CN113549690B - Long-chain non-coding RNA and application thereof as molecular marker of multiple myeloma cell - Google Patents

Abstract

The invention relates to a long-chain non-coding RNA and its application as a molecular marker of multiple myeloma cells. The long-chain non-coding RNA (lncRNA MSTRG.29039.1) has a transcript sequence shown in SEQ ID NO: 1. The present invention discovers for the first time a long-chain non-coding RNA, which is more highly expressed in multiple myeloma cells than normal plasma cells, and is highly expressed in multiple myeloma cell lines. Knockdown of lncRNA MSTRG.29039.1 in cell lines and knockdown of mRNA OSMR can increase cell apoptosis and reduce proliferation. It can be seen that the lncRNA MSTRG.29039.1 can be used as a molecular marker of multiple myeloma cells and is related to the occurrence and progression.

Description

A long-chain non-coding RNA and its application as a molecular marker of multiple myeloma cells

technical field

The invention relates to the field of monitoring the progression of multiple myeloma, in particular to a multiple myeloma cell molecular marker lncRNA MSTRG.29039.1, which can be applied to the occurrence, progression, disease assessment and targeted therapy of multiple myeloma, Provide accurate diagnosis and treatment to patients in order to achieve complete remission and long-term survival.

Background technique

Multiple myeloma (MM) is a plasma cell disease characterized by biological heterogeneity. Abnormal function, hypercalcemia, anemia, bone disease and a series of damage. With the use of proteasome inhibitors, the rise of autologous stem cell transplantation ASCT, immunomodulatory agents, monoclonal antibodies and cellular immunity and other immunotherapy, MM patients have achieved deeper remission and prolonged survival, but some patients are still unable to recover. Even many patients still relapse. Therefore, research on the underlying pathogenesis of multiple myeloma, monitoring of disease progression, and prevention of relapse is crucial. When evaluating the condition of multiple myeloma, molecular genetics is most closely related to the prognosis of multiple myeloma, such as P53 deletion, 1q21 amplification, 13q14 deletion, etc., most patients with multiple myeloma have poor prognosis. However, these detection techniques are not suitable for widespread implementation due to their high requirements, high price, and long time-consuming. Both gene mutations and chromosomal abnormalities affect the changes in gene expression levels in signaling pathways, and ultimately affect disease progression and prognosis. Therefore, the real-time fluorescent quantitative PCR technology based on the detection of gene expression has the advantages of short time-consuming, easy operation, repeatability, and low cost, and is more suitable for clinical detection.

With the rapid development of high-throughput technology, we have gradually discovered that non-coding RNA (ncRNA) plays an important role in the pathogenesis of myeloma. Long non-coding RNAs (lncRNAs) are RNAs longer than 200 nucleotides that have little or no ability to synthesize proteins. A large number of studies have shown that lncRNAs are also involved in the occurrence, development, metastasis and drug resistance of tumors, and are involved in a variety of biological functions, and are involved in the composition and regulation of cells, such as cell differentiation, proliferation, DNA replication, RNA transcription, Protein modification, nuclear transport and other important functions. MicroRNAs (miRNAs) are short non-coding RNAs of approximately 22 nucleotides in length that regulate gene expression at the post-transcriptional level, typically inhibit the expression of target genes, and have been shown to be dysfunctional or abnormally expressed in various cancers. A large number of experiments have shown that miRNAs can regulate proliferation, migration, and even regulate epigenetics to promote the occurrence and development of MM. Recent experiments suggest that ceRNAs are transcripts that can regulate each other at the post-transcriptional level by competing for binding to miRNAs, and they can be all transcripts that have a total binding site for miRNAs (also known as miRNA response elements (MREs)), including mirRNA, lncRNA, rRNA, tRNA, pseudogene RNA and circular RNA. The mode of action of ceRNA is that two transcripts containing the same MRE compete for binding to miRNAs, when one of the transcripts is up-regulated, it will bind more miRNAs, thereby inhibiting the binding of miRNAs to the other transcript and reducing the inhibit function. More and more experiments have demonstrated that ceRNA content and activity are dysregulated in tumors, and artificial miRNA sponges containing multiple MREs have been used to inhibit oncogenic miRNAs and show tumor suppressor functions.

Although the occurrence and progression of ncRNAs and multiple myeloma have been studied, few studies have dissected unknown lncRNAs through the interaction pathway of ceRNA, and studied their biological functions, pathogenic mechanisms, and evaluation of prognosis and progress in MM. In clinical and research, it is necessary to search for new molecular markers related to the progression of prognosis and targeted therapy.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a long-chain non-coding RNA and its application as a molecular marker of multiple myeloma cells.

The technical scheme adopted in the present invention is:

A long-chain non-coding RNA (lncRNA MSTRG.29039.1) whose transcript sequence is shown in SEQ ID NO:1.

AATGTATCTGTCTCAAATCACTAAAAATAAGACTTTGTATTGTTCAATGAAATCTCTAGTACATATAAAACATGATAGACTTTTTAGTATAGGTTACTCAAGAACTTTGAATGCAGAGGAAATATTTTCACAAAGATATACCTGATAAACCAGGCAGGGTTCTTGTTACATCCTCCTGAAAGAGACTTATGTTAAGATCTGATATCTACAAGAATCACTCACAGCATGGATTAGGAAGTGTACCCCACTTTTCAGGAAAGAATTTTCATAACCTGTGAAAAGGAGAGGGGGCAAGTGTCTTGACTTTAGTTAAGTGAGCCATTTTTGTTTCCTCTGAGGAAGAGTAGGCTGGCTGTGGCGTCTCTCACTCCATTGACATCTAGTGTTTTGTCTGCAGACTTTTCTGTCTGGGTGACTCCACTTCCTCCACCACCATACATCACAGGAGCCTGAGAACTCAATGTGTACATTGGAGACCAAAAGCAGATTTGCTTGATCCTTGAAAGAGGGAGAAGAGTTTTGTTTGTCTGATCCCTTTGGCCACCCTCTGTTCTGTTTTGGAAACACTGATTGTTGCAGAATGTTCTCAAAACCACACTCACCTTCACCCCTGAGTTATGCTTCACAAAAGAGACTGGTACAAAGCAACATTTAAAATGTTTTCACGTGATCAGTGGTTTCAATTTGGAATCTGGGGCTGGTTTATGGAAGAGATGAGAAGGAAATTTTTTCCTGTCCTGGGGATTTTGTATGTTTTTTCTGTCTCTTAGATTTTTCCCTTTAAAATGTGGTTGTGAAAAGCAGATGTTTAGTAAAGCTGAAAGTCTTGGAATATTCATCAATGCATCGCAACTCAATACCTTAGGGTAGACACTGTCTGTAGTAGCAAAACAGATCTTTTTTCTATGTCTACCATGAAATGATAGCCATTGAAATGATGCAAAATATGACCTCTATATTTAGTCTGTATTCAAATCCATTTATTGTTTAAAGAAACCAT GCAATGCCAATTAAAATAGTTTTTTTCTTTAACAAATCAACTGCATATTTATTTTGCTGACTAAAGAGAATTAGATAAAAACATCAAAAATTAAATTTCTGCCCTAAGTATCATAAAATGAATACCAAGGTCCTCCTAAAAGCTTAATGAGATCTCAACCTGGCCTCTAATAGCAGTGTCCGATGTCTGACATGTTGGCAATGTATAGTGAAATGTTATTATGACTGTTATTATGGTGTAGTACCACAGAGAGCTAGAAGTGCCCAGGACATCAATTGTATTCTATTGTTGTAAGGTGTCCTTAAACCTCATGTAAAGAGTTGCAGACATTCAAAGTTTGGATACTTACCTAGAAGAAACATTCAAAATATGGAGTGTTTGCTCAAATCATTGTTCTAGTGAAAATAGCATGTTGAAACCACTTTGATTCTCTGACATTTGTCTGTGTAAGGCATGTAATTCATTACACTGATTTTATATTGAAGAAATAAATTTGAAACAAACATTTTATTCCAGCAGAGGCTGATGTTCATAACTGTTTTCCTGAATACTAGGTAGCTAATCAGTCGTTGATATATTAACAAGTTATTCATGATTTTCTAGCTTCCACTATGAAGTAAATTAAAAGAGGCGGATTGGCAGGGGTGTCATGACATTGTAATGATACAGTGCTGATGATAACTCTATTGGGGAAATACAGTAGCTGTCTTTGCTTTCACCAAAATAATAAATAAGCAAACTTCCATCTTATATACAATCTTTTCTTAGTTTGTTACTTTCTGAAAATTGCTTTGAAAGTTTATTAGAACTGTGTAAAATACAGCATGCCATTTCCTAAGAATAGCACACAATGTGGAGACACTTTATCGAAAAGTAAAATGTTTATTTCACTGGCTAGATTTGGTAGCATTCTCCTACAGTGGCAACTCAGTCTTTCTTGAGTAACCCAGCCTCCCCACCCTTATTTAATCTTAAGTTTGTGAATTGAGCATTGTGGTTA TCTACTGCTGAATGTGTATTTGAGAGCTATTTCTTATTTCTAATTTTGGGGATATTATGTAGGCTTTTGTGTAATGCATATAATAACAAAGACTGTGCTTCCTCCCAGAATGTGTTCACTGGATGTCTTGTCACCCGATTTTACTAATCAACCAAATGTATTGTTCTACAGAGCTAGCCCCTTTAAGTCATAATTGCCCTATATTGAATTATCAAAATACAATTTCAGGCTAAATTAGCAATAATCCTTTTGGAAGCCTATGTATGCCAGAGAAAAATGAGGGCCATGTGTCTTCCATGCTATAGGCTGAGGAATCTTTCAGCTGTGTGGACAGCAAGGAAAGGGAGAGGCTGCAGCAAGTGGAAGGAGAGGCAAATACAGCTACAGAGGCAAAGGGAATAGGCCACAAAGGCAAAGGGTTAATGAGCTGTGTGGGTCTGATTTTATTGCATGATCAATATGaacctttttctagatgtcaggcttttgagtatctgattaaaactgtgactaatcctttaagtaaacgcaaatatacacaaacacaaaattgttgtgtaaaatttctagcaattcttaggtctctggaagccgcaggttaggaactcACACTGGGACCAATGGGGAGGGAGTTCAAAGCAGAAAAAAAGATGGAGATCAGCATATTAACCTACTAGGAATCTCTCAGAGAAGAAGATAATAAGTTCATAATAAGAGGAAGATGCAAAGGGGTTCACATAAGGCAAACTAATGGCATTGAAGCAATCACGGAAAGATTGAAAGTGAAATAGAACTGCAGAGAAATTATCTGGAAGGTTTGTTATTTTATGGGAATTAAACAGATGAGAAAATTGTTCAGGAAAACTGGAACTTTTTCGGAACTCACTCTAAAGGAGATACGATTTTGACGGATCTTGAGTTCTTGGCAGACTCTAAAAAGGTTATCTGGGTTGCTTGAGTTGCTGACTACATCAGAATCAGAAGGGATGTGGTTGCTCTT GGTAAGATAGAGTTTAGGAAAGTTAAAAATTTCATATCATTATGAATTTGAGATCTACTAAGAAAGCAAAAGCAGGGTACTGGAGTTGAGTTTAACAGGGAAGTGTATTTGGGTCATGGAAGAAAATGTGCAATTTTAAGTGTCCCTCAGCAATTCAGTGTAATGGGAACCATGGTGGGTGAAGGGTGGAGTCCTCAATTAAACCGAGGGCAGGACACTGCATGGTTAGAACAGCATGTGGTTATGCCCTGATGCTGGTTTGGTgacatgagaatcaagagactcaagtCccaatgttggatttaccacgagcttgtgaatttgagaaagttattttactactctggacctgtttttttcctgctcagaaaaggaccacaggaggcctaaataatctcgaagattccttccaactctagacatctatgatactGCAGAGAATGGAAATGAATAGAAAAAGAGCCTCAATACATCAACAATGTTTTACTTATTTTCAAAAAAGATTCCTCACTTTGATGCTGACTCAGATGCTGTGGATGTGTGATTAAGAATTTTAATTTCGTTAATATCATCAGTTTCCTGAAGTAGAAGAATGACAATTCTCCCAGGGAAATCTTGATGATTGAGTGTTTCCACTAGCAGTTGAGAGTCAGTATGTTAGTTAACATTTAAAAAAGCAAATACTTCATTTATTTACTCTGTGTGCTTTGTGGAAACTTACCACTGCCAGCTTCAGCCACAGAACATTCTATAAGAAACAGTGTGGTTTCCAGATGAGGGCAAGTGGATAGTGTTTTCCAAGGCTGCTTCAGGGAGCATCTGAGGCATTTCAGAACATTTTGCATCTTGGCACAGTGAACCTGCTATGAATTCTCTGTGCCTGGCTGTCTGTCTCCTACCCCAATCATTACCTTCTTATCTGGGGGGACAAGCACAACTGGGAACACGTGTGGAATGAAAGAGCTTGTTCCAGTGGTCTCAGCAATGCCTCATGTCTTCT GTTCAGTAGATTCTTGCAGAATGAAGCTGGAAGGGCCACTTAGAGGATCTGATTCTAACCTCTGCGTTAAGGAAAAATCCAGCTCCTTTTATTCCTTAACTGGAATCTAGGGTCTCATAGTGCCACTTATTTTTTTGTAAAGTGGAGTGAAGATTGTACCAATAAGAAATAAAGAATACTTATTTCGTGCATATTTAAAGCTGACATGTACTTTACATATTGTATTTAAACCTACAACTTGCTATGTGCAAACCTATTGGTCCATAGTTCTTGCCTAAAATTTCCAAGATAATTTCCCTATAGCATTTTAAATAAGAAATAATTATTTGACAATGACTGCGGTGGTGGGGGAAGACGCATTTGACAACAGTTGTGGTGGTAGGGAGAGGACACATCACATCTGAGACCTTTATTTCAATTGGTGGTTAAAATGTGGGATAAAATACTAAAAGCAATATGAATTTAGATTGAAAAAAGAGATGTGAGCTCATTCTGCTCTCCACTGGACAGGTACCATTCATGGCAATTCCCATCCTGGGTGTTTTGAATGTTTGGAGGAATTAATGTACAGAATTATTTAATAATATCTggcatgatggctcatgcctgtaatcccagcactttggaaagcccaggtgggagaattgtttagctcaggcattcaagaccactctgggctgtacatagtgagaccctgTctctaccaaaaaaaaataaaaattaattggacatgaaggtgtgcccctgtggtttcagatactcaggagggtgaagtgggaggatcgcttgagcccaggcattcaaggctacagtgagccaagattgcaccactgcactccagcctgTCTCTAAAAAGAAATAAATTTAAAAATCACAATATGTGTTACTCAAAATGTATAATAATTATTGCTTGTAGTTCAACTGAATAAAGTACAAAACTAACTAAAACATTTCTTAtttctgatttcaacaaatccacaacccacatctttatggtgtt cagattcagtcaccttctgttacaagggctcatggttcaagatgtggacaaacagtttgctcaggcctgaacagtctcaaGaacgagagaggatgtttggagttccacttcccatccagacagcactggaagcaggatcccatggcagaaatgcccatatgatcttcagtaaatgtatatgtgtcttcatttattttattttttaaattatactttaacttctgGgatacatgtgcagaaagtgcaagtttgttacataggtatacaagtgccatggcagtttgctggaccaaccaacccgtcatctacattaggtatttttcctaatgctagtgtcttttttttttaatgatatgtgggtcctctaaagtattaagacaaaggaagaagcctcttttgcttgtgcctaaATCAGACTGATGGACAACACTCAGATTTACTTGAAAAATATAGCATATTTTGAAGAGGAGAGATTTATAATGTCAGTGAATGCTATAACAGAGGAAAGAAATGTGATTTATCATGTGACTTAATATATGTATTTCTAAGTCCTAGAATATTATCCATTAGCATTATCATATTTATAGCAAAGTATATTTTTCTTCCTTTATCTAAAGTACCGAGTGCATTTAATTATTTATTTTCAAGTTGATATTTTCCTTAGTGACAGTTTTCATTGttaatatttatcaaaataaaaaatattttttatatatttttctattttttaCTATTCACATCATAACTATATTTTTCCTAAAATAAATTTATTAGAAAAATACTTCCTTACATCAATACTACTCCTGCTTCCCAATATAGAATATTACTCAATTACACTCACAAAGCAACAGCTCCTTTTCAAGGTAGTTCAATTTTTTCAGCTTTTTAGAACAGTTAGTAAATATGACATTATGAGTAATACTCCTCTAATTTTAAAATGAAAAATTAGGTGTTATAAATTACTAACCTCTTTAGTTATTTTATTAAGTTCAGTAGCAAA ATTACATGAATGGAAACTACAATTCAGTAGGTGTTCTGAAGCTGTTTTTCCTGGATTAATACTGCAATATAATTATAATAAAACAGCTTTCAACAGATTGTGGAGGTGTCTTAAGTATCATATAATGAGAGCACCACTGTTAATTTAATATATTGGATAGCAAACAGTTTGTGAGATGATAGTTGTGATTTCAATATAAGTTAATTTAGTGTTTTATTGTGTTTCTGCTAGGAGGGAAAGCATTCATTCAGATGCAAAAGTTACTTCTTATCATTGAAGGATTTTGTGGTAAAGGAGAATTGCTGTATTACGTCTGTTACTTTGGTAGTGGATGTTGCTTCTTTTAGAGGTAATAAAGTATATCAGTATCTAGAAATTATATTTATAGAATGAAATCAACCTACCAATTCTTTCTCTCTGCCCAGTCTCTTATGACATTGAATCTGTATAAATTATAAGCTATAAACTTTCAAAATTGGTGGTGAGTTAAAATTTTATTTTCTGTTTTATAATATCTTTGAAAATTTC。 (SEQ ID NO: 1)

The present invention also provides the application of the long-chain non-coding RNA as a molecular marker of multiple myeloma.

The present invention also provides the use of the long-chain non-coding RNA (lncRNA MSTRG.29039.1) in preparing a diagnostic reagent for multiple myeloma.

Specifically, the diagnostic reagent detects the expression of lncRNA MSTRG.29039.1 in the subject's multiple myeloma cells and compares it with the average value of the normal control to diagnose disease progression and evaluate the condition.

Among them, lncRNA MSTRG.29039.1 is highly expressed in multiple myeloma cells compared with normal plasma cells. Knockdown of lncRNA MSTRG.29039.1 in cell lines and knockdown of mRNA OSMR can increase cell apoptosis and reduce proliferation. It can be seen that the lncRNA MSTRG.29039.1 can be used as a molecular marker of multiple myeloma cells and is related to the occurrence and progression.

The present invention also provides a molecular marker, including the long-chain non-coding RNA.

The present invention also provides a primer set that can specifically identify the transcript DNA of the long-chain non-coding RNA, and the primer set includes the following primers:

Upstream primer (5'to3'): GACGGAAACAGCCCTGCCTTC (SEQ ID NO: 2);

Downstream primer (5'to3'): CCCTCTCCCTCTCCTGTTGTGG (SEQ ID NO:3).

The present invention also provides a method for preparing the long-chain non-coding RNA, comprising the steps of:

Use 1ml trizol to lyse cells, add 0.2ml chloroform, shake vigorously at 15-30°C for 15s, let stand for 3min; centrifuge at 12000g for 15 minutes at 4°C, add 0.5ml (0.8 times the volume) of isopropanol, and mix the liquid in the tube gently Evenly, let stand for 10 min at room temperature; centrifuge at 12000g for 15 min at 4°C, discard the supernatant. Add 1 ml of 75% ethanol (prepared with DEPC water or enzyme-free water), vortex, and gently wash the pellet for 2 min. Centrifuge at 7500g at 4°C for 15 minutes, discard the supernatant; air dry, add an appropriate amount of DEPC H2O (10-30 μl) to dissolve RNA, use Thermo NanoDrop 2000 to determine the concentration, and use agarose gel electrophoresis to detect the quality of RNA extraction.

The present invention also provides an auxiliary diagnosis kit for multiple myeloma, the kit includes:

(1), bone marrow mononuclear cell separation reagent (purchased from Solarbio company), multiple myeloma cell CD138 magnetic beads (purchased from Miltenyi Biotec company);

(2) Specific primers and probes for transcript DNA of lncRNA MSTRG.29039.1;

(3) Specific primers and probes for GAPDH internal reference markers;

The specific primers of lncRNA MSTRG.29039.1 are: upstream primer (5'to3'): ACGGAAACAGCCCTGCCTTC (SEQ ID NO:2); downstream primer (5'to3'): CCCTCTCCCTCTCCTGTTGTGG (SEQ ID NO:3).

The specific primers for the GAPDH internal reference marker are: upstream primer (5'to3'): TGATGACATCAAGAAGGTGGTGAAG (SEQ ID NO:4); downstream primer (5'to3'): TCCTTGGAGGCCATGTGGGCCAT (SEQ ID NO:5).

Further, the kit also includes:

(4), reverse transcriptase, qPCR Mix, 10mM dNT Ps, reverse transcription buffer, RNase inhibitor (purchased from Tiangen company), DEPC water;

(5), total RNA extraction reagent Trizol (purchased from InvitrogenTM), isopropanol, absolute ethanol, chloroform, PBS buffer.

The beneficial effects that the present invention has:

The present invention discovers for the first time a long-chain non-coding RNA, which is more highly expressed in multiple myeloma cells than normal plasma cells, and is highly expressed in multiple myeloma cell lines. Knockdown of lncRNA MSTRG.29039.1 and mRNAOSMR in cell lines can increase apoptosis and decrease proliferation. It can be seen that the lncRNA MSTRG.29039.1 can be used as a molecular marker of multiple myeloma cells and is related to the occurrence and progression.

The lncRNA MSTRG.29039.1 provided by the present invention is used as a diagnostic marker for the occurrence and progression of multiple myeloma, with simple operation, accurate detection, good repetition and low cost, and is suitable for mass screening and clinical application.

Description of drawings

Figure 1. Expression of novel lncRNAs in MM:

A. The location of Reads on chromosomes.

B. Volcano plot for differential gene expression analysis between samples.

C. Heat map of differential gene expression analysis between samples.

D. Expression of novel lncRNAs (FPKM).

E. Volcano plot of differential expression of novel lncRNAs.

F. Heatmap of differential expression of novel lncRNAs.

Figure 2. Screening predictions for novel lncRNAs and their target genes:

A. Interaction analysis diagram of lncRNA and mRNA.

B. Target gene KEGG pathway map.

C. Bubble plot of target gene KEGG pathway.

D. Target gene GO pathway map.

Figure 3. Screening of new lncRNA MSTRG.29039.1 and the expression of its target gene mRNA OSMR in MM.

A. The relative expression levels of MSTRG.29039.1 in patients and healthy controls were detected by real-time PCR.

B. Detect the relative expression of MSTRG.29039.1 in MM cell line and control.

Correlation analysis between the relative expression of C.MSTRG.29039.1 and β2-MG (P=0.0379).

D. Detect the relative expression of OSMR in patients and healthy controls (P=0.0352).

E. The expression of OSMR gene before and after knockdown of MSTRG.29039.1.

F. The expression of OSMR protein before and after knockdown of MSTRG.29039.1.

Figure 4 Knockdown of MSTRG.29039.1 promotes apoptosis and inhibits proliferation:

A. Transfection efficiency was examined after knockdown of MSTRG.29039.1 in AMO-1, U266 cell lines.

B. Flow cytogram of apoptosis after knockdown of MSTRG.29039.1 in AMO-1 cell line.

C. Flow cytogram of apoptosis after knockdown of MSTRG.29039.1 in U266 cell line.

D. Apoptosis rate after knockdown of MSTRG.29039.1 in AMO-1 cell line.

Apoptosis rate after knockdown of MSTRG.29039.1 in E.U266 cell line.

F. Cell viability after knockdown of MSTRG.29039.1 in AMO-1 and U266 cell lines.

Figure 5. Predicted miRNA and mRNA:

A. Veen plot of candidate miRNAs predicted to interact with the lncRNA MSTRG.29039.1.

B. lncRNA MSTRG.29039.1-miRNA-mRNA interaction map.

C. The relative expression levels of hsa-miR-12119 in patients and healthy controls were detected by real-time PCR (P=0.022).

D. The expression of hsa-miR-12119 gene before and after knockdown of MSTRG.29039.1.

Detailed ways

The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited.

Example 1

1. 8 cases of bone marrow from patients with multiple myeloma and 2 cases of normal bone marrow were received with 5ml of bone marrow each. After CD138 cells were extracted, the specimens were sent for inspection, and new lncRNAs were screened by RNA sequencing. The results are shown in Figure 1, and their target genes and target genes were predicted. GO function and KEGG biological pathway enrichment analysis (Fig. 2); candidate miRNAs and mRNAs that interact with the lncRNA MSTRG.29039.1 were predicted (Fig. 5A, B).

2. Put 3ml of lymphocyte separation solution in a 15ml centrifuge tube, add bone marrow fluid to it slowly, centrifuge at 20°C, 2000rpm for 20 minutes; aspirate the supernatant and collect the mononuclear cell layer (buffy coat) in PBS containing 10ml centrifuge at 1500 rpm for 5 minutes; discard the supernatant, resuspend, count, centrifuge, resuspend each 10 ⁷ MNCs with 80 μl PBS buffer, add 20 μl anti-CD138 antibody, mix well and incubate at 4°C in the dark for 20 minute. After rinsing the MScolumn fixed on the magnet twice with 500 μl PBS, add the cell suspension, collect the negative cells, add 500 μl PBS buffer to wash 1-2 times, remove the MS column from the magnetic field, add 500 μl PBS buffer and quickly put It was poured into a collection tube, the cells were counted, resuspended, and centrifuged. The bone marrow of 37 patients with multiple myeloma and the CD138 cells of 28 normal controls were extracted, the cells were lysed with 1 ml of trizol, 0.2 ml of chloroform was added, vigorously shaken at 15-30 °C for 15 s, and allowed to stand for 3 min; centrifuged at 12000g for 15 minutes at 4 °C, added 0.5ml (0.8 times the volume) of isopropanol, gently mix the liquid in the tube, let stand for 10min at room temperature; centrifuge at 12000g for 15min at 4°C, discard the supernatant. Add 1 ml of 75% ethanol (prepared with DEPC water or enzyme-free water), vortex, and gently wash the pellet for 2 min. Centrifuge at 7500g at 4°C for 15 minutes, discard the supernatant; air dry, add an appropriate amount of DEPC H ₂ O (10-30 μl) to dissolve RNA, use ThermoNanoDrop 2000 to determine the concentration, and agarose gel electrophoresis to detect RNA extraction quality.

3. Reverse transcription to obtain cDNA

Using Tiangen's reverse transcription reagent, the lncRNA MSTRG.29039.1 DNA removal transfection system was prepared as follows:

Composition Usage amount 5xgDNA Buffer 2μl Total RNA - RNase-Free Make up to 10μl

The gene reverse transcription reaction system was prepared as follows:

Composition Usage amount 10xKing RT Buffer 2μl FastKing RT Enzyme Mix 1μl FQ-RT Primer Mix 2μl RNase-Free ddH₂O Make up to 10μl

Mix the above liquids, incubate at 42°C for 3 minutes, 42°C for 15 minutes, 95°C for 3 minutes, cool at 4°C, and store the cDNA at -20°C.

4. For real-time quantitative PCR (Real-time Quantitative PCR, qPCR) detection, prepare the reaction solution according to the following system:

Composition Usage amount DEPC Water 10μl qPCR SYBR Green Master Mix 12.5μl Forward Primer (10μM) 0.75μl Reverse Primer (10μM) 0.75μl cDNA 1μl Total 25μl

lncRNA MSTRG.29039.1, OSMR, hsa-miR-12119, internal reference GAPDH, U6 qPCR primers, and probe fluorescence quantitative detection reagents were purchased from GenePharma Biological Company or entrusted to GenePharma Biological Company to synthesize. The above reaction solution was placed in an 8-connected tube (purchased from Tiangen Company), with three replicate wells for each sample, placed in an ABI7500 instrument, and the reaction conditions were set as: 95°C, 3min; 45S, 45 cycles; 70°C, 3min, 51 cycles; 4°C, 1min.

5. Standardization of data:

The average value of the three experiments is used as the Ct value of the gene. For the specific process, please refer to the product instructions for use. According to the measured Ct value of the gene in the sample, the internal reference is used as the reference, and the classical 2-△△Ct relative quantitative PCR is used to obtain the selected value. relative content of certain genes in CD138 cells;

In the present invention, bone marrow mononuclear cells from multiple myeloma patients and healthy controls were isolated, and CD138 cells were sorted. In CD138 cells of multiple myeloma patients, the RNA levels of lncRNA MSTRG.29039.1 (Fig. 3A) and OSMR (Fig. 3D) were significantly higher than those in healthy controls (lncRNA MSTRG.29039.1: MM vs HC 3.607±0.821vs2.196± 0.533; OSMR: MM vs HC 8.439±2.21vs 2.450±0.58), and its expression was also increased in cell lines; while the expression of hsa-miR-12119 (Fig. 5C) was decreased, indicating that lncRNA MSTRG.29039.1 may interact with ceRNA through ceRNA interaction. The diagnosis of multiple myeloid disease correlates with disease progression.

Table 2.

Clinical characteristics of MM patients and their correlation with the relative expression of IncRNA MSTRG.29039.1

Example 2

An auxiliary diagnosis kit for the progression of multiple myeloma, the kit includes:

(1), bone marrow mononuclear cell separation reagent (purchased from Solarbio company), multiple myeloma cell CD138 magnetic beads (purchased from Miltenyi Biotec company);

(2) Specific primers and probes for transcript DNA of lncRNA MSTRG.29039.1;

(3) Specific primers and probes for GapDH internal reference markers;

(4), reverse transcriptase, qPCR Mix, 10mM dNT Ps, reverse transcription buffer, RNase inhibitor (purchased from Tiangen company), DEPC water;

(5), total RNA extraction reagent Trizol (purchased from InvitrogenTM), isopropanol, absolute ethanol, chloroform, PB buffer.

The specific primers of lncRNA MSTRG.29039.1 are: upstream primer (5'to3'): ACGGAAACAGCCCTGCCTTC (SEQ ID NO:2); downstream primer (5'to3'): CCCTCTCCCTCTCCTGTTGTGG (SEQ ID NO:3).

The specific primers for the GAPDH internal reference marker are: upstream primer (5'to3'): TGATGACATCAAGAAGGTGGTGAAG (SEQ ID NO:4); downstream primer (5'to3'): TCCTTGGAGGCCATGTGGGCCAT (SEQ ID NO:5).

Example 3

Human MM cell lines (U266 and AMO-1 cells) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin-penicillin in a humidified 37°C with 5% CO 2 in the cell incubator. Logarithmically grown MM cells were seeded into ⁶ -well plates at approximately 1 x 10 cells per well, and cells were transiently transfected with Lipofectamine 3000 reagent according to the manufacturer's instructions. Positive, fluorescent and negative controls (NC) for lncRNA MSTRG.29039.1 (si-MSTRG.29039.1:sense(5'-3')GGAUCGGAAAUGAGAAAUUTT, antisense(5'-3')AAUUUCUCAUUUCCGAUCCTT) were purchased from GenePharma. The specific operations are as follows: Inoculate the cells in 500 μl of antibiotic-free medium, and let them grow to 30-50% confluence during transfection. 2. Preparation of transfection solution, the amount of cells per well is as follows: A. Use 50 μl of Opti-MEM low Dilute 20pmol siRNA in serum medium (or other serum-free medium) (the final concentration of siRNA during transfection is 33nM), and mix gently; B. Shake Lipofectamine 3000 gently before use, then take 1μl Lipofectamine 3000 in 50μl Opti-MEM Dilute in medium and incubate for 5 minutes at room temperature Note: Please proceed to the next step within 25 minutes; C. Mix the DNA diluted in the first two steps with Lipofectamine 3000 (to make the total volume 100 μl), mix gently, and place at room temperature 20 minutes (the solution may be turbid) 3. Add 100 μl of transfection solution to each well of cells, shake gently and culture at 37°C to detect gene expression. The medium can be replaced after 4-6 hours of transfection.

Example 4

Cell viability was measured at 24h, 48h and 72h after siRNA transfection using Cell Counting Kit-8 (Beyotime). 10 ⁴ MM cells/well were seeded into a 96-well plate, and 10 μl of CCK-8 reagent was added. After incubating for 3 hours in a cell incubator at 37°C and 5% CO ₂ , the absorbance (A) at a wavelength of 450 nm was measured using an automatic microplate reader, and the cell proliferation ability was expressed as the A value. Each group had 5 replicate wells and each experiment was replicated 3 times. It was found that the proliferation ability of multiple myeloma cells (U266, AMO-1) gradually decreased over time (U266: 24h vs 48h vs 72h 86.312%±3.712% vs 59.402%±7.802% vs 27.420%±1.810%; AMO-1: 24h vs 48h vs 72h 55.909% ± 4.511% vs 40.175% ± 2.305% vs 16.630% ± 0.340%) (Fig. 4F).

Example 5

The MM cells collected after transfection were washed with 1:9 prepared phosphate buffered saline (PBS; Corning Life Sciences), centrifuged at 1500 rpm for 5 minutes, and then collected, and resuspended by adding 100 μl of the prepared buffer. Add 5 μl of Annexin V-FITC and PI from PE Annexin V apoptosis detection kit (purchased from BD), mix gently, and incubate at room temperature for 15 min in the dark. Finally, apoptotic cells were distinguished by flow cytometry, and the apoptotic rate was calculated. Three replicate assays were performed. Cell apoptosis was detected at 24h, 48h, and 72h after siRNA transfection, and it was found that the apoptosis rate of tumor cells gradually increased. In U266 cell line, the apoptosis rates of NC vs siRNA at 24h, 48h, and 72h were: 19.515%±0.625 % vs 29.120% ± 0.91%; 19.075% ± 1.075% vs 28.180% ± 0.27%; 24.560% ± 0.66% vs 28.730% ± 0.55%; also increased in AMO-1 cell line (24h vs 48h vs 72h: 19.060% ± 0.57% vs 27.240% ± 0.06%, 32.845% ± 2.045% vs 40.600% ± 0.600%, 46.125% ± 0.165% vs 51.360% ± 0.74%) (Fig. 4D,E).

sequence listing

<110> Tianjin Medical University General Hospital

<120> A long non-coding RNA and its application as a molecular marker of multiple myeloma cells

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6528

<212> DNA

<213> Human (human)

<400> 1

aatgtatctg tctcaaatca ctaaaaataa gactttgtat tgttcaatga aatctctagt 60

acatataaaa catgatagac tttttagtat aggttactca agaactttga atgcagagga 120

aatattttca caaagatata cctgataaac caggcagggt tcttgttaca tcctcctgaa 180

agagacttat gttaagatct gatatctaca agaatcactc acagcatgga ttaggaagtg 240

taccccactt ttcaggaaag aattttcata acctgtgaaa aggagagggg gcaagtgtct 300

tgactttagt taagtgagcc atttttgttt cctctgagga agagtaggct ggctgtggcg 360

tctctcactc cattgacatc tagtgttttg tctgcagact tttctgtctg ggtgactcca 420

cttcctccac caccatacat cacaggagcc tgagaactca atgtgtacat tggagaccaa 480

aagcagattt gcttgatcct tgaaagaggg agaagagttt tgtttgtctg atccctttgg 540

ccaccctctg ttctgttttg gaaacactga ttgttgcaga atgttctcaa aaccacactc 600

accttcaccc ctgagttatg cttcacaaaa gagactggta caaagcaaca tttaaaatgt 660

tttcacgtga tcagtggttt caatttggaa tctggggctg gtttatggaa gagatgagaa 720

ggaaattttt tcctgtcctg gggattttgt atgtttttttc tgtctcttag atttttccct 780

ttaaaatgtg gttgtgaaaa gcagatgttt agtaaagctg aaagtcttgg aatattcatc 840

aatgcatcgc aactcaatac cttagggtag acactgtctg tagtagcaaa acagatcttt 900

tttctatgtc taccatgaaa tgatagccat tgaaatgatg caaaatatga cctctatatt 960

tagtctgtat tcaaatccat ttattgttta aagaaaccat gcaatgccaa ttaaaatagt 1020

ttttttcttt aacaaatcaa ctgcatattt attttgctga ctaaagagaa ttagataaaa 1080

acatcaaaaa ttaaatttct gccctaagta tcataaaatg aataccaagg tcctcctaaa 1140

agcttaatga gatctcaacc tggcctctaa tagcagtgtc cgatgtctga catgttggca 1200

atgtatagtg aaatgttatt atgactgtta ttatggtgta gtaccacaga gagctagaag 1260

tgcccaggac atcaattgta ttctattgtt gtaaggtgtc cttaaacctc atgtaaagag 1320

ttgcagacat tcaaagtttg gatacttacc tagaagaaac attcaaaata tggagtgttt 1380

gctcaaatca ttgttctagt gaaaatagca tgttgaaacc actttgattc tctgacattt 1440

gtctgtgtaa ggcatgtaat tcattacact gattttatat tgaagaaata aatttgaaac 1500

aaacatttta ttccagcaga ggctgatgtt cataactgtt ttcctgaata ctaggtagct 1560

aatcagtcgt tgatatatta acaagttatt catgattttc tagcttccac tatgaagtaa 1620

attaaaagag gcggattggc aggggtgtca tgacattgta atgatacagt gctgatgata 1680

actctattgg ggaaatacag tagctgtctt tgctttcacc aaaataataa ataagcaaac 1740

ttccatctta tatacaatct tttcttagtt tgttactttc tgaaaattgc tttgaaagtt 1800

tattagaact gtgtaaaata cagcatgcca tttcctaaga atagcacaca atgtggagac 1860

actttatcga aaagtaaaat gtttatttca ctggctagat ttggtagcat tctcctacag 1920

tggcaactca gtctttcttg agtaacccag cctccccacc cttatttaat cttaagtttg 1980

tgaattgagc attgtggtta tctactgctg aatgtgtatt tgagagctat ttcttatttc 2040

taattttggg gatattatgt aggcttttgt gtaatgcata taataacaaa gactgtgctt 2100

cctcccagaa tgtgttcact ggatgtcttg tcacccgatt ttactaatca accaaatgta 2160

ttgttctaca gagctagccc ctttaagtca taattgccct atattgaatt atcaaaatac 2220

aatttcaggc taaattagca ataatccttt tggaagccta tgtatgccag agaaaaatga 2280

gggccatgtg tcttccatgc tataggctga ggaatctttc agctgtgtgg acagcaagga 2340

aagggagagg ctgcagcaag tggaaggaga ggcaaataca gctacagagg caaagggaat 2400

aggccacaaa ggcaaagggt taatgagctg tgtgggtctg attttattgc atgatcaata 2460

tgaacctttt tctagatgtc aggcttttga gtatctgatt aaaactgtga ctaatccttt 2520

aagtaaacgc aaatatacac aaacacaaaa ttgttgtgta aaatttctag caattcttag 2580

gtctctggaa gccgcaggtt aggaactcac actgggacca atggggaggg agttcaaagc 2640

agaaaaaaag atggagatca gcatattaac ctactaggaa tctctcagag aagaagataa 2700

taagttcata ataagaggaa gatgcaaagg ggttcacata aggcaaacta atggcattga 2760

agcaatcacg gaaagattga aagtgaaata gaactgcaga gaaattatct ggaaggtttg 2820

ttattttatg ggaattaaac agatgagaaa attgttcagg aaaactggaa ctttttcgga 2880

actcactcta aaggagatac gattttgacg gatcttgagt tcttggcaga ctctaaaaag 2940

gttatctggg ttgcttgagt tgctgactac atcagaatca gaagggatgt ggttgctctt 3000

ggtaagatag agtttaggaa agttaaaaat ttcatatcat tatgaatttg agatctacta 3060

agaaagcaaa agcagggtac tggagttgag tttaacaggg aagtgtattt gggtcatgga 3120

agaaaatgtg caattttaag tgtccctcag caattcagtg taatgggaac catggtgggt 3180

gaagggtgga gtcctcaatt aaaccgaggg caggacactg catggttaga acagcatgtg 3240

gttatgccct gatgctggtt tggtgacatg agaatcaaga gactcaagtc ccaatgttgg 3300

atttaccacg agcttgtgaa tttgagaaag ttattttact actctggacc tgtttttttc 3360

ctgctcagaa aaggaccaca ggaggcctaa ataatctcga agattccttc caactctaga 3420

catctatgat actgcagaga atggaaatga atagaaaaag agcctcaata catcaacaat 3480

gttttactta ttttcaaaaa agattcctca ctttgatgct gactcagatg ctgtggatgt 3540

gtgattaaga attttaattt cgttaatatc atcagtttcc tgaagtagaa gaatgacaat 3600

tctcccaggg aaatcttgat gattgagtgt ttccactagc agttgagagt cagtatgtta 3660

gttaacattt aaaaaagcaa atacttcatt tatttactct gtgtgctttg tggaaactta 3720

ccactgccag cttcagccac agaacattct ataagaaaca gtgtggtttc cagatgaggg 3780

caagtggata gtgttttcca aggctgcttc agggagcatc tgaggcattt cagaacattt 3840

tgcatcttgg cacagtgaac ctgctatgaa ttctctgtgc ctggctgtct gtctcctacc 3900

ccaatcatta ccttcttatc tggggggaca agcacaactg ggaacacgtg tggaatgaaa 3960

gagcttgttc cagtggtctc agcaatgcct catgtcttct gttcagtaga ttcttgcaga 4020

atgaagctgg aagggccact tagaggatct gattctaacc tctgcgttaa ggaaaaatcc 4080

agctcctttt attccttaac tggaatctag ggtctcatag tgccacttat ttttttgtaa 4140

agtggagtga agattgtacc aataagaaat aaagaatact tatttcgtgc atatttaaag 4200

ctgacatgta ctttacatat tgtatttaaa cctacaactt gctatgtgca aacctattgg 4260

tccatagttc ttgcctaaaa tttccaagat aatttcccta tagcatttta aataagaaat 4320

aattatttga caatgactgc ggtggtgggg gaagacgcat ttgacaacag ttgtggtggt 4380

agggagagga cacatcacat ctgagacctt tatttcaatt ggtggttaaa atgtgggata 4440

aaatactaaa agcaatatga atttagattg aaaaaagaga tgtgagctca ttctgctctc 4500

cactggacag gtaccattca tggcaattcc catcctgggt gttttgaatg tttggaggaa 4560

ttaatgtaca gaattattta ataatatctg gcatgatggc tcatgcctgt aatcccagca 4620

ctttggaaag cccaggtggg agaattgttt agctcaggca ttcaagacca ctctgggctg 4680

tacatagtga gaccctgtct ctaccaaaaa aaaataaaaa ttaattggac atgaaggtgt 4740

gcccctgtgg tttcagatac tcaggagggt gaagtgggag gatcgcttga gcccaggcat 4800

tcaaggctac agtgagccaa gattgcacca ctgcactcca gcctgtctct aaaaagaaat 4860

aaatttaaaa atcacaatat gtgttactca aaatgtataa taattattgc ttgtagttca 4920

actgaataaa gtacaaaact aactaaaaca tttcttattt ctgatttcaa caaatccaca 4980

acccacatct ttatggtgtt cagattcagt caccttctgt tacaagggct catggttcaa 5040

gatgtggaca aacagtttgc tcaggcctga acagtctcaa gaacgagaga ggatgtttgg 5100

agttccactt cccatccaga cagcactgga agcaggatcc catggcagaa atgcccatat 5160

gatcttcagt aaatgtatat gtgtcttcat ttattttatt ttttaaatta tactttaact 5220

tctgggatac atgtgcagaa agtgcaagtt tgttacatag gtatacaagt gccatggcag 5280

tttgctggac caaccaaccc gtcatctaca ttaggtattt ttcctaatgc tagtgtcttt 5340

ttttttttaat gatatgtggg tcctctaaag tattaagaca aaggaagaag cctcttttgc 5400

ttgtgcctaa atcagactga tggacaacac tcagatttac ttgaaaaata tagcatattt 5460

tgaagaggag agatttataa tgtcagtgaa tgctataaca gaggaaagaa atgtgattta 5520

tcatgtgact taatatatgt atttctaagt cctagaatat tatccattag cattatcata 5580

tttatagcaa agtatatttt tcttccttta tctaaagtac cgagtgcatt taattattta 5640

ttttcaagtt gatattttcc ttagtgacag ttttcattgt taatatttat caaaataaaa 5700

aatatttttt atatatttttt ctatttttta ctattcacat cataactata tttttcctaa 5760

aataaattta ttagaaaaat acttccttac atcaatacta ctcctgcttc ccaatataga 5820

atattactca attacactca caaagcaaca gctccttttc aaggtagttc aattttttca 5880

gctttttaga acagttagta aatatgacat tatgagtaat actcctctaa ttttaaaatg 5940

aaaaattagg tgttataaat tactaacctc tttagttatt ttattaagtt cagtagcaaa 6000

attacatgaa tggaaactac aattcagtag gtgttctgaa gctgtttttc ctggattaat 6060

actgcaatat aattataata aaacagcttt caacagattg tggaggtgtc ttaagtatca 6120

tataatgaga gcaccactgt taatttaata tattggatag caaacagttt gtgagatgat 6180

agttgtgatt tcaatataag ttaatttagt gttttattgt gtttctgcta ggagggaaag 6240

cattcattca gatgcaaaag ttacttctta tcattgaagg attttgtggt aaaggagaat 6300

tgctgtatta cgtctgttac tttggtagtg gatgttgctt cttttagagg taataaagta 6360

tatcagtatc tagaaattat atttatagaa tgaaatcaac ctaccaattc tttctctctg 6420

cccagtctct tatgacattg aatctgtata aattataagc tataaacttt caaaattggt 6480

ggtgagttaa aattttattt tctgttttat aatatctttg aaaatttc 6528

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 2

gacggaaaca gccctgcctt c 21

<210> 3

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 3

ccctctccct ctcctgttgt gg 22

<210> 4

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 4

tgatgacatc aagaaggtgg tgaag 25

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 5

tccttggagg ccatgtgggc cat 23

Claims (5)

1. A long non-coding RNA characterized by: the sequence of the transcript is shown as SEQ ID NO. 1.

2. Use of a reagent for detecting the long non-coding RNA of claim 1 in the manufacture of a diagnostic product for multiple myeloma.

3. A primer set capable of specifically recognizing the transcript DNA of the long non-coding RNA according to claim 1, characterized in that: the primer group comprises the following primers:

an upstream primer: GACGGAAACAGCCCTGCCTTC, respectively;

a downstream primer: CCCTCTCCCTCTCCTGTTGTGG are provided.

4. An auxiliary diagnostic kit for multiple myeloma, which is characterized in that: the kit comprises:

(1) bone marrow mononuclear cell separating reagent, multiple myeloma cell CD138 magnetic bead;

(2) primers and probes for the specific detection of the transcript DNA of a long non-coding RNA according to claim 1;

(3) specific detection primers and probes for a GAPDH internal reference marker;

the primer for detecting the specificity of the long non-coding RNA in the claim 1 is: an upstream primer: GACGGAAACAGCCCTGCCTTC, respectively; a downstream primer: CCCTCTCCCTCTCCTGTTGTGG the flow of the air in the air conditioner,

wherein the specific detection primers of the GAPDH internal reference marker are as follows: an upstream primer: TGATGACATCAAGAAGGTGGTGAAG, respectively; a downstream primer: TCCTTGGAGGCCATGTGGGCCAT are provided.

5. The kit for the auxiliary diagnosis of multiple myeloma according to claim 4, wherein: the kit further comprises:

(4) reverse transcriptase, q PCR Mix, 10mM dNTPs, reverse transcription buffer, RNase inhibitor and DEPC water;

(5) the total RNA extraction reagent is Trizol, isopropanol, absolute ethyl alcohol, chloroform and PBS buffer solution.

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