Application of RNA-binding protein RBM15B in MLL fusion gene leukemia

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of the RNA-binding protein RBM15B in MLL fusion gene leukemia (MLL-R). Using qRT-PCR technology, this invention detected significant high expression of RBM15B in a large number of leukemia patient samples, revealing a significant negative correlation between high RBM15B expression and patient survival rate. Further validation at the adult level was achieved by constructing mouse leukemia models using mouse MLL-AF9 cells and human MLL fusion gene leukemia cell lines. The results showed that knocking down RBM15B expression significantly inhibited the survival of MLL fusion gene leukemia cells and significantly prolonged the lifespan of the model mice. This indicates that RBM15B holds promise as a diagnostic marker and / or therapeutic target in MLL fusion gene leukemia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of RNA-binding protein RBM15B in MLL fusion gene leukemia. Background Technology

[0002] MLL fusion gene leukemia, also known as MLL (mixed-lineage leukemia) gene rearrangement leukemia (MLL-rearranged leukemia, MLL-R leukemia), is a type of malignant tumor characterized by impaired differentiation and malignant proliferation of hematopoietic stem / progenitor cells. Its incidence increases with age, and it is often more prevalent in men than women, with an overall complete remission rate of only 30%. The pathogenesis of MLL fusion gene leukemia is complex, often resulting from the combined effects of multiple factors. Clinical symptoms mainly include fever, infection, bleeding, anemia, organ infiltration, and metabolic abnormalities. Without timely treatment, bone marrow failure can lead to death. Current treatment methods involve inducing remission with high-dose conventional chemotherapy, followed by subsequent treatments to enhance and maintain the therapeutic effect. For patients unsuitable for chemotherapy, those with refractory disease, or those requiring relapse prevention, hematopoietic stem cell transplantation or immunotherapy are used, but the prognosis and survival rate remain unsatisfactory. Therefore, it is necessary to adopt new methods to examine the biological characteristics of this type of leukemia, conduct in-depth research on the pathogenesis of acute leukemia, and thus find new early diagnosis and treatment methods to effectively improve the survival rate of MLL fusion gene leukemia patients.

[0003] In recent years, RNA-binding proteins (RBPs) have been found to participate in biological development and the pathogenesis of various cancers. As key players in post-transcriptional events, RBPs possess both the versatility of an RNA-binding domain and structural flexibility, enabling them to control the fate and metabolic processes of numerous transcripts and serve as ideal targets for small molecule drug development. RBPs establish highly dynamic interactions with other proteins, as well as coding and non-coding RNAs, generating functional units called ribonucleoprotein complexes (RNPs), which in turn regulate RNA splicing, polyadenylation, stability, localization, translation, and degradation. Therefore, deciphering the intricate network of interactions between RBPs and the series of cancer-related target RNAs they regulate will contribute to advancing our understanding of tumor biology and revealing new targets for cancer therapy. The important regulatory role of RNA-binding proteins in leukemia has been gradually discovered. RNA-binding proteins exhibit specific expression patterns in different leukemia subtypes, such as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), suggesting their crucial regulatory role across various leukemia subtypes. Therefore, identifying potential therapeutic targets for MLL fusion gene leukemia from RNA-binding proteins has significant theoretical and practical implications and can also provide valuable insights for the application of RNA-binding proteins in cancers of other leukemia patients. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides, on the one hand, the application of RNA-binding protein RBM15B as a diagnostic marker in the preparation of diagnostic products for MLL fusion gene leukemia, and on the other hand, the application of RNA-binding protein RBM15B as a therapeutic target in the preparation of drugs for treating MLL fusion gene leukemia.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of this invention provides the application of a reagent for detecting the expression level of RNA-binding protein RBM15B in the preparation of diagnostic products for MLL fusion gene leukemia.

[0007] This invention discovers an RNA-binding protein, RBM15B (Putative RNA-binding protein 15B; OTT3; Q8NDT2·RB15B_HUMAN; Q6PHZ5·RB15B_MOUSE), which is specifically highly expressed in MLL fusion gene leukemia but lowly expressed under normal physiological conditions and in other types of leukemia. The RBM15B gene locus is located on the positive DNA strand of human chromosome 3, and it can be transcribed into a 6624 nt mRNA with a coding region (CDS) of 2673 nt (NCBI Reference Sequence: NM_013286.5). The RBM15B protein has an amino acid length of 890 AA.

[0008] This study found that RBM15B is highly expressed in patients with MLL fusion gene leukemia, interestingly, RBM15 is not significantly expressed, indicating that RBM15B has a unique role in MLL fusion gene leukemia. Further ROC curve analysis confirmed that RBM15B can significantly distinguish between MLL fusion gene leukemia and normal human bone marrow samples; survival curve analysis showed that the five-year survival rate was significantly reduced with high RBM15B expression, suggesting that RBM15B has the potential clinical role as a classifier and prognostic indicator for MLL fusion gene leukemia.

[0009] Preferably, the reagent for detecting the expression level of the RNA-binding protein RBM15B is a primer for detecting the expression level of RBM15B, the sequence of which is shown in SEQ ID NO:1-2.

[0010] Preferably, the diagnostic product includes a diagnostic chip or a reagent kit.

[0011] The second aspect of this invention provides the use of a reagent that inhibits or reduces the expression level of the RNA-binding protein RBM15B in any of the following aspects 1)-4):

[0012] 1) To prepare products for the treatment of MLL fusion gene leukemia;

[0013] 2) To prepare products that inhibit the development and progression of MLL fusion gene leukemia;

[0014] 3) Prepare products that inhibit the proliferation of MLL fusion gene leukemia cells;

[0015] 4) Prepare products that promote the differentiation of MLL fusion gene leukemia cells.

[0016] This invention has confirmed that knocking down RBM15B using siRNA technology weakens the proliferation of MLL fusion gene leukemia cell lines, arrests cell cycle, increases differentiation, and reduces self-renewal capacity. Furthermore, NOD-SCID mouse model experiments showed that knocking down both mouse and human RBM15B with shRNA inhibited tumor growth in both mouse and human MLL fusion gene leukemia cell lines. This indicates that inhibiting RBM15B expression can suppress the growth of MLL fusion gene leukemia cells and tumors.

[0017] Preferably, the MLL fusion gene leukemia cells include MOLM-13 and MV4-11.

[0018] Preferably, the inhibition of MLL fusion gene leukemia development involves reducing the protein expression level of key proto-oncogenes in MLL fusion gene leukemia, wherein the key proto-oncogenes in MLL fusion gene leukemia include MYC, HOXA9, and BCL2.

[0019] More preferably, the reduction of protein expression level of key proto-oncogenes in MLL fusion gene leukemia is to reduce the translation efficiency of key proto-oncogene transcripts in MLL fusion gene leukemia.

[0020] The study also found that in MLL fusion gene leukemia cell lines, knocking down RBM15B inhibited the translation efficiency of key oncogenic proteins such as MYC, HOXA9, and BCL2, and significantly reduced their protein levels, confirming that RBM15B has an inhibitory effect on the progression of MLL fusion gene leukemia.

[0021] This invention directly and significantly reduces the protein expression level of MYC by regulating the expression level of the RNA-binding protein RBM15B through genetic engineering, which is of great value for the precision treatment of MLL fusion gene leukemia. Furthermore, it demonstrates the potential clinical value of RBM15B in indicative of the classification and prognosis of MLL fusion gene leukemia.

[0022] Preferably, the reagent used to inhibit or reduce the expression level of RNA-binding protein RBM15B is siRNA or shRNA that inhibits or reduces the expression level of RNA-binding protein RBM15B.

[0023] More preferably, the siRNA targeting human RBM15B is selected from one or more of siRNA-1 or siRNA-2, wherein the siRNA-1 sequence is shown in SEQ ID NO:6-7 and the siRNA-2 sequence is shown in SEQ ID NO:8-9.

[0024] More preferably, the shRNA targeting human RBM15B is selected from shRNA-1, the sequence of which is shown in SEQ ID NO:10-11; and the shRNA targeting mouse RBM15B is selected from shRNA-2, the sequence of which is shown in SEQ ID NO:12-13.

[0025] A third aspect of the present invention provides a therapeutic agent for MLL fusion gene leukemia, said agent comprising an agent that inhibits or reduces the expression level of RNA-binding protein RBM15B.

[0026] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0027] More preferably, the excipients are functional pharmaceutical excipients available in the pharmaceutical field, including (but not limited to) surfactants, suspending agents, emulsifiers, and some novel pharmaceutical polymers, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid-polylactic acid copolymer (PLGA), hyaluronic acid, etc.

[0028] Preferably, the dosage form of the drug includes injection, powder, granules, capsules, and tablets.

[0029] Preferably, the method of administration of the drug includes injection or oral administration.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] This invention, using qRT-PCR technology to analyze a large number of leukemia patient samples, found that the RNA-binding protein RBM15B is highly expressed in MLL fusion gene leukemia, and that high RBM15B expression is significantly negatively correlated with patient survival. Experiments in two mouse leukemia models showed that knocking down RBM15B expression significantly inhibited the progression of MLL fusion gene leukemia and significantly prolonged the survival of the model mice. This indicates that RBM15B can function as a diagnostic marker and / or therapeutic target in MLL fusion gene leukemia, suggesting that RBM15B holds promise for indicative of the diagnosis and prognosis of this type of leukemia, as well as for its treatment. Furthermore, this invention elucidates that RBM15B affects the progression of MLL fusion gene leukemia by regulating the expression levels of proto-oncoproteins such as MYC, providing a new theoretical basis for developing drug targets for MLL fusion gene leukemia. Attached Figure Description

[0032] Figure 1To evaluate the expression and clinical significance of RNA-binding protein RBM15B in MLL fusion gene leukemia; (A) qRT-PCR was used to detect that RBM15B mRNA was specifically highly expressed in MLL fusion gene leukemia samples, while RBM15 showed no change; (B) Log-rank (Mantel-Cox) survival curves were used to distinguish between high and low RBM15B expression levels by the optimal cutoff point (0.001292), with high RBM15B expression associated with a lower leukemia-free survival rate.

[0033] Figure 2 To investigate the cellular function of the RNA-binding protein RBM15B in MLL fusion gene leukemia; (A) the knockdown effect of RBM15B in MOLM-13 cells was detected using qRT-PCR and Western blotting techniques; (B) the knockdown effect of RBM15B in MOLM-13 cells was detected using qRT-PCR and Western blotting techniques. (C) Blot analysis showed the knockdown effect of RBM15B in MV4-11 cells; (D) CCK-8 assay showed that the proliferation level of MLL fusion gene leukemia cell lines was significantly reduced when RBM15B was knocked down; (E) Flow cytometry analysis showed that the proportion of differentiated cells in MLL fusion gene leukemia cell lines was significantly increased when RBM15B was knocked down; (F) Clonogenic assay showed that the self-renewal capacity of MLL fusion gene leukemia cells was significantly weakened when RBM15B was knocked down; (G) Flow cytometry analysis showed that the proportion of differentiated cells in primary MLL fusion gene leukemia patient samples was increased when RBM15B was knocked down. All figures in this figure use the mean ± standard deviation of three replicates. An asterisk indicates that the difference between the two groups was statistically significant when compared using a t-test (*P<0.05; **P<0.01; ***P<0.001).

[0034] Figure 3 This study established an animal model of MLL fusion gene leukemia regulated by RBM15B from human cell lines. (A) In a mouse hematologic malignancy model induced by tail vein injection, flow cytometry showed that the tumor infiltration rate in various tissues and organs was significantly lower in the group inoculated with RBM15B-knockdown MLL fusion gene leukemia cells than in the group inoculated with NC cells. (B) In a mouse hematologic malignancy model induced by tail vein injection, flow cytometry showed that the differentiation degree of tumor cells in the bone marrow of mice inoculated with RBM15B-knockdown cells was significantly higher than that in the group inoculated with NC cells. (C) Survival curves showed that the survival time of mice inoculated with RBM15B-knockdown MOLM-13 cells was significantly prolonged. All figures use the mean ± standard deviation of three replicates. An asterisk indicates that the difference between the two groups was statistically significant using a t-test (*p<0.05, **, p<0.01; ***, p<0.001).

[0035] Figure 4 This study aims to establish an animal model of MLL fusion gene leukemia derived from the Rbm15b-regulated mouse cell line (MLL-AF9 AML). Specifically, (A) a mouse hematologic malignancy model induced by tail vein injection, combined with flow cytometry, showed that the tumor infiltration rate in various tissues and organs was significantly lower in the RBM15B-knockdown MLL fusion gene leukemia cell group than in the NC cell group; (B) a mouse hematologic malignancy model induced by tail vein injection showed that the spleen size and weight were significantly lower in the RBM15B-knockdown MLL fusion gene leukemia cell group than in the NC cell group; and (C) a mouse hematologic malignancy model induced by tail vein injection, combined with… Flow cytometry showed that the tumor cell differentiation degree was significantly higher in the group inoculated with RBM15B knockdown MLL fusion gene leukemia cells than in the group inoculated with NC cells; (D) Survival curves showed that the survival time of mice was significantly prolonged after inoculation with RBM15B knockdown MLL fusion gene leukemia cells; All figures in this figure use the mean ± standard deviation of three replicates, and the asterisk indicates that the difference between the two groups is statistically significant when compared by t test (*p<0.05,**,p<0.01;***,p<0.001).

[0036] Figure 5 RBM15B regulates the expression of key proto-oncogenes in MLL fusion gene leukemia cells. (A) Western blotting showed that the levels of MYC, HOXA9, and BCL2 were significantly reduced in RBM15B knockdown MLL fusion gene leukemia cell lines; (B) Western blotting showed that the levels of MYC, HOXA9, and BCL2 were significantly increased in RBM15B overexpression MLL fusion gene leukemia cell lines; (C) Polysome profiling showed that the translation efficiency of MYC, HOXA9, and BCL2 was significantly weakened in RBM15B knockdown MLL fusion gene leukemia cells. All figures use the mean ± standard deviation of three replicates. An asterisk indicates that the difference between the two groups was statistically significant using a t-test (*p<0.05, **, p<0.01; ***, p<0.001). Detailed Implementation

[0037] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0038] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0039] Example 1: RBM15B Expression Analysis and Clinical Value Assessment

[0040] This invention discovers an RNA-binding protein, RBM15B (Putative RNA-binding protein 15B; OTT3; Q8NDT2·RB15B_HUMAN; Q6PHZ5·RB15B_MOUSE), which is specifically highly expressed in MLL fusion gene leukemia but lowly expressed under normal physiological conditions and in other types of leukemia. Interestingly, RBM15 (RNA-binding protein 15; OTT1; NM_022768.5; Q96T37·RBM15_HUMAN) does not exhibit this variation. This suggests that RBM15B plays an important role in MLL fusion gene leukemia. The RBM15B gene locus is located on the positive strand of human chromosome 3 and can be transcribed into a 6624 nt mRNA, with a coding region (CDS) of 2673 nt (NCBI Reference Sequence: NM_013286.5; CCDS33764.1, 116-2788). The protein amino acid length of RBM15B is 890 aa (SEQ ID NO: 1):

[0041] MKRQSERDSSPSGRGSSSSAKRPREREREAEAGGRRAAHKASGGAKHPVPARARDKPRGSGSGGGGHRDGRGTGDANHRASSGRSSGSGAGGGGRGGKASGDPGASGMSPRASPLPPPPPPPGAEPACPGSSAAAPEYKTLLISSLSPALPAEHLEDRLFHQFKRFGEISLRLSHTPELGRVAYVNFRHPQDAREARQHALARQLLLYDRPLKVEPVYLRGGGGSSRRSSSSSAAASTPPPGPPAPADPLGYLPLHGGYQYKQRSLSPVAAPPLREPRARHAAAAFALDAAAAAAVGLSRERALDYYGLYDDRGRPYGYPAVCEEDLMPEDDQRATRNLFIGNLDHSVSEVELRRAFEKYGIIEEVVIKRPARGQGGAYAFLKFQNLDMAHRAKVAMSGRVIGRNPIKIGYGKANPTTRLWVGGLGPNTSLAALAREFDRFGSIRTIDHVKGDSFAYIQYESLDAAQAACAKMRGFPLGGPDRRLRVDFAKAEETRYPQQYQPSPLPVHYELLTDGYTRHRNLDADLVRDRTPPHLLYSDRDRTFLEGDWTSPSKSSDRRNSLEGYSRSVRSRSGERWGADGDRGLPKPWEERRKRRSLSSDRGRTTHSPYEERSRTKGSGQQSERGSDRTPERSRKENHSSEGTKESSSNSLSNSRHGAEERGHHHHHHEAADSSHGKKARDSERNHRTTEAEPKPLEEPKHETKKLKNLSEYAQTLQLGWNGLLVLKNSCFPTSMHILEGDQGVISSLLKDHTSGSKLTQLKIAQRLRLDQPKLDEVTRRIKQGSPNGYAVLLATQATPSGLGTEGMPTVEPGLQRRLLRNLVSYLKQKQAAGVISLPVGGSKGRDGTGMLYAFPPCDFSQQYLQSALRTLGKLEEEHMVIVIVRDTA。

[0042] To further determine the expression specificity of RBM15B in MLL fusion gene leukemia, this embodiment re-collected a batch of bone marrow samples from patients at the First Affiliated Hospital of Sun Yat-sen University for analysis. This included 163 samples of MLL wild-type (MLL-wt) leukemia and 53 samples of MLL-r leukemia. The collection of all samples was approved by the Ethics Committee of Sun Yat-sen University and informed consent was obtained from the patients. RNA was extracted and qRT-PCR was used to specifically detect RBM15B. The primer sequences used in this qRT-PCR process are as follows:

[0043] The forward primer sequence of RBM15B is: 5'-GAGAACCACTCCAGTGAAGGG-3' (SEQ ID NO: 2);

[0044] The reverse primer sequence of RBM15B is: 5'-GCTGACTGGAGGTACTGCTG-3' (SEQ ID NO: 3);

[0045] The forward primer sequence of RBM15 is: 5'-TCCCACCTTGTGAGTTCTCC-3' (SEQ ID NO: 4);

[0046] The reverse primer sequence for RBM15 is: 5'-GTCAGCGCCAAGTTTTCTCT-3' (SEQ ID NO: 5).

[0047] The results showed that RBM15B was significantly overexpressed in MLL-R leukemia compared to MLL wild-type leukemia samples (p<0.001), while RBM15 showed no significant difference. Figure 1 A). Furthermore, this embodiment also found that normal samples had lower RBM15B expression levels, indicating a possible correlation between RBM15B expression levels and disease malignancy. To further explore the clinical significance of RBM15B in MLL-r leukemia, this embodiment subsequently used Log-rank (Mantel-Cox) survival curves in 136 samples (bone marrow samples from MLL fusion gene leukemia patients, collected from the First Affiliated Hospital of Sun Yat-sen University, with informed consent from the patients) to examine whether RBM15B has an indicative role in the prognosis of MLL-r leukemia. The experimental analysis results are as follows: Figure 1 As shown in Figure B, high expression of RBM15B was associated with a lower leukemia-free survival rate. These results suggest that RBM15B has the potential to differentiate between MLL-r and MLL-wt leukemia, and that high expression of RBM15B is also a risk factor in leukemia and a potential target for leukemia treatment.

[0048] Example 2: Functional identification of RBM15B in MLL fusion gene leukemia

[0049] To address the core issue of RBM15B's involvement in the regulation of MLL fusion gene leukemia, this study aims to further investigate the function of RBM15B in MLL fusion gene leukemia. The MLL fusion gene leukemia cell lines MOLM-13 and MV4-11 were selected as the research subjects. Two different siRNA sequences were designed for RBM15B using siRNA interference technology.

[0050] Forward sequence of siRNA-1: 5'-CCAACUGUAUGGAAAGUUGUCAGUAdTdT-3' (SEQ ID NO: 6);

[0051] The reverse sequence of siRNA-1: 5'-UACUGACAACUUUCCAUACAGUUGG dTd-3' (SEQ ID NO: 7);

[0052] Forward sequence of siRNA-2: 5'-CCCAGCGUCAUGUUUGUGU dTdT-3' (SEQ ID NO: 8);

[0053] The reverse sequence of siRNA-2: 5'-ACACAAACAUGACGCUGGG dTd-3' (SEQ ID NO: 9).

[0054] In the two MLL fusion gene leukemia cell lines mentioned above, RBM15B was knocked out, and cell proliferation was then assessed using a CCK-8 assay. First, qRT-PCR and Western blotting were used to detect the knockdown effect of RBM15B in MOLM-13 and MV4-11 cells. Figure 2 A, B). Experimental results are as follows: Figure 2 As shown in Figure C, knockdown of RBM15B significantly reduced cell proliferation in both MOLM-13 and MV4-11 cells. This indicates that RBM15B plays a crucial role in maintaining the proliferation of MLL-r leukemia cells. Simultaneously, RBM15B was knocked down using siRNA interference, and cell differentiation was assessed by flow cytometry. The experimental results are as follows: Figure 2 As shown in Figure D, the percentage of differentiated cells was significantly increased in both MOLM-13 and MV4-11 cells when RBM15B was knocked down. Further investigation was conducted to examine the effect of RBM15B on the self-renewal capacity of MLL fusion gene leukemia cells. Using clonogenic assays, it was found that the self-renewal capacity of MLL fusion gene leukemia cells was significantly reduced when RBM15B was knocked down. Figure 2E); Flow cytometry analysis showed that, in the case of RBM15B knockdown, the cell differentiation rate of primary MLL fusion gene leukemia patient samples increased ( Figure 2 F). Therefore, through cell proliferation, differentiation and clonogenic experiments, it is speculated that RBM15B has a potential regulatory role in the occurrence and development of MLL fusion gene leukemia.

[0055] Next, we will further verify the regulatory role of human RBM15B in MLL fusion gene leukemia at the adult level. We will also design an RBM15B shRNA sequence using the aforementioned siRNA sequence.

[0056] The forward sequence of shRNA-1:

[0057] 5'-GATCCCCAACTGTATGGAAAGTTGTCAGTACTTCCTGTCAGATACTGACAACTTTCCATACAGTTGGTTTTTG-3' (SEQ ID NO: 10);

[0058] The reverse sequence of shRNA-1:

[0059] 5'-AATTCAAAAACCAACTGTATGGAAAGTTGTCAGTATCTGACAGGAAGTACTGACAACTTTCCATACAGTTGGG-3' (SEQ ID NO: 11).

[0060] Using the expression vector pGreenPuro from System Biosciences TM A stable RBM15B knockdown cell line was constructed using the shRNA Cloning and Expression Vector lentiviral expression system, and the MOLM-13RBM15B knockdown cell line was obtained through puromycin selection. Subsequently, the validated stable MOLM-13RBM15B cell line was expanded and cultured, and then injected into 5-week-old NOD / SCID mice via tail vein injection: a total of 35 mice were divided into 3 groups (PBS, sh-NC, sh-RBM15B-1), with 5 mice in the PBS group, and 15 mice in each of the sh-NC and sh-RBM15B groups (150 μL per mouse, cell injection volume 1×10⁻⁶). 6 (Each animal). After 3 weeks, the tumor formation was assessed using flow cytometry. Figure 3 As shown in Figure A, mice subcutaneously inoculated with sh-RBM15B MOLM-13 exhibited lower tumor infiltration in the bone marrow, peripheral blood, spleen, and liver; moreover, statistical analysis revealed that the differentiation degree of tumor cells was higher in the NC group than in the NC group. Figure 3B). These results indicate that RBM15B promotes tumorigenesis in MLL fusion gene leukemia and affects the growth of these leukemia cells, suggesting that RBM15B may be a potential target for the treatment of this type of leukemia. Further analysis using Log-rank (Mantel-Cox) test survival curves revealed that the survival rate of mice with RBM15B knockdown was higher than that of the control group. Figure 3 C). This suggests that specifically targeting highly expressed RBM15B may be a potential strategy for treating MLL fusion gene leukemia.

[0061] Furthermore, this embodiment utilizes murine RBM15B to investigate the function of RBM15B in a mouse MLL fusion gene leukemia model. First, the following shRNA sequence targeting murine RBM15B was designed:

[0062] The forward sequence of shRNA-2:

[0063] 5'-GATCCCTAACTGTATGGAAAATTGTCAGTACTTCCTGTCAGATACTGACAATTTTCCATACAGTTAGTTTTTG-3' (SEQ ID NO: 12);

[0064] The reverse sequence of shRNA-2:

[0065] 5'-AATTCAAAAACTAACTGTATGGAAAATTGTCAGTATCTGACAGGAAGTACTGACAATTTTCCATACAGTTAGG-3' (SEQ ID NO: 13).

[0066] Using the expression vector pGreenPuro from System Biosciences TM A stable RBM15B knockdown cell line was constructed using the shRNA Cloning and Expression Vector lentiviral expression system. The successfully knocked-down MOLM-13RBM15B cell line was then injected via tail vein into 5-week-old NOD / SCID mice: a total of 35 mice were divided into three groups (PBS, sh-NC, and sh-RBM15B-2), with 5 mice in the PBS group, and 15 mice in each of the sh-NC and sh-RBM15B groups (150 μL per mouse, cell injection volume 1 × 10⁻⁶). 5 (Each animal). After two weeks, the tumor formation was assessed using flow cytometry. Figure 4As shown in Figure A, mice subcutaneously inoculated with sh-RBM15B exhibited lower levels of leukemia cell infiltration in the bone marrow, peripheral blood, spleen, and liver. Furthermore, statistical analysis revealed that the spleen size and weight of mice inoculated with sh-RBM15B were significantly lower than those in the NC group. Figure 4 B); furthermore, the differentiation degree of mouse leukemia cells inoculated with sh-RBM15B was significantly higher than that in the NC group (B); Figure 4 C). These results indicate that murine RBM15B also promotes the tumorigenesis progression of mouse MLL fusion gene leukemia and affects the growth of leukemia cells. Finally, further Log-rank (Mantel-Cox) test survival curve analysis revealed that the survival rate of mice with RBM15B knockdown was higher than that of the control group ( Figure 4 D) This further illustrates that specifically targeting highly expressed RBM15B is a potentially effective strategy for treating MLL fusion gene leukemia.

[0067] Example 3: RBM15B regulates the expression of key proto-oncogenes in leukemia caused by MLL fusion genes.

[0068] Both cellular and in vitro mouse experiments have demonstrated that RBM15B significantly affects the function of MLL fusion gene leukemia cells. So, how does RBM15B participate in the regulation of this type of disease? To investigate this question, this study used siRNA (siRNA-1 and siRNA-2) and shRNA (shRNA-1) interference techniques to inhibit or overexpress RBM15B in MOLM-13 cells, and then examined the protein expression levels of key proto-oncogenes in MLL fusion gene leukemia. The results showed that knocking down RBM15B in MLL fusion gene leukemia cell lines significantly reduced the protein levels of MYC, HOXA9, and BCL2; while overexpression of RBM15B (pCDH-CMV-MCS-EF1-Puro-copGFP plasmid, Addgene#72263) led to a significant increase in the protein levels of MYC, HOXA9, and BCL2. Figure 5 A and B) suggest that RBM15B regulates the expression of key proto-oncoproteins (MYC, HOXA9, BCL2) in MLL fusion gene leukemia. Finally, through polysome profiling experiments, it was verified in the MLL fusion gene leukemia cell lines MOLM-13 and MV4-11 that RBM15B mediates protein expression levels by regulating the translation efficiency of proto-oncogene transcripts. Figure 5 C).

[0069] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Application of reagents for detecting the expression level of RNA-binding protein RBM15B in the preparation of diagnostic products for MLL fusion gene leukemia.

2. The application according to claim 1, characterized in that, The reagent used to detect the expression level of the RNA-binding protein RBM15B is a primer for detecting the expression level of RBM15B, and its sequence is shown in SEQ ID NO:2-3.

3. The application of a reagent that inhibits or reduces the expression level of the RNA-binding protein RBM15B in the preparation of products for treating MLL fusion gene leukemia, characterized in that, The reagent that inhibits or reduces the expression level of the RNA-binding protein RBM15B is a siRNA or shRNA that inhibits or reduces the expression level of the RNA-binding protein RBM15B; the siRNA targeting human RBM15B is selected from one or both of siRNA-1 and siRNA-2, wherein the siRNA-1 sequence is shown in SEQ ID NO:6-7 and the siRNA-2 sequence is shown in SEQ ID NO:8-9; the shRNA targeting human RBM15B is selected from shRNA-1, wherein the shRNA-1 sequence is shown in SEQ ID NO:10-11; the shRNA targeting mouse RBM15B is selected from shRNA-2, wherein the shRNA-2 sequence is shown in SEQ ID NO:12-13.

4. The application according to claim 3, characterized in that, The inhibition or reduction of the expression level of RNA-binding protein RBM15B is to inhibit the occurrence and development of MLL fusion gene leukemia, or to inhibit the proliferation of MLL fusion gene leukemia cells, or to promote the differentiation of MLL fusion gene leukemia cells.

5. The application according to claim 4, characterized in that, The MLL fusion gene leukemia cells include MOLM-13 and MV4-11.

6. The application according to claim 4, characterized in that, The inhibition of MLL fusion gene leukemia development involves reducing the protein expression level of key proto-oncogenes in MLL fusion gene leukemia, including MYC, HOXA9, and BCL2.

7. The application according to claim 6, characterized in that, The reduction of protein expression levels of key proto-oncogenes in MLL fusion gene leukemia is intended to reduce the translation efficiency of key proto-oncogene transcripts in MLL fusion gene leukemia.

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