Application of KLF4 gene in preparation of medicine for treating myelofibrosis

Abstract

The invention discloses an application of a KLF4 gene in preparation of a medicine for treating myelofibrosis. The KLF4 gene can be used as a detection marker of myelofibrosis which is a rare hematological tumor. Specifically, through flow cytometry and GEO big data analysis, an upstream molecular mechanism for regulating KLF4 activity is analyzed, and it is found that the KLF4 promoter is highly methylated and the expression level is reduced in cells of patients with myelofibrosis; further, the specific molecular mechanism of KLF4-mediated myelofibrosis progress is comprehensively explored from three levels, namely a cellular level, a bone marrow microenvironment level and an animal in-vivo level by constructing KLF4 overexpressed myelofibrosis model cells, co-culturing mesenchymal cells and a humanized xenotransplantation mouse model; and the occurrence and development of the myelofibrosis mediated by the KLF4-RELN-AKT1-CDK20 signal axis are clear. The discovery provides a new thought for analyzing the complex pathogenesis of myelofibrosis, and provides a new target for clinical early diagnosis and intervention treatment of myelofibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of the KLF4 gene in the preparation of drugs for treating myelofibrosis. Background Art

[0002] Myelofibrosis is a myeloproliferative neoplasm caused by collagen hyperplasia in the bone marrow hematopoietic tissue, where fibrous tissue severely affects hematopoietic function. The root cause is the malignant proliferation of bone marrow hematopoietic stem / progenitor cells (CD34 + cells), which is a rare blood tumor. Currently, the diagnosis of myelofibrosis mainly relies on traditional means such as bone marrow aspiration and biopsy. However, these methods have certain limitations. Bone marrow aspiration is invasive, causing pain to patients, and the accuracy of the results is greatly affected by the operator's technical level; the selection of biopsy samples may also be biased, resulting in some pathological conditions not being detected in a timely manner. Due to the complex pathogenesis and difficult clinical diagnosis of this disease, patients are extremely prone to missed diagnosis and misdiagnosis, thus missing the "golden stage" of disease intervention and treatment. Moreover, the existing treatment methods mainly focus on relieving symptoms and delaying the progression of the disease, and it is difficult to cure the disease fundamentally. Therefore, it is urgent to explore the pathogenesis of this disease in more depth and detail to find new detection indicators and potential treatment targets to achieve the goal of "early detection and early treatment".

[0003] In the prior art, Patent CN110437311B discloses the application of a polypeptide containing the structural domains Pro-Arg-Cys-X-Y-Gly-Glu, where X is Trp or Tyr; Y is Arg or Cys; and Arg-Gly-Ala-Asp-Arg-Ala; or a sequence after mutating any amino acid in the above structural domains, which can treat various fibrotic diseases and conditions, including myelofibrosis. Patent CN115175937A discloses a combination therapy comprising a TIM-3 inhibitor and a TGF-β inhibitor. The combination can be used for treating or preventing cancerous diseases and disorders, including myelofibrosis or myelodysplastic syndrome. Patent CN117512110A provides the application of PD-L1 as a molecular marker in the preparation of an auxiliary diagnostic kit for myeloproliferative neoplasms, which can detect the expression level of PD-L1 in the bone marrow of patients, thereby quickly and conveniently detecting the expression levels of PD-L1 in patients with myeloproliferative neoplasms of the bone marrow and normal controls.

[0004] KLF4 is one of the key transcription factors for inducing iPSCs. It regulates cell proliferation and differentiation by controlling cell cycle-related genes, participates in various life activities, and exhibits tumor-suppressive or carcinogenic activities in different solid tumors in a tissue-dependent manner. However, in the hematopoietic system, the upstream signals regulating KLF4 expression and activity have been poorly studied so far, and there are few studies on the roles and functions of the KLF4 gene. Only a few literatures have shown that KLF4 can inhibit B-cell non-Hodgkin lymphoma, Hodgkin lymphoma, and multiple myeloma, indicating that KLF4 may play a role as a "tumor suppressor factor" in hematological malignancies. However, its role and specific mechanism in hematological malignancies such as myelofibrosis have not been clarified, and its potential as a detection indicator and therapeutic target has not been fully explored, which limits the further development of myelofibrosis diagnosis and treatment technologies. Summary of the Invention

[0005] Based on the above-mentioned drawbacks in the prior art, the objective of this application is to provide the use of the KLF4 gene in the preparation of drugs for treating myelofibrosis. In the early stage of this invention, bone marrow hematopoietic stem cells of myelofibrosis patients were obtained and isolated. Flow cytometry detection found that KLF4 was significantly downregulated in CD34 + cells of myelofibrosis patients. At the same time, by combining bioinformatics analysis of the gene expression profiles of myelofibrosis patients in the GEO database, it was found that the expression level of KLF4 in the hematopoietic stem cells of patients was generally decreased, suggesting that it may be an important factor for the abnormal proliferation of malignant CD34 + cells, leading to the occurrence and development of myelofibrosis. Based on the above findings, we analyzed the possibility of KLF4 as a potential therapeutic target in the rare disease of myelofibrosis at three levels: the cell level, the bone marrow microenvironment level, and the in vivo level of mice.

[0006] Based on the above objective, this application provides the following technical solutions:

[0007] One of the technical solutions of this application provides the use of the KLF4 gene in the preparation of drugs for treating myelofibrosis. The nucleic acid sequence of the KLF4 gene is as shown in SEQ.ID.No.1.

[0008] Furthermore, the KLF4 gene is highly expressed in normal hematopoietic stem cells, but significantly downregulated in the hematopoietic stem cells of myelofibrosis patients.

[0009] Furthermore, the downstream target of the KLF4 gene is the RELN gene, and the targeted binding of the KLF4 gene upregulates the expression level of the RELN gene and / or RELN protein; the downstream targeted pathway of the RELN protein is the PI3K / AKT pathway, and the PI3K / AKT pathway is related to the progression of myelofibrosis. Inhibiting the PI3K / AKT pathway can inhibit the progression of myelofibrosis.

[0010] Furthermore, at the molecular level, cellular level, and in vivo level of mice, the present invention evaluated the possibility of the KLF4 gene as a potential therapeutic target for myelofibrosis from two aspects: the degree of cell malignant proliferation and the release level of inflammatory factors in the bone marrow microenvironment, providing a new basis for the clinical diagnosis and drug target of myelofibrosis.

[0011] The second technical solution of the present invention provides a drug for treating myelofibrosis. The drug includes an active ingredient and an excipient.

[0012] Furthermore, the active ingredient is any one of a DNA methyltransferase inhibitor, an activator of the RELN protein which is a downstream target of the KLF4 gene, a KLF4 gene therapy vector, or a PI3K / AKT pathway inhibitor. The DNA methyltransferase inhibitor is selected from any one of 5-Azacytidine or RG108; the activator of the RELN protein which is a downstream target of the KLF4 gene is a small molecule compound, an antibody, a peptide compound, or a nucleic acid molecule. The excipient is a pharmaceutically acceptable carrier for improving the processing performance of the active ingredient. When in use, the active ingredient is mixed with at least one pharmaceutically acceptable carrier. The dosage form of the drug is any one of an oral preparation, an intravenous injection preparation, or a muscle injection preparation.

[0013] Furthermore, the pharmaceutically acceptable carrier includes at least any one of a filler, a diluent, a lubricant, a binder, or a disintegrant.

[0014] The third technical solution of the present invention provides a method for constructing a myelofibrosis model cell with overexpression of KLF4, and the method includes the following steps:

[0015] 1) Amplify KLF4 cDNA from human myelofibrosis model cell cDNA, and ligate the KLF4 cDNA sequence with the linearized vector pIRES to obtain a KLF4 overexpression vector;

[0016] The human myelofibrosis model cell is selected from any one of hel92.1.7 or set2;

[0017] 2) Transfect the KLF4 overexpression vector obtained in step 1) into the cell line of human myelofibrosis model cells, and after screening, a myelofibrosis model cell with overexpression of KLF4 is obtained.

[0018] Furthermore, the specific steps of the screening in step 2) are: adding G418 antibiotic for screening for 3 weeks, and then sorting GFP-positive cells using a flow cytometer.

[0019] Furthermore, the human myelofibrosis model cells with overexpressed KLF4 gene obtained in step 3) are subjected to cell expansion culture, and then total cellular RNA and total proteins are extracted. The overexpression of mRNA level is detected by qPCR, and the overexpression of cellular protein level is detected by Western Blot.

[0020] The fourth technical solution of the present invention provides a method for establishing an animal model of myelofibrosis, which comprises the following steps:

[0021] (1) Expand and culture the myelofibrosis model cells described in the above technical solution three, collect the cells, adjust the cell suspension concentration to 1.25×10 7 / mL, and place on ice;

[0022] (2) Inject the myelofibrosis model cell suspension obtained in step 1) into an animal, and raise it to obtain an animal model of myelofibrosis.

[0023] Furthermore, in some specific embodiments of the present application, the myelofibrosis animal is a mouse: 4-week-old M-NSG immunodeficient mice are raised for two weeks and used for injection at 6 weeks of age; 200 μL of the model cell suspension obtained in step 1) is injected into the tail vein of each mouse to obtain an animal model of myelofibrosis.

[0024] Furthermore, the M-NSG immunodeficient mice are raised in an SPF-class animal house; after continued culture for 8 weeks, the mice are sacrificed, and their spleens, bone marrows, and peripheral blood are taken for physiological and biochemical tests to evaluate whether the animal model is successfully constructed.

[0025] The fifth technical solution of the present application provides a biomarker combination for clinical detection of myelofibrosis. The biomarker combination includes at least two of KLF4, RELN, AKT1, and CDK20.

[0026] Furthermore, the expression level of KLF4 includes: the protein expression level of KLF4, the expression level of the KLF4 gene, or the methylation level of the KLF4 gene promoter.

[0027] Specifically, when the protein expression level of KLF4 is high, the protein expression level of RELN is high, the serine phosphorylation level at position 473 of AKT1 protein is decreased, and the expression level of CDK20 is decreased, it indicates a possible risk of myelofibrosis, and further bone marrow aspiration biopsy screening is required.

[0028] The sixth technical solution of the present application provides a kit for detecting myelofibrosis, and the kit detects the biomarker combination as described in the technical solution five.

[0029] Seventh technical solution of the present application provides an application of a RELN gene knockout mouse model, in which the RELN gene of the mouse model is knocked out for studying myelofibrosis. The promoter of the RELN gene is targeted and regulated by KLF4, and the targeted binding of KLF4 upregulates the expression level of RELN, thereby inhibiting the PI3K / AKT pathway and suppressing the progression of myelofibrosis.

[0030] Furthermore, the mouse model is used for screening drugs for treating myelofibrosis.

[0031] Compared with the prior art, the present application has at least the following beneficial effects:

[0032] (1) The present invention first discovers and clarifies that in myelofibrosis, the KLF4 promoter is inhibited by methylation, resulting in a decrease in the expression level of KLF4 at the pathological level. Through qPCR, flow cytometry detection and GEO data analysis of CD34 + cells of myelofibrosis patients, combined with the experiment of treating with DNA methyltransferase inhibitor, the association between the down-regulation of KLF4 expression and the methylation of its promoter is clarified, the upstream signal research on regulating the expression and activity of KLF4 is improved, and a new perspective is provided for understanding the pathogenesis of myelofibrosis, breaking through the limitations of the previous understanding of the pathogenesis of this disease.

[0033] (2) The present invention first proves at the cellular and animal levels that KLF4 can inhibit the progression of myelofibrosis. At the cellular level, a myelofibrosis model cell line with specifically up-regulated KLF4 expression is constructed. By means of EDU cell proliferation experiment, colony formation experiment and ELISA detection, it is found that overexpression of KLF4 can inhibit the proliferation of malignant hematopoietic cells and reduce the release level of inflammatory factors; at the animal level, by constructing a mouse xenograft model of myelofibrosis and detecting indicators such as spleen size, blood biochemistry and immunohistochemistry, it is proved that high expression of KLF4 can delay the progression of myelofibrosis. This multi-level research provides a solid experimental basis for the application of KLF4 in the treatment of myelofibrosis.

[0034] (3) The present invention first clarifies the specific molecular mechanism by which KLF4 inhibits the progression of myelofibrosis. By screening the differential genes of myelofibrosis model cells after high expression of KLF4 by RNA-seq, it is found that the expression level of RELN gene is significantly up-regulated and the differential genes are mainly enriched in the PI3K / AKT signaling pathway. KLF4 can specifically bind to the promoter of RELN gene, target and up-regulate the expression of RELN, and then inhibit the PI3K / AKT signaling pathway, and finally inhibit the progression of myelofibrosis. This discovery lays a theoretical foundation for the development of targeted therapeutic drugs against KLF4.

[0035] (4) The research results of the present invention indicate that KLF4 and its related regulatory mechanisms play a key role in the occurrence and development of myelofibrosis, which provides a new biomarker for the early diagnosis of myelofibrosis and also provides a direction for the development of treatment methods targeting KLF4, and is expected to improve the current situation of difficult diagnosis and limited treatment means of myelofibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The expression level of KLF4 was significantly decreased in patients with myelofibrosis and model cells;

[0037] Figure 1 A. The expression level of KLF4 in CD34 + cells of patients with myelofibrosis was significantly decreased (mRNA level); Figure 1 B. The expression level of KLF4 in CD34 + cells of patients with myelofibrosis was significantly decreased (protein level); Figure 1 C. Based on the analysis of the GEO database, the expression level of KLF4 in patients with myelofibrosis was significantly decreased compared with that of healthy volunteers; Figure 1 D. The expression level of KLF4 in the myelofibrosis model cell line was significantly decreased compared with the expression of KLF4 in the plasma of healthy volunteers;

[0038] Figure 2 After the demethylation of the KLF4 gene promoter, the proliferation and colony formation ability of malignant tumor cells can be inhibited;

[0039] Figure 2 A. GEO data analysis showed that the KLF4 promoter was hypermethylated in CD34 + cells of patients with myelofibrosis; Figure 2 B. After demethylation with the DNA methyltransferase inhibitor 5-Azacytidine, the expression level of KLF4 in malignant CD34 + cells of patients with myelofibrosis was up-regulated in a dose-dependent manner; Figure 2 C. After demethylation with the DNA methyltransferase inhibitor RG108, the expression level of KLF4 in the myelofibrosis model cell line was up-regulated; Figure 2 D. After demethylation with the DNA methyltransferase inhibitor 5-Azacytidine, the colony formation ability of malignant CD34 + cells of patients with myelofibrosis was inhibited; Figure 2 E. After demethylation with the DNA methyltransferase inhibitor RG108, the colony formation ability of the myelofibrosis model cell line was inhibited; Figure 2F. After demethylation with the DNA methyltransferase inhibitor RG108, the expression of a series of proliferation-related genes in the myelofibrosis model cell line changed significantly, and cell proliferation was inhibited;

[0040] Figure 3 To specifically up-regulate the expression of KLF4 and inhibit the clonal proliferation ability of malignant tumor cells at the cellular level;

[0041] Figure 3 A. Up-regulate the expression level of KLF4 in the myelofibrosis model cells (hel92.1.7 cell line). The EDU detection results show that the proliferation ability of malignant tumor cells is inhibited; Figure 3 B. Up-regulate the expression level of KLF4 in the myelofibrosis model cells (set2 cell line). The EDU detection results show that the proliferation ability of malignant tumor cells is inhibited; Figure 3 C. Up-regulate the expression level of KLF4 in the myelofibrosis model cells (hel92.1.7 cell line). The colony formation assay results show that the colony formation ability of malignant tumor cells is significantly inhibited; Figure 3 D. Up-regulate the expression level of KLF4 in the myelofibrosis model cells (set2 cell line). The colony formation assay results show that the colony formation ability of malignant tumor cells is significantly inhibited;

[0042] Figure 4 Specifically up-regulating the expression of KLF4 can inhibit the release of inflammatory factors in the bone marrow microenvironment;

[0043] Figure 4 A: Co-culture protocol of myelofibrosis model cells (hel92.1.7 cell line, set2 cell line) and mesenchymal stem cell line (HS5 cell line) in vitro; Figure 4 B: After co-culturing the set2 cell line and the HS5 cell line, the ELISA detection results show that after targetedly up-regulating the expression level of KLF4, the expression levels of a series of pro-inflammatory factors decrease; Figure 4 C: After co-culturing the hel92.1.7 cell line and the HS5 cell line, the Luminex detection results show that after targetedly up-regulating the expression level of KLF4, the expression levels of a series of pro-inflammatory factors decrease;

[0044] Figure 5 Overexpressing KLF4 at the in-vivo level can reduce the burden of myelofibrosis in mice;

[0045] Figure 5 A: Procedure for constructing a myelofibrosis pathological model by xenotransplanting the human-derived hel92.1.7 cell line into NSG mice; Figure 5B: Spleen sizes of mice in the normal saline group (sham group), the group injected with the control hel92.1.7 cell line (IRES group), and the group injected with the hel92.1.7 cell line overexpressing KLF4 (KLF4 group); Figure 5 C: Spleen / body weight ratios of xenograft mice under different injection conditions. After overexpressing KLF4, the spleen burden of mice was significantly reduced; Figure 5 D: Peripheral blood indexes of xenograft mice under different injection conditions; Figure 5 E: Masson staining results of spleens of xenograft mice under different injection conditions; Figure 5 F: Reticular fiber staining results of spleens of xenograft mice under different injection conditions; Figure 5 G: Proportion of malignant white blood cells in the spleen (upper) and bone marrow (lower) of xenograft mice under different injection conditions;

[0046] Figure 6 For the specific molecular mechanism by which KLF4 regulates the proliferation of hematopoietic cells in myelofibrosis;

[0047] Figure 6 A: KEGG pathway enrichment map of RNA-seq differential genes after targetedly upregulating KLF4 expression in the myelofibrosis model cell line. The results show that the differential genes are mainly enriched in the PI3K / AKT signaling pathway; Figure 6 B: Western Blot verification results show that after the upregulation of KLF4 expression, there is no significant difference in the total protein level of AKT, but the phosphorylation level of serine at site 473 is significantly reduced; Figure 6 C: qPCR verification results show that after the upregulation of KLF4 expression, the expression level of the downstream target gene CDK20 of the PI3K / AKT signaling pathway is significantly downregulated; Figure 6 D: Volcano plot of differential genes after targetedly upregulating KLF4 expression, in which the expression of RELN is significantly upregulated; Figure 6 E: Combined analysis results of the TCGA database and the GTEx database show that the expression level of RELN is generally downregulated in patients with myeloid leukemia; Figure 6 F: Analysis results of the JASPAR and UCSC databases show that KLF4 is a direct transcription factor of the RELN gene; Figure 6 G: Analysis results of CHIP-seq data of KLF4 in the CistromeDB database show that KLF4 directly binds to the promoter region of the RELN gene; Figure 6 H: Dual-luciferase reporter gene assay results confirm that KLF4 is a direct transcription factor of the RELN gene (left), and targetedly upregulates the expression of RELN (right). Figure 6 I: The above results confirm that there is a KLF4-RELN-AKT1-CDK20 signaling axis in hematopoietic cells of patients with myelofibrosis to regulate the malignant proliferation of hematopoietic cells. Detailed implementation methods

[0048] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can be made. These all belong to the protection scope of the present invention.

[0049] For all raw materials of the present invention, there are no special restrictions on their sources, and those purchased on the market or prepared according to conventional methods well-known to those skilled in the art are all acceptable. Specifically, see Tables M1 to M4.

[0050] Table M1. Reagent purchase manufacturers and catalog numbers

[0051]

[0052]

[0053] Table M2. Reagent preparation

[0054]

[0055] Table M3. Experimental instruments

[0056]

[0057]

[0058] Table M4. Experimental consumables

[0059]

[0060]

[0061] In the first stage of the present invention, the expression level of KLF4 in hematopoietic cells of patients with myelofibrosis was explored. Quantitative polymerase chain reaction (qPCR) and flow cytometry were performed on bone marrow hematopoietic stem cells (CD34 + cells) of patients with myelofibrosis, and it was found that the expression levels of KLF4 mRNA and protein in CD34 + cells of patients with myelofibrosis were down-regulated compared with those of healthy volunteers. Further, the results of GEO data analysis showed that the expression of KLF4 was significantly down-regulated in patients with myelofibrosis. The results of this stage indicate that KLF4 is generally pathologically down-regulated in malignant hematopoietic cells of patients with myelofibrosis.

[0062] In the second stage of the present invention, the reason for the down-regulation of KLF4 in hematopoietic cells of patients with myelofibrosis was explored. After demethylating the patient's CD34 + cells and the myelofibrosis model cell line with a DNA methyltransferase inhibitor (RG108 / 5-Azacytidine), the ability of malignant hematopoietic cell clone formation could be inhibited, and at the same time, the expression level of KLF4 was up-regulated. The results of this stage indicate that the KLF4 promoter is inhibited by methylation in malignant hematopoietic cells.

[0063] In the third stage of the present invention, the effect of KLF4 expression level on myelofibrosis was explored at the cellular level. First, a myelofibrosis model cell line with specifically up-regulated KLF4 expression was constructed. Further, through the EDU cell proliferation experiment and the clone formation experiment, the proliferation ability of malignant hematopoietic cells was detected. The results showed that the proliferation and cloning ability of malignant hematopoietic cells were inhibited after overexpression of KLF4; by co-culturing the myelofibrosis model cell line with the mesenchymal cell line HS-5 to simulate the bone marrow microenvironment, and ELISA was used to detect the release level of inflammatory factors. The results showed that the release level of inflammatory factors decreased after overexpression of KLF4. The results of this stage indicate that overexpression of KLF4 at the cellular level can alleviate the progression of myelofibrosis by inhibiting cell proliferation and inhibiting the release of inflammatory factors.

[0064] In the fourth stage of the present invention, the effect of KLF4 expression level on myelofibrosis was explored at the in vivo level of animals. First, a mouse xenograft myelofibrosis model was constructed. Further, by detecting indicators such as mouse spleen size, blood biochemistry, and immunohistochemistry, the effects of transplanting myelofibrosis cell lines with different KLF4 expression levels on mice were clarified. The results of this stage indicate that high expression of KLF4 at the in vivo level can delay the progression of myelofibrosis.

[0065] In the fifth stage of the present invention, the specific molecular mechanism by which KLF4 affects the progression of myelofibrosis was explored. First, differential genes of myelofibrosis model cells after high expression of KLF4 were screened by RNA-seq. The results of KEGG enrichment analysis showed that the differential genes were mainly enriched in the PI3K / AKT signaling pathway. Further, Western Blot experiments confirmed that after the up-regulation of KLF4 expression, the phosphorylation level of serine at position 473 of AKT1 protein decreased, the activity of the AKT1 pathway was inhibited, and the expression of its downstream target gene CDK20 was down-regulated; on the other hand, the expression of RELN among the differential genes was significantly up-regulated. Through the analysis of CHIP-seq data results and dual-luciferase reporter gene experiments, it was confirmed that KLF4 could specifically bind to the promoter of the RELN gene and target the up-regulation of RELN expression. Previous studies have confirmed that RELN can regulate the activity of the PI3K / AKT signaling pathway. Therefore, we believe that there is a KLF4-RELN-AKT1-CDK20 signaling axis to mediate the malignant proliferation of hematopoietic cells, thereby regulating the progression of myelofibrosis.

[0066] More specifically, the present invention will be described in detail in combination with the following examples:

[0067] Example 1. Exploration of the expression level of KLF4 in hematopoietic cells of patients with myelofibrosis.

[0068] (1) Detection of the expression level of KLF4 in hematopoietic stem cells of patients with myelofibrosis.

[0069] 1. Detection of the expression level of KLF4 mRNA in hematopoietic stem cells of patients with myelofibrosis by qPCR.

[0070] 1) RNA extraction. Hematopoietic stem cells of patients with myelofibrosis were collected into 1.5 mL EP tubes, centrifuged at 1000 rpm for 5 min, the supernatant was aspirated and discarded, washed twice with PBS, then 1 mL of Trizol was added, shaken on a normal temperature shaker for 10 min, repeatedly pipetted, 0.2 mL of chloroform was added, shaken vigorously and mixed evenly, and left to stand at room temperature for 3 min, then centrifuged at 12000 rpm for 10 min in a 4 °C centrifuge. Carefully aspirate the supernatant (about 500 μL) into a new 1.5 mL EP tube, add 0.5 mL of isopropanol, gently invert and mix evenly, leave at room temperature for 10 min, then transfer to a 4 °C centrifuge and centrifuge at 12000 rpm for 10 min. Aspirate and discard the supernatant, add 1 mL of 75% ethanol, gently invert and mix evenly, then transfer to a 4 °C centrifuge and centrifuge at 8500 rpm for 5 min. Aspirate and discard the supernatant, dry the precipitate in a fume hood, add 30 μL of DEPC water, and place it in a 55 °C metal bath for 10 min to dissolve the precipitate. Finally, aspirate 1.5 μL of the product and measure the concentration with a nanodrop for standby.

[0071] 2) Reverse transcription of RNA into cDNA.

[0072] First, remove genomic DNA. After preparing a mixture of the following system, react at 42 °C for 2 min:

[0073] Table 1. RNA Removal of Genomic DNA

[0074] Reagent Volume 5X gDNA Eraser Buffer 2.0 μL gDNA Eraser 1.0 μL The above Total RNA 1000 ng <![CDATA[RNase Free ddH20]]> To 10 μL

[0075] Further perform RNA reverse transcription. After preparing the system in Table 2, perform reverse transcription according to the procedure described in Table 3:

[0076] Table 2. RNA Reverse Transcription Reaction System

[0077] Reagent Volume The reaction solution in Table 1 above 10 μL Prime Script RT Enzyme Mix1 1.0 μL RT Primer Mix 1.0 μL 5X Prime Script Buffer 2 4.0 μL <![CDATA[RNase Free ddH20]]> 4.0 μL

[0078] Table 3. RNA Reverse Transcription Reaction Procedure

[0079]

[0080]

[0081] 3) Detect the level of KLF4 mRNA by qPCR.

[0082] Using GAPDH as an internal reference and SYBR as a fluorescent dye, the reaction system is 10 μL, and the reaction is carried out in a 96-well plate. Statistical calculations are performed using the 2 -ΔΔc(t) method. Prepare the reaction solution according to the system in Table 4 and perform the reaction according to the procedure described in Table 5:

[0083] Table 4. qPCR Reaction System

[0084] Reagent Volume qPCR SYBR Green Master 5 μL Forward Primer(10 μM) 0.2 μL Reverse Primer(10 μM) 0.2 μL cDNA 0.8 μL <![CDATA[ddH2O]]> 3.8 μL

[0085] Table 5. qPCR Reaction Procedure

[0086]

[0087] 2. Detect the expression level of KLF4 protein in hematopoietic stem cells of patients with myelofibrosis by flow cytometry.

[0088] Collect hematopoietic stem cells from patients with myelofibrosis, and centrifuge at 1000 rpm for 5 min to collect cell pellets. Add 1 mL of PBS and gently pipette to wash the cells. Filter the cell suspension through a 40 μM cell strainer and transfer it to a 15 mL centrifuge tube, then centrifuge at 1000 rpm for 5 min. Add 1 mL of PBS to count the cells. Select 100,000 cells, fix them with 4% paraformaldehyde, and wash three times with PBS. Then permeabilize the cells with 0.1% Triton X-100 for 10 - 20 min, wash once with PBS, and block with 1% BSA for 1 h. After washing three times with PBS, incubate overnight at 4°C in the dark with KLF4 antibody. The next day, aspirate the antibody and wash three times with PBS. Subsequently, incubate with the secondary antibody at room temperature for 2 h, wash three times with PBS, resuspend the cells with 0.5 mL of flow cytometry loading buffer, and transfer them to a BD flow cytometry tube for detection on a flow cytometer.

[0089] (2) Bioinformatics analysis of the expression level of KLF4 in hematopoietic stem cells of patients with myelofibrosis in the GEO database.

[0090] Select a group of samples of patients with myelofibrosis (GSE9827) in the GEO database and analyze the expression level of KLF4 among the differentially expressed genes in the samples. The GEO (Gene Expression Omnibus) database is a public gene expression data repository maintained by the National Center for Biotechnology Information (NCBI) of the United States and is also one of the largest public databases of gene expression data globally. It collects a large amount of gene expression profiles, microarray data, high-throughput sequencing data, etc. submitted by researchers from all over the world, covering research data on various species, tissues, and disease states. Researchers can obtain data from this database for free for further bioinformatics analysis, verification of research hypotheses, etc.

[0091] (3) Detect the expression level of KLF4 mRNA in peripheral blood of healthy volunteers and myelofibrosis model cell lines by qPCR.

[0092] Refer to the specific steps in the above result (1) for the experimental procedure, which will not be elaborated here. Specifically, the cells used are peripheral blood of healthy volunteers and myelofibrosis model cell lines.

[0093] The specific experimental results are as Figure 1 shown. Figure 1 A shows that the expression of KLF4 at the mRNA level in CD34 + cells of patients with myelofibrosis is significantly decreased; Figure 1 B shows that the expression of KLF4 at the protein level in CD34 + cells of patients with myelofibrosis is significantly decreased; Figure 1 C shows that based on the analysis of the GEO database, the expression level of KLF4 in patients with myelofibrosis is significantly decreased compared with that of healthy volunteers;Figure 1 D indicates that the expression level of KLF4 in the myelofibrosis model cell line is significantly lower than that in the plasma of healthy volunteers. In summary, the KLF4 gene is significantly downregulated in patients with myelofibrosis and myelofibrosis cell lines.

[0094] Example 2. Exploration of the methylation level of the KLF4 promoter in hematopoietic cells of patients with myelofibrosis.

[0095] (1) Bioinformatics analysis of the methylation level of the KLF4 promoter in hematopoietic stem cells of patients with myelofibrosis in the GEO database.

[0096] Select a group of methylation sequencing samples (GSE42721) of patients with myelofibrosis in the GEO database and analyze the methylation level of the KLF4 promoter in the samples.

[0097] (2) Detection of the effect of demethylating inhibitors on the colony-forming ability of hematopoietic stem cells / bone marrow fibrosis model cells of patients with myelofibrosis.

[0098] 1. Preparation of methylcellulose medium

[0099] Purchase MethoCult TM H4435 medium and thaw and aliquot it according to the instructions. Note that it needs to be gently mixed to avoid generating bubbles.

[0100] 2. Pretreatment of cells

[0101] The isolated cells from patients with myelofibrosis were cultured with 0 nM, 200 nM, and 500 nM of 5-Azacytidine respectively and made into cell suspensions with a concentration of 10,000 cells / mL for standby; the myelofibrosis model cells were pretreated with 0 μM and 20 μM of RG108 and made into cell suspensions with a concentration of 10,000 cells / mL for standby.

[0102] 3. Seeding of cells

[0103] The cell suspension (100 μL) and the methylcellulose medium (1 mL) were gently pipetted and mixed in a 1:10 ratio, inoculated into a 6-well plate, and the culture dish was gently shaken to make the mixture evenly distributed. Let it stand for 5 - 10 minutes, and after the mixture solidifies, place the culture dish in an incubator at 37 °C and 5% CO2.

[0104] 4. Colony counting and analysis

[0105] After culturing for 10 - 14 days, use an inverted microscope to count the colonies. The colony-forming efficiency CFE (%) = number of colonies / number of inoculated cells × 100%, and compare the number of colonies between different experimental groups.

[0106] (3) Detect the effect of the demethylating inhibitor on the expression level of KLF4 in hematopoietic stem cells of patients with myelofibrosis / myelofibrosis model cells by qPCR.

[0107] The steps of cell RNA extraction, reverse transcription and qPCR refer to Example 1 and will not be elaborated here. In particular, the cells used here are hematopoietic stem cells of patients with myelofibrosis and myelofibrosis model cells respectively.

[0108] The experimental results are as Figure 2 shown. Figure 2 A shows that GEO data analysis shows that the KLF4 promoter in CD34+ cells of patients with myelofibrosis is highly methylated; Figure 2 B shows that after demethylation with the DNA methyltransferase inhibitor 5-Azacytidine, the expression level of KLF4 in malignant CD34 + cells of patients with myelofibrosis is up-regulated in a dose-dependent manner; Figure 2 C shows that after demethylation with the DNA methyltransferase inhibitor RG108, the expression level of KLF4 in the myelofibrosis model cell line is up-regulated; Figure 2 D shows that after demethylation with the DNA methyltransferase inhibitor 5-Azacytidine, the colony formation ability of malignant CD34 + cells of patients with myelofibrosis is inhibited; Figure 2 E shows that after demethylation with the DNA methyltransferase inhibitor RG108, the colony formation ability of the myelofibrosis model cell line is inhibited; Figure 2 F shows that after demethylation with the DNA methyltransferase inhibitor RG108, the expression of a series of proliferation-related genes in the myelofibrosis model cell line is significantly changed and cell proliferation is inhibited. In summary, the KLF4 promoter in CD34 + cells of patients with myelofibrosis and the myelofibrosis model cell line is inhibited by methylation, resulting in a decrease in KLF4 expression under pathological conditions. After demethylating the KLF4 promoter region with a DNA methyltransferase inhibitor and up-regulating the KLF4 expression level, the proliferation and colony formation ability of malignant tumor cells are significantly inhibited.

[0109] Example 3. Explore the effect of KLF4 expression level on the proliferation of myelofibrosis cells at the cellular level.

[0110] (1) Construct a myelofibrosis model cell with overexpressed KLF4.

[0111] 1. Construct a vector for overexpressing the human KLF4 gene.

[0112] After extracting the total RNA of the myelofibrosis model cells (hel 92.1.7 cell line / set2 cells) and reverse transcribing it into cDNA, using the cDNA as a template, PCR amplification was performed with the primer sequences shown in Table 6 below to obtain the human KLF4 sequence. The specific amplification system and PCR reaction program are shown in Table 7 and Table 8 below.

[0113] Table 6. Primer sequences for human KLF4 amplification

[0114] Primer Name Primer Sequence h-KLF4-Fw cgGAATTCATGAGGCAGCCACCTGGCGAGT h-KLF4-Rv cgcGGATCCTTAAAAATGCCTCTTCATGT

[0115] Table 7. PCR reaction system

[0116] Reagent Volume h-KLF4-Fw(10 μM) 2 μL h-KLF4-Rv(10 μM) 2 μL cDNA 1 μL 2x Q5 PCR mix 25 μL <![CDATA[ddH2O]]> 20 μL

[0117] Table 8. PCR reaction program

[0118]

[0119] After identifying and purifying the above DNA products by agarose gel electrophoresis, they were mixed with the PIRES vector according to the system shown in Table 9 respectively, and then digested at 37°C for 3 h:

[0120] Table 9. Digestion system

[0121] Reagent Volume DNA product / PIRES vector 1 μg EcoRI endonuclease 1 μL BamHⅠ endonuclease 1 μL cutsmart buffer 5 μL <![CDATA[ddH2O]]> up to 50 μL

[0122] After purifying and recovering the above digestion products by agarose gel electrophoresis, they were mixed according to the system shown in Table 10 below, and then ligated at 16°C for 16 h:

[0123] Table 10. Ligation system

[0124] Reagent Volume Linearized vector after digestion 50 ng DNA product after digestion 15 ng T4 ligase 1 μL 10×ligase buffer 2 μL <![CDATA[ddH2O]]> up to 20 μL

[0125] After that, the ligation products were transformed into DH5α and plated (kanamycin resistance). After 16 h, single colonies were picked for colony PCR identification, and positive bacterial solutions were selected for sanger sequencing. If the sequence was correct, it indicated that the vector construction was successful, and subsequent preservation and plasmid extraction (as an overexpression vector) were carried out.

[0126] 2. Construct myelofibrosis model cells (hel 92.1.7 cell line and set2 cell line) overexpressing the human KLF4 gene.

[0127] (1) Resuscitation of hel 92.1.7 cell line / set2 cell line: Take out the cells in the liquid nitrogen tank and transfer them to a cryopreservation tube, gently shake the cryopreservation tube in a 37°C water bath. Take it out when there is still a small amount of crystallization in the tube, disinfect it with 75% ethanol and transfer it to a laminar flow hood; transfer all the cell suspension in the cryopreservation tube to a 15 mL centrifuge tube containing 4 mL of complete medium; centrifuge at 1000 rpm for 5 min, disinfect the centrifuge tube with 75% ethanol and transfer it to the laminar flow hood; discard the supernatant, resuspend the cell pellet with complete medium; aspirate and discard the complete medium in the six-well plate, and transfer the SRA01 / 04 cells to the six-well plate culture dish, then place it in the cell culture incubator.

[0128] (2) Plasmid transfection: When the cell density of the hel 92.1.7 cell line / set2 cell line grows to 60%-70%, transfect the plasmid constructed in step (1) into the hel 92.1.7 cell line / set2 cell line, and change the medium after 8 h and continue culturing.

[0129] (3) Subculture of hel 92.1.7 cell line / set2 cell line: When the hel 92.1.7 cell line / set2 cell line grows to an appropriate density, pipette the cell suspension and transfer it to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, disinfect the centrifuge tube with 75% ethanol and transfer it to the laminar flow hood. Discard the supernatant, add the corresponding volume of REMP-1640 complete medium to the cell pellet, and transfer the hel 92.1.7 cell line / set2 cell line to a new six-well plate culture dish according to the ratio of 1 well:6 wells, and continue suspension culture.

[0130] (4) G418-resistant clone screening: First, screen the hel 92.1.7 cell line / set2 cell line transfected with the plasmid with G418 antibiotic for two weeks, then use a flow cytometer to sort out GFP-positive cells for expansion culture. Subsequently, use qPCR and western Blot to detect the expression level of KLF4 in the cells. The results show that the expression level of KLF4 has increased by 50-100 times, indicating that the stable transfected cell line is successfully constructed.

[0131] (2) Methylcellulose colony formation ability experiment: Detect the colony formation ability of bone marrow fibrosis model cells with different expression levels of KLF4.

[0132] For the specific steps of detecting the methylcellulose colony formation ability, refer to Example 2. Different cells used here are the above constructed hel 92.1.7-IRES control cell line, hel 92.1.7-KLF4 overexpression cell line; set2-IRES control cell line, set2-KLF4 overexpression cell line.

[0133] (3) The EDU experiment was used to detect the proliferation ability of bone marrow fibrosis model cells with different expression levels of KLF4.

[0134] 1. Cell seeding

[0135] The hel 92.1.7-IRES control cell line and the hel 92.1.7-KLF4 overexpression cell line (or the set2-IRES control cell line and the set2-KLF4 overexpression cell line) were seeded in 6-well plates at a concentration of 1×10 5 cells / well and cultured in an incubator at 37°C and 5% CO2 until the logarithmic growth phase. The medium containing EDU was prepared according to the concentration recommended in the instruction manual. After replacing the cell medium with the EDU medium, the cells were incubated in the incubator for 2 h.

[0136] 2. Flow cytometry detection

[0137] According to the steps in the instruction manual, the cells were collected and fixed with paraformaldehyde, neutralized with glycine, and permeabilized with Triton X-100. The APOLLO reaction solution was prepared, and the cells were resuspended with the reaction solution. After incubating in the dark at room temperature for 10 minutes, the cells were washed and resuspended with the flow cytometry loading buffer for detection on the machine.

[0138] The experimental results are as Figure 3 shown. Figure 3 A shows that upregulating the expression level of KLF4 in bone marrow fibrosis model cells (hel92.1.7 cell line), the EDU detection results show that the proliferation ability of malignant tumor cells is inhibited; Figure 3 B shows that upregulating the expression level of KLF4 in bone marrow fibrosis model cells (set2 cell line), the EDU detection results show that the proliferation ability of malignant tumor cells is inhibited; Figure 3 C shows that upregulating the expression level of KLF4 in bone marrow fibrosis model cells (hel92.1.7 cell line), the results of the colony formation experiment show that the colony formation ability of malignant tumor cells is significantly inhibited; Figure 3 D shows that upregulating the expression level of KLF4 in bone marrow fibrosis model cells (set2 cell line), the results of the colony formation experiment show that the colony formation ability of malignant tumor cells is significantly inhibited. In summary, specifically upregulating the expression of KLF4 significantly inhibits the proliferation and cloning ability of bone marrow fibrosis model cell lines, demonstrating that upregulating the expression of KLF4 at the cellular level can inhibit the proliferation of malignant tumor cells.

[0139] Example 4. Explore the effect of KLF4 expression level on the inflammatory factor release ability of bone marrow fibrosis cells at the cellular level.

[0140] (1) Co-culture of bone marrow fibrosis model cells (hel 92.1.7 cell line / set2 cell line) and mesenchymal cell line HS5.

[0141] Inoculate the HS5 cell line into the lower chamber of a six-well trans-well plate at a concentration of 2×10 5 cells / well, and then inoculate the hel 92.1.7-IRES control cell line and the hel 92.1.7-KLF4 overexpressing cell line (or the set2-IRES control cell line and the set2-KLF4 overexpressing cell line) into the upper chamber of the six-well trans-well plate at a concentration of 2×10 5 cells / well, and culture them in an incubator at 37°C and 5% CO2 for 24 h.

[0142] (2) ELISA was used to detect the release levels of inflammatory factors in the co-culture supernatant.

[0143] Centrifuge the co-cultured cells described in step (1) at 1000 rpm for 5 min at 4°C, collect the supernatant, and discard the precipitate. Then centrifuge again at 12000 rpm for 5 min at 4°C, discard the precipitate, and aliquot the supernatant into 200 μL EP tubes at a system of 100 μL / tube, and store at -80°C for later use. Subsequently, send the above-aliquoted supernatant to Shanghai Youningwei Company for multi-factor Luminex detection to obtain the release levels of inflammatory factors during co-culture of different cell lines.

[0144] The experimental results are as Figure 4 shown. Figure 4 A is the in vitro co-culture protocol of the myelofibrosis model cells (hel92.1.7 cell line, set2 cell line) and the mesenchymal stem cell line (HS5 cell line); Figure 4 B shows that after co-culture of the set2 cell line and the HS5 cell line, the ELISA detection results show that after targeting and upregulating the expression level of KLF4, the expression levels of a series of pro-inflammatory factors decrease; Figure 4 C shows that after co-culture of the hel92.1.7 cell line and the HS5 cell line, the Luminex detection results show that after targeting and upregulating the expression level of KLF4, the expression levels of a series of pro-inflammatory factors decrease. In summary, specifically upregulating the expression of KLF4 significantly reduces the expression of a series of pro-inflammatory factors during in vitro co-culture of the myelofibrosis model cell line and the bone marrow mesenchymal cell line HS5, demonstrating that upregulating the expression of KLF4 at the cellular level can inhibit the release of inflammatory factors in the bone marrow microenvironment.

[0145] Example 5. Explore the effect of KLF4 expression level on myelofibrosis at the in vivo level of animals.

[0146] (1) Establish a mouse xenograft model of myelofibrosis

[0147] Purchase 4-week-old mice from Model Animal Research Center of Shanghai, and place them in a SPF animal room for two weeks of feeding (until 6 weeks old). Then collect the cultured myelofibrosis model cells, the hel 92.1.7-IRES control cell line and the hel 92.1.7-KLF4 overexpression cell line, and set aside. Divide the mice into three groups, with four parallel controls in each group. The three groups of mice are injected with 200 μL of normal saline (sham group), 2.5*10 6 / 200 μL of hel 92.1.7-IRES control cells (IRES group) and 2.5*10 6 / 200 μL of hel 92.1.7-KLF4 overexpression cells (KLF4 group), and continue to culture for 8 weeks, then sacrifice the mice to detect pathological indicators.

[0148] (2) Explore the effect of KLF4 expression level on myelofibrosis

[0149] 1. Blood biochemistry of mice

[0150] Before sacrificing the mice, weigh the mice and record. Then anesthetize the mice with anesthetic, and collect 300 μL of blood from the orbital sinus into an EDTA anticoagulation tube, store at 4°C and send it to Shanghai Sevier Biotechnology Monitoring Company on the same day, and measure the blood biochemical indicators of the mice with a mouse blood routine analyzer.

[0151] 2. Immunohistochemistry

[0152] Immediately sacrifice and dissect the mice after collecting blood from the eye socket, completely remove the spleen of the mice, measure the size and weight of the spleen of the mice and take pictures for record. Subsequently, simply process the spleen, liver and bone marrow of the mice and soak them in Sevier universal tissue fixative for more than 24 hours, and send them to Sevier Company for subsequent masson staining, HE staining, reticular fiber staining and immunohistochemical detection.

[0153] 3. Flow cytometry

[0154] Immediately sacrifice and dissect the mice after collecting blood from the eye socket, completely remove the spleen of the mice, gently grind the spleen to make a single cell suspension of the spleen, filter the suspension with a 40 μM cell strainer, adjust the cell concentration to 10,000 cells / 100 μL, and then add 5 μL of CD45 + / CD33 + antibody, incubate in the dark on ice for 30 minutes, wash once with PBS, resuspend with 500 μL of flow cytometry sample loading buffer, and immediately perform flow cytometry on the machine for detection.

[0155] Immediately after eye blood collection, the mice were sacrificed and dissected. The tibia and femur of the mice were removed, and the bone marrow cells were flushed out with a 1 mL syringe and pipetted to mix evenly into a bone marrow single cell suspension. The suspension was filtered through a 40 μM cell strainer, and the cell concentration was adjusted to 10,000 cells / 100 μL. Subsequently, 5 μL of CD45 + / CD33 + antibody was added. After incubating in the dark on ice for 30 min, it was washed once with PBS and resuspended with 500 μL of flow cytometry sample loading buffer, and immediately subjected to flow cytometry analysis on the machine.

[0156] The experimental results are as Figure 5 shown. Figure 5 A shows the process of constructing a pathological model of myelofibrosis by xenotransplanting the human-derived hel92.1.7 cell line into NSG mice; Figure 5 B shows the spleen sizes of mice in the normal saline group (sham group), the group injected with the control hel92.1.7 cell line (IRES group), and the group injected with the hel92.1.7 cell line overexpressing KLF4 (KLF4 group); Figure 5 C shows the spleen / body weight ratio of xenotransplanted mice under different injection conditions, indicating that the spleen burden of mice is significantly reduced after overexpressing KLF4; Figure 5 D shows the peripheral blood indexes of xenotransplanted mice under different injection conditions, and the related indexes are all reduced; Figure 5 E shows the results of masson staining of the spleen of xenotransplanted mice under different injection conditions; Figure 5 F shows the results of reticular fiber staining of the spleen of xenotransplanted mice under different injection conditions; Figure 5 G shows the proportion of malignant white blood cells in the spleen (upper) and bone marrow (lower) of xenotransplanted mice under different injection conditions. In summary, a xenograft model mouse of myelofibrosis was constructed by injecting the hel92.1.7 cell line. The results showed that compared with the mice injected with the ordinary hel92.1.7 cell line (hel92.1.7-IRES), the myelofibrosis burden of the mice injected with the hel92.1.7 cell line overexpressing KLF4 (hel92.1.7-KLF4) was significantly reduced, confirming that KLF4 can also inhibit the progression of myelofibrosis at the in vivo level.

[0157] Example 6. Exploring the downstream molecular mechanism by which KLF4 affects the progression of myelofibrosis using RNA-seq.

[0158] (I) Identification of differentially expressed genes by RNA-seq of myelofibrosis model cells

[0159] 1. RNA library construction and sequencing: Total RNA was extracted from hel 92.1.7-IRES control cells and hel92.1.7-KLF4 overexpressing cells using Trizol. The RNA quality was detected by Agilent 2200 and stored at -80 °C. RNA with RIN (RNA integrity) greater than 7.0 was used to construct cDNA libraries. Poly-A containing mRNA was purified from 1 μg of total RNA using oligo(dT) magnetic beads and fragmented into 200 - 600 bp fragments by incubation with divalent cations at 85 °C for 6 min. The fragmented RNA was used for the synthesis of first-strand and second-strand complementary DNA (cDNA). The cDNA fragments were end-repaired, A-tailed and ligated to indexed adapters. The purified first-strand cDNA was enriched by PCR to create cDNA libraries. The libraries were quality-controlled by Agilent 2200 and sequenced in paired-end 150 bp runs on the NovaSeq 6000.

[0160] 2. Data analysis: Hisat2 was used to align clean reads to the human reference genome (GRCh38, Ensembl104). HTseq was used to obtain gene counts and RPKM to determine gene expression levels. The DESeq2 algorithm was used for significance analysis, and P-values and FDRs (Fold Change > 2 or < 0.5; P-value < 0.05, FDR < 0.05) were used to identify differentially expressed genes (DEGs). KEGG pathway annotation was performed on the differentially expressed genes to precisely identify the key pathways and signal transduction pathways involved, thereby elucidating the biological significance of the differentially expressed genes.

[0161] 3. After the above data analysis, the results showed that the expression of RELN was significantly upregulated after KLF4 overexpression; the KEGG pathway enrichment results showed that the differentially expressed genes were mainly enriched in the PI3K / AKT signaling pathway.

[0162] (2) Dual-luciferase reporter gene assay confirmed the direct binding of KLF-RELN.

[0163] 1. Plasmid transfection: Plate HEK293T cells, inoculate the cells into a 12-well plate at 70% confluence. After 16 h, transfect the RELN-luciferase reporter gene plasmid and the KLF4 overexpression target gene plasmid. Set 3 replicates for each sample. Prepare the DNA and transfection reagent. The ratio for each well is target gene plasmid: reporter gene plasmid = 2:1, that is, KLF4-OE: RELN-Firefly-Renilla reporter gene plasmid = 0.7 μg: 0.35 μg. Dilute the DNA with 50 μL of DMEM and mix it with 4 μL of transfection reagent (PEI), incubate at room temperature for 15 - 20 min, add the mixture evenly to the cell samples. After 12 h of transfection, change to fresh complete medium. Conduct subsequent detection 36 - 48 h after transfection.

[0164] 2. Dual-luciferase reporter gene assay: Lyse the cells, add 300 μL of reporter gene cell lysis buffer. After sufficient lysis, centrifuge at 10000 - 15000 rpm for 3 - 5 min, and take the supernatant for determination.

[0165] 3. When measuring each sample, take 20 - 100 μL of the sample (keep the usage amount of the same batch of samples consistent), take an equal volume of reporter gene cell lysis buffer as the blank control. Add 100 μL of firefly luciferase detection working solution to each sample, and use a multifunctional microplate reader with the function of detecting chemiluminescence to measure the RLU (Relative light unit).

[0166] 4. Add 100 μL of Renilla luciferase detection working solution, mix well and then measure the RLU.

[0167] 5. Using Renilla luciferase as the internal reference, divide the RLU value obtained by measuring firefly luciferase by the RLU value obtained by measuring Renilla luciferase. Compare the activation degree of the target reporter gene among different samples according to the obtained ratio. The detection results confirm that KLF4 can directly target and bind to the promoter of the RELN gene.

[0168] The experimental results are as Figure 6 shown. Figure 6 A is the volcano plot of differentially expressed genes by RNA-seq after targetedly upregulating the expression of KLF4 in the myelofibrosis model cell line hel92.1.7; Figure 6 B shows the results of KEGG enrichment analysis. After targetedly upregulating the expression of KLF4, the differentially expressed genes are mainly enriched in the PI3K-AKT signaling pathway; Figure 6 C are the mainly upregulated differentially expressed genes after targetedly upregulating the expression of KLF4, among which the expression of RELN is significantly upregulated; Figure 6 D shows the combined analysis results of the TCGA database and the GTEx database. The expression level of RELN is generally downregulated in patients with myeloid leukemia; Figure 6Analysis results of JASPAR and UCSC databases in E show that KLF4 is a direct transcription factor of the RELN gene; Figure 6 Results of the dual-luciferase reporter gene assay in F confirm that KLF4 is a direct transcription factor of the RELN gene (left), and targets to up-regulate the expression of RELN (right). In summary, after the expression of KLF4 is targeted and up-regulated, the PI3K-AKT signaling pathway is inhibited, and the downstream target protein RELN of KLF4 is activated.

[0169] The present invention provides a new potential diagnostic and therapeutic target for myelofibrosis, KLF4. In hematopoietic stem cells of patients with myelofibrosis, the KLF4 promoter is hypermethylated and its expression level is significantly reduced. Activating the expression of KLF4 can inhibit the proliferation of malignant hematopoietic stem cells, inhibit the release of pro-inflammatory factors in the bone marrow microenvironment, and contain the progression of myelofibrosis from multiple pathways. In addition, KLF4 can activate the expression of the downstream target gene RELN, further inhibiting the proliferation of malignant hematopoietic cells, which provides a new therapeutic target for the treatment of myelofibrosis.

[0170] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

[0171] SEQ.ID.No.1 (Nucleic acid sequence of the KLF4 gene):

[0172]

[0173] Table 11. qPCR primers

[0174]

Claims

1. Use of the KLF4 gene in the preparation of a medicament for treating myelofibrosis, characterized in that, The nucleic acid sequence of the KLF4 gene is as shown in SEQ.ID.No.

1.

2. A drug for treating myelofibrosis, characterized in that, The drug comprises an active ingredient and an excipient; the active ingredient is any one of a DNA methyltransferase inhibitor, an activator of the RELN protein which is a downstream target of the KLF4 gene, a KLF4 gene therapy vector, or a PI3K / AKT pathway inhibitor.

3. The drug for treating myelofibrosis according to claim 2, characterized in that, The DNA methyltransferase inhibitor is selected from any one of 5-Azacytidine or RG108; The activator of the RELN protein which is a downstream target of the KLF4 gene is a small molecule compound, an antibody, a peptide compound or a nucleic acid molecule.

4. A drug for treating myelofibrosis according to claim 2, characterized in that, The excipient is a pharmaceutically acceptable carrier; The dosage form of the drug is any one of an oral preparation, an intravenous injection or a muscle injection preparation.

5. A method for constructing a myelofibrosis model cell with overexpression of KLF4, characterized in that, The method comprises the following steps: 1) Amplify KLF4 cDNA from human myelofibrosis model cell cDNA, and ligate the KLF4 cDNA sequence with the linearized vector pIRES to obtain a KLF4 overexpression vector; The human myelofibrosis model cell is selected from any one of hel92.1.7 or set2; 2) Transfect the KLF4 overexpression vector obtained in step 1) into the cell line of the human myelofibrosis model cell, and after screening, the myelofibrosis model cell with overexpressed KLF4 is obtained.

6. A method for establishing an animal model of myelofibrosis, characterized in that, The method comprises the following steps: (1) Culture the myelofibrosis model cell with overexpressed KLF4 as claimed in claim 5, collect the cells, adjust the cell suspension concentration, and place on ice; (2) Inject the myelofibrosis model cell suspension obtained in step (1) into an animal, and raise it to obtain a myelofibrosis animal model.

7. A biomarker combination for clinical detection of myelofibrosis, characterized in that, The biomarker combination comprises at least two of KLF4, RELN, AKT1 and CDK20.

8. A kit for detecting myelofibrosis, characterized in that, The kit detects the biomarker combination as claimed in claim 7.

9. Use of a RELN gene knockout mouse model, characterized in that, The RELN gene of the mouse model is knocked out for studying myelofibrosis; the promoter of the RELN gene is targeted and regulated by KLF4, and the targeted binding of KLF4 up-regulates the expression level of RELN, thereby inhibiting the PI3K / AKT pathway and inhibiting the progression of myelofibrosis.

10. Use of a RELN gene knockout mouse model according to claim 9, characterized in that, The mouse model is used for screening drugs for treating myelofibrosis.

Images