Application of orphan nuclear receptor nr5a2 in anti-multiple myeloma drug screening

Abstract

The application discloses application of orphan nuclear receptor NR5A2 in anti-multiple myeloma drug screening and preparation of a multiple myeloma prognosis evaluation preparation, wherein overexpression of the orphan nuclear receptor NR5A2 indicates poor prognosis of multiple myeloma and can be used as a drug inhibition target.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmacy and biological detection technology, and relates to the application of orphan nuclear receptor NR5A2 in anti-multiple myeloma drug screening and preparation of multiple myeloma prognosis evaluation preparation. BACKGROUND

[0002] Multiple myeloma (MM) is the second most common hematological malignancy, mainly the proliferation of abnormal monoclonal plasma cells. In the past few decades, the survival rate of MM patients has been significantly improved due to the emergence of new therapies, but most MM patients will eventually experience relapse. The characteristics of disease progression and relapse are the evolution of malignant plasma cell subclone and the increasing drug resistance, and eventually the patients will face the situation of "no available drugs". Exploring MM drug resistance must thoroughly understand the molecular mechanisms that drive MM progression and drug resistance. Cytogenetic abnormalities-chromosomal structural abnormalities and certain specific gene mutations are the fundamental causes of promoting the growth of malignant drug-resistant plasma cells, eventually leading to relapse and refractory MM patients. Chromosome 1q amplification (1q+) is the most common secondary molecular genetic abnormality in MM, and its detection rate shows a clear increasing trend as the MM disease progresses: about 20% of monoclonal gammopathy of unknown significance (MGUS), 35-45% of smoldering myeloma (SMM) and newly diagnosed MM, and 50-80% of RRMM patients can detect 1q+. 1q+ MM patients are clinically and biologically heterogeneous. The underlying molecular mechanisms are still under study, and there is an urgent need to identify targets for effective treatment of this subgroup of MM patients.

[0003] The latest diagnostic criteria for MM are: 1. The proportion of confirmed monoclonal plasma cells in bone marrow is ≥10% or histological biopsy confirms the presence of bone or extramedullary plasmacytoma; 2. At the same time, any one of the following events is accompanied: (1) MM events exist: ① hypercalcemia: serum calcium level exceeds normal upper limit 0.25 mmol / L or total concentration > 2.75 mmol / L; ② renal dysfunction: creatinine clearance rate < 40 mL / min or serum creatinine total concentration > 177 μmol / L; ③ anemia: hemoglobin is lower than the normal lower limit 20 g / L or total concentration < 100 g / L; ④ bone destruction: X-ray, CT or PET / CT finds osteolytic bone damage; (2) early disease progression biomarkers exist: ⑤ the proportion of monoclonal plasma cells in BM is ≥60%; ⑥ serum free light chain in involved / non-involved is ≥100, and involved FLC is ≥100 mg / L; ⑦ MRI finds at least 1 local bone damage (lesion diameter is more than 5 mm).

[0004] MM is highly heterogeneous and currently considered incurable. Despite the new drug era, including proteasome inhibitors represented by bortezomib, immunomodulatory agents represented by lenalidomide, and monoclonal antibodies represented by daratumumab, and the combination of autologous stem cell transplantation (ASCT) or chimeric antigen receptor T cell immunotherapy (CAR-T), the response to treatment and the final outcome of MM patients are different.

[0005] In order to better determine the prognosis of patients, the international staging system (ISS) composed of two simple and routine laboratory indicators (serum albumin and β-2 microglobulin) is widely used. Among the many prognostic indicators of MM, the most important factor affecting the prognosis of tumor cells is their gene mutation and gene expression profile (GEP). A large number of studies have shown that cytogenetic abnormalities are always significantly related to worse overall survival (OS) or progression-free survival (PFS). The cytogenetic abnormalities (CAs) commonly associated with MM patients include: (1) deletion of 17p13 (del(17p13)), (2) deletion of 13q14 (del(13q14)), (3) 1q21 gain / amplification (1q21 Gain / Amp), (4) t(4;14), (5) t(6;14), (6) t(11;14), (7) t(14;16), and (8) t(14;20). Among them, the latter five can only be detected by fluorescence in situ hybridization (FISH) specific probes, while the former three can be detected by both FISH and traditional cytogenetic chromosome karyotype analysis. Therefore, when the two methods cannot be performed simultaneously, FISH can provide more information and is therefore the preferred method for detecting CAs.

[0006] 1q21 Gain / Amp is one of the most common CAs in MM with 1q amplification (1q+ MM). In recent years, researchers have divided MM patients with 1q21 Gain / Amp into two subgroups, namely: Gain group with 3 1q21 copy numbers (CNs) and Amplification group (Amp group) with at least 4 1q21 CNs. The results showed that the median PFS (mPFS) of Gain group (3 CNs) patients was 28.0 months, and the mPFS of Amp group (>3 CNs) patients was 17.6 months compared with patients with normal copy numbers (2 CNs). The results of OS were similar, and the 3-year OS rate of Amp group patients decreased from 73% to 52% compared with Gain group. However, it is still unclear whether 1q21 Gain / Amp is a cause or a result of poor prognosis in MM, and the deep molecular mechanisms related to 1q21 Gain / Amp still need further exploration.

[0007] The increased metabolic demand of hyperproliferative tumor cells triggers the reprogramming of multiple metabolic pathways, including fatty acid oxidation, glycolysis, mitochondrial energy metabolism, etc. In our previous work, we found that the overall survival time of obese MM patients under 65 years old was significantly lower than that of the normal control group. Lipid metabolism plays a core role in ferroptosis. Ferroptosis is a cell regulated death driven by iron-dependent phospholipid (PLs) peroxidation, with the final manifestation of plasma membrane rupture. PLs are an important component of the cell membrane or organelle membrane, mainly composed of two fatty acyl chains (sn-1 and sn-2) and a head group. The sn-1 position is occupied by saturated fatty acids or monounsaturated fatty acids, and the sn-2 position can be SFA, MUFA or polyunsaturated fatty acids (PUFA). In animal cells, the abundant arachidonic acid (AA, 20:4) and adrenal acid (AdA, 22:4) both contain two diallyl groups. The diallyl group (-CH=CH-CH2-CH=CH-) is most prone to lipid peroxidation due to the weak hydrogen bonding force of the connecting central methylene group. Not all lipids can cause ferroptosis. Phosphatidylethanolamine with 1 AA or 22:4 PUFA tail, PLs containing 2 PUFAs or PUFA-containing ether esters are specific lipids that drive ferroptosis. Exogenous treatment with low-dose AA in a mouse plasmacytoma model can reduce tumor burden. Currently, the relationship between lipid metabolism, ferroptosis and MM cell drug resistance is still in the unknown stage. Understanding the mechanisms of ferroptosis is very valuable for basic biology and disease treatment. Tumor cells may use various ferroptosis monitoring mechanisms to escape ferroptosis, and understanding these mechanisms can provide inspiration for developing new combination therapies. SUMMARY

[0008] In order to better guide the clinical treatment of MM patients, especially 1q+ MM patients, it is urgent to determine more reliable prognostic evaluation indicators and treatment methods.

[0009] Our team found in the research on the occurrence and development mechanism of 1q+ MM that orphan nuclear receptor NR5A2 can inhibit MM cell ferroptosis, promote MM cell proliferation, invasion and drug resistance by regulating phospholipid remodeling through MBOAT1 and MBOAT2. The results provide a new direction for understanding the pathogenesis of MM and developing new MM treatment targets. Based on the findings, the present application includes the following technical solutions.

[0010] One aspect of the present application provides the use of orphan nuclear receptor NR5A2 in anti-multiple myeloma (MM) drug screening and preparation of multiple myeloma prognosis evaluation preparation, wherein the orphan nuclear receptor NR5A2 is used as a drug inhibition target; accordingly, the drug is an NR5A2 inhibitor.

[0011] Preferably, the above-mentioned multiple myeloma (MM) is chromosome 1q amplified (1q+) multiple myeloma, i.e. 1q+ MM.

[0012] In one embodiment, the above-mentioned drug is used to inhibit the expression of NR5A2.

[0013] In another embodiment, the above-mentioned drug is used to down-regulate or block the expression of the NR5A2 gene, or inactivate the NR5A2 gene.

[0014] Further, the above-mentioned drug can also inhibit the expression of MBOAT1 (Membrane-bound O-acyltransferase 1, membrane-bound o-acyltransferase 1) and / or MBOAT2 (Membrane-bound O-acyltransferase 2, membrane-bound o-acyltransferase 2).

[0015] Alternatively, the above-mentioned drug can also down-regulate or block the expression of the MBOAT1 and / or MBOAT2 gene, or inactivate the MBOAT1 and / or MBOAT2 gene.

[0016] In a preferred embodiment, the above-mentioned drug has little or no drug resistance. Only such a drug can overcome the defect of existing MM treatment drugs that drug resistance has already occurred.

[0017] For example, the above-mentioned drug resistance can refer to the resistance to dexamethasone (Dexa). To this end, the screened drug can maintain or increase the sensitivity of the MM cell line to dexamethasone.

[0018] The specific form of the multiple myeloma prognosis evaluation preparation described above can be a kit for evaluating the prognosis of multiple myeloma by detecting the expression level of NR5A2 in a biological sample.

[0019] When using the kit, the detection of the expression level of NR5A2 in the biological sample is carried out by detecting the level (content) of NR5A2 protein in the biological sample; or by detecting the DNA copy number of NR5A2 gene in the biological sample; or by detecting the mRNA level (content) of NR5A2 gene in the biological sample.

[0020] Preferably, the kit described above can also detect the RNA or protein level of MBOAT1 and / or MBOAT2 in the biological sample.

[0021] Correspondingly, the kit detects the expression level of MBOAT1 and / or MBOAT2 in the biological sample by detecting the level (content) of MBOAT1 and / or MBOAT2 protein in the biological sample; or by detecting the mRNA level (content) of MBOAT1 and / or MBOAT2 gene in the biological sample.

[0022] The biological sample detected by the kit described above can be selected from the group consisting of tissue sampling cells, tissue fluid, whole blood, plasma, serum, saliva, oral mucosa, nasopharyngeal secretions, body fluids, and urine.

[0023] Preferably, when the DNA copy number of NR5A2 gene in the biological sample is detected using the kit described above, the detection can be carried out by fluorescence in situ hybridization (FISH) method.

[0024] When the DNA copy number of NR5A2 gene in the biological sample is detected by fluorescence in situ hybridization (FISH) method, a PCR method is used, and the corresponding kit contains fluorescence in situ hybridization probes for detecting NR5A2 gene, respectively.

[0025] The present application first discovers that the orphan nuclear receptor NR5A2 promotes the proliferation and invasion of multiple myeloma, and discovers that the mechanism is that NR5A2 inhibits MM cell ferroptosis by mediating phospholipid remodeling through membrane-bound o-acyltransferase MBOAT1 and MBOAT2, suggesting that NR5A2 can be used as a new biomarker for chromosome 1q amplified multiple myeloma (1q+MM) to improve the risk stratification of this subgroup of patients, and provide a new target for targeted therapy for this subgroup of patients. Accordingly, NR5A2 can be used as a drug target to develop and screen anti-multiple myeloma drugs, and drugs that are less likely to develop drug resistance. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The results of screening differential transcription factors in 1q amplified multiple myeloma are shown. Among them, A shows the comparison of the amplification and deletion ratio of 22 pairs of autosomal genes in 47 cases of NDMM, 5 cases of SMM, 7 cases of MGUS and 17 cases of RRMM patients; B shows the amplification and deletion ratio of chromosome 1q segment genes in the above 4 groups of patients; C shows the screening of differential transcription factors in 1q segment; D shows that NR5A2 is highly expressed in mRNA in 1q amplified patients (p=0.020); E shows that high expression of NR5A2 mRNA indicates worse PFS of MM patients; F shows that high expression of NR5A2 mRNA indicates worse OS of MM patients.

[0027] Figure 2 The expression and overexpression of NR5A2 in HMCLs and the construction of knockdown stable strains are shown. Among them, A shows the verification of mRNA expression and protein level of NR5A2 in HMCLs in the laboratory; B shows the verification of mRNA level and protein level of AMO1-NR5A2-OE and RPMI-8226-NR5A2-OE NR5A2 overexpression stable strains, C shows the verification of mRNA level and protein level of NCI-H929-NR5A2-KD and U266-NR5A2-KD NR5A2 knockdown stable strains.

[0028] Figure 3 It is shown that high expression of NR5A2 can promote the proliferation and invasion of HMCLs, and inhibit the apoptosis of HMCLs. Among them, A shows that CCK8 detection experiment confirms that high expression of NR5A2 can promote the proliferation of MM cells, B shows that Tanswell invasion experiment shows that NR5A2 promotes the invasion of HMCLs, C shows that flow cytometry apoptosis experiment confirms that high expression of NR5A2 can inhibit the apoptosis of MM cells, D shows that mouse xenotransplant tumor experiment shows that NR5A2 promotes the proliferation of MM.

[0029] Figure 4The phenomenon that NR5A2 overexpression regulates phospholipid metabolism is shown. Among them, A shows AMOl-NR5A2-NC and AMOl-NR5A2-OE cell proteomic pathway analysis, B shows lipid metabolism data suggesting that after NR5A2 overexpression, the lipid changes, C shows oil red O staining detection photos, and D shows oil red O staining detection data statistical results.

[0030] Figure 5 The case that NR5A2 overexpression inhibits ferroptosis is shown. Among them, A shows that after NR5A2 OE / KD, i.e., NR5A2 overexpression / knockdown, the mitochondrial cristae structure in the cell is clearer / broken (disappeared) under transmission electron microscope observation, B shows the fluorescence microscope observation photos after NR5A2 OE / KD, C shows that after NR5A2 OE / KD, ROS production is reduced / increased, D shows that after NR5A2 OE / KD, ATP production is increased / decreased, E shows that after NR5A2 OE / KD, Fe 2+ shows the fluorescence microscope observation photos after Fe 2+ D shows that after NR5A2 OE / KD, ATP production is increased / decreased, E shows that after NR5A2 OE / KD, Fe

[0031] Figure 6 MBOAT1 and MBOAT2 are shown to be downstream target genes of NR5A2. Among them, A shows that after intersection of KEGG enrichment pathways, NR5A2 downstream regulation network is confirmed, B shows that PCR verifies that MBOAT1 and MBOAT2 and NR5A2 change in the same direction at the mRNA level, C shows that WB verifies the downstream situation, D shows that CUT-Tag verifies that NR5A2 is a transcription factor of MBOAT1 and MBOAT2, E shows the changes in transcription level and protein level after overexpression or knockdown of MBOAT1 and MBOAT2.

[0032] Figure 7 It is shown that in vitro experiments prove that overexpression of MBOAT1 and MBOAT2 can promote HMCLs proliferation. Among them, A shows that CCK8 detection finds that high expression of MBOAT1 and MBOAT2 can promote HMCLs proliferation, B shows that Transwell confirms that knockdown of MBOAT1 and MBOAT2 can inhibit HMCLs invasion, C and D show that flow cytometry apoptosis confirms that high expression of MBOAT1 and MBOAT2 can inhibit HMCLs apoptosis.

[0033] Figure 8The results showed that overexpression of MBOAT1 and MBOAT2 could inhibit ferroptosis in HMCLs. Specifically, A shows, under TEM observation, that the intracellular mitochondrial cristae structure was more clearly defined after high expression of MBOAT1 and MBOAT2; B and C show the decrease / increase in ROS production after MBOAT1 OE / KD and MBOAT2 OE / KD; D shows the decrease / increase in ATP production after MBOAT1 OE / KD and MBOAT2 OE / KD; and E and F show the decrease in Fe production after MBOAT1 OE / KD and MBOAT2 OE / KD. 2+ G indicates a decrease / increase in MDA production after MBOAT1OE / KD and MBOAT2OE / KD, while H and I indicate a significant increase / decrease in OCR after MBOAT1OE / KD and MBOAT2OE / KD.

[0034] Figure 9 The study showed a decrease in ferroptosis-specific lipid substrates after NR5A2 overexpression. Specifically, A shows changes in various lipids in phosphatidylcholine (PC) after NR5A2 overexpression; B shows changes in various lipids in phosphatidylethanolamine (PE) after NR5A2 overexpression; C shows changes in various fatty acids after NR5A2 overexpression; and D shows changes in PUFA-containing ether lipids after NR5A2 overexpression.

[0035] Figure 10 The results show that NR5A2 regulates MBOAT1 / MBOAT2 to inhibit ferroptosis in HMCLs via a non-GPX4 pathway. In A, GPX4 mRNA levels remain unchanged after NR5A2 OE / KD; in B, GPX4 protein levels remain unchanged after NR5A2 OE / KD.

[0036] Figure 11Figure 6 shows that NR5A2 makes MM cells resistant to dexamethasone by inhibiting ferroptosis of HMCLs. A shows that overexpression of NR5A2 can make HMCLs resistant to dexamethasone, and knockdown of NR5A2 can make HMCLs sensitive to dexamethasone; B shows that in vivo administration of NR5A2 inhibitor ML-180 can significantly inhibit the growth of mouse tumors; C shows that after 100 μmol dexamethasone for 48H, the overexpression of NR5A2 group produces less ROS than the NC group, and the knockdown of NR5A2 group produces more ROS than the NC group; D shows that after 100 μmol dexamethasone for 48H, the overexpression of NR5A2 group shows less ROS than the NC group, and the knockdown of NR5A2 group shows more ROS than the NC group; E shows that after 100 μmol dexamethasone for 48H, the overexpression of NR5A2 group produces less MDA than the NC group, and the knockdown of NR5A2 group produces more MDA than the NC group.

[0037] Figure 12 Figure 7 shows that inhibition of the expression of NR5A2 can increase the sensitivity of MM cell lines to dexamethasone. A shows the effect of NR5A2 inhibitor ML-180 and dexamethasone on the proliferation of mouse tumors in vivo; B shows fatty acid co-culture experiments, including SFA (14:0), MUFA (18:1) and PUFA (20:4); C shows that selinexor can inhibit NR5A2 transcription and translation; D shows that selinexor can antagonize the dexamethasone resistance of NR5A2 overexpressed MM cells. DETAILED DESCRIPTION

[0038] MM with 1q+ is usually clinically and biologically heterogeneous. Our research group combined WES detection data of MM patients' bone marrow plasma cells with the molecular genetic characteristics of MM (chromosome 1q amplification), and for the first time proposed that orphan nuclear receptor NR5A2 (located in chromosome 1q32) participates in the proliferation and invasion of MM cells by regulating lipid metabolism reprogramming. Through lipid metabolism reprogramming, ferroptosis and other aspects, we deeply explored and explained the molecular mechanism of orphan nuclear receptor NR5A2 in MM. This research achievement can help understand the pathogenesis of MM patients and find new therapeutic targets.

[0039] NR5A2 gene is located on chromosome 1q32, encoding protein LRH-1 is a zinc finger protein transcription factor, belonging to the nuclear hormone receptor family, the protein is involved in the regulation of bile acid synthesis, cholesterol balance, triglyceride synthesis gene expression and maintaining phospholipid diversity in the liver, NR5A2 is a transcriptional regulator of various lipid metabolism. In 2005, Holly A. Ingraham team published in CELL journal that phospholipid (PLs) can directly bind to NR5A2 as a ligand to promote its gene expression and activity. In T lymphocyte leukemia, NR5A2 can inhibit glucocorticoid receptor leading to glucocorticoid resistance, and NR5A2 inhibitor can make T cell leukemia cells re-sensitized to glucocorticoid-induced apoptosis. In prostate cancer, NR5A2 can promote tumor cell resistance to darolutamide by regulating HSD3B1. But there is no relevant research report on the relationship between NR5A2 and MM cell resistance. According to the previous results that 1q+ RRMM is closely related to high expression of NR5A2, we further found that NR5A2 has the phenotype of promoting MM proliferation and invasion. MM cells overexpressing NR5A2 have significantly increased resistance to dexamethasone, and this resistance can be reversed by AA or AdA and promoted by SFA (14:0) or MUFA (18:1). At the same time, MM cells overexpressing NR5A2 have significantly reduced ferroptosis, which is manifested as increased Fe2+ and reactive oxygen species (ROS) in cells, altered mitochondrial morphology and function loss. Through lipid non-target mass spectrometry combined with proteomic analysis, we found that NR5A2 can mediate phospholipid remodeling through MBOAT1 and MBOAT2, and inhibit MM cell ferroptosis. Among them, MBOAT2 is one of the key enzymes in the Lands cycle of phospholipid remodeling--LPCAT 4. In 2023, CELL journal published a study confirming that MBOAT1 / 2 can directly inhibit ferroptosis independently of glutathione peroxidase 4 (GPX4) under the regulation of sex hormone receptors. But in MM, the relationship between NR5A2 and MBOAT superfamily, between NR5A2 and cholesterol metabolism, and between NR5A2 and ferroptosis has not been reported.

[0040] In this paper, for the sake of convenience in description, the name of a certain gene (DNA) NR5A2 is sometimes mixed with its encoded protein such as transcription factor LRH-1, and those skilled in the art should understand that they represent different substances in different descriptions, and their meanings can be easily understood according to the context and context.

[0041] The expression levels of NR5A2, MBOAT1 and MBOAT2 in CD138+ plasma cells of MM patients with 1q+ were further detected by the research group to further predict the prognosis of the patients. The discovery of NR5A2 can help clinicians better judge the prognosis of MM patients with 1q+, and at the same time provide a new therapeutic target and possible new drug for this relapsed and refractory MM patient. Selinexor, NR5A2 inhibitor ML-180 (CAS NO: 863588-32-3), arachidonic acid (AA) as an inhibitor of this new target for patients in this subgroup, can further design clinical trials to determine the effect of the drug and be applied to the treatment of MM patients with 1q+.

[0042] The application will be further described below in conjunction with specific examples. It should be understood that these examples are for illustrative purposes only and are not limiting to the application.

[0043] Examples

[0044] The addition amount, content and concentration of various substances involved in the present application are all mass percentage content unless otherwise specified.

[0045] In the examples herein, if no specific description is made for the reaction temperature or operating temperature, the temperature generally refers to room temperature (15-35°C).

[0046] The detection samples used in the examples were derived from the Department of Hematology, Zhongshan Hospital Affiliated to Fudan University.

[0047] The detection of NR5A2, MBOAT1 and MBOAT2 genes and proteins was performed according to the kit instructions.

[0048] The NR5A2 gene qRT-PCR detection kit was purchased from Takara Company, Japan.

[0049] The qRT-PCR primers were ordered from Shanghai Sungene Biotech Co., Ltd., and the gene sequences are listed in the following table:

[0050]

[0051] The antibody information is shown in the following table

[0052]

[0053] Part of the equipment information is as follows:

[0054] Rotary microtome, Leica, model RM2235;

[0055] In situ hybridization instrument, Abbott, model TermoBrite;

[0056] Fluorescence microscope, Olympus, model BX43.

[0057] Example 1: NR5A2 is highly expressed in 1q+ MM patients and is a marker of poor clinical prognosis in MM patients

[0058] By analyzing the WES data of 47 cases of newly diagnosed multiple myeloma (NDMM), 5 cases of SMM, 7 cases of MGUS, and 17 cases of RRMM patients in our center, it was found that compared with MGUS and SMM, the frequency of gene amplification (Amplification: copy number > 3, Gain: copy number = 3, Deletion: copy number = 1, Loss: copy number = 0) or deletion gradually increased in the clonal evolution process of MM, and the abnormalities of chromosome 1 were more significant Figure 1 Fig. 1A). Further analysis of the 1q segment showed that there were more gene amplifications in the 1q segment of NDMM and RRMM patients than in MGUS and SMM patients Figure 1 Fig. 1B).

[0059] Screening of differential transcription factors in the 1q segment, the following transcription factors were screened: NHLH1, GON4L, USF1, PRRX1, NR5A2, PIAS3, ETV3, NR1I3, ATF6, TBX19, BLZF1, ZBTB37, LHX4, ZNF648, ZBTB41, LHX9, ZNF124, ZNF496, ZNF672, ZNF692 Figure 1 Fig. 1C). Using the MMRF CoMMpass database of 574 MM patients with fluorescence in situ hybridization (FISH) data, the expression of the above-mentioned transcription factors in 1q- and 1q+ and the relationship between the mRNA expression and the survival prognosis of MM patients were explored. The results showed that NR5A2 was highly expressed in 1q+ MM patients Figure 1 Fig. 1D, p = 0.020); the mRNA expression of NR5A2 was divided into high, medium and low three groups, compared with the low expression group, the NR5A2 high expression group had worse progression free survival (PFS) and overall survival (OS) of MM patients.

[0060] Example 2: High expression of NR5A2 promotes the proliferation and invasion of MM cell lines

[0061] Using HMCLs (Human myeloma cell lines) preserved in our laboratory, we performed PCR and Western blot analysis to verify the expression of NR5A2 at both the mRNA and protein levels. NR5A2 was expressed at relatively low levels in AMO1 and RPMI-8226 cells, but at relatively high levels in NCI-H929 and U266 cells. Figure 2 (A) AMO1 and RPMI-8226, which have relatively low NR5A2 expression, were selected to construct stable NR5A2 overexpression (OE) transgenic lines, while NCI-H929 and U266, which have relatively high NR5A2 expression, were selected to knock down NR5A2 (KD).

[0062] qRT-PCR results showed that the mRNA levels of NR5A2 overexpression in AMO1 and RPMI-8226 cells (AMO1-NR5A2-OE and RPMI-8226-NR5A2-OE) were significantly upregulated compared to the negative control (NC). Figure 2 In the NC group (B), the expression level of NR5A2 knocked down in NCI-H929 and U266 cells (NCI-H929-NR5A2-KD and U266-NR5A2-KD) was significantly lower than that in the NC group. Figure 2 Similarly, Western blot analysis showed that the NR5A2 protein expression level in AMO1-NR5A2-OE and RPMI-8226-NR5A2-OE cells was significantly higher than that in the NC group, while the NR5A2 protein expression level in NCI-H929-NR5A2-KD and U266-NR5A2-KD cells was significantly lower than that in the NC group. Figure 2 (C)

[0063] Based on the verification results at the mRNA and protein levels, the stable transgenic cells for NR5A2 knockdown were NCI-H929-NR5A2-SH3 and U266-NR5A2-SH3, which will be referred to as NCI-H929-NR5A2-KD and U266-NR5A2-KD respectively, when conducting the following phenotypic experiments.

[0064] MM cell proliferation was assessed using the CCK8 assay in AMO1-NR5A2-OE and RPMI-8226-NR5A2-OE overexpression stable transgenic cells and NCI-H929-NR5A2-KD and U266-NR5A2-KD knockdown stable transgenic cells. CCK8 data at each time point showed that the OD values ​​of AMO1-NR5A2-OE and RPMI-8226-NR5A2-OE were significantly higher than those of the NC group. Figure 3NCI-H929-NR5A2-KD and U266-NR5A2-KD groups were significantly lower than the NC group at each time point (Fig. 3A). Figure 3 NCI-H929-NR5A2-KD and U266-NR5A2-KD groups were significantly lower than the NC group at each time point (Fig. 3A).

[0065] To detect the effect of NR5A2 on the invasion ability of HMCLs, cell invasion experiments were performed. Three time points of 24 hours, 48 hours and 72 hours were set for observation, and the results showed that AMO1-NR5A2-OE and RPMI-8226-NR5A2-OE overexpression stable strains penetrated more cells at each time point than the NC group (Fig. 4A). Figure 3 NCI-H929-NR5A2-KD and U266-NR5A2-KD groups penetrated fewer cells at each time point than the NC group (Fig. 4B). Figure 3 To observe the effect of NR5A2 on the apoptosis of HMCLs, flow cytometry was used. The percentage of apoptotic cells in AMO1-NR5A2-OE (p = 0.005) and RPMI-8226-NR5A2-OE (p = 0.0003) overexpression stable strains was significantly lower than that in the NC group (Fig. 5A). Figure 3 NCI-H929-NR5A2-KD (p = 0.0021) and U266-NR5A2-KD (p < 0.0001) knockdown stable strains had a significantly higher percentage of apoptotic cells than the NC group (Fig. 5B). Figure 3 To verify the effect of NR5A2 on MM proliferation in vivo, mouse xenograft experiments were performed. 6-8 week old NOG-SCID mice were randomly divided into groups of 5, and 3x10 6 AMO1-NR5A2-OE cells and AMO1-NR5A2-NC cells were injected subcutaneously, and the tumor size was recorded regularly. The results showed that the tumor volume in the AMO1-NR5A2-OE group was significantly larger than that in the AMO1-NR5A2-NC group, and the difference was statistically significant (Fig. 6D). Figure 3

[0066] Example 3: NR5A2 participates in the regulation of phospholipid metabolism and ferroptosis in MM cells

[0067] Previous studies have shown that NR5A2 acts as a key regulator of lipid metabolism by regulating the expression of genes involved in cholesterol balance and triglyceride synthesis. Phospholipids (PLs) can directly bind to NR5A2 as ligands. To further explore the mechanism of NR5A2 promoting the proliferation and invasion of HMCLs, we used AMO1-NR5A2-NC and AMO1-NR5A2-OE cells for TMT labeled quantitative proteomics and non-targeted lipidomics research.

[0068] ​KEGG pathway analysis of proteomic data from AMO1-NR5A2-NC and AMO1-NR5A2-OE cells revealed significant enrichment of glycerophospholipid metabolism and phospholipid metabolism pathways in the NR5A2 overexpression group. Figure 4 (A). Next, non-targeted lipid metabolomics was used to detect lipid metabolism in the AMO1-NR5A2-NC and AMO1-NR5A2-OE groups. According to the lipid classification bubble chart, the AMO1-NR5A2-OE group showed the most significant change in glycerophospholipids (GP) compared to the AMO1-NR5A2-NC group. Figure 4 (B) To further verify the effect of NR5A2 expression on phospholipid metabolism in HMCLs, Oil Red O staining was performed. The results showed that the phospholipid content in AMO1-NR5A2-OE cells was significantly higher than that in the AMO1-NR5A2-NC group (p<0.0001); the phospholipid content in RPMI-8226-NR5A2-OE cells was significantly higher than that in the NC group (p<0.0001). Knockdown of NR5A2 in NCI-H929-NR5A2-KD (p<0.0001) and U266-NR5A2-KD (p<0.0001) cells significantly reduced phospholipid content. Figure 4 (C, D)

[0069] Previous studies have shown that ferroptosis is a regulated cell death process driven by iron-dependent phospholipid (PL) peroxidation, ultimately manifested as plasma membrane rupture. To further clarify whether NR5A2-regulated phospholipid metabolism in MM cells affects ferroptosis, we first performed TEM observation on AMO1-NR5A2-NC, AMO1-NR5A2-OE, NCI-H929-NR5A2-NC, and NCI-H929-NR5A2-KD cells. The results showed that the cristae structure inside the mitochondria in HMCLs was clearer after NR5A2 overexpression compared to the control group. Figure 5 (A); The cristae structure inside the mitochondria in HMCLs after NR5A2 knockdown was less clear than that in the control group. Figure 5 (A). Ferroptosis is often accompanied by an increase in ROS, so the ROS levels of cells in each group were measured. The results showed that the AMO1-NR5A2-OE group had fewer bright cells than the AMO1-NR5A2-NC group (p<0.0001); the NCI-H929-NR5A2-KD group showed more bright cells than the NCI-H929-NR5A2-NC group (p<0.0001). Figure 5(B, C) suggests that NR5A2 overexpression can inhibit ferroptosis in MM cells. To further clarify whether NR5A2 affects mitochondrial function in addition to its influence on mitochondrial structure, ATP levels were measured in each group of cells. The results showed that the ATP level in the AMO1-NR5A2-OE group was significantly higher than that in the AMO1-NR5A2-NC group (p<0.0001). The ATP level in the NCI-H929-NR5A2-KD group was significantly lower than that in the control group (p<0.0001). Figure 5 D). Fe 2+ As a substrate for ferroptosis, it is an important marker of ferroptosis. Fe was analyzed in each group of cells. 2 + After staining, the cells were observed and photographed under a fluorescence microscope. The results showed that the AMO1-NR5A2-OE group had fewer bright cells than the AMO1-NR5A2-NC group (p<0.0001); the NCI-H929-NR5A2-KD group had more bright cells than the NCI-H929-NR5A2-NC group (p<0.0001). Figure 5 E, F), suggesting that NR5A2 overexpression followed by Fe 2+ Fe content decreased and then knocked down 2+ The content decreased significantly but increased significantly. Malondialdehyde (MDA) is one of the main products of membrane lipid peroxidation, and its content can be used as an indicator of lipid peroxidation to reflect the level of cell membrane lipid peroxidation. The MDA content of each group of cells was detected using a kit. The results showed that the MDA content of the AMO1-NR5A2-OE group was significantly lower than that of the AMO1-NR5A2-NC group; the MDA content of the NCI-H929-NR5A2-KD group was significantly higher than that of the NCI-H929-NR5A2-NC group. Figure 5Fig. 6C, suggesting that the level of cell membrane lipid peroxidation decreased after overexpression of NR5A2, while the level of cell membrane lipid peroxidation significantly increased after knockdown of NR5A2. Seahorse XF Cell Mito Stress Test Kit was used to reflect the function of cell mitochondria by measuring the oxygen consumption rate (OCR). The results showed that basal respiration (p=0.000763), ATP-linked respiration (p=0.000242), spare respiratory capacity (p=0.000105), non-mitochondrial oxygen consumption (p=0.000472), proton leak (p=0.018647) and maximal respiration (p=0.000006) were significantly increased in the AMOl-NR5A2-OE group compared with the AMOl-NR5A2-NC group. While proton leak (p=0.014559) and maximal respiration (p=0.023006) were significantly decreased in the NCI-H929-NR5A2-KD group compared with the NCI-H929-NR5A2-NC group. Figure 5 Fig. 6H, I. To further observe, we collected 522 MM patients with FISH results in our center, of which 244 were 1q- and 278 were 1q+. We compared the differences of iron death and lipid metabolism related clinical indicators in 1q- and 1q+ MM patients, respectively. The results showed that compared with 1q- MM patients, 1q+ MM patients had significantly higher ferritin content (p=0.0088); significantly lower high-density lipoprotein cholesterol content (p=0.0033); significantly lower apolipoprotein Al content (p=0.0001); and significantly lower apolipoprotein (a) content (p=0.0301). Figure 5

[0070] Example 4: MBOAT1 and MBOAT2 are downstream target genes of NR5A2, and the NR5A2-MBOAT1 / 2 axis inhibits MM cell ferroptosis by regulating phospholipid remodeling

[0071] To further explore the downstream regulatory network of NR5A2, we found GPAT3, MBOAT1 and MBOAT2 by intersecting the significantly differentially expressed molecules in the glycerophospholipid metabolism and phospholipid metabolism pathways enriched by KEGG. Figure 6 ​MBOAT1 and MBOAT2 mRNA expression levels were significantly higher in AMOl-NR5A2-OE cells than in AMOl-NR5A2-NC cells (p = 0.0002 and p = 0.0405, respectively), and MBOAT1 and MBOAT2 mRNA expression levels were significantly lower in H929-NR5A2-KD cells than in H929-NR5A2-NC cells (p = 0.0004 and p < 0.0001, respectively). However, there was no significant change in GPAT3 mRNA expression levels in AMOl-NR5A2-OE and H929-NR5A2-KD cells compared with the NC group. Figure 6 MBOAT1 and MBOAT2 protein expression levels were positively correlated with NR5A2 in AMOl-NR5A2-NC, AMOl-NR5A2-OE, H929-NR5A2-NC, and H929-NR5A2-KD cells. Figure 6

[0072] To further verify the relationship between NR5A2 and MBOAT1 and MBOAT2, a CUT-Tag kit (TD904) was used for the experiment. The experiment was performed in AMOl-NR5A2-NC and AMOl-NR5A2-OE cells. The results showed that, in AMOl-NR5A2-NC cells, when the internal reference DNA-SPIKE-IN was balanced and had no difference, the experimental group NR5A2 group MBOAT1 (p = 0.001193) and MBOAT2 (p = 0.000124) were significantly higher than the negative control IgG group; in AMOl-NR5A2-OE cells, when the internal reference DNA-SPIKE-IN was balanced and had no difference, the experimental group NR5A2 group MBOAT1 (p = 0.000006) and MBOAT2 (p = 0.000015) were also significantly higher than the negative control IgG group. Compared with AMOl-NR5A2-NC cells, the CT values of MBOAT1 and MBOAT2 in the experimental group NR5A2 group of AMOl-NR5A2-OE cells were significantly reduced. Figure 6 MBOAT1 and MBOAT2 mRNA expression levels were significantly higher in AMOl-NR5A2-OE cells than in AMOl-NR5A2-NC cells (p = 0.0002 and p = 0.0405, respectively), and MBOAT1 and MBOAT2 mRNA expression levels were significantly lower in H929-NR5A2-KD cells than in H929-NR5A2-NC cells (p = 0.0004 and p < 0.0001, respectively). However, there was no significant change in GPAT3 mRNA expression levels in AMOl-NR5A2-OE and H929-NR5A2-KD cells compared with the NC group. Figure 6 ​MBOAT1 and MBOAT2 mRNA levels were significantly higher in MBOAT1-OE than in NC group (p<0.0001) and MBOAT2-OE than in NC group (p<0.0001) in H929-NR5A2-KD cells. In AMOl-NR5A2-OE cells, MBOAT1 mRNA levels were significantly lower in MBOAT1-OE than in NC group (p<0.0001) and MBOAT2 mRNA levels were significantly lower in MBOAT2-OE than in NC group (p<0.0001).

[0073] MBOAT1 and MBOAT2 knockdown was performed in AMOl-NR5A2-OE cells and MBOAT1 and MBOAT2 overexpression was performed in NCI-H929-NR5A2-KD cells. To detect the effect of MBOAT1 and MBOAT2 on the proliferation of HMCLs, CCK8 detection was performed. The data results of OD values at each time point showed that MBOAT1-KD (48H: p<0.0001; 72H: p<0.0001) and MBOAT2-KD (24H: p<0.0001; 48H: p<0.0001; 72H: p<0.0001) were significantly lower than NC group (Fig. 2A); MBOAT1-OE (48H: p<0.0001; 72H: p<0.0001) and MBOAT2-OE (24H: p<0.0001; 48H: p<0.0001; 72H: p<0.0001) were significantly higher than NC group (Fig. 2A). Figure 7 Figure 7

[0074] To detect the effect of MBOAT1 and MBOAT2 on the invasion ability of HMCLs, cell invasion experiments were performed after MBOAT1 and MBOAT2 knockdown transfection in AMOl-NR5A2-OE cells. Three time points of 24 hours, 48 hours and 72 hours were set for observation, respectively. The results showed that MBOAT1-KD (24H: p=0.002066; 48H: p<0.0001; 72H: p=0.000376) and MBOAT2-KD (24H: p=0.002243; 48H: p=0.000172; 72H: p=0.000108) were less than NC group at each time point (Fig. 3B), that is, MBOAT1 and MBOAT2 knockdown can weaken the invasion ability of HMCLs. Figure 7

[0075] ​​​The effect of MBOAT1 and MBOAT2 on the apoptosis of HMCLs was observed by flow cytometry. Transient transfection of MBOAT1 and MBOAT2 knockdown was performed in AMO1-NR5A2-OE cells, and transient transfection of MBOAT1 and MBOAT2 overexpression was performed in NCI-H929-NR5A2-KD cells. The results showed that the proportion of HMCL cell apoptosis after MBOAT1 overexpression was significantly lower than that of the NC group (p<0.0001), and the proportion of HMCL cell apoptosis after MBOAT2 overexpression was significantly lower than that of the NC group (p<0.0001); the proportion of HMCL cell apoptosis after MBOAT1 knockdown was significantly higher than that of the NC group (p<0.0001), and the proportion of HMCL cell apoptosis after MBOAT2 knockdown was significantly higher than that of the NC group (p=0.0007). That is, after knocking down the expression of MBOAT1 and MBOAT2, the apoptosis of HMCLs can be significantly promoted Figure 7 Fig. 9C.

[0076] TEM observation was performed after MBOAT1 and MBOAT2 knockdown in AMO1-NR5A2-OE cells and MBOAT1 and MBOAT2 overexpression in NCI-H929-NR5A2-KD cells. The results showed that the mitochondrial inner ridge structure of H929-NR5A2-KD-MBOAT1-OE and H929-NR5A2-KD-MBOAT2-OE was clearer than that of H929-NR5A2-KD-MBOAT1-NC and H929-NR5A2-KD-MBOAT2-NC Figure 8 Fig. 10A. The mitochondrial inner ridge structure of AMO1-NR5A2-OE-MBOAT1-KD and AMO1-NR5A2-OE-MBOAT2-KD showed more broken and missing than that of AMO1-NR5A2-OE-MBOAT1-NC and AMO1-NR5A2-OE-MBOAT2-NC Figure 8 Fig. 10A.

[0077] The ROS levels of each group of cells were then detected. The results showed that H929-NR5A2-KD-MBOAT1-OE and H929-NR5A2-KD-MBOAT2-OE cells showed fewer high-light cells under a fluorescence microscope than H929-NR5A2-KD-MBOAT1-NC and H929-NR5A2-KD-MBOAT2-NC cells; AMO1-NR5A2-OE-MBOAT1-KD and AMO1-NR5A2-OE-MBOAT2-KD showed more high-light cells under a fluorescence microscope than AMO1-NR5A2-OE-MBOAT1-NC and AMO1-NR5A2-OE-MBOAT2-NC groups. Statistical analysis of the mean fluorescence intensity of each group of cells using Image J and Prism found that the mean fluorescence intensity of H929-NR5A2-KD-MBOAT1-OE (p = 0.0002) and H929-NR5A2-KD-MBOAT2-OE (p < 0.0001) was significantly lower than that of H929-NR5A2-KD-MBOAT1-NC and H929-NR5A2-KD-MBOAT2-NC groups; the mean fluorescence intensity of AMO1-NR5A2-OE-MBOAT2-KD (p < 0.0001) and AMO1-NR5A2-OE-MBOAT2-KD (p < 0.0001) was significantly higher than that of AMO1-NR5A2-OE-MBOAT1-NC and AMO1-NR5A2-OE-MBOAT2-NC groups Figure 8 B, C) of the present application.

[0078] To further clarify whether MBOAT1 and MBOAT2 affect mitochondrial function in addition to affecting mitochondrial structure. ATP detection was performed on each group of cells, and the results showed that the ATP level of H929-NR5A2-KD-MBOAT1-OE group cells was significantly higher than that of H929-NR5A2-KD-MBOAT1-NC group cells (p < 0.0001); the ATP level of H929-NR5A2-KD-MBOAT2-OE group cells was significantly higher than that of H929-NR5A2-KD-MBOAT2-NC group cells (p < 0.0001). The ATP level of AMO1-NR5A2-OE-MBOAT1-KD group cells was significantly lower than that of AMO1-NR5A2-OE-MBOAT1-NC group (p < 0.0001). The ATP level of AMO1-NR5A2-OE-MBOAT2-KD group cells was significantly lower than that of AMO1-NR5A2-OE-MBOAT2-NC group (p = 0.0051) Figure 8MDA content was detected in each group of cells, and the results showed that the MDA content of the H929-NR5A2-KD-MBOAT1-OE group was significantly lower than that of the H929-NR5A2-KD-MBOAT1-NC group (p = 0.0106); the MDA content of the H929-NR5A2-KD-MBOAT2-OE group was significantly lower than that of the H929-NR5A2-KD-MBOAT2-NC group (p = 0.0201); the MDA content of the AMO1-NR5A2-OE-MBOAT1-KD group was significantly higher than that of the AMO1-NR5A2-OE-MBOAT1-NC group (p = 0.0072), and the MDA content of the AMO1-NR5A2-OE-MBOAT2-KD group was significantly higher than that of the AMO1-NR5A2-OE-MBOAT2-NC group (p = 0.0384) Figure 8 MDA content was detected in each group of cells, and the results showed that the MDA content of the H929-NR5A2-KD-MBOAT1-OE group was significantly lower than that of the H929-NR5A2-KD-MBOAT1-NC group (p = 0.0106); the MDA content of the H929-NR5A2-KD-MBOAT2-OE group was significantly lower than that of the H929-NR5A2-KD-MBOAT2-NC group (p = 0.0201); the MDA content of the AMO1-NR5A2-OE-MBOAT1-KD group was significantly higher than that of the AMO1-NR5A2-OE-MBOAT1-NC group (p = 0.0072), and the MDA content of the AMO1-NR5A2-OE-MBOAT2-KD group was significantly higher than that of the AMO1-NR5A2-OE-MBOAT2-NC group (p = 0.0384) 2+ After staining, the cells were observed and photographed under a fluorescence microscope. The results showed that the H929-NR5A2-KD-MBOAT1-OE group and the H929-NR5A2-KD-MBOAT1-OE group had fewer high-light cells than the NC group; while the AMO1-NR5A2-OE-MBOAT1-KD group and the AMO1-NR5A2-OE-MBOAT2-KD group had more high-light cells than the NC group. Statistical analysis of the mean fluorescence intensity of each group of cells using Image J and Prism showed that the mean fluorescence intensity of H929-NR5A2-KD-MBOAT1-OE (p < 0.0001) and H929-NR5A2-KD-MBOAT2-OE (p < 0.0001) was significantly lower than that of H929-NR5A2-KD-MBOAT1-NC and H929-NR5A2-KD-MBOAT2-NC; the mean fluorescence intensity of AMO1-NR5A2-OE-MBOAT2-KD (p < 0.0001) and AMO1-NR5A2-OE-MBOAT2-KD (p < 0.0001) was significantly higher than that of AMO1-NR5A2-OE-MBOAT1-NC and AMO1-NR5A2-OE-MBOAT2-NC Figure 8 MDA content was detected in each group of cells, and the results showed that the MDA content of the H929-NR5A2-KD-MBOAT1-OE group was significantly lower than that of the H929-NR5A2-KD-MBOAT1-NC group (p = 0.0106); the MDA content of the H929-NR5A2-KD-MBOAT2-OE group was significantly lower than that of the H929-NR5A2-KD-MBOAT2-NC group (p = 0.0201); the MDA content of the AMO1-NR5A2-OE-MBOAT1-KD group was significantly higher than that of the AMO1-NR5A2-OE-MBOAT1-NC group (p = 0.0072), and the MDA content of the AMO1-NR5A2-OE-MBOAT2-KD group was significantly higher than that of the AMO1-NR5A2-OE-MBOAT2-NC group (p = 0.0384)

[0079] OCR levels in each group of cells were detected using Seahorse XF Cell Mito Stress Test Kit. The experimental results showed that the H929-NR5A2-KD-MBOAT1-OE group and the H929-NR5A2-KD-MBOAT1-NC group were significantly increased in basal respiration (p=0.003454), non-mitochondrial oxygen consumption (p<0.0001), and proton leakage (p=0.000209); the H929-NR5A2-KD-MBOAT2-OE group and the H929-NR5A2-KD-MBOAT2-NC group were significantly increased in non-mitochondrial oxygen consumption (p=0.010307). Compared with the AMO1-NR5A2-OE-MBOAT1-NC group cells, the AMO1-NR5A2-OE-MBOAT1-KD group cells were significantly reduced in spare respiratory capacity (p=0.022414) and non-mitochondrial oxygen consumption (p<0.0001); compared with the AMO1-NR5A2-OE-MBOAT2-NC group cells, the AMO1-NR5A2-OE-MBOAT2-KD group cells were significantly reduced in basal respiration (p<0.0001), ATP-related respiration (p=0.000445), non-mitochondrial oxygen consumption (p=0.00138), proton leakage (p=0.000895), and maximal respiration (p=0.048329). Figure 8 H, I).

[0080] Previous studies have shown that PUFA-containing lipids on membranes are key substrates for ferroptosis. MUFA-containing membrane lipids inhibit lipid peroxidation. Specific lipids driving ferroptosis include: phosphatidylethanolamine (PE), which contains one arachidonic acid (AA, 20:4) and one adrenaline acid (AdA, 22:4), is more prone to ferroptosis; PLs containing two PUFAs or PUFA-containing ether esters are specific lipids driving ferroptosis. To further explore the mechanism by which NR5A2 inhibits ferroptosis in HMCLs, lipid metabolism data were analyzed. Phosphatidylcholine (PC) and PE are phospholipids that mainly perform biological functions on the cell membrane and are closely related to cellular lipid peroxidation and ferroptosis. Therefore, the changes in PC and PE after NR5A2 overexpression were first analyzed, and the results are as follows: Figure 9 As shown ( Figure 9 (A, B) suggests that NR5A2 overexpression increases SFA expression levels while decreasing PUFA expression abundance in PC and PE.

[0081] Further investigation was conducted into the changes in fatty acids in phospholipids after NR5A2 overexpression. The expression abundance of each fatty acid was calculated based on the structural formulas of various phospholipid metabolites, and the fatty acids showing statistically significant changes after NR5A2 overexpression were summarized. Figure 9 The results showed that PEs with one arachidonic acid (AA, 20:4) and one adrenaline acid (AdA, 22:4) both decreased after NR5A2 overexpression, with the decrease in arachidonic acid being more significant. Figure 9 (C). Statistical analysis of changes in ether lipids in lipid metabolism data showed that the expression abundance of PUFA-containing ether esters was significantly reduced after NR5A2 overexpression. These included the following ether lipids: PC(16:0e / 20:4) (p = 0.0003), PC(32:4e) (p = 0.0051), PE(31:5e) (p = 0.0164), PE(35:5e) (p = 0.0002), PS(38:2e) (p = 0.0236), PS(40:4e) (p < 0.0001), and PS(42:5e) (p = 0.0159). Figure 9 (D). The above lipid metabolism results all indicate that the expression abundance of most specific lipids driving ferroptosis was significantly reduced after NR5A2 overexpression, further confirming that NR5A2 overexpression inhibits ferroptosis in HMCLs.

[0082] Previous studies have shown that there are two classic regulatory mechanisms of ferroptosis: glutathione peroxidase 4 (GPX4) and human fibroblast-specific protein 1 (FSP1). Previous studies have found that MBOAT1 and MBOAT2 are highly expressed in prostate cancer and breast cancer to regulate phospholipid remodeling and inhibit non-GPX4-dependent ferroptosis, so this study was conducted to explore. Verification was carried out in AMO1-NR5A2-NC, AMO1-NR5A2-OE, RPMI-8226-NR5A2-NC, RPMI-8226-NR5A2-OE, NCI-H929-NR5A2-NC, NCI-H929-NR5A2-KD and U266-NR5A2-NC, U266-NR5A2-KD cells. The experimental results suggest that the expression of NR5A2 does not affect the transcription and translation of GPX4. That is, the NR5A2-MBOAT1 / 2 axis regulates phospholipid remodeling to inhibit MM cell ferroptosis through a non-GPX4-dependent pathway Figure 10

[0083] Example 5: NR5A2 promotes dexamethasone resistance in MM cell lines

[0084] Previous studies have reported that in T lymphocyte leukemia, NR5A2 can compete with glucocorticoid receptor to cause glucocorticoid resistance. As a cornerstone drug for MM treatment, glucocorticoids, there is no literature to support the correlation between NR5A2 and dexamethasone resistance in MM cells. This embodiment explores the correlation between NR5A2 and dexamethasone resistance in MM cells.

[0085] ​The sensitivity of HMCLs to dexamethasone was detected in AMO1-NR5A2-NC, AMO1-NR5A2-OE, RPMI-8226-NR5A2-NC, RPMI-8226-NR5A2-OE overexpression stable strains and NCI-H929-NR5A2-NC, NCI-H929-NR5A2-KD, U266-NR5A2-NC, U266-NR5A2-KD knockdown stable strains, respectively. The following dexamethasone (Dexa) concentrations were set: 0 μmol, 0.2 μmol, 1 μmol, 2.5 μmol, 5 μmol, 10 μmol, 25 μmol, 100 μmol, 250 μmol, 1000 μmol. The inhibition rate was calculated based on the OD value of each group compared with the 0 μmol group. The results showed that AMO1-NR5A2-OE and RPMI-8226-NR5A2-OE had significantly lower inhibition rate than the NC group at each gradient Dexa concentration, while NCI-H929-NR5A2-KD and U266-NR5A2-KD had significantly higher inhibition rate than the NC group at each gradient Dexa concentration, suggesting that high expression of NR5A2 promotes the drug resistance of HMCLs to Dexa, and knocking down NR5A2 can improve the sensitivity of HMCLs to Dexa Figure 11 A) in the middle.

[0086] A mouse in vivo tumor transplantation experiment was performed to verify the effect of NR5A2 inhibitor ML-180 on MM proliferation in vivo. 6-8 week old NOG-SCID mice were randomly divided into groups of 6, and 3x10 6 AMO1-NR5A2-OE cells or AMO1-NR5A2-NC cells were injected subcutaneously, and after the mice formed tumors, AMO1-NR5A2-NC-ML-180 and AMO1-NR5A2-OE-ML-180 groups were fed (30 mg / kg) and the tumor size was regularly recorded. The results showed that the NR5A2 small molecule inhibitor ML-180 can significantly inhibit the proliferation of tumors in mice, and the inhibition effect of the AMO1-NR5A2-OE group is more significant Figure 11(B) The following assays were performed on cells in AMO1-NR5A2-NC, AMO1-NR5A2-OE, NCI-H929-NR5A2-NC, and NCI-H929-NR5A2-KD groups: ROS levels were measured after 100 μmol Dexa was applied to each group of cells for 48 hours. The results showed that the AMO1-NR5A2-OE group had fewer green highlight cells observed under a fluorescence microscope than the AMO1-NR5A2-NC group (p = 0.002), while the NCI-H929-NR5A2-KD group had more green highlight cells observed under a fluorescence microscope than the NCI-H929-NR5A2-NC group (p < 0.0001). Figure 11 (C). That is, under the action of 100 μmol Dexa, the NR5A2 overexpression group produced less ROS, while the NR5A2 knockdown group produced more ROS.

[0087] The following detections were performed on AMO1-NR5A2-NC, AMO1-NR5A2-OE, NCI-H929-NR5A2-NC, and NCI-H929-NR5A2-KD cells: 100 μmol Dexa was used to treat each group of cells for 48 hours, followed by Fe... 2+ Detection. Detection of Fe in cells of each group. 2+ The results showed that the AMO1-NR5A2-OE group had fewer red highlight cells observed under a fluorescence microscope than the AMO1-NR5A2-NC group (p<0.0001), while the NCI-H929-NR5A2-KD group had more red highlight cells observed under a fluorescence microscope than the NCI-H929-NR5A2-NC group (p=0.0014). Figure 11 (D). That is, under the action of 100 μmol Dexa, the NR5A2 overexpression group showed less Fe. 2+ The NR5A2 knockdown group showed more Fe. 2+ Subsequent MDA analysis showed that, under the influence of 100 μmol Dexa, the AMO1-NR5A2-OE group produced less MDA than the AMO1-NR5A2-NC group (p = 0.0002); the NCI-H929-NR5A2-KD group produced more MDA than the NCI-H929-NR5A2-NC group (p = 0.0003). Figure 11 (E).

[0088] Example 6: Inhibiting NR5A2 expression increases the sensitivity of MM cell lines to dexamethasone.

[0089] In vivo xenograft experiments were performed to explore the effect of NR5A2 inhibitor ML-180 and dexamethasone on MM proliferation in the presence of NR5A2 overexpression. 6-8 week old NOG-SCID mice were randomly divided into groups, 5 mice in each group, and each mouse was subcutaneously injected with 3x10 6 AMO1-NR5A2-OE cells. After the mice were tumor-bearing, AMO1-NR5A2-OE-Dexa, AMO1-NR5A2-OE-ML-180, and AMO1-NR5A2-OE-Dexa+ML-180 groups were fed (ML-180: 30 mg / kg; Dexa: 10 mg / kg), and the tumor size was regularly recorded. Statistical analysis of tumor size showed that NR5A2 small molecule inhibitor ML-180 can significantly inhibit the proliferation of tumor in mice in vivo, and can synergize with Dexa to significantly inhibit the growth of tumor in mice in vivo Figure 12 A) in mice.

[0090] Further analysis of lipid metabolism data, found that AMO1-NR5A2-OE than AMO1-NR5A2-NC group, the most significant changes in SFA, MUFA, PUFA were myristic acid (Myristic acid, MA, 14:0), oleic acid (Oleic acid, OA, 18:1) and arachidonic acid (Arachidonic acid, AA, 20:4). To explore whether the above fatty acids have an impact on the sensitivity of HMCLs to Dexa, the following experiments were designed. In AMO1-NR5A2-NC and AMO1-NR5A2-OE groups, the following groups were designed: AMO1-NR5A2-NC, AMO1-NR5A2-NC+25μmol Dexa, AMO1-NR5A2-NC+25μmol Dexa+10μmol MA / OA / AA, AMO1-NR5A2-NC+25μmol Dexa+25μmol MA / OA / AA, AMO1-NR5A2-NC+25μmol Dexa+50μmol MA / OA / AA, AMO1-NR5A2-NC+25μmol Dexa+100μmol MA / OA / AA; AMO1-NR5A2-OE, AMO1-NR5A2-OE+25μmol Dexa, AMO1-NR5A2-OE+25μmol Dexa+10μmol AA, AMO1-NR5A2-OE+25μmol Dexa+25μmol AA, AMO1-NR5A2-OE+25μmol Dexa+50μmol AA, AMO1-NR5A2-OE+25μmol Dexa+100μmol AA. The results showed that each concentration gradient of MA had no effect on the sensitivity of AMO1-NR5A2-NC group to dexamethasone; each concentration gradient of OA inhibited the sensitivity of AMO1-NR5A2-NC group cells to dexamethasone; each concentration gradient of AA promoted the sensitivity of AMO1-NR5A2-NC group cells to dexamethasone. In addition, to explore whether AA can reverse the drug resistance of MM cells to dexamethasone caused by high expression of NR5A2, AMO1-NR5A2-OE cells were used for exploration, and the results suggested that AA co-culture can significantly reverse the drug resistance of MM cells to dexamethasone caused by high expression of NR5A2 Figure 12 B) of the middle.

[0091] XPO1 is the most critical nuclear export protein, also known as CRM1 (chromosome maintenance protein 1), is the only nuclear export receptor of several tumor suppressor proteins (TSP), growth regulatory proteins (GRP), and is the most widely studied nuclear export protein. Selective XPO1 inhibitor: Selinexor binds to XPO1, activates tumor suppressor proteins, inactivates oncogenic proteins, activates GR pathways, and restores hormone sensitivity. NR5A2 as an orphan nuclear receptor, in T lymphocyte leukemia, NR5A2 can compete with glucocorticoid receptor to cause glucocorticoid resistance. In summary, it is explored whether Selinexor can antagonize Dexa resistance caused by high expression of NR5A2 by inhibiting NR5A2. The experimental results show that Selinexor can significantly inhibit the transcription (p=0.0405) and translation of NR5A2 Figure 12 In the middle C). The following Dexa concentrations were set on AMO1-NR5A2-OE cells: 0 μmol, 0.2 μmol, 1 μmol, 2.5 μmol, 5 μmol, 10 μmol, 25 μmol, 100 μmol, 250 μmol, 1000 μmol. The inhibition rate was calculated by comparing the OD value of each group with the 0 μmol group. The results show that AMO1-NR5A2-OE-Selinexor has a significantly higher inhibition rate than AMO1-NR5A2-OE-Control at each gradient Dexa concentration, that is, Selinexor can antagonize Dexa resistance caused by high expression of NR5A2 by inhibiting NR5A2 Figure 12 In the middle D).

[0092] The above description of the embodiments is to facilitate those of ordinary skill in the art to understand and use the present application. Those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described herein to other embodiments, without having to go through creative labor. Therefore, the present application is not limited to the above embodiments. Those skilled in the art can make improvements and modifications to the present application without departing from the scope of the present application.

Claims

1. Use of the orphan nuclear receptor NR5A2 as a target in the screening of anti- multiple myeloma drugs, characterized in that, The multiple myeloma is chromosome 1q amplified multiple myeloma, i.e. 1q+ MM, and the orphan nuclear receptor NR5A2 is used as a drug inhibition target.

2. Use according to claim 1, wherein The drug inhibits the expression of NR5A2.

3. Use according to claim 2, wherein the compound is ###0002### The drug down-regulates or blocks the expression of, or inactivates, the NR5A2 gene.

4. The use according to claim 2, wherein the compound is ###0002### The drug also inhibits the expression of MBOAT1 and / or MBOAT2.

5. The use according to claim 4, wherein the compound is ###0002### The drug also down-regulates or blocks the expression of, or inactivates, the MBOAT1 and / or MBOAT2 gene.

6. The use according to claim 1, wherein The drug does not produce drug resistance.

7. Use of an agent that detects the orphan nuclear receptor NR5A2 for the manufacture of a preparation for the prognosis of multiple myeloma, characterized in that, The multiple myeloma is chromosome 1q amplified multiple myeloma, i.e. 1q+ MM.

8. Use according to claim 7, wherein the compound is ###0002### The preparation is a kit for evaluating the prognosis of multiple myeloma, which is chromosome 1q amplified multiple myeloma, i.e. 1q+ MM, by detecting the expression level of NR5A2 in a biological sample.

9. The kit of claim 8, wherein The detection of the expression level of NR5A2 in a biological sample is implemented by detecting the level of NR5A2 protein in the biological sample.

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