Application of RNA binding protein RBM15B in MLL fusion gene leukemia

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of RNA binding protein RBM15B in MLL fusion gene leukemia (MLL-R). A large number of leukemia patient samples are detected through the qRT-PCR technology, it is found that RBM15B in MLL fusion leukemia patients is remarkably and highly expressed, and the highly expressed RBM15B is remarkably and negatively correlated with the survival rate of the patients; furthermore, a mouse leukemia model is constructed through a mouse MLL-AF9 cell source and a human MLL fusion leukemia cell line for adult level verification, and it is found that the survival of MLL fusion gene leukemia cells can be remarkably inhibited by knocking down the expression of RBM15B, and the life cycle of a model mouse is remarkably prolonged. The result shows that RBM15B is expected to be used as a diagnostic marker and / or a therapeutic target to play a role in MLL fusion leukemia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to application of RNA binding protein RBM15B in MLL fusion gene leukemia. Background Art

[0002] MLL fusion gene leukemia, i.e. MLL (mixed-lineage leukemia) gene rearrangement leukemia (MLL-rearranged leukemia, MLL-R leukemia), is a type of malignant tumor characterized by blocked differentiation and malignant proliferation of hematopoietic stem / progenitor cells. Its incidence rate increases with age, and the incidence rate in men is often higher than that in women, while the total complete remission rate is only 30%. The pathogenesis of MLL fusion gene leukemia is complex, often caused by the combined action of multiple factors. The clinical symptoms are mainly fever, infection, bleeding, anemia, organ infiltration and metabolic abnormalities. If not treated in time, the bone marrow will rapidly fail and lead to death. The current treatment method is to induce remission with high-dose conventional chemotherapy first, and then carry out subsequent treatment after complete remission, so as to achieve the effect of strengthening and maintaining treatment. For some patients who are not suitable for chemotherapy, refractory or relapse prevention, hematopoietic stem cell transplantation or immunotherapy is used for treatment, but the prognosis and survival rate are still not very ideal. Therefore, it is necessary to adopt new methods to examine the biological characteristics of this type of leukemia and conduct in-depth research on the pathogenesis of acute leukemia, so as to find new early diagnosis and treatment methods to effectively improve the survival rate of patients with MLL fusion gene leukemia.

[0003] In recent years, RNA-binding proteins (RBPs) have been found to be involved in biological development and the pathogenesis of various cancers. RNA-binding proteins are key players in post-transcriptional events. They have the versatility and structural flexibility of RNA-binding domains, which enables RBPs to control the fate and metabolic processes of a large number of transcripts and become ideal targets for the development of small molecule drugs. RNA-binding proteins establish highly dynamic interactions with other proteins and coding and non-coding RNAs to produce functional units called ribonucleoprotein complexes (RNPs), which in turn regulate RNA splicing, polyadenylation, stability, localization, translation, and degradation. Therefore, deciphering the intricate interaction network between RBPs and a series of cancer-related target RNAs that they regulate will help advance the understanding of tumor biology and help reveal new targets for cancer treatment. The important regulatory role of RNA binding proteins in leukemia has also been gradually discovered. In different leukemia subtypes such as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), RNA binding proteins have specific expression patterns, suggesting that RNA binding proteins have important regulatory roles in different types of leukemia. Therefore, it is of great theoretical and practical significance to explore potential therapeutic targets for MLL fusion gene leukemia from RNA binding proteins, and it can also provide a positive reference for the application of RNA binding proteins in other leukemia cancers. Summary of the invention

[0004] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides, on the one hand, the use of RNA-binding protein RBM15B as a diagnostic marker in the preparation of MLL fusion gene leukemia diagnostic products, and on the other hand, provides the use of RNA-binding protein RBM15B as a therapeutic target in the preparation of drugs for the treatment of MLL fusion gene leukemia.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides the use of a reagent for detecting the expression level of RNA binding protein RBM15B in the preparation of a product for diagnosing MLL fusion gene leukemia.

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

[0008] The present invention has found through research that RBM15B is highly expressed in patients with MLL fusion gene leukemia. Interestingly, RBM15 has no significant difference, indicating that RBM15B has a unique role in MLL fusion gene leukemia. Further ROC curve analysis confirmed that RBM15B can significantly distinguish MLL fusion gene leukemia and normal human bone marrow samples; survival curve analysis showed that the five-year survival rate of patients with high expression of RBM15B was significantly reduced, indicating that RBM15B has a potential clinical role as a classifier and prognostic indicator for MLL fusion gene leukemia.

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

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

[0011] The second aspect of the present invention provides the use of an agent for inhibiting or reducing the expression level of RNA binding protein RBM15B in any of the following aspects 1)-4):

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

[0013] 2) Prepare products that inhibit the occurrence and development of MLL fusion gene leukemia;

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

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

[0016] The present invention has been confirmed through research that after knocking down RBM15B using siRNA technology, the proliferation of MLL fusion gene leukemia cell lines is weakened, the cycle is blocked, differentiation is increased, and the self-renewal ability is weakened. Moreover, the NOD-SCID mouse model experiment shows that after shRNA knocks down mouse RBM15B and human RBM15B, both mouse MLL fusion gene leukemia cells and human MLL fusion gene leukemia cell lines have the effect of inhibiting tumor growth. It shows that inhibiting the expression of RBM15B can inhibit 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 the occurrence and development of MLL fusion gene leukemia is to reduce the protein expression level of key oncogenes in MLL fusion gene leukemia, and the key oncogenes in MLL fusion gene leukemia include MYC, HOXA9, and BCL2.

[0019] More preferably, the reducing the protein expression level of the key oncogene in MLL fusion gene leukemia is reducing the translation efficiency of the key oncogene transcript in MLL fusion gene leukemia.

[0020] The present invention also found through research that in the MLL fusion gene leukemia cell line, after knocking down RBM15B, the translation efficiency of key cancer-promoting proteins such as MYC, HOXA9, and BCL2 was inhibited, and the protein level was significantly reduced, confirming that RBM15B has an inhibitory effect on the progression of MLL fusion gene leukemia.

[0021] The present invention regulates the expression level of RNA binding protein RBM15B by genetic engineering means to directly and significantly reduce the protein expression level of MYC, which is of great value for the precise treatment of MLL fusion gene leukemia. At the same time, it also confirms that RBM15B has potential clinical practical value in indicating the classification and prognosis of MLL fusion gene leukemia.

[0022] Preferably, the agent for inhibiting or reducing 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, 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, and the sequence of the shRNA-1 is shown in SEQ ID NO:10-11; the shRNA targeting mouse RBM15B is selected from shRNA-2, and the sequence of the shRNA-2 is shown in SEQ ID NO:12-13.

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

[0026] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[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 new pharmaceutical polymer materials, 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, granule, capsule, tablet.

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

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention detects a large number of leukemia patient samples through qRT-PCR technology, and finds that the RNA binding protein RBM15B is highly expressed in MLL fusion gene leukemia, and the highly expressed RBM15B is significantly negatively correlated with the patient's survival rate; experiments on two types of mouse leukemia models show that knocking down the expression of RBM15B can significantly inhibit the progression of MLL fusion gene leukemia and significantly prolong the survival period of model mice. It shows that RBM15B can play a role in MLL fusion gene leukemia as a diagnostic marker and / or therapeutic target, indicating that RBM15B is expected to be used to indicate the diagnosis and prognosis of this type of leukemia, as well as for the treatment of MLL fusion gene leukemia. In addition, the present invention also clarifies that RBM15B affects the progression of MLL fusion gene leukemia by regulating the expression level of proto-oncoproteins such as MYC, which can provide a new theoretical basis for the development of drug action targets for MLL fusion gene leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1To evaluate the expression and clinical significance of RNA binding protein RBM15B in MLL fusion gene leukemia; (A) qRT-PCR technology detected that RBM15B mRNA was specifically highly expressed in MLL fusion gene leukemia samples; while RBM15 did not change; (B) Log-rank (Mantel-Cox) Test survival curve was used to distinguish the high and low expression levels of RBM15B through the optimal cut-off point (0.001292), and high expression of RBM15B had a lower leukemia-free survival rate.

[0033] Figure 2 RNA binding protein RBM15B regulates the cytological function of MLL fusion gene leukemia; (A) qRT-PCR and Western Blot techniques were used to detect the knockdown effect of RBM15B in MOLM-13 cells; (B) qRT-PCR and Western Blot techniques were used to detect the knockdown effect of RBM15B in MOLM-13 cells. Blot technology was used to detect the knockdown effect of RBM15B in MV4-11 cells; (C) CCK-8 technology was used to detect that when RBM15B was knocked down, the proliferation level of the MLL fusion gene leukemia cell line was significantly reduced; (D) Flow cytometry technology was used to detect that when RBM15B was knocked down, the proportion of differentiated cells in the MLL fusion gene leukemia cell line was significantly increased; (E) Using cloning technology to detect that when RBM15B was knocked down, the self-renewal ability of MLL fusion gene leukemia cells was significantly weakened; (F) Flow cytometry technology was used to detect that when RBM15B was knocked down, the proportion of differentiated cells in the primary MLL fusion gene leukemia patient sample was increased; All figures are the mean ± standard deviation of three replicates, and asterisks indicate that the difference between the two groups is statistically significant when compared by t-test (*P<0.05; **P<0.01; ***P<0.001).

[0034] Figure 3 It is an animal model of RBM15B-regulated human cell line-derived MLL fusion gene leukemia; among them, (A) the mouse blood tumor model injected by tail vein combined with flow cytometry technology showed that in the group of MLL fusion gene leukemia cells inoculated with RBM15B knockdown, the tumor infiltration rate of various tissues and organs was significantly lower than that in the group inoculated with NC cells; (B) The mouse blood tumor model injected by tail vein combined with flow cytometry technology showed that in the bone marrow of mice inoculated with RBM15B knockdown cells, the degree of differentiation of tumor cells was significantly higher than that in the group inoculated with NC cells; (C) The survival curve was used to count the mice inoculated with MOLM-13 with RBM15B knockdown, and the survival period of the mice was significantly prolonged; the mean ± standard deviation of three repetitions was used in this figure, and the asterisk indicates that the difference between the two groups was statistically significant by t-test (*p<0.05, **, p<0.01; ***, p<0.001).

[0035] Figure 4 The animal model of MLL fusion gene leukemia derived from the Rbm15b-regulated mouse cell line (MLL-AF9 AML); (A) the mouse blood tumor model injected by tail vein combined with flow cytometry showed that the tumor infiltration rate of various tissues and organs in the group inoculated with MLL fusion gene leukemia cells with RBM15B knockdown was significantly lower than that in the group inoculated with NC cells; (B) the mouse blood tumor model injected by tail vein showed that the spleen size and weight in the MLL fusion gene leukemia cells with RBM15B knockdown were significantly lower than those in the NC cell group; (C) the mouse blood tumor model injected by tail vein combined with Flow cytometry showed that the degree of tumor cell differentiation in the group inoculated with MLL fusion gene leukemia cells with RBM15B knockdown was significantly higher than that in the group inoculated with NC cells; (D) The survival curve showed that the survival period of mice inoculated with MLL fusion gene leukemia cells with RBM15B knockdown was significantly prolonged; the mean ± standard deviation of three replicates was used in this figure, and the asterisk indicates that the difference between the two groups was 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 oncogenes in MLL fusion gene leukemia cells; (A) Western Blot experiments showed that the levels of MYC, HOXA9, and BCL2 were significantly reduced in MLL fusion gene leukemia cell lines with RBM15B knockdown; (B) Western Blot experiments showed that the levels of MYC, HOXA9, and BCL2 were significantly increased in MLL fusion gene leukemia cell lines with RBM15B overexpression; (C) Polysome profiling technology showed that the translation efficiency of MYC, HOXA9, and BCL2 was significantly weakened in MLL fusion gene leukemia cells with RBM15B knockdown; The figures all use the mean ± standard deviation of three replicates, and the asterisk indicates that the difference between the two groups is statistically significant by t-test (*p<0.05, **, p<0.01; ***, p<0.001). DETAILED DESCRIPTION

[0037] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0039] Example 1: RBM15B expression analysis and clinical value assessment

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

[0041] MKRQSERDSSPSGRGSSSSAKRPREREREAEAGGRRAAHKASGGAKHPVPARARDKPRGSGSGGGGHRDGRGTGDANHRASSGRSSGSGAGGGGRGGKASGDPGASGMSPRASPLPPPPPPPGAEPACPGSSAAAPEYKTLLISSLSPALPAEHLEDRLFHQFKRFGEISLRLSHTPELGRVAYVNFRHPQDAREARQHALARQLLLYDRPLKVEPVYLRGGGGSSRRSSSSSAAASTPPPGPPAPADPLGYLPLHGGYQYKQRSLSPVAAPPLREPRARHAAAAFALDAAAAAAVGLSRERALDYYGLYDDRGRPYGYPAVCEEDLMPEDDQRATRNLFIGNLDHSVSEVELRRAFEKYGIIEEVVIKRPARGQGGAYAFLKFQNLDMAHRAKVAMSGRVIGRNPIKIGYGKANPTTRLWVGGLGPNTSLAALAREFDRFGSIRTIDHVKGDSFAYIQYESLDAAQAACAKMRGFPLGGPDRRLRVDFAKAEETRYPQQYQPSPLPVHYELLTDGYTRHRNLDADLVRDRTPPHLLYSDRDRTFLEGDWTSPSKSSDRRNSLEGYSRSVRSRSGERWGADGDRGLPKPWEERRKRRSLSSDRGRTTHSPYEERSRTKGSGQQSERGSDRTPERSRKENHSSEGTKESSSNSLSNSRHGAEERGHHHHHHEAADSSHGKKARDSERNHRTTEAEPKPLEEPKHETKKLKNLSEYAQTLQLGWNGLLVLKNSCFPTSMHILEGDQGVISSLLKDHTSGSKLTQLKIAQRLRLDQPKLDEVTRRIKQGSPNGYAVLLATQATPSGLGTEGMPTVEPGLQRRLLRNLVSYLKQKQAAGVISLPVGGSKGRDGTGMLYAFPPCDFSQQYLQSALRTLGKLEEEHMVIVIVRDTA。

[0042] In order to further determine the expression specificity of RBM15B in MLL fusion gene leukemia, this example recollected a batch of patient bone marrow samples from the First Affiliated Hospital of Sun Yat-sen University for detection and analysis, including 163 MLL wild-type (MLL-wt) leukemia samples and 53 MLL-r leukemia samples. The collection of all samples was approved by the Ethics Committee of Sun Yat-sen University and the informed consent of the patients was obtained. RBM15B was specifically detected by extracting RNA and using qRT-PCR technology. The primer sequences of qRT-PCR used in this process are as follows:

[0043] Forward primer sequence for RBM15B: 5′-GAGAACCACTCCAGTGAAGGG-3′ (SEQ ID NO: 2);

[0044] Reverse primer sequence for RBM15B: 5′-GCTGACTGGAGGTACTGCTG-3′ (SEQ ID NO: 3);

[0045] Forward primer sequence of RBM15: 5′-TCCCACCTTGTGAGTTCTCC-3′ (SEQ ID NO: 4);

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

[0047] The results showed that RBM15B was significantly overexpressed in MLL-R leukemia samples compared with MLL wild-type leukemia samples (p<0.001), while RBM15 showed no significant difference ( Figure 1 A). Moreover, this example also found that normal samples have lower RBM15B expression levels, indicating that the expression level of RBM15B may be related to the malignancy of the disease. In order to further explore the clinical significance of RBM15B in MLL-r leukemia, this example then used the Log-rank (Mantel-Cox) Test survival curve to test 136 samples (bone marrow samples of MLL fusion gene leukemia patients, collected from the First Affiliated Hospital of Sun Yat-sen University, with informed consent from the patients) to find out whether RBM15B has an indicative effect on the prognosis of MLL-r leukemia. The experimental analysis results are as follows Figure 1 As shown in Figure B, it was found that high expression of RBM15B had a lower leukemia-free survival rate. The above results indicate that RBM15B has the potential to distinguish between MLL-r leukemia and MLL-wt leukemia, and high expression of RBM15B is also a risk factor in leukemia and a potential target for the treatment of leukemia.

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

[0049] In order to solve the core problem of RBM15B's involvement in the regulation of MLL fusion gene leukemia, this example intends to further study the function of RBM15B on MLL fusion gene leukemia. MLL fusion gene leukemia cell lines MOLM-13 and MV4-11 were selected as research objects. 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] 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] Reverse sequence of siRNA-2: 5'-ACACAAACAUGACGCUGGG dTd-3' (SEQ ID NO: 9).

[0054] In the above two MLL fusion gene leukemia cell lines, RBM15B was knocked out respectively, and then the cell proliferation was detected by CCK-8 experiment. First, qRT-PCR technology and Western Blot technology were used to detect the knockdown effect of RBM15B in MOLM-13 and MV4-11 cells ( Figure 2 A, B). The experimental results are as follows Figure 2 As shown in C, when RBM15B was knocked down, cell proliferation was significantly reduced in both MOLM-13 and MV4-11. This indicates that RBM15B plays an important role in maintaining the proliferation of MLL-r leukemia cells. At the same time, RBM15B was knocked down by siRNA interference technology, and cell differentiation was detected by flow cytometry. The experimental results are shown in Figure 2 As shown in D, when RBM15B was knocked down, the differentiation cell ratio of both MOLM-13 and MV4-11 increased significantly. The effect of RBM15B on the self-renewal ability of MLL fusion gene leukemia cells was further tested. Using cloning technology to detect the knockdown of RBM15B, the self-renewal ability of MLL fusion gene leukemia cells was significantly weakened ( Figure 2E); Flow cytometry technology was used to detect that when RBM15B was knocked down, the proportion of cell differentiation in primary MLL fusion gene leukemia patient samples increased ( Figure 2 F). Therefore, through cell proliferation, differentiation and clone formation experiments, it is speculated that RBM15B has a potential regulatory role in the occurrence and development of MLL fusion gene leukemia.

[0055] Next, the regulatory effect of human RBM15B on MLL fusion gene leukemia was further verified at the adult level. The shRNA sequence of RBM15B was designed using the above siRNA sequence.

[0056] Forward sequence of shRNA-1:

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

[0058] Reverse sequence of shRNA-1:

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

[0060] Using the expression vector pGreenPuro from System Biosciences TM The shRNA Cloning and Expression Vector lentiviral expression system was used to construct a stable expression strain that knocked down RBM15B, and the MOLM-13RBM15B knockdown cell line was obtained by puromycin selection. Subsequently, the validated stable MOLM-13RBM15B cell line was expanded and cultured, and then inoculated into 5-week-old NOD / SCID mice by tail vein injection: a total of 35 mice, divided into 3 groups (PBS, sh-NC, sh-RBM15B-1), 5 mice in the PBS group, 15 mice in each sh-NC and sh-RBM15B group (150 μL per mouse, the cell injection volume was 1×10 6 After 3 weeks, the tumor formation was detected by flow cytometry. Figure 3 As shown in A, mice inoculated subcutaneously with sh-RBM15B MOLM-13 had lower tumor infiltration in the bone marrow, peripheral blood, spleen, and liver; moreover, statistically, the degree of differentiation of tumor cells was higher than that of the NC group ( Figure 3B). These results indicate that RBM15B promotes the formation of MLL fusion gene leukemia tumors and affects the growth of such leukemia cells, indicating that RBM15B is expected to be a potential target for the treatment of such leukemia. Subsequently, further Log-rank (Mantel-Cox) Test survival curve analysis revealed that the survival rate of the RBM15B knockdown group 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 the treatment of MLL fusion gene leukemia.

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

[0062] Forward sequence of shRNA-2:

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

[0064] Reverse sequence of shRNA-2:

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

[0066] Using the expression vector pGreenPuro from System Biosciences TM shRNA Cloning and Expression Vector Lentivirus expression system was used to construct a stable expression cell line knocking down RBM15B. Subsequently, the MOLM-13RBM15B cell line that was successfully knocked down was injected into 5-week-old NOD / SCID mice through the tail vein: a total of 35 mice, divided into 3 groups (PBS, sh-NC, sh-RBM15B-2), 5 mice in the PBS group, 15 mice in each sh-NC and sh-RBM15B group (150 μL per mouse, the cell injection volume was 1×10 5 After 2 weeks, the tumor formation was detected by flow cytometry. Figure 4As shown in A, mice inoculated subcutaneously with sh-RBM15B had lower infiltration of leukemia cells in the bone marrow, peripheral blood, spleen, and liver. At the same time, statistics showed that the spleen size and weight of mice inoculated with sh-RBM15B were significantly lower than those in the NC group ( Figure 4 B); Moreover, the differentiation degree of leukemia cells in mice inoculated with sh-RBM15B was significantly higher than that in the NC group ( Figure 4 C). The above results indicate that mouse RBM15B also has the effect of promoting the progression of mouse MLL fusion gene leukemia tumorigenesis and affecting the growth of leukemia cells. Finally, further Log-rank (Mantel-Cox) Test survival curve analysis found that the survival rate of the RBM15B knockdown mouse group was higher than that of the control group ( Figure 4 D), further indicating that specifically targeting highly expressed RBM15B is an effective strategy for the potential treatment of MLL fusion gene leukemia.

[0067] Example 3: RBM15B regulates the expression of key oncogenes in MLL fusion gene leukemia

[0068] Both at the cellular level and in vitro mouse experiments have shown that RBM15B can significantly affect the function of MLL fusion gene leukemia cells. So, how does RBM15B participate in the regulation of this type of disease? In order to study this issue, this example uses siRNA (siRNA-1 and siRNA-2) and shRNA (shRNA-1) interference technology in MOLM-13 cells to inhibit the expression of RBM15B or overexpress RBM15B, and then detects the protein expression levels of key oncogenes in MLL fusion gene leukemia. The experimental results show that after knocking down RBM15B in the MLL fusion gene leukemia cell line, the protein levels of MYC, HOXA9, and BCL2 are significantly reduced; while overexpression of RBM15B (pCDH-CMV-MCS-EF1-Puro-copGFP plasmid, Addgene#72263) will cause the protein levels of MYC, HOXA9, and BCL2 to increase significantly ( Figure 5 A and B), suggesting that RBM15B regulates the expression of key oncoproteins (MYC, HOXA9, BCL2) in MLL fusion gene leukemia. Finally, through polysome profiling experiments, it was verified in MLL fusion gene leukemia cell lines MOLM-13 and MV4-11 that RBM15B mediates its protein expression level by regulating the translation efficiency of oncogene transcripts ( Figure 5 C).

[0069] The embodiments of the present invention are 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 of these embodiments are made without departing from the principles and spirit of the present invention, and 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 MLL fusion gene leukemia diagnostic products.

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

3. Use of an agent for inhibiting or reducing the expression level of RNA binding protein RBM15B in any of the following aspects 1)-4): 1) Preparation of products for the treatment of MLL fusion gene leukemia; 2) Prepare products that inhibit the occurrence and development of MLL fusion gene leukemia; 3) Preparation of products that inhibit the proliferation of MLL fusion gene leukemia cells; 4) Prepare products that promote differentiation of MLL fusion gene leukemia cells.

4. The use according to claim 3, characterized in that: The MLL fusion gene leukemia cells include MOLM-13 and MV4-11.

5. The use according to claim 3, characterized in that: The method of inhibiting the occurrence and development of MLL fusion gene leukemia is to reduce the protein expression level of key oncogenes in MLL fusion gene leukemia, and the key oncogenes in MLL fusion gene leukemia include MYC, HOXA9, and BCL2.

6. The use according to claim 5, characterized in that: The method of reducing the protein expression level of the key oncogene in MLL fusion gene leukemia is to reduce the translation efficiency of the key oncogene transcript in MLL fusion gene leukemia.

7. The use according to claim 3, characterized in that: The reagent for inhibiting or reducing the expression level of RNA binding protein RBM15B is siRNA or shRNA that inhibits or reduces the expression level of RNA binding protein RBM15B.

8. The use according to claim 7, characterized in that: The siRNA targeting human RBM15B is selected from one or more of siRNA-1 or siRNA-2, 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.

9. The use according to claim 7, characterized in that: The shRNA targeting human RBM15B is selected from shRNA-1, and the sequence of the shRNA-1 is shown in SEQ ID NO:10-11; the shRNA targeting mouse RBM15B is selected from shRNA-2, and the sequence of the shRNA-2 is shown in SEQ ID NO:12-13.

10. A therapeutic drug for MLL fusion gene leukemia, characterized in that: The drug includes an agent that inhibits or reduces the expression level of RNA binding protein RBM15B.

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