Application of JunD gene in regulating erythropoiesis

Abstract

The invention discloses application of a JunD gene in regulating erythropoiesis, and belongs to the technical field of biological medicines. The invention finds that the JunD gene has the function of regulating erythropoiesis, JunD expression is inhibited by constructing a JunD shRNA plasmid packaging lentiviral vector, and the differentiation and maturation process of an erythroid pedigree is promoted. The invention can provide an effective method for in vitro manufacturing of erythrocytes and research and development of drugs for promoting erythropoiesis of targeted JunD.

Description

Technical Field

[0001] The present invention relates to the application of JunD gene in regulating erythropoiesis, specifically including: 1) producing erythrocytes in vitro, 2) developing drugs promoting erythropoiesis based on JunD as a target, and belongs to the field of biomedicine technology. Background Art

[0002] Low red blood cell (RBC) production is one of the most common subhealth conditions in adults. With age, the body's ability to produce red blood cells gradually weakens due to physiological aging and pathological factors, such as anemia, myelodysplastic syndrome, leukemia, or other diseases of impaired immune function. Low red blood cell count is also closely related to a variety of clinically relevant diseases, including cardiovascular disease, cognitive impairment, insomnia, depression, and physical decline. Currently, in addition to blood transfusions and nutritional supplements, recombinant erythropoietin (EPO) and its analogs have become the main first-line treatment options for anemia caused by chronic kidney disease and cancer patients receiving chemotherapy.

[0003] In recent years, gene editing technology has been applied to treat congenital anemias such as β-thalassemia. However, there is still a lack of effective medical solutions for adult patients with chronic low red blood cell counts. Therefore, studying the targets and related mechanisms that regulate erythropoiesis is of great significance for studying treatment strategies for patients with chronic low red blood cell counts. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides an application of the JunD gene in regulating erythropoiesis, wherein the JunD lentivirus is transformed into a mouse erythroleukemia cell line (MEL) and a megakaryotic erythroid progenitor cell (MEP) to reduce the expression of the JunD gene or protein. Inhibition of JunD expression increases the proportion of Ter119-positive cells in MEL cells, upregulates the transcription levels of α / β-globin and GYPA, and also increases the erythroid colony-forming ability of MEP, and the effect is significant, indicating that the reduction of JunD expression promotes the differentiation and maturation process of the erythroid lineage. The present invention can provide an effective method for in vitro production of erythrocytes and the development of drugs targeting JunD to promote erythropoiesis.

[0005] The first object of the present invention is to provide an application of JunD gene in regulating erythropoiesis.

[0006] In one embodiment, the use promotes erythropoiesis by inhibiting JunD gene expression.

[0007] In one embodiment, the regulating erythropoiesis is promoting differentiation and maturation of the mammalian erythroid lineage.

[0008] In one embodiment, the mammals include but are not limited to humans and mice.

[0009] In one embodiment, the inhibition of JunD gene expression includes but is not limited to RNA interference, antisense nucleic acid technology, chemical induction, gene knockout, and CRISPR / Cas9 gene editing technology.

[0010] In one embodiment, the JunD gene expression is inhibited by constructing a JunD shRNA plasmid packaged lentiviral vector to infect cells and knock down the JunD gene, and the specific steps are:

[0011] (1) A shRNA targeting the knockdown of the JunD gene is constructed into an expression vector, and a mixture of the expression vector and the packaging plasmid is added to virus packaging cells for culture. After the culture, the cell supernatant is collected and filtered to obtain a lentivirus containing the expression vector.

[0012] (2) Infecting cells or cell lines with lentivirus, washing the virus after infection, and screening to obtain the JunD knockdown cell lines or cells.

[0013] In one embodiment, the shRNA sequence for targeting and knocking down the JunD gene is shown as SEQ ID NO.1-2.

[0014] SEQ ID NO.1:5'-GGAGGACCTGCACAAGAAAG-3'

[0015] SEQ ID NO.2:5'-GGCTGATCATCCAGTCCAACG-3'

[0016] In one embodiment, the virus packaging cells are 293T cells.

[0017] In one embodiment, the lentiviral vector is used to construct JunD knockdown mouse erythroleukemia cell line (MEL) and megakaryoerythroid progenitor cell (MEP).

[0018] In one embodiment, the mouse erythroleukemia cell line can be induced to differentiate into erythroid cells.

[0019] The second object of the present invention is to provide a JunD gene knockdown cell or cell line capable of differentiating into erythroid cells.

[0020] In one embodiment, the cell or cell line type includes but is not limited to MEL, MEP.

[0021] In one embodiment, the cells or cell lines are derived from, but not limited to, humans and mice.

[0022] In one embodiment, the MEL is stably transfected with an expression vector that targets and knocks down the JunD gene in a mouse erythroleukemia cell line.

[0023] In one embodiment, the MEP is stably transfected with an expression vector for targeted knockdown of the JunD gene in megakaryocyte progenitor cells.

[0024] The third object of the present invention is to provide a JunD gene knockdown cell or cell line capable of differentiating into erythroid cells for use in studying erythropoiesis.

[0025] In one embodiment, the applications include, but are not limited to, erythrocyte differentiation, hemoglobin synthesis, and drug screening.

[0026] A fourth object of the present invention is to provide a method for promoting erythropoiesis by inhibiting JunD gene expression to promote differentiation and maturation of erythroid lineage.

[0027] In one embodiment, the inhibition of JunD gene expression includes but is not limited to RNA interference, antisense nucleic acid technology, chemical induction, gene knockout, and CRISPR / Cas9 gene editing technology.

[0028] In one embodiment, the method involves constructing a JunD shRNA plasmid to package a lentiviral vector to infect cells, including but not limited to a mouse erythroleukemia cell line (MEL) and a megakaryocyte progenitor cell (MEP).

[0029] In one embodiment, the method promotes erythropoiesis by increasing the proportion of Ter119-positive cells, the transcription levels of α / β-globin and GYPA in MEL cells.

[0030] In one embodiment, the method promotes erythropoiesis by enhancing the erythroid colony-forming ability of MEPs.

[0031] Advantages and effects of the present invention:

[0032] The application of the JunD gene provided by the present invention in regulating erythropoiesis, for the first time discovered that inhibiting the JunD gene can promote the differentiation and maturation of the erythroid lineage. The present invention can increase the proportion of Ter119-positive cells in MEL cells by constructing a lentiviral vector to knock down the JunD gene, upregulate the transcription levels of α / β-globin and GYPA, and increase the erythroid colony-forming ability of primary MEPs. This discovery is expected to achieve in vitro production of erythrocytes by targeting JunD, and provide a new target for the treatment of diseases related to erythropoiesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1The results of successfully constructing a JunD-knockdown MEL cell line. A is qRT-PCR detection of JunD expression after virus infection of cells; B is immunoblotting detection of JunD protein expression after virus infection of cells. sh Scramble: control plasmid; shJund-1: JunD knockdown plasmid 1; shJund-2: JunD knockdown plasmid 2. n = 3, values ​​are expressed as Mean ± SD, ** represents P value < 0.01, *** represents P value < 0.001.

[0034] Figure 2 The results of JunD gene knockdown in the differentiation of erythroid progenitor cells into more mature erythrocytes. A is the number of Ter119-positive cells detected by flow cytometry before and after MEL-induced differentiation; B is the mRNA levels of globin and GYPA detected by qRT-PCR. Before differentiation: before induction of differentiation; sh Scramble: control plasmid; shJund-1: JunD knockdown plasmid 1. n = 3, values ​​are expressed as Mean ± SD, * represents P value < 0.05, ** represents P value < 0.01, *** represents P value < 0.001, **** represents P value < 0.0001.

[0035] Figure 3 The results of the enhanced colony formation ability of MEP cells after JunD gene knockdown are shown in Figure 1. A is the expression of JunD detected by qRT-PCR after virus infection; B is the result of CFU colony assay. sh Scramble: control plasmid; shJund-1: JunD knockdown plasmid 1. n = 3, values ​​are expressed as Mean ± SD, * represents P value < 0.05, ** represents P value < 0.01. DETAILED DESCRIPTION

[0036] definition

[0037] The terms used in this specification generally have their ordinary meanings in the art in the context of the present invention and in the specific context in which each term is used. Some terms used to describe the present invention are discussed below, or elsewhere in this specification, to provide practitioners with additional guidance on the description of the present invention. For convenience, some terms are emphasized, such as using italics and / or brackets. The use of these emphasizing does not affect the scope and meaning of these terms. Whether emphasized or not, in the same context, the scope and meaning of a term are the same. It should be understood that the same thing can be expressed in different ways. Therefore, different languages ​​and synonyms can be used for any one or more terms discussed herein, and a term is not listed according to whether it is elaborated or discussed in detail herein. Synonyms for certain terms are provided. One or more synonyms do not exclude the use of other synonyms. The use of embodiments anywhere in this specification containing examples of any term discussed herein is only illustrative and does not limit the scope and meaning of the present invention or any exemplary term. Similarly, the present invention is not limited to the various embodiments given in this specification.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. In case of conflict, the present text including the definitions will control.

[0039] Example

[0040] The apparatus, devices, methods and their associated results according to embodiments of the present invention are given below, but are not intended to limit the scope of the present invention. Please note that titles or subtitles may be used in the examples for the convenience of the reader and are not intended to limit the scope of the present invention. Furthermore, some theories are proposed and disclosed herein; however, whether right or wrong, as long as the present invention is implemented according to the present invention without regard to any particular implementation theory and scheme, they do not limit the scope of the present invention.

[0041] The present invention provides an application of a JunD gene in regulating erythropoiesis, wherein the application regulates erythropoiesis by inhibiting the expression of the JunD gene.

[0042] Optionally, the regulating erythropoiesis is promoting differentiation and maturation of the mammalian erythroid lineage.

[0043] Optionally, the mammals include but are not limited to humans and mice.

[0044] Optionally, the inhibition of JunD gene expression includes but is not limited to RNA interference, antisense nucleic acid technology, chemical induction, gene knockout, and CRISPR / Cas9 gene editing technology.

[0045] Optionally, the inhibition of JunD gene expression is to knock down JunD gene by constructing JunD shRNA plasmid packaging lentiviral vector to infect cells, and the specific steps are:

[0046] (1) A shRNA targeting the knockdown of the JunD gene is constructed into an expression vector, and a mixture of the expression vector and the packaging plasmid is added to virus packaging cells for culture. After the culture, the cell supernatant is collected and filtered to obtain a lentivirus containing the expression vector.

[0047] (2) Infecting cells or cell lines with lentivirus, washing the virus after infection, and screening to obtain the JunD knockdown cell lines or cells.

[0048] Optionally, the shRNA sequence for targeting and knocking down the JunD gene is shown in SEQ ID NO.1-2.

[0049] SEQ ID NO.1:5'-GGAGGACCTGCACAAGAAAG-3'

[0050] SEQ ID NO.2:5'-GGCTGATCATCCAGTCCAACG-3'

[0051] Optionally, the virus packaging cells are 293T cells.

[0052] Optionally, the lentiviral vector is used to construct JunD knockdown mouse erythroleukemia cell line (MEL) and megakaryoerythroid progenitor cell (MEP).

[0053] Optionally, the mouse erythroleukemia cell line can be induced to differentiate into erythroid cells.

[0054] The present invention also provides a JunD gene knockdown cell or cell line capable of differentiating into erythroid cells.

[0055] Optionally, the cell or cell line type includes but is not limited to MEL, MEP, and the cell or cell line source includes but is not limited to human and mouse.

[0056] Optionally, the MEL is stably transfected with an expression vector for targeted knockdown of the JunD gene in a mouse erythroleukemia cell line.

[0057] Optionally, the MEP is stably transfected with an expression vector for targeted knockdown of the JunD gene in megakaryocyte progenitor cells.

[0058] The present invention also provides the use of JunD gene knocked-down cells or cell lines capable of differentiating into erythroid cells in studying erythropoiesis.

[0059] Optionally, the applications include but are not limited to erythrocyte differentiation, hemoglobin synthesis, and drug screening.

[0060] The present invention also provides a method for promoting erythropoiesis, wherein the method promotes the differentiation and maturation of erythroid lineage by inhibiting the expression of JunD gene.

[0061] Optionally, the inhibition of JunD gene expression includes but is not limited to RNA interference, antisense nucleic acid technology, chemical induction, gene knockout, and CRISPR / Cas9 gene editing technology.

[0062] Optionally, the method involves constructing a JunD shRNA plasmid to package a lentiviral vector to infect cells, including but not limited to mouse erythroleukemia cell line (MEL) and megakaryocyte progenitor cells (MEP).

[0063] Optionally, the method promotes erythropoiesis by increasing the proportion of Ter119-positive cells, the transcription levels of α / β-globin and GYPA in MEL cells.

[0064] Optionally, the method promotes erythropoiesis by enhancing the erythroid colony-forming ability of MEPs.

[0065] The materials and methods involved in the following examples are as follows:

[0066] CFU colony assay: obtaining GFP by sorting + MEP cells; take a cell suspension containing 6600 cells, 33μL of 100× double antibody and PBS with serum (300μL for the three in total) and add them to 3mL of methylcellulose; after fully inverting and mixing, let it stand for 5min until the bubbles are basically above the liquid surface; use a 1mL syringe to draw 1mL of methylcellulose and seed it into a 35mm dish, and seed two replicate wells for each sample; place the 35mm dish in a large colony plate. In order to avoid water loss in the culture medium, an open culture dish filled with sterile water is generally placed in the colony plate, and culture is carried out in an incubator at 37°C and 5% CO2; after 10 to 14 days of culture, observe the formation of BFU-E colonies, count the number of colonies formed, and take pictures of the colonies under a microscope.

[0067] MEL cell line: The MEL cell line (Murine Erythroleukemia cell line) is derived from a leukemia-like disease in mice and is widely used to simulate and study the erythrocyte maturation process, the role of regulatory factors, hemoglobin synthesis, and the pathogenesis of related diseases. The MEL cell line in the embodiments of the present invention is a tool cell line in hematological research.

[0068] MEP cells: MEP cells (Megakaryocyte-Erythroid Progenitors) are multipotent progenitor cells in the bone marrow that can differentiate into megakaryocytes and erythrocytes. The mouse MEP cells in the present embodiment are obtained by the following method: magnetic bead sorting of MEP cells (Lin － ,Sca-1 － ,c-Kit + ,CD34 － ,CD16 / 32 － ).

[0069] Complete medium: RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS).

[0070] Methylcellulose-based culture medium (M3434 medium) was purchased from StemCell Technologies (Cat. No. 03434).

[0071] Example 1: Construction and identification of JunD lentivirus

[0072] 1. Virus packaging

[0073] (1) When 293T cells reach 80% to 90% confluence, they are subcultured into new culture dishes;

[0074] (2) Pour off the culture medium and wash the cells twice with 1 mL of PBS;

[0075] (3) Add 1 mL of Trypsin-EDTA solution, mix well, and incubate at 37°C for 2–3 min.

[0076] (4) Aspirate the trypsin solution, add 2 mL of DMEM culture medium containing 10% FBS, and pipette the cells to form a single-cell suspension;

[0077] (5) The cell suspension was transferred into a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 min.

[0078] (6) Add 1 mL of DMEM culture medium containing 10% FBS and pipette the cells to form a single-cell suspension;

[0079] (7) Inoculate the cell suspension into a new culture dish, add 9 mL of DMEM culture medium containing 10% FBS, mix well and culture in a 37°C, 5% CO2 incubator. When the cell density grows to 70-80%, prepare for transfection;

[0080] (8) Add 0.5 mL of sterile 0.9% NaCl solution to a sterile 1.5 mL centrifuge tube, add the shuttle plasmid and packaging plasmid containing the JunD shRNA sequence (SEQ ID NO.1-2) in proportion, mix well, take another 1.5 mL centrifuge tube, add 0.41 mL of sterile 0.9% NaCl solution, and then add 90 μL of PEI-4000K transfection reagent, mix well, and add the solution with transfection reagent to the plasmid solution. Finally, mix the two tubes of liquid and let stand at room temperature for 20 min;

[0081] (9) Add the transfection mixture dropwise to the culture dish, gently shake the culture dish back and forth to mix the complex, and incubate in a 37°C, 5% CO2 incubator for 8 to 12 hours;

[0082] (10) Remove the transfection solution by aspiration, add 10 mL of DMEM culture medium containing 10% FBS, and continue to culture in a 37°C, 5% CO2 incubator for 48 h. Collect the supernatant, which is the virus solution.

[0083] 2. Virus collection and concentration

[0084] (1) The cell supernatant was transferred into a 50 mL centrifuge tube and centrifuged at 4000 rpm for 4 min at 4°C.

[0085] (2) After low-speed centrifugation, pour the supernatant into a 50-mL syringe and filter with a 0.22-μm filter;

[0086] (3) ultracentrifuging the filtrate at 4°C, 25,000 rpm for 1 h 30 min;

[0087] (4) Collecting the virus concentrate and packaging it;

[0088] (5) Label the aliquoted virus solution and store it in a -80°C refrigerator.

[0089] 3. Virus titer detection

[0090] (1) When 293T cells reached 80-90% confluence, the culture medium was discarded and the cells were washed twice with PBS;

[0091] (2) Add 1 mL of Trypsin-EDTA solution, mix well, carefully remove the trypsin solution, and incubate at 37°C for 3–5 min.

[0092] (3) Add 2 mL of DMEM culture medium containing 10% FBS and pipette the cells to form a single-cell suspension;

[0093] (4) The single cell suspension was transferred into a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 min.

[0094] (5) According to 3×10 4 The cells were seeded into 96-well plates at a concentration of 10 cells / well, mixed, and cultured for 24 h;

[0095] (6) Dilute 10 μL of the lentiviral stock solution with DMEM containing 10% FBS;

[0096] (7) Aspirate the culture medium in the 96-well plate, add 100 μL of diluted virus solution to each well, and culture for 24 h;

[0097] (8) Aspirate the medium in the 96-well plate, add 100 μL of complete medium to each well, and continue culturing for 72 h;

[0098] (9) Use a flow cytometer to count fluorescent cells and calculate the virus titer based on the dilution factor.

[0099] Example 2: Construction of JunD knockdown MEL cell line

[0100] The present invention uses the JunD lentivirus prepared in Example 1 to treat the MEL cell line to knock down the JunD gene. The treatment steps are as follows:

[0101] (1) Diluting the virus: diluting the lentiviral stock solution with culture medium at a ratio of 1:20 to obtain a virus suspension;

[0102] (2) Cell counting: Count the cells at 20×10 5 Cells were taken out from the well and centrifuged at 1000 rpm for 5 min. The precipitate was resuspended with 500 μL of the virus suspension diluted in step (1) and placed in an incubator for culture. At the same time, a Jund blank plasmid was established as a control;

[0103] (3) After 12 to 24 h of culture, the cell culture medium was removed by centrifugation, 500 μL of complete culture medium was added, and the culture was continued overnight;

[0104] (4) Observe the results under an inverted fluorescence microscope 48 hours after virus infection;

[0105] (5) 72 hours after virus infection, the positive rate of virus infection was detected using flow cytometer.

[0106] qRT-PCR and immunoblotting results showed that the transcription and protein expression levels of JunD were significantly reduced compared with the control group, indicating that the JunD gene was successfully knocked down ( Figure 1 AB).

[0107] MEL cells can be induced to differentiate into erythrocytes by adding chemical inducers (such as DMSO), showing more erythrocyte characteristics and functions, which makes them a powerful model for studying erythrocyte differentiation and related gene regulatory networks. In the process of inducing MEL cell differentiation with 0.5% DMSO, it was found that inhibiting JunD gene expression significantly increased the proportion of Ter119 positive cells ( Figure 2 A), suggesting that JunD protein and its encoding gene may negatively regulate the maturation of erythroid progenitor cells.

[0108] In order to further study the effect of JunD on erythroid differentiation, qRT-PCR was used to detect the expression of α-globins, β-globins, the main protein chains of hemoglobin, and glycophorin-A (Gypa), the main sialic acid glycoprotein on the erythrocyte membrane, in JunD gene knockdown cells. The results showed that compared with MEL cells infected with JunD blank plasmid, the expression levels of α-globins, β-globins and glycophorin-A (Gypa) in JunD knockdown MEL cells were significantly increased. The above data show that JunD knockdown promotes the differentiation of erythroid cells into mature erythrocytes ( Figure 2 B).

[0109] Example 3: Construction of JunD knockdown MEP cells

[0110] After the mouse MEP cells are isolated in the present invention, the MEP cells are infected with the concentrated JunD lentivirus, and the specific steps are as follows:

[0111] (1) Virus dilution: dilute the lentiviral stock solution with culture medium at a ratio of 1:30;

[0112] (2) Cell counting: Count the cells in 1×10 5 Cells / well were removed and centrifuged at 500 g for 5 min. The pellet was resuspended with 100 μL of diluted virus suspension and placed in an incubator for culture. A control was also established;

[0113] (3) After 12 to 24 hours, the cell culture medium was removed by centrifugation, 100 μL of complete culture medium was added, and the culture was continued overnight;

[0114] (4) Observe the results under an inverted fluorescence microscope 48 hours after virus infection;

[0115] (5) 72 hours after virus infection, the positive rate of virus infection was detected using flow cytometer.

[0116] qRT-PCR results showed that infection of MEP cells with concentrated JunD lentivirus could reduce the transcription level of JunD ( Figure 3A). Through CFU colony assay, 2000 mouse MEP cells transfected with JunD sh Scramble and JunD shRNA were cultured in complete methylcellulose-based medium for 10 to 14 days. The results showed that JunD gene knockdown increased the colony formation ability of MEP ( Figure 3 B), indicating that the erythroid differentiation process of MEPs is enhanced.

[0117] The present invention relates to the sequence:

[0118] SEQ ID NO.1:5'-GGAGGACCTGCACAAGAAAG-3'

[0119] SEQ ID NO.2:5'-GGCTGATCATCCAGTCCAACG-3'

[0120] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. The use of JunD gene in regulating erythropoiesis, characterized in that: By inhibiting JunD gene expression, erythropoiesis is promoted. This application is not directly used for diagnosis or treatment.

2. The use according to claim 1, characterized in that: The application is to promote the differentiation and maturation of the erythroid lineage of mammals, and the mammals include humans and mice.

3. The use according to claim 1, characterized in that: The inhibition of JunD gene expression includes RNA interference, antisense nucleic acid technology, chemical induction, gene knockout, and CRISPR / Cas9 gene editing technology.

4. The use according to claim 1 or 3, characterized in that: The inhibition of JunD gene expression is to knock down JunD gene by infecting cells with JunDshRNA plasmid-packaged lentiviral vector, comprising the following steps: (1) constructing shRNA targeting the knockdown of JunD gene into an expression vector, adding the expression vector and packaging plasmid mixture to virus packaging cells for culture, collecting the cell supernatant after culture, and filtering to obtain the lentivirus containing the expression vector; (2) Infecting cells or cell lines with lentivirus, washing the virus after infection, and screening to obtain the JunD knockdown cell lines or cells.

5. The use according to claim 4, characterized in that: The lentiviral vector is used to construct JunD knockdown mouse erythroleukemia cell lines or megakaryocyte progenitor cells.

6. A cell or cell line with JunD gene knockdown capable of differentiating into erythroid cells, characterized in that: The types of cells or cell lines include erythroleukemia cell lines and megakaryocyte progenitor cells, and the sources of cells or cell lines include humans and mice.

7. The cell or cell line according to claim 6, characterized in that The erythroleukemia cell line is stably transfected with an expression vector for targeted knockdown of the JunD gene in the mouse erythroleukemia cell line.

8. The cell or cell line according to claim 6, characterized in that The megakaryotic erythroid progenitor cells are stably transfected with an expression vector for targeted knockdown of the JunD gene.

9. Use of the JunD gene knockdown cell or cell line according to claim 6 in studying erythropoiesis, characterized in that: The applications include erythrocyte differentiation, hemoglobin synthesis, and drug screening.

10. A method for promoting erythropoiesis, characterized in that: The method promotes the differentiation and maturation of erythroid lineage by inhibiting JunD gene expression.

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