siRNA targeting HMGB1 gene sequence, HMGB1 knockdown cell model and its construction method and application

Abstract

The present invention belongs to the field of gene editing technology, and specifically relates to a siRNA targeting an HMGB1 gene sequence, an HMGB1 knockdown cell model, and a construction method and application thereof, wherein the siRNA sequence is shown in SEQ ID NO: 1 to SEQ ID NO: 6. The siRNA can target the HMGB1 gene and knock it down, and the knockdown efficiency can reach 71.89%.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene editing, and specifically relates to a siRNA targeting an HMGB1 gene sequence, an HMGB1 knockdown cell model, and a construction method and application thereof. Background Art

[0002] High mobility group protein B1 (HMGB1) is an abundant and ubiquitously expressed nuclear protein that is widely involved in biological processes such as DNA repair and gene transcription. In recent years, studies have found that HMGB1 plays a key role in immune inflammatory responses, especially in acute inflammatory diseases such as sepsis; specifically, HMGB1 activates immune cells through receptors (such as TLR4 or RAGE), leading to excessive inflammatory responses; therefore, knocking down the expression of the HMGB1 gene may effectively regulate the immune response and reduce inflammatory damage.

[0003] Based on the above, constructing a cell model with HMGB1 gene knockdown is of great significance for studying acute inflammatory diseases such as sepsis. At present, mouse bone marrow-derived macrophages (BMDM) are an in vitro model for studying immune response and inflammatory mechanisms, and they play an important role in studying immunopathological processes such as sepsis. In addition, BMDM can better simulate the function of macrophages in vivo and can be widely used to study immune response and inflammatory response. Therefore, constructing a bone marrow-derived macrophage model with HMGB1 gene knockdown is helpful for the study of acute inflammatory diseases such as sepsis.

[0004] Small interfering RNA (siRNA) is a highly effective gene silencing tool that can specifically inhibit the expression of target genes. Using siRNA to knock down HMGB1 in BMDM can further explore its role in immune inflammation and provide a more reliable cell model for the study of diseases such as sepsis. Summary of the invention

[0005] The present invention designs three siRNAs targeting the HMGB1 gene sequence and uses them in the construction of an HMGB1 knockdown cell model. The siRNA can effectively knock out the HMGB1 gene sequence in cells, and the knockdown efficiency is above 60%, and can reach up to 71.89%.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides an siRNA targeting the HMGB1 gene sequence, selected from any one of the following siRNAs:

[0008] A first siRNA, the sequence of which is shown in SEQ ID NO: 1 and SEQ ID NO: 2; or

[0009] A second siRNA, the sequences of which are shown in SEQ ID NO: 3 and SEQ ID NO: 4; or

[0010] The third siRNA, its sequence is shown in SEQ ID NO:5 and SEQ ID NO:6.

[0011] In another aspect, the present invention provides a method for constructing an HMGB1 knockdown cell model, comprising: using the siRNA of the present invention to knockdown the HMGB1 gene sequence in the cell to obtain the HMGB1 knockdown cell model.

[0012] Preferably, the construction method of the present invention comprises: (1) vortexing the transfection reagent; (2) mixing the treated transfection reagent and siRNA to obtain a transfection complex; (3) mixing the transfection mixture with cells to obtain an HMGB1 knockdown cell model.

[0013] Preferably, the construction method of the present invention satisfies one or more of the following conditions: (i) the transfected cells are primary cells; (ii) the transfected cells are macrophages; (iii) the transfection reagent is INTERFERin transfection reagent.

[0014] More preferably, the above-mentioned macrophages are derived from animal bone marrow.

[0015] More preferably, the above-mentioned animal is a mouse.

[0016] Preferably, the above-mentioned method for extracting macrophages from animal bone marrow comprises: (a) placing the tibia and / or femur of the animal limbs in an environment containing pre-cooled PBS, flushing the bone marrow cavity, and obtaining a precipitate after centrifugation of the flushing fluid; (b) adding the precipitate to a culture medium to suspend the precipitate and culture the macrophages; wherein the culture medium contains GM-CSF.

[0017] More preferably, in the above step (a), the bone marrow cavity is flushed using a syringe with a needle aperture of 22; and / or in step (b), the culture medium is DMEM complete culture medium; and / or the concentration of GM-CSF is 8ng / ml-12ng / ml.

[0018] In another aspect, the present invention provides an HMGB1 knockdown cell model, which is constructed by the construction method of the present invention.

[0019] In another aspect, the present invention provides a use of the HMGB1 knockdown cell model of the present invention as or in the preparation of a sepsis cell model, a tumor cell model or an autoimmune disease cell model.

[0020] The beneficial effects of the present invention include:

[0021] (1) The siRNAs provided in the present invention can all target the HMGB1 gene and knock it down, among which the first siRNA (hereinafter also referred to as siRNA mHMGB1-216 or si-HMGB1-216) with a sequence as shown in SEQ ID NO: 1 and SEQ ID NO: 2 has the highest knockdown efficiency, which can reach 71.89%; followed by the second siRNA (hereinafter also referred to as siRNA mHMGB1-547 or si-HMGB1-547) as shown in SEQ ID NO: 3 and SEQ ID NO: 4, whose knockdown efficiency can reach 69.73%; and finally, the third siRNA (hereinafter also referred to as siRNA mHMGB1-797 or si-HMGB1-797) as shown in SEQ ID NO: 5 and SEQ ID NO: 6, whose knockdown efficiency can reach 62.12%.

[0022] (2) The method for constructing an HMGB1 knockdown cell model based on siRNA in the present invention is simple, quick, and takes a short time to construct. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a flowchart for the extraction and culture of mouse bone marrow-derived macrophages;

[0024] Figure 2 The cell morphology of mouse bone marrow-derived macrophages (BMDMs) was observed under a light microscope (40x);

[0025] Figure 3 Flow cytometry was used to detect the cell purity of mouse bone marrow-derived macrophages (BMDMs);

[0026] Figure 4 To screen the optimal siRNA transfection concentration;

[0027] Figure 5 The knockdown efficiency of HMGB1 by different siRNAs is shown in Figure 2. DETAILED DESCRIPTION

[0028] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0029] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0030] The present invention provides an siRNA targeting the HMGB1 gene sequence, which is selected from any one of the following siRNAs:

[0031] A first siRNA, the sequence of which is shown in SEQ ID NO: 1 and SEQ ID NO: 2; or

[0032] A second siRNA, the sequences of which are shown in SEQ ID NO: 3 and SEQ ID NO: 4; or

[0033] The third siRNA, its sequence is shown in SEQ ID NO:5 and SEQ ID NO:6.

[0034] It should be noted that the three groups of siRNA pairs in the present invention can target the HMGB1 gene and knock it down, among which the first siRNA (hereinafter also referred to as siRNA mHMGB1-216 or si-HMGB1-216) with a sequence as shown in SEQ ID NO: 1 and SEQ ID NO: 2 has the highest knockdown efficiency, which can reach 71.89%; followed by the second siRNA (hereinafter also referred to as siRNA mHMGB1-547 or si-HMGB1-547) as shown in SEQ ID NO: 3 and SEQ ID NO: 4, whose knockdown efficiency can reach 69.73%; finally, the third siRNA (hereinafter also referred to as siRNA mHMGB1-797 or si-HMGB1-797) as shown in SEQ ID NO: 5 and SEQ ID NO: 6 has a knockdown efficiency of 62.12%.

[0035] The embodiment of the present invention further provides a method for constructing an HMGB1 knockdown cell model, comprising: using the siRNA of the present invention to knockdown the HMGB1 gene sequence in the cell to obtain the HMGB1 knockdown cell model.

[0036] It should be noted that the overexpression of the HMGB1 gene is associated with some existing inflammatory diseases. Inflammatory diseases can be studied by constructing an HMGB1 knockdown cell model. Therefore, in the present invention, an HMGB1 knockdown cell model can be constructed based on siRNA.

[0037] In some specific examples, the construction method of the present invention includes: (1) vortexing the transfection reagent; (2) mixing the treated transfection reagent and siRNA to obtain a transfection complex; (3) mixing the transfection mixture with cells, homogenizing, and reacting to obtain an HMGB1 knockdown cell model.

[0038] It should be noted that the "vortex" in the present invention is a conventional operation in the art and has no specific meaning. For example, vortex and centrifugation can be performed by vortex and centrifugal equipment; in addition, the reaction is generally carried out at 37°C-40°C.

[0039] In some specific examples, the construction method in the present invention satisfies one or more of the following conditions:

[0040] (i) The transfected cells are primary cells; specifically, primary cells have stronger physiological and immune responses and are more suitable for the construction of cell models;

[0041] (ii) The transfected cells are macrophages; specifically, macrophages can better study immune responses and inflammatory mechanisms;

[0042] (iii) The transfection reagent is INTERFERin transfection reagent; specifically, the transfection reagent is well known in the art, and INTERFERin transfection reagent is preferred in the present invention, because the transfection effect in the present invention is higher.

[0043] It should be noted that the above construction method may satisfy only one of the above conditions, but preferably satisfies all three of the above conditions at the same time, so that a primary cell HMGB1 knockdown macrophage model can be prepared.

[0044] In some specific examples, the macrophages are derived from animal bone marrow, especially mouse bone marrow.

[0045] It should be noted that mouse bone marrow-derived macrophages (BMDM) come from mice themselves, can more accurately simulate the immune response in vivo, and have phenotypes and functions that are closer to the physiological state; they can exhibit more immune phenotypes and functions and have a wider range of immune response capabilities, especially in the face of complex inflammatory responses (such as sepsis), and can simulate immune and inflammatory response processes that are closer to the body.

[0046] In some specific examples, the above-mentioned method for extracting macrophages from animal bone marrow includes: (a) placing the tibia and / or femur of the animal limbs in an environment containing pre-cooled PBS, flushing the bone marrow cavity, and obtaining a precipitate after centrifugation of the flushing fluid; (b) adding the precipitate to a culture medium to suspend the precipitate and culture macrophages; wherein the culture medium contains GM-CSF.

[0047] In some specific examples, in the above step (a), the bone marrow cavity is flushed with a syringe, and the needle aperture of the syringe is 22; and / or in step (b), the culture medium is DMEM complete culture medium; and / or the concentration of GM-CSF is 8ng / ml-12ng / ml, such as 9ng / ml, 10ng / ml or 11ng / ml, etc.

[0048] The embodiment of the present invention further provides an HMGB1 knockdown cell model, which is constructed by the construction method of the present invention.

[0049] The embodiment of the present invention also provides a use of the HMGB1 knockdown cell model of the present invention as or in preparing a sepsis cell model, a tumor cell model or an autoimmune disease cell model.

[0050] It should be noted that the HMGB1 knockdown cell model constructed in the present invention can be used in applications in sepsis, related middle inflammation and related autoimmune diseases.

[0051] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0052] 1. Extraction and culture of mouse bone marrow-derived macrophages

[0053] The extraction and culture process of mouse bone marrow-derived macrophages can be referred to Figure 1 The details are as follows: 6-8 week old C57BL / 6J male mice were selected, and the tibia and femur of the limbs were separated under sterile conditions. The two ends of the bones were cut and immersed in 75% alcohol for 5 minutes; after removing the muscle tissue, they were placed in a culture dish containing pre-cooled PBS and the bone marrow cavity was repeatedly flushed with a syringe needle with a bore of 22 until it was transparent. After the flushing fluid was centrifuged, the supernatant was discarded, and DMEM complete medium (DMEM high glucose basal medium + 10% FBS + 1% penicillin-streptomycin) containing 10ng / ml GM-CSF (granulocyte-macrophage colony stimulating factor) was added to suspend the precipitate, plate it, and culture it at a constant temperature for 7 days to obtain primary bone marrow macrophages.

[0054] After extracting mouse bone marrow cells, they were co-cultured with GM-CSF (granulocyte-macrophage colony stimulating factor) in DMEM complete medium (GM-CSF concentration was 10 ng / mL) under 5% CO2 and 37°C incubator. Under an optical microscope, it was observed that on the third day, the cells were basically completely attached to the wall and the cell morphology changed. On the fifth day, the cell volume increased significantly and the cell morphology was irregular or pseudopodia-like. After seven days of culture, flow cytometry was performed to detect cell maturity and purity ( Figure 2 ); In addition, flow cytometry results showed that the proportion of cells with double positive expression of mouse macrophage surface markers F4 / 80 and CD11b was 96% ( Figure 3 ), indicating that the macrophages were successfully cultured and their purity could meet the requirements of subsequent experiments.

[0055] 2. Transfection of BMDMs with HMGB1 knockdown plasmid

[0056] (I) Screening of optimal transfection concentration

[0057] FAM-NC (Shanghai Biotechnology) carries a green fluorescent marker. Different concentrations of FAM-NC have different fluorescent marker intensities in BMDM cells. The FAM-NC concentration corresponding to the highest fluorescence intensity is selected as the optimal transfection concentration, thereby determining the optimal transfection concentration of siRNA in the present invention. In the present invention, the FAM-NC group is set with a concentration gradient, which are control (cells + culture medium), 1nmol, 5nmol, 10nmol, 20nmol, 30nmol, 40nmol and 50nmol, and INTERFERin transfection reagent is used for transfection, as follows:

[0058] (1) Use a 24-well plate. For each well, dilute 6 pmol (84 ng) of siRNA duplex into 100 μL Opti-MEM (from Gibco) and mix by pipetting up and down.

[0059] (2) vortex the INTERFERin reagent (3000 rpm / min) for 5 seconds;

[0060] (3) adding 4 μL of vortexed INTERFERin reagent to 100 μL of siRNA duplex (containing siRNA duplex), and then immediately vortexing (3000 rpm / min) for 10 seconds to homogenize the mixture;

[0061] (4) The mixture was reacted at 25°C for 10 minutes to form a transfection complex between the siRNA duplex and INTERFERin; during the complex formation process, the growth medium of the 24-well plate was removed, and 0.5 mL of fresh pre-warmed DMEM basal medium (from Gibco) was added to each well;

[0062] (5) 100 μL of transfection complex was added to the above-extracted and cultured macrophages, and the plate was gently rotated for homogenization. The volume of the final system (including macrophages and transfection complex) was 600 μL, and the siRNA concentrations were control, 1 nmol, 5 nmol, 10 nmol, 20 nmol, 30 nmol, 40 nmol, and 50 nmol;

[0063] (6) The plate was incubated at 37°C and the cell fluorescence intensity was observed after 24 hours.

[0064] The results are as follows Figure 4 As shown (in the figure, FNC-1 is the 1 nmol group, FNC-2 is the 2 nmol group, and so on), the results show that FNC-10 has the strongest fluorescence intensity, indicating that the optimal transfection concentration is 10 nmol.

[0065] (II) siRNA knockdown efficiency test

[0066] In the following examples, the sequences of different siRNAs are shown in Table 1.

[0067] Table 1 siRNA sequences for HMGB1 knockdown

[0068]

[0069] (1) Use a 24-well plate. For each well, dilute 6 pmol (84 ng) of siRNA duplex into 100 μL Opti-MEM (from Gibco) and mix by pipetting up and down.

[0070] (2) vortex the INTERFERin reagent (3000 rpm / min) for 5 seconds;

[0071] (3) adding 4 μL of vortexed INTERFERin reagent to 100 μL of siRNA duplex (containing siRNA duplex), and then immediately vortexing (3000 rpm / min) for 10 seconds to homogenize the mixture;

[0072] (4) The mixture was reacted at 25°C for 10 minutes to form a transfection complex between the siRNA duplex and INTERFERin; during the complex formation process, the growth medium was removed and 0.5 mL of fresh pre-warmed DMEM basal medium (from Gibco) was added to each well;

[0073] (5) 100 μL of transfection complex was added to the above-extracted and cultured macrophages, and the plate was gently rotated for homogenization. The volume of the final system (including macrophages and transfection complex) was 600 μL, and the siRNA concentration was 10 nM;

[0074] (6) The plate was reacted at 37°C, and q-PCR was used to detect the RNA expression of HMGB1 after 24 h (q-PCR was used to obtain the mRNA expression of different knockdown sequences), and graphpad was used for statistical analysis to determine the knockdown efficiency of HMGB1 by the siRNA duplex.

[0075] According to the above method, the knockdown efficiency of HMGB1 by siRNA mHMGB1-547 (si-HMGB1-547), siRNA mHMGB1-216 (si-HMGB1-216) and siRNA mHMGB1-797 (si-HMGB1-797) was tested respectively; si-NC group and control group were set up at the same time; the test results are shown in Figure 5 As shown, the results showed that the three small interfering RNAs all knocked down HMGB1 to a certain extent, and the knockdown efficiency of siRNA mHMGB1-216 was the most significant.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. siRNA targeting HMGB1 gene sequence, characterized in that: Select any of the following siRNAs: A first siRNA, the sequence of which is shown in SEQ ID NO: 1 and SEQ ID NO: 2; or A second siRNA, the sequences of which are shown in SEQ ID NO: 3 and SEQ ID NO: 4; or The third siRNA, its sequence is shown in SEQ ID NO:5 and SEQ ID NO:

6.

2. A method for constructing a HMGB1 knockdown cell model, characterized in that: include: The HMGB1 gene sequence in the cell is knocked down using the siRNA described in claim 1 to obtain an HMGB1 knockdown cell model.

3. The construction method according to claim 2, characterized in that: include: (1) vortexing the transfection reagent; (2) mixing the treated transfection reagent and siRNA to obtain a transfection complex; (3) mixing the transfection mixture with cells to obtain an HMGB1 knockdown cell model.

4. The construction method according to claim 2 or 3, characterized in that: The construction method meets one or more of the following conditions: (i) The transfected cells are primary cells; (ii) the transfected cells are macrophages; (iii) The transfection reagent is INTERFERin transfection reagent.

5. The construction method according to claim 4, characterized in that: Macrophages originate from animal bone marrow.

6. The construction method according to claim 5, characterized in that: The animals are mice.

7. The construction method according to claim 5 or 6, characterized in that: Methods for extracting macrophages from animal bone marrow include: (a) placing the tibia and / or femur of the animal's limbs in an environment containing pre-cooled PBS, flushing the bone marrow cavity, and obtaining a precipitate after centrifugation of the flushing fluid; (b) adding the precipitate to a culture medium to suspend the precipitate and culture macrophages; wherein the culture medium contains GM-CSF.

8. The construction method according to claim 7, characterized in that: In step (a), the bone marrow cavity is flushed using a syringe, the needle diameter of the syringe is 22; and / or in step (b), the culture medium is DMEM complete culture medium; and / or the concentration of GM-CSF is 8ng / ml-12ng / ml. 9.HMGB1 knockdown cell model, characterized in that Constructed by the construction method described in any one of claims 1 to 8.

10. Use of the HMGB1 knockdown cell model according to claim 9 as or in the preparation of a sepsis cell model, a tumor cell model or an autoimmune disease cell model.

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