Genes for treating acute myeloid leukemia and uses thereof

Abstract

The application relates to the technical field of medicines, and discloses application of an lncRNA AC098613.1 gene in preparation of a medicine for treating leukemia, and provides an LncRNA AC098613.1 gene for treating acute myeloid leukemia, an expression vector of the AC098613.1 gene, and an AC098613.1 gene overexpression cell strain. The application also provides application of a medicine for treating acute myeloid leukemia, and the medicine comprises an overexpression plasmid or a small-molecule compound capable of enhancing expression of the lncRNA AC098613.1, wherein the AC098613.1 is a long-chain non-coding RNA located on chromosome 3, the AC098613.1 overexpression is applied by inhibiting proliferation of acute myeloid leukemia cells, the AC098613.1 overexpression is applied by maintaining CDC5L stability, affecting ADAP1-NRD1-CDK1, the AC098613.1 overexpression is applied by inducing cell cycle arrest of acute myeloid leukemia cells, and the AC098613.1 overexpression is applied by inducing differentiation of acute myeloid leukemia cells.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of the lncRNA AC098613.1 gene in the preparation of drugs for treating leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the clonal proliferation of abnormally differentiated or undifferentiated myeloid cells in the bone marrow and peripheral blood. Despite recent significant advancements in AML treatment, long-term survival remains low due to drug resistance in most patients after chemotherapy and a high relapse rate. Finding new therapeutic targets to improve the clinical outcomes of AML treatment remains a pressing issue. With the deepening of molecular biology research, studies have revealed that the development and progression of leukemia are related to the abnormal expression, activation, and mutation of molecules in the body. A comprehensive understanding of the functions and regulatory mechanisms of these related molecules can fundamentally elucidate the molecular mechanisms of AML and provide more effective targets for its treatment.

[0003] Long non-coding RNAs (lncRNAs) play a crucial role in gene expression regulation, and their abnormal expression is associated with various diseases. They can act as proto-oncogenes or tumor suppressor genes involved in tumorigenesis and development. A growing number of researchers are finding that regulating gene expression is a viable approach to cancer treatment. Studies have found that AC098613.1 can serve as a prognostic marker for bladder urothelial carcinoma, but its application in leukemia has not been studied, and it has not been shown to inhibit leukemia progression.

[0004] Based on the above analysis, the problems and shortcomings of the existing technology are as follows:

[0005] (1) Currently, most patients develop drug resistance after chemotherapy and have a high relapse rate.

[0006] (2) Currently, AC098613.1 has not been studied in leukemia, and there is no application for inhibiting the progression of leukemia. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides the application of the LncRNA AC098613.1 gene in the preparation of drugs for treating acute myeloid leukemia.

[0008] The present invention is achieved as follows: a LncRNA AC098613.1 gene for the treatment of acute myeloid leukemia.

[0009] Another object of the present invention is to provide an expression vector for the AC098613.1 gene.

[0010] Another objective of this invention is to provide a cell line overexpressing the AC098613.1 gene.

[0011] Another object of the present invention is to provide an application of preparing a drug for treating acute myeloid leukemia, characterized in that: the drug comprises an overexpression plasmid or small molecule compound capable of enhancing the expression of lncRNA AC098613.1.

[0012] Another object of the present invention is to provide a specific antibody against the AC098613.1 gene prepared by immunoassay.

[0013] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0014] First, differentiation-inducing therapy for leukemia has become a highly regarded treatment strategy in recent years, with highly effective and low-toxicity differentiation-inducing agents being the core of this strategy. The success of all-trans retinoic acid in the clinical treatment of acute promyelocytic leukemia has greatly promoted research on differentiation-inducing therapy for leukemia. Phorbol12-myristate 13-acetate (PMA) has been shown to induce AML cell differentiation. U937, HL-60, and THP-1 cells treated with PMA all exhibited differentiation phenotypes such as growth arrest, enhanced NBT reducing capacity, enhanced cell adhesion, and increased expression of the differentiation marker protein CD11B. In this study, we used whole-transcriptome sequencing to analyze the lncRNA expression profiles in ATRA and PMA-treated THP-1 cells. By screening for differentially expressed lncRNAs shared by both groups of cells, we aimed to discover lncRNAs involved in regulating AML cell differentiation and to find new targets for differentiation therapy of AML. This invention further analyzes the regulatory role of lncRNA in cell differentiation and the molecular mechanisms by which it exerts this regulatory role using techniques such as RNA pull-down, mass spectrometry, and gene knockdown / overexpression. To achieve the above research objectives, this invention is implemented as follows:

[0015] THP-1 cell differentiation was induced using ATRA and PMA. Transcriptome sequencing was used to screen for significantly differentially expressed (|log2(fold change)|>2.0, P<0.05) lncRNAs with consistent trends between the two treatment groups. Based on factors such as expression level, length, location, and degree of differential expression, lncRNAs were screened, and the expression of target lncRNAs was verified by qRT-PCR. AC098613.1, associated with AML cell differentiation, was selected.

[0016] The tetracycline (Dox)-induced lentiviral overexpression vector plvx-Teton-puro-AC098613.1 / plvx-Teton-puro-NC was designed and synthesized, and transfected into THP-1 / HL-60 cells. Stable cell lines were selected using puromycin. Subsequently, the effects of AC098613.1 high expression on differentiation indicators such as cell proliferation activity, NBT reduction capacity, CD11b expression level, and cell morphology were analyzed using CCK8 assays, NBT reduction assays, Western blot, and microscopic observation, demonstrating that high expression of AC098613.1 can promote AML cell differentiation.

[0017] The target proteins of AC098613.1 were analyzed by combining RNA pull-down and LC-MS / MS. The target protein CDC5L was evaluated and screened based on indicators such as protein confidence (FDR), abundances, and score sequestration (HT). The binding sites of AC098613.1 and CDC5L were predicted by molecular docking, and AC098613.1-mut was synthesized by site-directed mutagenesis of the binding sites. Through RNA pull-down, western blot, and fluorescence microscopy co-localization, it was found that AC098613.1 can target and bind to CDC5L, while AC098613.1-mut cannot bind. The lentiviral overexpression vector plvx-Teton-puro-AC098613.1-mut was designed and synthesized, and transfected into THP-1 / HL-60 cells to construct a stable THP-1 / HL-60 cell line overexpressing AC098613.1-mut. Through CCK8 assay, NBT reduction assay, Western blot, and microscopic observation, it was found that AC098613.1-mut overexpression did not affect the differentiation indicators of AML cells, thus proving that AC098613.1 regulates AML cell differentiation by binding to CDC5L.

[0018] High expression of AC098613.1 significantly increased CDC5L protein levels in cells, while high expression of AC098613.1-mut had no significant effect on CDC5L protein levels. This invention demonstrated that high expression of AC098613.1 significantly enhanced the stability of CDC5L protein in THP-1 cells by treating THP-1 cells with the protein synthesis inhibitor CHX; and further demonstrated that AC098613.1 significantly reduced CDC5L protein degradation by inhibiting the ubiquitin-proteasome system (UPS) and autophagy-lysosome pathways using protein degradation inhibitors. Simultaneously, the distribution level of CDC5L in the cell nucleus was found to be significantly increased. This invention further demonstrates that AC098613.1 positively regulates ADAP1 expression by enhancing CDC5L stability. A siRNA (si-ADAP1) for ADAP1 knockdown expression was designed and synthesized, and transfected into AC098613.1 stably transfected THP-1 / HL-60 cells. ADAP1 knockdown expression significantly inhibited cell differentiation, demonstrating that ADAP1 positively regulates AML cell differentiation.

[0019] In human neuroblastoma cells, highly expressed ADAP1 promotes cell differentiation by positively regulating the expression of nardilysin (NRD1).

[27] However, the regulation of NRD1 on leukemia cell differentiation has not been reported to date. In AC098613.1 cells stably transfected with THP-1 and HL-60, NRD1 expression was significantly upregulated, CDK1 expression was significantly downregulated, and significant G0 / G1 cell cycle arrest occurred. Transfection with si-ADAP1 significantly reduced NRD1 expression and increased CDK1 expression in AC098613.1 cells stably transfected with THP-1 and HL-60, and significantly alleviated cell cycle arrest. Based on these results, this invention demonstrates that ADAP1 positively regulates NRD1 expression, induces G0 / G1 cell cycle arrest, and thus affects cell differentiation. si-NRD1 was synthesized, and transfection of AC098613.1 cells stably transfected with THP-1 significantly reduced NRD1 expression, which significantly inhibited cell differentiation, demonstrating that NRD1 positively regulates AML cell differentiation.

[0020] A nude mouse model of HL-60 tumor-bearing cells stably transfected with AC098613.1 was constructed. In vivo, it was demonstrated that AC098613.1 can induce AML cell differentiation by affecting CDC5L stability and regulating ADAP1-NRD1-CDK1. The role of AC098613.1 as a tumor suppressor gene in AML was further evaluated by recruiting newly diagnosed AML patients (M4 stage) and patients with non-hematologic malignancies.

[0021] Second, in view of the problems existing in the prior art, the present invention provides an application of the LncRNA AC098613.1 gene in the preparation of a drug for treating acute myeloid leukemia.

[0022] Furthermore, the drug comprises an overexpression plasmid or small molecule compound capable of enhancing the expression of AC098613.1.

[0023] Furthermore, AC098613.1 is a long non-coding RNA located on chromosome 3.

[0024] Furthermore, the overexpression of AC098613.1 is applied by inhibiting the proliferation of acute myeloid leukemia cells.

[0025] Furthermore, the overexpression of AC098613.1 affects the application of ADAP1-NRD1-CDK1 by maintaining the stability of CDC5L.

[0026] Furthermore, the overexpression of AC098613.1 is applied through inducing cell cycle arrest in acute myeloid leukemia.

[0027] Furthermore, the overexpression of AC098613.1 is applied to induce differentiation of acute myeloid leukemia cells.

[0028] Third, the technical solution of this invention fills a technological gap in the domestic and international industry: the abnormal expression of the long non-coding RNA AC098613.1 has been found to be used for risk assessment of the prognosis of bladder urothelial carcinoma. However, the expression and function of AC098613.1 in acute myeloid leukemia (AML) are still unclear. In this invention, we found that the expression of AC098613.1 was significantly upregulated in THP-1 cells treated with PMA or ATRA. Functional studies showed that, in both in vitro and in vivo conditions, the upregulation of AC098613.1 expression inhibited AML cell proliferation and induced cell differentiation. Mechanistic studies revealed that AC098613.1 enhances CDC5L stability by targeting and binding to CDC5L, thereby increasing the intracellular level of CDC5L and its distribution in the cell nucleus. CDC5L is a transcriptional regulator. High levels of CDC5L can activate ADAP1 transcription, thereby positively regulating nardilysin (NRD1) expression and affecting NRD1's transcriptional regulation of the cell cycle regulator CDK1, thus influencing cell cycle progression and promoting cell differentiation. Knockdown of both ADAP1 and NRD1 effectively inhibited cell cycle arrest and differentiation induced by AC098613.1 overexpression, demonstrating that AC098613.1 regulates AML cell cycle progression and differentiation through the ADAP1-NRD1-CDK1 signaling pathway. This invention reveals for the first time the regulatory role and molecular mechanism of AC098613.1 in AML cell differentiation, providing a theoretical basis for identifying therapeutic targets for AML differentiation therapy.

[0029] Leukemia differentiation-inducing therapy has become a highly regarded treatment strategy in recent years, with highly effective and low-toxicity differentiation-inducing agents being the core of this strategy. This invention reveals that AC098613.1 overexpression induces AML cell differentiation, and provides the application of AC098613.1 as a target gene in the preparation or screening of AML differentiation-inducing therapeutic drugs or biological reagents. These drugs contain overexpression plasmids or small molecule compounds capable of enhancing AC098613.1 expression. Furthermore, in this invention, AC098613.1 acts as a target-binding molecule of CDC5L, positively regulating CDC5L stability. ADAP1, as a downstream regulator of CDC5L, is regulated by CDC5L. This invention discovers and analyzes the influence of the ADAP1-NRD1-CDK1 signaling pathway on AML cell differentiation, providing new insights for the development of multi-target (AC098613.1, ADAP1, and NRD1) AML differentiation-inducing therapeutic drugs and improving drug resistance issues encountered during treatment. Attached Figure Description

[0030] Figure 1Expression and functional analysis of AC098613.1. AC098613.1 was highly expressed in ATRA and PMA-treated THP-1 cells through whole-transcriptome sequencing. Tetracycline-induced AC098613.1 overexpression was used to construct stable THP-1 and HL-60 cells, and the effect of AC098613.1 on cell differentiation was analyzed. A: Heatmap, differentially expressed lncRNAs in ATRA and PMA-treated THP-1 cells; B: Venn diagram, 50 differentially expressed lncRNAs in ATRA and PMA-treated THP-1 cells; C: qRT-PCR analysis of AC098613.1 expression in ATRA and PMA-treated THP-1 cells; D: qRT-PCR detection of AC098613.1 expression level in stable THP-1 and HL-60 cells; E: CCK-8 assay of cell proliferation activity, cells with high AC098613.1 expression showed significantly higher proliferation activity. F: NBT reduction assay analysis showed that AC098613.1 significantly enhanced the NBT reduction capacity of cells; G: CD11b expression analysis showed that CD11b expression was significantly increased in AC098613.1-stably converted to THP-1 and HL-60 cells; H: Statistical analysis of CD11b expression levels; I, J: Cell morphology observation showed that AC098613.1-stably converted to THP-1 and HL-60 cells had increased volume and decreased nucleocytoplasmic ratio; K, L: Cell localization of AC098613.1, mainly distributed in the cytoplasm. n=3; **P<0.01.

[0031] Figure 2 Screening of target proteins for AC098613.1. Pull-down assays were performed using biotin-labeled AC098613.1 probes. A: SDS-PAGE analysis of the pulled-down protein samples; B: Mass spectrometry analysis of the pulled-down protein samples, using indicators such as FDR < 0.01, Score Sequest HT > 50, and Abundances > 335,000 to screen for proteins specific to the AC098613.1 pull-down group; C: Western blot analysis of CDC5L protein abundance in the pulled-down samples; the abundance of CDC5L in the AC098613.1 pull-down group was significantly higher than that in the control group; D, E: Immunofluorescence co-localization; AC098613.1 and CDC5L co-localized in THP-1 and HL-60 cells.

[0032] Figure 3Functional analysis of the target protein AC098613.1. Molecular docking was used to predict the binding site of AC098613.1 and CDC5L. Based on site-directed mutagenesis of the AC098613.1 binding site, the role of targeted binding of AC098613.1 and CDC5L in AML cell differentiation was analyzed. A: AC098613.1 model constructed using RNA Composer; B: Schematic diagram and electrostatic surface map of AC098613.1 binding to CDC5L; red ribbons represent CDC5L, brown sticks represent AC098613.1, and yellow dashed lines in the magnified image represent hydrogen bonds or salt bridges; C: Binding site of AC098613.1 and CDC5L; D: Pull-down experiments were performed using a biotin-labeled AC098613.1-mut probe. Western blot analysis was used to analyze the abundance of CDC5L in the pull-down samples. E: No CDC5L was detected in the pull-down sample; E: The AC098613.1-mut overexpression vector pcDNA3.1(+)-AC098613.1-mut was constructed and transfected into THP-1 and HL-60 cells. The expression level of AC098613.1-mut in the cells was detected by qRT-PCR; F: Effect of AC098613.1-mut on cell proliferation activity. The proliferation activity of cells with high expression of AC098613.1-mut was not significantly changed; G: A Effects of AC098613.1-mut on NBT reduction capacity: Cells with high AC098613.1-mut expression showed no significant change in NBT reduction capacity; H, I: Effects of AC098613.1-mut on CD11b expression: Cells with high AC098613.1-mut expression showed no significant change in CD11b expression; J: Effects of AC098613.1-mut on cell morphology: Cells with high AC098613.1-mut expression showed no significant change in morphology. n=3; **P<0.01.

[0033] Figure 4Effects of AC098613.1 on CDC5L stability and nuclear distribution. THP-1 cells were treated with protein synthesis inhibitors (CHX) and protein degradation inhibitors (MG132 and NH4Cl) to observe the effect of AC098613.1 on CDC5L degradation. A, B: Western blot analysis of CDC5L protein levels in THP-1 cells with high expression of AC098613.1 or AC098613.1-mut; C, D: THP-1 cells with high expression of AC098613.1 or AC098613.1-mut were treated with CHX (50 μg / mL) for 0, 2, 4, and 6 h, and then Western blot analysis of CDC5L protein levels; E, F: THP-1 cells with AC098613.1 knockdown expression were treated with MG132 (10 μM / L) and NH4Cl (10 mM / L), and then Western blot analysis of CDC5L protein levels. Blot analysis of CDC5L protein levels in cells; G, H: Cytoplasmic and nuclear proteins were extracted from AC098613.1 or AC098613.1-mut overexpressing THP-1 cells, respectively, and Western blot analysis was performed to analyze the abundance of CDC5L in each protein sample. The content of CDC5L in both cytoplasmic and nuclear proteins overexpressed by AC098613.1 was significantly upregulated; I: Immunofluorescence co-localization was used to observe the effect of AC098613.1 or AC098613.1-mut overexpression on CDC5L expression and cell distribution in THP-1 cells. The content of CDC5L was significantly increased in AC098613.1 overexpressing cells, and the distribution of the protein in the cell nucleus was also significantly increased. n=3; *P<0.01, **P<0.01.

[0034] Figure 5The regulatory role of CDC5L in ADAP1 expression. Differentially expressed mRNAs shared by ATRA and PMA-treated THP-1 cells were obtained through transcriptome sequencing. Combined with CDC5L chip-seq results, shared genes were further screened, and the regulatory role of CDC5L in ADAP1 expression was analyzed. A: Heatmap, differentially expressed mRNAs in ATRA and PMA-treated THP-1 cells; B: Venn diagram, 209 differentially expressed mRNAs shared by ATRA and PMA-treated THP-1 cells; C: Venn diagram, two target genes were screened from the shared differentially expressed mRNAs using CDC5L chip-seq; D: qRT-PCR analysis of ADAP1 mRNA expression levels in cells with high expression of AC098613.1 or AC098613.1-mut; E, F: Western blotting. Blot analysis was performed on the protein expression level of ADAP1 in AC098613.1 or AC098613.1-mut cells with high expression. G: AC098613.1 cells stably transfected with si-CDC5L were transfected into THP-1 cells, and qRT-PCR was used to analyze the expression level of CDC5L in the cells. Si-CDC5L transfection significantly reduced the expression level of CDC5L in the cells. H: AC098613.1 cells stably transfected with si-CDC5L were transfected into THP-1 cells, and qRT-PCR was used to analyze the expression level of ADAP1 in the cells. Si-CDC5L transfection significantly inhibited the increase in ADAP1 mRNA expression level caused by AC098613.1 high expression. I, J: AC098613.1 cells stably transfected with si-CDC5L were transfected into THP-1 cells, and Western blot analysis was performed on the expression level of ADAP1 in the cells. Si-CDC5L transfection significantly inhibited the increase in ADAP1 protein expression level caused by AC098613.1 high expression. n = 3; *P < 0.05; **P < 0.01.

[0035] Figure 6The regulatory role of ADAP1 in AML cell differentiation. A: Three ADAP1 siRNAs were designed and synthesized. After transfecting AC098613.1 stably transfected THP-1 and HL-60 cells, the expression level of ADAP1 in the cells was analyzed by qRT-PCR. All three siRNAs significantly reduced the expression of ADAP1 in the cells. si-ADAP1-2, which had the highest knockdown rate, was selected and transfected into AC098613.1 stably transfected THP-1 and HL-60 cells. B: CCK-8 assay was used to analyze the effect of ADAP1 knockdown on cell proliferation activity. ADAP1 knockdown significantly inhibited the cell proliferation inhibition caused by AC098613.1 overexpression and significantly improved cell proliferation activity. C, D: Western blot analysis was used to analyze the effect of ADAP1 knockdown on CD11b expression in the cells. ADAP1 knockdown significantly inhibited the increase in CD11b expression caused by AC098613.1 overexpression. E: Cell morphology was observed under a microscope. ADAP1 knockdown significantly inhibited the cell morphological changes caused by AC098613.1 overexpression. n = 3; *P < 0.05; **P < 0.01.

[0036] Figure 7 The regulatory role of ADAP1 in AML cell cycle progression. A, B: Western blot analysis of the effect of ADAP1 knockdown on NRD1 protein levels. ADAP1 knockdown significantly inhibited the increase in NRD1 expression induced by AC098613.1 overexpression and significantly reduced the level of NRD1 expression in cells. C, D: Western blot analysis of the effect of ADAP1 knockdown on CDK1 protein levels. ADAP1 knockdown significantly inhibited the decrease in CDK1 expression induced by AC098613.1 overexpression and significantly increased the level of CDK1 expression in cells. E, F: Flow cytometry analysis of cell cycle. ADAP1 knockdown significantly inhibited G0 / G1 cell cycle arrest induced by AC098613.1 overexpression. n=3; *P<0.05; **P<0.01.

[0037] Figure 8The regulatory role of NRD1 in AML cell differentiation. A: Three NRD1 siRNAs were designed and synthesized. After transfecting AC098613.1 cells stably transfected with THP-1, qRT-PCR analysis showed that the expression level of NRD1 in the cells was significantly reduced by all three siRNAs. si-NRD1-2, which had the highest knockdown rate, was selected and transfected into AC098613.1 cells stably transfected with THP-1. B: CCK-8 assay showed that NRD1 knockdown significantly inhibited the cell proliferation inhibition caused by AC098613.1 high expression and significantly improved cell proliferation activity. C: NBT reduction assay showed that NRD1 knockdown significantly inhibited the increase in NBT reduction capacity caused by AC098613.1 high expression. D, E: Western blotting... Blot analysis of CD11b expression in cells showed that NRD1 knockdown significantly inhibited the increase in CD11b expression induced by AC098613.1 overexpression. F: Microscopic observation of cell morphology showed that NRD1 knockdown significantly inhibited the morphological changes induced by AC098613.1 overexpression. n=3; *P<0.05; **P<0.01.

[0038] Figure 9 Effect of AC098613.1 on HL-60 cell differentiation in vivo. A nude mouse model of AC098613.1 stably converted to HL-60 cells was constructed. Mice were treated with Dox (10 mg / kg) intraperitoneally for 2 weeks, after which the mice were sacrificed and tumors were harvested. A: Photographs of the tumors: HL-60 cell tumor (Model), NC stably converted to HL-60 cells tumor (NC), AC098613.1 stably converted to HL-60 cells without Dox induction (Saline), and Dox-induced AC098613.1 stably converted to HL-60 cells tumor (DOX); B: Tumor volume (long axis × short axis). 2 C: Tumor weight; D: Ki-67 immunohistochemical analysis in tumor cells; E: CD11b immunohistochemical analysis in tumor cells; F: Expression analysis of AC098613.1 in tumor cells; G, H: Expression analysis of CDC5L, ADAP1, NRD1, CDK1 and CD11b in tumor cells. n=6; *P<0.05; **P<0.01.

[0039] Figure 10Expression levels of AC098613.1 and related proteins in AML patients. Bone marrow mononuclear cells were isolated from patients with non-malignant hematological diseases and AML patients, and the expression levels of AC098613.1, CDC5L, ADAP1, and NRD1 in the cells were detected. A: qRT-PCR analysis of AC098613.1 expression level; B: qRT-PCR analysis of ADAP1 expression level; C: qRT-PCR analysis of NRD1 expression level; D, E: Western blot analysis of CDC5L, ADAP1, and NRD1 expression levels. n=3; *P<0.05; **P<0.01. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The technical solutions in the embodiments of this invention will be clearly and completely described and discussed below with reference to the accompanying drawings. Obviously, what is described here is only a part of the examples of this invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the protection scope of this invention.

[0041] To facilitate understanding of the embodiments of the present invention, further explanations and descriptions will be provided below with reference to the accompanying drawings and specific embodiments, and these embodiments do not constitute a limitation on the embodiments of the present invention.

[0042] Aberrant expression of AC098613.1 has been found to be useful for risk assessment in the prognosis of bladder urothelial carcinoma. However, the expression and function of AC098613.1 in acute myeloid leukemia (AML) remain unclear, and there are no targeted drugs associated with AC098613.1. In this invention, we found that AC098613.1 expression was significantly upregulated in THP-1 cells treated with PMA or ATRA. Functional studies revealed that upregulation of AC098613.1 expression inhibited AML cell proliferation and induced cell differentiation in both in vitro and in vivo conditions. Mechanistic studies showed that AC098613.1 enhances CDC5L stability by targeting and binding to CDC5L, increasing intracellular CDC5L levels and its distribution in the nucleus. High levels of CDC5L can activate ADAP1 transcription, thereby positively regulating nardilysin (NRD1) expression, affecting NRD1's transcriptional regulation of the cell cycle regulator CDK1, and thus influencing cell cycle progression and promoting cell differentiation. Both ADAP1 and NRD1 knockdown effectively inhibited cell cycle arrest and differentiation induced by AC098613.1 overexpression, demonstrating that AC098613.1 regulates AML cell cycle progression and differentiation by modulating the ADAP1-NRD1-CDK1 signaling pathway. This invention identifies AC098613.1 as a target for differentiation-inducing therapy in acute myeloid leukemia (AML), providing a new approach for the development of clinical therapeutic targets for AML. Furthermore, this invention discovers that cell cycle progression and differentiation in leukemia cells can be controlled by regulating the ADAP1-NRD1-CDK1 signaling pathway.

[0043] Example 1: Expression and Functional Analysis of AC098613.1

[0044] 1. Experimental Design:

[0045] (I) Analysis of differentially expressed lncRNAs in THP-1 cells treated with ATRA and PMA

[0046] (1) Transcriptome sequencing analysis of THP-1 cells: THP-1 cells in the logarithmic growth phase were cultured under normal conditions for 24 h and divided into three groups: ① Control group: blank control group, THP-1 cells cultured in RPMI-1640; ② ATRA group: THP-1 cells cultured in RPMI-1640 containing different concentrations of ATRA; ③ PMA group: THP-1 cells cultured in RPMI-1640 containing different concentrations of PMA. After culturing the cells in the above three groups for 96 h, differentiation indicators such as cell morphology, proliferation activity, NBT reduction capacity and CD11b expression level were analyzed, and transcriptome sequencing was performed.

[0047] (2) Screening and verification of lncRNAs: Screening for differentially expressed lncRNAs in cells treated with ATRA and PMA, verifying the expression of lncRNAs by qRT-PCR, selecting lncRNAs consistent with the transcriptome sequencing results, and further screening for target lncRNAs based on factors such as expression level, length and degree of differential expression.

[0048] (II) Analysis of the regulatory role of AC098613.1 in the differentiation of THP-1 / HL-60 cells.

[0049] AC098613.1 / NC cells stably transformed into THP-1 / HL-60 cells in the logarithmic growth phase were harvested at a rate of 8 × 10⁻⁶. 3 / wells were seeded in 96-well plates and cultured statically in RPMI-1640 medium for 24 h. Then, 400 ng / ml Dox was added. The experiment was divided into two groups: ① vector group: negative control group, NC cells stably transfected with THP-1 / HL-60 cells; ② AC098613.1 group: AC098613.1 cells stably transfected with THP-1 / HL-60 cells. Each group was set up with 3 replicates. After culturing for 96 h, cell differentiation indicators were detected to analyze the effect of AC098613.1 on the differentiation of THP-1 / HL-60 cells.

[0050] 2. Experimental methods:

[0051] 2.1 THP-1 Cell Culture: THP-1 cells were suspension cells obtained from passaged cells in our laboratory. The complete culture medium used for cell culture was RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-dextrose antibody. Cells were seeded into T25 cell flasks and incubated statically at 37°C in an incubator containing 5% CO2.

[0052] 2.2 CCK-8 assay for THP-1 cell proliferation activity: THP-1 cells in logarithmic growth phase were collected and divided into 8 × 10⁸ cells per well. 3 Cells were seeded into 96-well plates and incubated statically for 24 hours. Different concentrations of ATRA (4, 8, 16, 32 μM) and PMA (25, 50, 75, 100 ng / mL) were added to the culture medium. After another 96 hours of incubation, 10 μL of CCK-8 solution was added, and the plates were incubated statically until the solution turned orange-yellow. The optical density (OD) was then measured at 450 nm. 450 nm Calculate cell viability. Cell viability = (Experimental wells - Blank wells) / (Control wells - Blank wells)

[0053] 2.3 Microscopic observation of THP-1 cell morphology: THP-1 cells in the logarithmic growth phase were taken and divided into 5 × 10⁶ cells per well. 5Cells were seeded into 6-well plates and incubated statically for 24 hours. Then, 8 μM ATRA or 25 ng / mL PMA was added to the culture medium. After culturing for another 96 hours, cell morphology was observed and photographed under an inverted microscope.

[0054] 2.4 NBT Reduction Assay to Detect the NBT Reduction Capacity of Cells: THP-1 cells in logarithmic growth phase were used, and the cells were divided into groups of 8 × 10⁸ cells per well. 3 Cells were seeded in 96-well plates and incubated statically for 24 hours. NBT (1 mg / mL) and TPA (20 ng / mL) were added to the culture medium. After incubation at 37°C for 30 minutes, the cells were collected by centrifugation, washed, and resuspended in 1×PBS. The OD value was measured at 570 nm, and the NBT reducing capacity of the cells was calculated.

[0055] 2.5 Western blot analysis of CD11b expression levels in cells: THP-1 cells in logarithmic growth phase were used, and 5 × 10⁶ cells were extracted per well. 5 Cells were seeded into 6-well plates and incubated statically for 24 h. Then, 8 μL of MATRA or 25 ng / mL PMA was added to the culture medium, and the cells were cultured for another 96 h before collection. Cells were washed with pre-cooled 1×PBS, and 200 μL of RIPA protein lysis buffer was added. The mixture was gently vortexed at 4°C for 30 min, centrifuged, and the supernatant was collected. 5×SDS-PAGE protein loading buffer was added at a 1:4 ratio, and the mixture was boiled at 100°C for 20 min. After the protein samples cooled to room temperature, SDS-PAGE electrophoresis was performed. The samples were transferred to nitrocellulose membranes and blocked in 5% skim milk powder at room temperature for 2 h. Primary antibody was added and incubated overnight at 4°C. After washing with 1×TBST, secondary antibody was added and incubated at room temperature for 2 h. After washing with 1×TBST, ECL developing solution was added, and the protein bands were observed using a FluorChem FC3 System and ImageJ.

[0056] 2.6 Whole transcriptome sequencing and result analysis: THP-1 cells in logarithmic growth phase were used, and the sequencing was performed at a density of 5 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates and cultured statically for 24 hours. Then, 8 μM ATRA or 25 ng / mL PMA was added to the culture medium, and the cells were cultured for another 48 hours. Cells were then collected, and RNA was extracted from the cells by adding Trizol lysis buffer. The whole transcriptome of THP-1 cells was sequenced using the Illumina sequencing platform. The expression of lncRNA in cells of each treatment group was analyzed using the EBseq algorithm and Ballgown software.

[0057] 2.7 qRT-PCR Analysis of lncRNA Expression: Total RNA was extracted using TRIzol according to the reagent instructions, dissolved in DEPC water, and the concentration was determined using NanoDrop 2000. cDNA was synthesized using the TransGen Biotech TransScript II One-Step qDNA Removal and cDNA Synthesis Super Mix reverse transcription kit according to the product instructions. The cDNA was diluted 4-fold and used as a template for qRT-PCR to detect lncRNA expression levels. The reaction program was set as follows: 95℃ pre-denaturation for 10 min; 40 cycles: 95℃ denaturation for 20 s, 58℃ annealing for 30 s, and 72℃ extension for 30 s. β-actin was used as an internal control gene. -ΔΔCt The method is used to analyze the results.

[0058] 2.8 Construction and Screening of Stable Transfected Cells with AC098613.1: The lentiviral vector plvx-Teton-puro provided by Shanghai Jikai Biotechnology Co., Ltd. was selected, and after enzyme digestion, the AC098613.1 / NC sequence was ligated to construct the plvx-Teton-puro-AC098613.1 / NC recombinant vector. The recombinant vector was packaged with lentivirus and infected HEK-293T cells. After cloning assay, lentivirus was extracted from the cells. 24 hours before lentiviral transfection, THP-1 / HL-60 cells were seeded into 96-well plates, with a cell number of approximately 8 × 10⁶ cells / well. 3 / well, when the cell density reached 40%-60%, were infected with the virus. After cell selection using puromycin, the expression of AC098613.1 was detected by qRT-PCR. Cells were treated according to the experimental protocol, and changes in morphology, proliferation activity, NBT reducing ability, and CD11b expression level of AC098613.1 / NC stably transformed THP-1 / HL-60 cells were detected to analyze the regulatory role of AC098613.1 in the differentiation process of THP-1 / HL-60 cells.

[0059] 3. Experimental Results:

[0060] This study used ATRA and PMA to induce THP-1 cell differentiation and identified 50 lncRNAs with differential expression (|log2(fold change)|>2.0, P<0.05) in both groups of differentiated cells through whole transcriptome sequencing. Figure 1 A, B); among them, AC098613.1 was found to be significantly upregulated in THP-1 cells treated with ATRA and PMA (A, B); Figure 1C). Previous studies have reported that aberrant expression of AC098613.1 can be used for risk assessment of prognosis in bladder urothelial carcinoma; however, the expression and function of AC098613.1 in AML cells remain unclear. To investigate the biological function of AC098613.1 in AML cells, this study selected THP-1 and HL-60 cells and constructed a tetracycline-induced stable transfection cell line overexpressing AC098613.1. Compared with the control group cells, the expression of AC098613.1 in the stable transfection cells was significantly upregulated (C). Figure 1 D), indicating successful construction of stable transgenic cells. After tetracycline induction, proliferation and differentiation indices were measured. The results showed that high expression of AC098613.1 significantly inhibited the proliferation of THP-1 and HL-60 cells, with inhibition rates of 30.14% and 27.78%, respectively. Figure 1 E); The NBT reduction capacity of stably transformed THP-1 and HL-60 cells was enhanced ( Figure 1 F), upregulation of the differentiation-related protein CD11B (F) Figure 1 G, H), the cells showed obvious morphological changes, mainly manifested as increased cell volume and decreased nucleocytoplasmic ratio (NCR). Figure 1 I, J). This demonstrates that high expression of AC098613.1 can promote AML cell differentiation. Furthermore, cellular localization analysis of AC098613.1 showed that it is mainly distributed in the cytoplasm (I, J). Figure 1 K, L).

[0061] Example 2: Screening of target proteins for AC098613.1

[0062] 1. Experimental Design:

[0063] Screening of AC098613.1 target proteins: RNA pull-down experiments were performed using biotin-labeled AC098613.1 / NC probes. The experiment was divided into two groups: ① NC group: negative control group, with the complementary sequence of biotin-labeled AC098613.1 as the probe; ② AC098613.1 group: biotin-labeled AC098613.1 as the probe. After collecting the products, LC-MS / MS protein proteometry was performed, and the colocalization of AC098613.1 and target proteins in cells was observed by fluorescence microscopy to screen for AC098613.1 target proteins.

[0064] 2. Experimental methods:

[0065] 2.1 RNA pull-down: Based on a mass-to-length ratio of 1 μg / 1000 nt, biotin-labeled AC098613.1 / NC probes were prepared. The prepared probes were incubated at 90°C for 2 min, then 50 μL of RNA Structure Buffer and 20 μL of RNase-free water were added, mixed, and incubated at room temperature for 20 min. 40 μL of streptaridin magnetic beads were washed with 100 μL of 1×TES and collected. The magnetic beads were mixed with the prepared AC098613.1 / NC probes, 100 μL of RNase-free water and 200 μL of 1×TES were added, and the mixture was incubated at room temperature for 30 min by rotation. After incubation on a magnetic rack for 1 min, the supernatant was discarded and the magnetic beads were collected. 500 μL of 1×TES was added to the magnetic beads, and after incubation for 1 min, the supernatant was discarded and the magnetic beads were collected. 800 μL of protein sample and 5 μL of RNase were taken. Inhibitor and 5 μL poly(dI.dc) were mixed and added to magnetic beads. The beads were gently rotated and bound at room temperature for 2 h. After standing for 1 min, the supernatant was removed and the magnetic beads were collected. The magnetic beads were washed with 1 mL of pre-cooled NT2 buffer at 4 °C for 5 min by rotation. After standing for 1 min, the supernatant was removed and the washing was repeated 4 times. 60 μL of protein elution buffer and 0.6 μL of DTT were added to the magnetic beads and incubated at 37 °C for 2 h for elution. The supernatant was collected for LC-MS / MS mass spectrometry analysis.

[0066] 2.2 Fluorescence microscopy colocalization observation: Cell slides were prepared from well-functioning AC098613.1 cells stably transfected with THP-1 / HL-60. The prepared slides were soaked in methanol for 30 min, washed three times with 1×PBS, treated with 0.5% Triton for 5 min, and washed twice with 1×PBS. Prehybridization solution was added, and the slides were incubated at 37℃ for 2 h. The AC098613.1 probe labeled with cy5 was added, and the slides were incubated at 37℃ for 16 h. The slides were washed three times with 2×SSC, blocked in 5% BSA for 30 min, incubated overnight with primary antibody at 4℃, washed three times with 1×PBS, incubated with secondary antibody (Alexa Fluor 488, anti-Rabbit) at room temperature for 1 h, washed three times with 1×PBS, 50 μL DLI was added, and the slides were incubated at room temperature in the dark for 5 min, washed three times with 1×PBS, mounted with resin, and observed and photographed under a laser confocal microscope.

[0067] 3. Experimental Results:

[0068] To investigate the molecular mechanism by which AC098613.1 regulates AML cell differentiation, a biotin-labeled AC098613.1 probe was constructed. The probe was then used to extract proteins interacting with AC098613.1 via RNA pull-down assay. Figure 2 A). LC-MS / MS mass spectrometry analysis of the samples revealed 309 unique protein molecules in the AC098613.1 pull-down group. Based on indicators such as Protein FDR Confidence (FDR < 0.01), Abundances (Abundances > 335,000), and ScoreSequest HT (ScoreSequest HT > 50), ACTN1, EZR, NME2, and CDC5L were finally screened. Figure 2 B), among which the regulatory role of CDC5L in leukemia cells has not been reported. CDC5L (cell division cycle 5-like protein) is a cell division cycle protein involved in regulating the cell cycle and plays an essential role in mitosis. Studies have found that CDC5L is highly expressed in tumor cells and is closely related to tumor development and progression, and is considered a potential target for tumor therapy. Therefore, this study analyzed the regulatory role of CDC5L in AML cell differentiation. This study first verified the targeted binding of AC098613.1 to CDC5L. The protein abundance of CDC5L in the AC098613.1 pull-down group was significantly higher than that in the control group ( Figure 2 C); In addition, immunofluorescence co-localization showed that AC098613.1 and CDC5L could co-localize in the cytoplasm of THP-1 and HL-60 cells (C); Figure 2 (D, E) The results above all demonstrate that AC098613.1 can target and bind to CDC5L.

[0069] Example 3: Functional analysis of the target protein AC098613.1

[0070] 1. Experimental Design:

[0071] (1) Binding analysis of AC098613.1 and CDC5L: The binding sites of AC098613.1 and CDC5L were predicted by molecular docking technology, and AC098613.1-mut was synthesized by mutating the binding sites. The binding of AC098613.1 / AC098613.1-mut to CDC5L was analyzed by RNA pull-down and Western blot. The experiment was divided into three groups: ①NC group: negative control group, biotin-labeled complementary sequence of AC098613.1 as probe; ②AC098613.1 group: biotin-labeled AC098613.1 as probe; ③AC098613.1-mut group: biotin-labeled AC098613.1-mut as probe; Western blot was used to detect the abundance of CDC5L in each group of samples and to analyze the effect of the binding site on the binding of AC098613.1 to CDC5L.

[0072] (2) Effect of AC098613.1 binding with CDC5L on THP-1 / HL-60 cell differentiation: THP-1 / HL-60 cells stably transfected with AC098613.1-mut / NC in logarithmic growth phase were cultured statically in RPMI-1640 medium for 24 h, and then 400 ng / ml of CDC5L was added. The experiment was conducted in two groups: ① vector group: negative control group, NC cells stably transfected with THP-1 / HL-60 cells; ② AC098613.1-mut group: AC098613.1-mut cells stably transfected with THP-1 / HL-60 cells. Each group was set up with 3 replicates. After culturing for 96 hours, the morphology, proliferation activity, NBT reduction capacity and CD11b expression level of AC098613.1-mut / NC cells stably transfected with THP-1 / HL-60 cells were detected to analyze the effect of AC098613.1-mut on cell differentiation and explore the role of AC098613.1 and CDC5L in regulating the differentiation process of THP-1 / HL-60 cells.

[0073] 2. Experimental methods:

[0074] 2.1 Molecular docking analysis: The structure of lncRNA was predicted using RNA Composer and a nucleic acid model was constructed. The protein structure was processed in the Molecular Operations Environment (MOE 2019.1) platform with Amber10 as the field. This included removing water and ions, protonation, adding missing atoms and completing missing groups, and minimizing protein energy. Using HDOCK software, CDC5L was set to rigid, the docking contact site was set to full surface, and 100 conformations were generated after docking. The conformation with the most negative energy was selected using a scoring function and visualized using Pymol 2.1 software.

[0075] 3. Experimental Results:

[0076] The target binding site of AC098613.1 to CDC5L was predicted using molecular docking. The structure of AC098613.1 was predicted using RNA Composer, and a model was constructed. Figure 3 A) The crystal structure of CDC5L was downloaded directly from the RCSB PDB protein crystal database. Molecular docking was performed using HDOCK and Pymol 2.1 software, and the resulting conformations were visualized and analyzed. The docking complex with the lowest binding energy (-309.49 kcal / mol) was selected. Figure 3 B). The binding sites of CDC5L include amino acid residues such as LYS-255, GLN-233, ASN-231, LYS-135, VAL-10, ARG-12, and GLU-66; the binding sites of AC098613.1 include bases such as C-343, C-400, G-408, C-313, A-311, C-310, and G-432. Figure 3 C). Electrostatic surface mapping showed that AC098613.1 and CDC5L had good surface matching, forming a stable complex through various interactions such as salt bridges, hydrogen bonds, and hydrophobicity. To investigate the role of targeted binding of AC098613.1 and CDC5L in AML cell differentiation, a mutated binding site AC098613.1 (AC098613.1-mut) was synthesized. CDC5L was not detected in the AC098613.1-mut pull-down sample. Figure 3 D) indicates that the binding site mutation can significantly inhibit the binding of AC098613.1 to CDC5L. AC098613.1-mut overexpression cell lines were constructed by transfecting THP-1 and HL-60 cells with the AC098613.1-mut overexpression vector, and the expression of AC098613.1-mut in these cells was significantly upregulated. Figure 3 E). Meanwhile, analysis of cell differentiation markers showed no significant changes in cell proliferation activity, NBT reduction capacity, CD11B expression, or morphology. Figure 3 FJ), thus proving that AC098613.1 regulates AML cell differentiation by binding to CDC5L.

[0077] Example 4: Effects of AC098613.1 on the stability and nuclear distribution of CDC5L cells.

[0078] 1. Experimental Design:

[0079] (1) Take AC098613.1 / AC098613.1-mut / NC stable THP-1 cells in the logarithmic growth phase and administer at a dose of 5×10⁻⁶. 5The cells were seeded in 6-well plates and incubated statically for 24 hours. 50 μg / mL Dox was added. The experiment was divided into three groups: ① vector group: negative control group, NC cells stably transfected with THP-1; ② AC098613.1 group: AC098613.1 cells stably transfected with THP-1; ③ AC098613.1-mut group: AC098613.1-mut cells stably transfected with THP-1. After culturing for 2, 4, and 6 hours, total protein was extracted from each group, and the expression of CDC5L in the protein samples was detected to analyze the effect of AC098613.1 on the stability of CDC5L.

[0080] (2) Take THP-1 cells transfected with si-NC / si-AC098613.1 and administer at a dose of 5×10⁻⁶. 5 Cells were seeded in 6-well plates and incubated statically for 24 hours. MG132 (10 μM / L) or NH4Cl (10 mM / L) was added. The experiment was divided into three groups: ① Untreated group: negative control group, without MG132 or NH4Cl; ② MG132 group: 10 μM / L MG132 added; ③ NH4Cl group: 10 mM / L NH4Cl added. Cells were cultured for another 48 hours. Total protein was extracted from each group, and the expression of CDC5L in the protein samples was detected to analyze the effect of AC098613.1 on CDC5L degradation.

[0081] 2. Experimental methods:

[0082] siRNA transfection: The nucleic acid sequence of lncRNA was obtained based on the transcriptome sequencing results; siRNA was designed based on the nucleic acid sequence and synthesized by Shanghai Jikai Company. THP-1 cells were seeded into 96-well plates (8×10⁶ cells / wells). 3 Cells were cultured statically in a cell culture incubator until they reached a density of approximately 90%. Then, transfection experiments were performed according to the experimental groups. The transfection reagent was Lipofectamine 3000; specific experimental procedures were followed according to the reagent instructions. Transfected cells were cultured for another 96 hours, and lncRNA expression was detected by qRT-PCR.

[0083] Cells were treated according to the experimental protocol, and total protein, nuclear protein, and cytoplasmic protein were extracted from AC098613.1 / AC098613.1-mut / NC stable THP-1 cells using the Aid Lab nuclear / plasmic protein extraction kit. The distribution of CDC5L in each group of samples was analyzed by Western blot and fluorescence microscopy colocalization.

[0084] 3. Experimental Results:

[0085] High expression of AC098613.1 significantly increased the level of CDC5L protein in cells, while high expression of AC098613.1-mut had no significant effect on the level of CDC5L protein. Figure 4 A, B). To elucidate the mechanism by which AC098613.1 regulates CDC5L protein levels, THP-1 cells were treated with the protein synthesis inhibitor CHX, and CDC5L protein levels were analyzed at 0, 2, 4, and 6 h. With prolonged CHX treatment, CDC5L protein levels significantly decreased in the control group and the AC098613.1-mut high-expression group, but there was no significant change in CDC5L protein levels in the AC098613.1 high-expression group. Figure 4 (C, D) This indicates that high expression of AC098613.1 significantly enhances the stability of CDC5L protein in THP-1 cells. The main protein degradation systems in eukaryotic cells include the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP). In this study, both MG-132 (a specific proteasome inhibitor) and ammonium chloride (a lysosome inhibitor) significantly inhibited CDC5L degradation induced by AC098613.1 knockdown, indicating that both UPS and ALP are involved in regulating CDC5L protein degradation, and AC098613.1 increases the level of CDC5L in cells by affecting CDC5L degradation. Furthermore, the nuclear distribution of CDC5L was significantly increased in cells with high AC098613.1 expression, while high expression of AC098613.1-mut had no significant effect on the nuclear distribution of CDC5L. Figure 4 (G, H) suggests that AC098613.1 regulates the cellular distribution of CDC5L by affecting CDC5L stability. In immunofluorescence co-localization experiments, CDC5L protein levels were also found to be significantly increased in THP-1 cells with high AC098613.1 expression, and its distribution in the nucleus was also significantly enhanced. Figure 4 I).

[0086] Example 5: The regulatory effect of CDC5L on ADAP1 expression

[0087] 1. Experimental Design:

[0088] AC098613.1 cells stably transformed into THP-1 / HL-60 cells in the logarithmic growth phase were harvested at a rate of 5 × 10⁻⁶. 5The cells were seeded in 6-well plates and cultured under normal conditions for 24 h. Then, 400 ng / mL Dox was added, and the seeded cells were divided into four groups: ① Vector group: blank control group, AC098613.1 cells stably transfected with THP-1 / HL-60 under normal culture conditions; ② AC098613.1 group: negative control group, AC098613.1 cells stably transfected with THP-1 / HL-60 under Dox treatment; si-NC group: negative control group, AC098613.1 cells stably transfected with THP-1 / HL-60 transfected with si-NC; si-CDC5L group: AC098613.1 cells stably transfected with THP-1 / HL-60 transfected with si-CDC5L. Each group had three replicates. After 96 h of culture, the expression level of ADAP1 in each group was detected to analyze the effect of CDC5L on ADAP1 expression.

[0089] 2. Experimental methods:

[0090] Si-CDC5L for CDC5L knockdown expression was designed and synthesized, and AC098613.1 stably transfected THP-1 / HL-60 cells were transfected according to the aforementioned transfection method. After treating the cells according to the experimental protocol, the mRNA and protein expression levels of ADAP1 in the cells were detected by qRT-PCR and Western blot.

[0091] 3. Experimental Results

[0092] Previous studies have identified 155 CDC5L target genes using chip-seq analysis. In this study, we further screened downstream target genes of CDC5L by combining differentially expressed mRNAs common to both ATRA and PMA-treated THP-1 cells. A total of 209 common differentially expressed mRNAs were found in ATRA and PMA-treated THP-1 cells. Figure 5 A, B), where ADAP1 and CNOT3 were identified as CDC5L target genes in Chip-seq analysis. Figure 5 C). ADAP1 is an Arf GTPase activator protein (GAP) highly expressed in the nervous system, involved in regulating neuronal differentiation and neurodegeneration; however, the regulatory role of ADAP1 in leukemia cells has not been reported to date. This study found that in AC098613.1 stably transfected THP-1 and HL-60 cells, ADAP1 expression was significantly upregulated at both the mRNA and protein levels, while in AC098613.1-mut overexpression cells, ADAP1 expression showed no significant change. Figure 5DF). Furthermore, si-CDC5L for CDC5L knockdown expression was designed and synthesized, and transfected into AC098613.1 stably transfected THP-1 cells. CDC5L knockdown significantly inhibited the increase in ADAP1 expression levels induced by AC098613.1 high expression. Figure 5 (GJ). The above results indicate that CDC5L can positively regulate ADAP1 expression.

[0093] Example 6: The regulatory effect of ADAP1 on AML cell differentiation

[0094] 1. Experimental Design:

[0095] AC098613.1 / NC cells in logarithmic growth phase that have been stably transformed into THP-1 / HL-60 cells were harvested at a rate of 5 × 10⁻⁶. 5 Cells were seeded in 6-well plates and cultured under normal conditions for 24 h. Then, 400 ng / mL Dox was added, and the cells were divided into four groups: ① vector group: THP-1 / HL-60 cells stably transfected with NC; ② AC098613.1 group: AC098613.1 cells stably transfected with THP-1 / HL-60; ③ AC098613.1+si-NC group: AC098613.1 cells stably transfected with si-NC and THP-1 / HL-60; ④ AC098613.1+si-ADAP1 group: AC098613.1 cells stably transfected with si-ADAP1 and THP-1 / HL-60. Each group had three replicates. After 96 h of culture, differentiation indicators such as cell proliferation activity, CD11b expression level, and cell morphology were detected to analyze the effect of ADAP1 on THP-1 / HL-60 cell differentiation.

[0096] 2. Experimental methods:

[0097] Si-ADAP1, used to knock down ADAP1 expression, was designed and synthesized. AC098613.1 stable THP-1 / HL-60 cells were transfected according to the aforementioned transfection method. After treating the cells according to the experimental protocol, the expression level of ADAP1 in the cells and its effect on cell differentiation indicators were detected by qRT-PCR, CCK-8 and western blot.

[0098] 3. Experimental Results:

[0099] In this study, three si-ADAP1 molecules were synthesized and transfected into AC098613.1 stably transfected THP-1 and HL-60 cells, all of which significantly reduced the expression level of ADAP1 in the cells. Figure 6A). si-ADAP1-2, which showed the most significant knockdown effect, was selected for subsequent cell transfection. Compared with the control group transfected with si-NC, the proliferation activity of cells in the si-ADAP1-2 transfection group was significantly increased ( Figure 6 B), the expression level of CD11B in cells was significantly reduced ( Figure 6 (C, D) Cell volume increases and nucleocytoplasmic ratio decreases significantly ( Figure 6 E) demonstrates that ADAP1 is involved in regulating AML cell differentiation.

[0100] Example 7: The regulatory role of ADAP1 in AML cell cycle progression

[0101] 1. Experimental Design:

[0102] AC098613.1 / NC cells in logarithmic growth phase that have been stably transformed into THP-1 / HL-60 cells were harvested at a rate of 5 × 10⁻⁶. 5 Cells were seeded in 6-well plates and cultured under normal conditions for 24 h. Then, 400 ng / mL Dox was added, and the cells were divided into four groups: ① vector group: THP-1 / HL-60 cells stably transfected with NC; ② AC098613.1 group: AC098613.1 cells stably transfected with THP-1 / HL-60; ③ AC098613.1+si-NC group: AC098613.1 cells stably transfected with si-NC and THP-1 / HL-60; ④ AC098613.1+si-ADAP1 group: AC098613.1 cells stably transfected with si-ADAP1 and THP-1 / HL-60. Each group had three replicates. After 96 h of culture, NRD1 expression and cell cycle progression were detected to analyze the effect of ADAP1 on the cell cycle progression of THP-1 / HL-60 cells.

[0103] 2. Experimental methods:

[0104] Cells in the logarithmic growth phase were collected and treated according to the experimental protocol. After culturing for 96 hours, the cells were collected, washed with pre-cooled 1×PBS, and fixed in pre-cooled 70% anhydrous ethanol for 4 hours. The cells were collected, washed with pre-cooled 1×PBS, resuspended in 500 L of staining solution, incubated at 37°C in the dark for 30 minutes, and the cell cycle was detected by flow cytometry within 1 hour.

[0105] 3. Experimental Results:

[0106] Studies have found that high expression of ADAP1 promotes retinoic acid (RA)-induced differentiation of human neuroblastoma cells. Highly expressed ADAP1 promotes cell differentiation by positively regulating nardilysin (NRD1) expression. NRD1 is a metallopeptidase of the M16 family. Located in the cell nucleus, NRD1 acts as a transcriptional co-regulator, positively or negatively regulating gene transcription by binding to dimethylated H3K4 protein. Previous studies have used high-throughput Chip-seq analysis to identify 448 candidate NRD1 target genes, among which cell cycle-related genes were significantly enriched. RNA-seq confirmed 10 cell cycle-related direct target genes, including CDK1, Fbxo5, Brca1, Hmga2, Cdt1, Src, Ajuba, Dscc1, Ccnf, and Ccnd1. Cell differentiation requires permanent cell cycle arrest; when cells enter a differentiated state, cell cycle arrest inevitably occurs. Given the regulatory roles of ADAP1 and NRD1 in cell cycle and differentiation, this study investigated the effects of ADAP1 knockdown on NRD1 and CDK1 expression and cell cycle progression. The results showed that, compared to control cells, AC098613.1 cells stably transfected with THP-1 and HL-60 showed significantly upregulated NRD1 expression, significantly downregulated CDK1 expression, and marked G0 / G1 cell cycle arrest. Transfection with si-ADAP1 significantly reduced NRD1 expression in AC098613.1 cells stably transfected with THP-1 and HL-60. Figure 7 A, B), increase CDK1 expression level ( Figure 7 C, D), and significantly alleviated cell cycle arrest (C, D), Figure 7 Based on the above results, this study proposes that ADAP1 induces cell G0 / G1 cycle arrest by positively regulating NRD1 expression, thereby affecting the cell differentiation process.

[0107] Example 8: The regulatory role of NRD1 in AML cell differentiation

[0108] 1. Experimental Design:

[0109] AC098613.1 / NC cells in logarithmic growth phase that have been stably transformed into THP-1 / HL-60 cells were harvested at a rate of 5 × 10⁻⁶. 5Cells were seeded in 6-well plates and cultured under normal conditions for 24 h. Then, 400 ng / mL Dox was added, and the cells were divided into four groups: ① vector group: THP-1 / HL-60 cells stably transfected with NC; ② AC098613.1 group: AC098613.1 cells stably transfected with THP-1 / HL-60; ③ AC098613.1+si-NC group: AC098613.1 cells stably transfected with si-NC and THP-1 / HL-60; ④ AC098613.1+si-NRD1 group: AC098613.1 cells stably transfected with si-NRD1 and THP-1 / HL-60. Each group had three replicates. After 96 h of culture, cell proliferation activity, NBT reduction capacity, CD11b expression, and morphology were measured to analyze the effect of NRD1 on THP-1 / HL-60 cell differentiation.

[0110] 2. Experimental methods:

[0111] After treating the cells according to the experimental protocol, transfection experiments were conducted using the aforementioned experimental methods, and differentiation indicators such as cell morphology, proliferation activity, NBT reduction capacity, and CD11b expression level were detected to analyze the effect of NRD1 on the differentiation of THP-1 / HL-60 cells.

[0112] 3. Experimental Results:

[0113] In this study, si-NRD1 was synthesized and transfected into AC098613.1 stably transfected THP-1 cells, resulting in a significant decrease in NRD1 expression in the cells. Figure 8 A). Compared with AC098613.1 cells stably transfected with THP-1 and transfected with si-NC, the proliferation activity of cells transfected with si-NRD1 was significantly increased. Figure 8 B) The NBT reduction capacity of cells was significantly reduced. Figure 8 C) The expression level of CD11B in cells was significantly reduced. Figure 8 D, E), cell volume increased and nucleocytoplasmic ratio decreased significantly ( Figure 8 F) demonstrates that NRD1 is involved in regulating THP-1 cell differentiation.

[0114] Differentiation-inducing therapy for leukemia has become a highly regarded treatment strategy in recent years, with highly effective and low-toxicity differentiation-inducing agents being the core of this strategy. In recent years, non-coding RNAs (ncRNAs), such as miRNAs and long non-coding RNAs (lncRNAs), have been found to play a regulatory role in hematopoiesis and the development and progression of leukemia. The high conservation and tissue specificity of lncRNAs have made them widely studied in disease treatment and drug development. Research on lncRNAs in leukemia has also increased in recent years, and some lncRNAs have been found to have clinical application potential in the diagnosis, prognosis, and treatment of leukemia. Abnormal expression of the long non-coding RNA AC098613.1 has been found to be useful for risk assessment in the prognosis of bladder urothelial carcinoma. However, the expression and function of AC098613.1 in acute myeloid leukemia (AML) remain unclear. In this invention, we found that high expression of AC098613.1 can significantly inhibit the proliferation activity of THP-1 and HL-60 cells, increase CD11b expression levels and NBT reduction capacity in cells (see Example 1 for details). Furthermore, the effect of AC098613.1 on AML cell differentiation in vivo was further investigated using a subcutaneous tumorigenesis experiment in nude mice. The results showed that tumor volume and mass were significantly reduced in nude mice bearing tumors with high AC098613.1 expression, Ki-67 expression was significantly decreased, and CD11B expression was significantly upregulated, thus confirming the positive regulatory effect of AC098613.1 on AML cell differentiation in vivo (see Example 9 for details). Based on these results, both in vitro and in vivo studies demonstrate that upregulation of AC098613.1 expression can inhibit AML cell proliferation and induce cell differentiation.

[0115] Example 9, Effect of AC098613.1 on HL-60 cell differentiation in vivo.

[0116] 1. Experimental Design:

[0117] A nude mouse model of tumor bearing AC098613.1 / NC stably transfected HL-60 cells was constructed until the tumor grew to approximately 6 mm. 3The tumor-bearing nude mice were divided into four groups: ① Control group: HL-60 cell-bearing nude mice, intraperitoneally injected with Dox (10 mg / kg); ② NC1 group: NC stable-transfer HL-60 cell-bearing nude mice, intraperitoneally injected with Dox (10 mg / kg); ③ NC2 group: AC098613.1 stable-transfer HL-60 cell-bearing nude mice, intraperitoneally injected with saline; ④ AC098613.1 group: AC098613.1 stable-transfer HL-60 cell-bearing nude mice, intraperitoneally injected with saline. Dox (10 mg / kg) was injected into mice; 15 replicates were set up in each group, and the injections were carried out once a day for 2 consecutive weeks. After the experiment, the weight of the mice was measured and the tumors were removed by killing the mice. The weight and volume of the tumors, the expression of AC098613.1, ADAP1, NRD1, CDK2 in the tumor cells, the cell distribution of CDC5L, and the expression of the proliferation marker protein Ki-67 and the differentiation marker protein CD11b were analyzed to explore the effect of AC098613.1 on cell differentiation in vivo.

[0118] 2. Experimental methods:

[0119] 2.1 Construction of tumor-bearing nude mouse model: AC098613.1 / NC cells stably transfected with HL-60 in the logarithmic growth phase were taken and suspended in 100 μL of RPMI-1640 incomplete culture medium. 100 μL of matrix gel was added, mixed well, and placed on ice. Each nude mouse was inoculated with 1 × 10⁶ cells / mL. 5 Cells were inoculated from the skin of the neck and back of 4-week-old nude mice; the nude mice were fed under SPF conditions and treated according to the above experimental protocol.

[0120] 2.2 Immunohistochemical analysis: After washing the tumor with 1×PBS, a 3mm sample was taken. 3 After fixing in tissue fixative at 4°C for 12-16 h, the tissues were dehydrated, cleared, embedded, and sectioned. After gradient rehydration, the sections were treated with 3% H2O2 for 10 min, blocked with 5% BSA at room temperature for 20 min, incubated overnight at 4°C with primary antibody, and then rinsed three times in 1×TBS for 5 min each time. Biotin-labeled goat anti-rabbit secondary antibody was added and incubated at room temperature for 30 min, followed by three rinses in 1×TBS. The SABC complex was added and incubated at room temperature for 20 min, developed with DAB-H2O2 chromogenic solution, and counterstained with hematoxylin for 15 s. After gradient dehydration to xylene with alcohol, the sections were mounted with neutral resin and observed and photographed under a microscope.

[0121] The extracted tumors were washed twice with 1×PBS, weighed, and their volume was measured. The tumors were then digested with trypsin to prepare a single-cell suspension. The expression of AC098613.1, ADAP1, NRD1, and CDK2 in the cells was analyzed by qRT-PCR and Western blot.

[0122] 3. Experimental Results:

[0123] To investigate the effect of AC098613.1 on AML cell differentiation in vivo, this study conducted a subcutaneous tumorigenesis experiment in nude mice. Tumor growth was observed after intraperitoneal injection of Dox (10 mg / kg) into tumor-bearing nude mice. Compared with HL-60 cell-bearing nude mice, there was no significant difference in tumor volume and mass between NC-stably converted HL-60 cell-bearing nude mice and AC098613.1-stably converted HL-60 cell-bearing nude mice without Dox induction. However, the tumor volume and mass of AC098613.1-stably converted HL-60 cell-bearing nude mice were significantly reduced. Figure 9 AC098613.1 can inhibit the proliferation of tumor cells in vivo. Immunohistochemistry showed that the expression level of Ki-67 in cells was significantly reduced, and the expression level of CD11B was significantly upregulated. Figure 9 (D, E). Furthermore, compared to tumor cells from nude mice with NC-stably converted HL-60 cells and untreated AC098613.1-stably converted HL-60 cells, the expression levels of AC098613.1, CDC5L, ADAP1, and NRD1 were significantly upregulated, while CDK1 expression was significantly downregulated in the tumor cells of AC098613.1-stably converted HL-60 cells. Figure 9 (FH). Thus, in vivo, it was demonstrated that AC098613.1 can induce AML cell differentiation by affecting the stability of CDC5L and regulating the expression of ADAP1 / NRD1 / CDK1.

[0124] Example 10: Expression levels of AC098613.1 and related proteins in AML patients.

[0125] 1. Experimental Design:

[0126] Bone marrow samples were collected from patients with non-malignant hematological diseases and newly diagnosed AML patients (M4 stage), with a collection volume of 2 mL per patient. The experiment was divided into two groups: ①NC group: negative control group, bone marrow from patients with non-malignant hematological diseases; ②AML group: bone marrow from patients with AML. Each group consisted of 12 patients. Bone marrow samples were processed with lymphocyte separation medium to obtain bone marrow mononuclear cells. The expression of AC098613.1, CDC5L, ADAP1 and NRD1 in the cells was detected to explore the correlation of the expression of AC098613.1, CDC5L, ADAP1 and NRD1 in AML patients.

[0127] 2. Experimental methods:

[0128] Bone marrow mononuclear cell isolation: Bone marrow samples were stored in heparin buffer (10 IU / mL), and an equal volume of lymphocyte separation medium was added. The samples were centrifuged at room temperature for 20 min, and bone marrow mononuclear cells were collected from the interface between the plasma and lymphocyte separation medium. The expression levels of AC098613.1, CDC5L, ADAP1, and NRD1 in each group of samples were analyzed using qRT-PCR and Western blot to explore their correlation with AML patients.

[0129] 3. Experimental Results:

[0130] To validate the expression pattern of AC098613.1 in AML, this study recruited 12 newly diagnosed AML patients (M4 stage) and 12 patients with non-malignant hematological diseases to evaluate the clinical significance of AC098613.1 (Appendix 4). Figure 10 As shown in Figure A, compared with patients with non-malignant hematological diseases, the expression level of AC098613.1 was significantly reduced in AML patients. Figure 10 A) validated that AC098613.1 plays a tumor suppressor role in AML. Compared with patients with non-malignant hematological malignancies, the expression levels of CDC5L, ADAP1, and NRD1 were also significantly reduced in AML patients. Figure 10 The results (BE) were consistent with those of cell experiments, verifying the regulatory effect of AC098613.1 on AML cell differentiation.

[0131] This invention relates to the field of pharmaceutical technology, and more particularly to the application of the lncRNA AC098613.1 gene in the preparation of drugs for treating leukemia. Through whole transcriptome sequencing, this invention revealed that AC098613.1 is expressed at low levels in acute myeloid leukemia (AML) THP-1 cells, while its expression level is significantly upregulated in cells treated with ATRA and PMA. This invention utilizes an AC098613.1 overexpression plasmid to enhance the expression level of AC098613.1 in AML THP-1 and HL-60 cells; AC098613.1 overexpression in THP-1 and HL-60 cells resulted in reduced proliferation, significant cell cycle arrest, and significantly upregulated cell differentiation. This invention provides a new target for the clinical treatment of AML and has significant clinical applications.

[0132] The following are six specific embodiments provided by the present invention:

[0133] 1. Preparation of AC098613.1 gene siRNA: First, an siRNA sequence specifically targeting the AC098613.1 gene needs to be designed and synthesized. Then, the synthesized siRNA is transfected into acute myeloid leukemia cells, and its inhibitory effect is evaluated using quantitative real-time PCR.

[0134] 2. Construction of expression vector for AC098613.1 gene: The expression vector for AC098613.1 gene can be constructed using conventional molecular biology techniques (such as PCR, restriction endonuclease digestion, ligase reaction, etc.), and introduced into acute myeloid leukemia cells by transfection or transient transfection to evaluate its effect on cell proliferation and differentiation.

[0135] 3. Construction of AC098613.1 gene overexpression cell lines: CRISPR / Cas9 gene editing technology can be used to design sgRNA sequences that can specifically overexpress the AC098613.1 gene and introduce them into acute myeloid leukemia cells. Cell lines that overexpress the AC098613.1 gene can be screened and their impact on the occurrence of acute myeloid leukemia can be evaluated.

[0136] 4. Small molecule compound screening technology: Through high-throughput screening technology, compounds that have an agonistic effect on the AC098613.1 gene are screened from a large number of small molecule compounds, and then validated and optimized in vivo animal models.

[0137] 5. Combination therapy: The expression vector of the AC098613.1 gene was used in combination with other drugs for the treatment of acute myeloid leukemia, such as doxorubicin and apataro, to evaluate their synergistic effects and efficacy.

[0138] 6. Antibody Development: Using immunotherapy, specific antibodies against the AC098613.1 gene were prepared, and validated and optimized in in vivo animal models, ultimately for the treatment of acute myeloid leukemia.

[0139] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of LncRNA AC098613.1 overexpression plasmid in the preparation of drugs for treating acute myeloid leukemia, characterized in that: The drug can enhance the expression of lncRNA AC098613.1.

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