th17 cells and is essential for the development and immune response of B cells. The Hippo pathway plays an important role in maintaining the Treg cell pool and regulating the generation of Th17 cells and the function of Treg. Therefore, MBD-related peripheral blood exosomal circular RNA may originate from immune cells in the bone marrow microenvironment and participate in the pathological process of MBD by regulating the PI3K-Akt pathway and the Hippo pathway.

[0082] The inventors further analyzed the interaction between monocytes / macrophages and other cell types in the MM bone marrow microenvironment at the single-cell level, and explored other potential signaling pathways. Monocytes / macrophages interact with both tumor cells and immune cells, and the main pathways involved are the MIF pathway. MIF exerts its biological effects by binding to CXCR or CD74 receptors. The binding of MIF to CD74 induces its phosphorylation and the recruitment of CD44, leading to the phosphorylation of ERK1 / 2 and the sustained activation of the ERK-MAPK pathway. MIF binding to CXCR can also induce inflammation and immune cell chemotaxis. The role of the MIF signaling pathway has not yet been clearly defined in MBD, but there is increasing evidence that the MIF pathway is involved in the development of various bone diseases. Targeting MIF inhibits osteoclastogenesis and reduces bone loss caused by ovarian resection. In addition, the MIF-CXCR4 axis is a key pathway for inducing osteoclast precursors to home to peripheral osteolytic lesions. Therefore, the MIF pathway ligand-receptor interaction between monocytes / macrophages and tumor cells and immune cells may be an important factor in the occurrence of MBD.

[0083] In summary, this study, based on exosome sequencing data and scRNA-seq data, preliminarily identified the bone marrow microenvironment source cells of MBD-related peripheral blood exosome circular RNA, and portrayed the communication map between osteoclast precursor cells and the bone marrow microenvironment at the single-cell level. The MIF pathway may be a potential diagnostic and therapeutic target for MBD.

[0084] Example 2, Application of a Material for Detecting Exosome Circular RNA Markers in Peripheral Blood

[0085] This embodiment provides one or more applications including the following:

[0086] a. Use in the preparation of products for diagnosing multiple myeloma bone disease;

[0087] b. Application in the preparation of a product for assessing the prognosis of bone disease in multiple myeloma;

[0088] The exosomal circular RNA marker is one or more nucleotide sequences as shown in SEQ ID No.1 to SEQ ID No.20.

[0089] In some embodiments, the product can be a product for diagnosing multiple myeloma bone disease by detecting the expression levels of SEQ ID No. 1 to SEQ ID No. 20 through qRT-PCR, real-time quantitative PCR, or high-throughput sequencing platform.

[0090] The detection is for an in vitro sample, and the in vitro sample is a peripheral blood sample.

[0091] In some embodiments, the exosomal circular RNA markers shown in the sequence table as SEQ ID No. 1 to SEQ ID No. 10 are significantly upregulated in peripheral blood samples of multiple myeloma bone disease.

[0092] The exosomal circular RNA markers shown in the sequence table as SEQ ID No. 11 to SEQ ID No. 20 were significantly downregulated in the peripheral blood samples of multiple myeloma bone disease.

[0093] The product can be used as a reagent for detecting the expression level of a sequence list such as SEQ ID No. 1 to SEQ ID No. 20, or atopy recognition, or detecting the content of SEQ ID No. 1 to SEQ ID No. 20.

[0094] The product may be a primer for detecting the expression amount of SEQ ID No. 1 to SEQ ID No. 20, or a primer that specifically recognizes SEQ ID No. 1 to SEQ ID No. 20; or a reagent set or a kit containing the primer.

[0095] Example 3, application of exosomal circular RNA in peripheral blood as a target for treating multiple myeloma bone disease.

[0096] Based on the above research, it can be seen that the exosomal circular RNA shown in the sequence table such as SEQ ID No. 11 to SEQ ID No. 20 can be used as a target for the treatment of multiple myeloma bone disease.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of exosomal circular RNA in peripheral blood as a biomarker for bone disease in multiple myeloma, characterized in that: The exosomal circular RNA in the peripheral blood is selected from one or more of the following: The nucleotide sequence is as shown in SEQ ID No. 1, chr2: 2744264-2744443+; The nucleotide sequence is as shown in SEQ ID No. 2: chr2: 2744228-2744407+; The nucleotide sequence is shown in SEQ ID No. 3 as chr7:75847812-75847976+; The nucleotide sequence is shown in SEQ ID No. 4 as chr19:42853602-42853758-; The nucleotide sequence is shown in SEQ ID No.5 as chr7:33923121-33923277-; The nucleotide sequence is shown in SEQ ID No.6 as chr1:181379313-181379498-; The nucleotide sequence is shown in SEQ ID No.7 as chr11:85831034-85831205-; The nucleotide sequence is shown in SEQ ID No. 8 as chr12:69748686-69748849-; The nucleotide sequence is shown in SEQ ID No.9 as chr3:3830938-3831112+; The nucleotide sequence is shown in SEQ ID No. 10 as chr7:30728074-30728244+; The nucleotide sequence is shown in SEQ ID No. 11 as chr7:151946961-151948051-; The nucleotide sequence is shown in SEQ ID No. 12 as chr11: 108046973-108047817-; The nucleotide sequence is shown in SEQ ID No. 13: chr5: 138699448-138700432+; The nucleotide sequence is shown in SEQ ID No. 14 as chr6:18236683-18258636-; The nucleotide sequence is shown in SEQ ID No. 15 as chr8:71126138-71128999-; The nucleotide sequence is shown in SEQ ID No. 16 as chr9:79996892-80022523+; The nucleotide sequence is shown in SEQ ID No. 17 as chr2:89100616-89104394+; The nucleotide sequence is shown in SEQ ID No. 18 as chr17:30267305-30315516+; The nucleotide sequence is shown in SEQ ID No. 19: chrX:76907604-76912143-; The nucleotide sequence is shown in SEQ ID No.20 as chr19:23541232-23545527-.

2. An application of a substance for detecting exosome circular RNA markers in peripheral blood, characterized in that: Includes one or more of the following applications: a. Use in the preparation of products for diagnosing multiple myeloma bone disease; b. Application in the preparation of a product for evaluating the prognosis of bone disease in multiple myeloma.

3. The use of the substance according to claim 2, characterized in that The exosomal circular RNA marker is one or more nucleotide sequences as shown in SEQ ID No.1 to SEQ ID No.

20.

4. The use of the substance according to claim 2, characterized in that The product is a product for diagnosing multiple myeloma bone disease by detecting the expression levels of SEQ ID No. 1 to SEQ ID No. 20 through qRT-PCR, real-time quantitative PCR, or high-throughput sequencing platform.

5. Use of the substance according to claim 2, characterized in that The detection is for an in vitro sample; the in vitro sample is a peripheral blood sample.

6. Use of the substance according to claim 2, characterized in that In the application, the exosomal circular RNA markers shown in the sequence table as SEQ ID No. 1 to SEQ ID No. 10 are significantly upregulated in the peripheral blood samples of multiple myeloma bone disease.

7. Use of the substance according to claim 2, characterized in that The exosomal circular RNA markers shown in the sequence table as SEQ ID No. 11 to SEQ ID No. 20 were significantly downregulated in the peripheral blood samples of multiple myeloma bone disease.

8. Use of the substance according to claim 2, characterized in that The product is a reagent for detecting the expression amount of a sequence list such as SEQ ID No. 1 to SEQ ID No. 20, or atopy recognition, or detecting the content of SEQ ID No. 1 to SEQ ID No.

20.

9. Use of the substance according to claim 2, characterized in that The product is a primer for detecting the expression amount of SEQ ID No. 1 to SEQ ID No. 20, or a primer for specifically recognizing SEQ ID No. 1 to SEQ ID No. 20; or a reagent set or a kit containing the primer.

10. Application of exosomal circular RNA in peripheral blood as a target for the treatment of multiple myeloma bone disease.

Images