Application of Hacl1 gene in the preparation of drugs for treating RNA virus infection-related diseases and maintaining innate immune homeostasis

Abstract

The present invention discloses the use of the Hacl1 gene in the preparation of drugs for treating RNA virus infection-related diseases and maintaining innate immune homeostasis, and relates to the field of biomedicine technology. The present invention finds that the transcription level of Hacl1 is significantly upregulated during the infection of eukaryotic cells by RNA viruses represented by NDV and VSV. After knocking down Hacl1 using siRNA, viral proliferation was significantly inhibited, while overexpression of HACL1 had a significant promoting effect on the proliferation of NDV virus in eukaryotic cells. Further testing found that knocking down Hacl1 could increase the transcription levels of IFNβ, ISG15, IFIT2 and MX1 in cells, thereby inhibiting the replication of the virus in cells. The above suggests that the HACL1 protein plays an important role in RNA virus-related infectious diseases and innate immune regulation, providing a new basis for the clinical participation of HACL1 in regulating innate immune responses, and has broad application prospects in the field of RNA virus vaccine development.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of the Hacl1 gene in preparing drugs for treating RNA virus infection-related diseases and maintaining innate immune homeostasis. Background Art

[0002] Fatty acids are compounds composed of carbon, hydrogen, and oxygen. The main chain is a repeating methylene sequence, and the end carries a carboxyl group. According to their length, they can be divided into short-chain fatty acids, medium-chain fatty acids, long-chain fatty acids, and very long-chain fatty acids; according to the number of carbon atoms, they can be divided into even-numbered fatty acids and odd-numbered fatty acids. Under aerobic conditions, fatty acids can be oxidized and decomposed to release energy. This process is called fatty acid oxidation (FAO). Fatty acid β-oxidation is the main pathway of fatty acid oxidation in the body and can supply a large amount of energy needed by the body. In addition, there are some special forms of fatty acid oxidation. For example, long-chain even-numbered fatty acids are catalyzed by monooxygenase and decarboxylase in microsomes to produce α-hydroxy fatty acids (2OH-FA), and the process in which 2OH-FA continues to oxidize and decarboxylate to produce odd-numbered carbon fatty acids is called α-oxidation of fatty acids (FAO). Figure 1 ).

[0003] Even-numbered fatty acids undergo alpha-oxidation in peroxisomes, catalyzed by endoplasmic reticulum-associated NAD(P)H-dependent monooxygenases and fatty acid alpha-hydroxylases (FA2H) to form 2OH-FAs. These 2OH-FAs are incorporated into sphingolipids, important components of cerebrosides and sulfatides. After activation to 2OH-acyl-CoA, these 2OH-FAs are cleaved by HACL1 and HACL2 to produce formyl-CoA and n-1 fatty aldehydes, which can be oxidized to n-1 fatty acids. Therefore, 2OH-FAs are considered intermediates in the generation of odd-chain fatty acids, and peroxisomes may contribute to this process. 2OH-FAs are endogenous in this process, and studies have reported that HACL1 contributes to the synthesis of odd-chain fatty acids in Chinese hamster ovary (CHO) cells. With increasing understanding of HACL1, HACL1 is considered the primary enzyme for the alpha-oxidation of straight-chain fatty acids in vivo.

[0004] Fatty acid oxidation is crucial for the balance between cell proliferation and function, as the carbon content of fatty acids provides a large supply of tricarboxylic acid (TCA) for the synthesis of aspartic acid (a nucleotide precursor) and uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates), ultimately leading to DNA synthesis. Reduced fatty acid oxidation depletes cellular stores of deoxynucleoside triphosphates and further impairs the synthesis of new nucleotides required for DNA replication. Furthermore, as cellular components, lipids are essential for all stages of the viral life cycle. They enter cells as part of the virus and are used for viral replication (e.g., remodeling the cell membrane, modifying viral protein maturation, and producing mature enveloped virus particles and infectivity). Lipids also provide an energy source for viral replication through fatty acid oxidation. To exploit host fatty acid metabolism, viruses employ diverse mechanisms to influence fatty acid biosynthesis. For example, viral infection induces reprogramming of lipid metabolism in immune cells, thereby promoting viral replication and immune evasion. Numerous viruses manipulate host fatty acid metabolism (e.g., cholesterol metabolism, lipid peroxidation, and utilization of fatty acids in post-translational protein modification) to suppress host innate immune responses and establish persistent infection. Studies have reported that myristic acid (C14:0), a naturally occurring long-chain saturated fatty acid, participates in a variety of intracellular physiological activities, and that herpes simplex virus 1 (HSV-1) infection selectively reduces intracellular levels of C14:0. C14:0 inhibits IFN-β secretion and mRNA levels induced by HSV-1 infection and interferon-stimulated DNA (ISD) mediated by cGAS. However, IFN-β expression induced by Sendai virus (SeV), an RNA virus recognized by RIG-I, is unaffected by C14:0. Unlike DNA viruses, RNA viruses are characterized by poor genomic stability and prone to mutation, allowing them to rapidly adapt to environmental changes and resulting in their enhanced transmissibility. Diseases caused by RNA viruses, such as COVID-19, pose a serious threat to human life and health, hindering normal life and socioeconomic development. A growing number of reports focus on how RNA viruses alter host fatty acid metabolism to promote their own proliferation. For example, enveloped RNA viruses, such as hepatitis C virus (HCV), can alter lipid homeostasis, enhancing viral replication and infectivity.

[0005] HACL1 is known to be the primary enzyme for α-oxidation of straight-chain fatty acids and is closely related to the endogenous production of odd-chain fatty acids. However, there are currently no reports on whether α-oxidation of straight-chain fatty acids or odd-chain fatty acids can affect RNA virus infection. This study investigated the effect of HACL1 expression in cells by altering its expression. The study found that increasing HACL1 expression promoted viral replication, while knocking down HACL1 inhibited viral replication. This suggests that enhancing α-oxidation of straight-chain fatty acids or increasing the content of odd-chain fatty acids can affect RNA virus infection. This provides a key target for the development of drugs to treat diseases related to RNA virus infection and maintain innate immune homeostasis. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the use of the Hacl1 gene in the preparation of drugs for treating RNA virus infection-related diseases and maintaining innate immune homeostasis.

[0007] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0008] As a first aspect of the present invention, the use of the Hacl1 gene in the preparation of a drug for treating diseases related to RNA virus infection and maintaining innate immune homeostasis is provided. By knocking down the Hacl1 gene, the transcription and protein expression of HACL1 in cells are inhibited, thereby inhibiting the proliferation of RNA viruses in cells, clearing RNA virus infection, and maintaining innate immune homeostasis.

[0009] A further improvement is that the Hacl1 gene is derived from human or mouse, the NCBI accession number of the human Hacl1 gene is NM_012260.4, and the sequence of the encoded HACL1 is shown as SEQ ID No. 1, and the NCBI accession number of the mouse Hacl1 gene is NM_019975.3, and the sequence of the encoded HACL1 is shown as SEQ ID No. 2.

[0010] A further improvement is that knocking down the Hacl1 gene can increase the transcription levels of IFNβ, ISG15, IFIT2 and MX1 in the intracellular antiviral response.

[0011] A further improvement is that the RNA virus is Newcastle disease virus NDV or vesicular stomatitis virus VSV.

[0012] As a second aspect of the present invention, a siRNA for knocking down the expression of Hacl1 gene is also disclosed. The sequences of the upstream primer and the downstream primer of the siRNA for knocking down the expression of Hacl1 gene are shown as SEQ ID No.11 and SEQ ID No.12.

[0013] As a third aspect of the present invention, also disclosed is the use of the above-described siRNA for knocking down Hacl1 gene expression in the preparation of drugs for treating RNA virus infection-related diseases and maintaining innate immune homeostasis.

[0014] A further improvement is that the RNA virus infection-related disease refers to an immune disease caused by RNA virus infection, and maintaining innate immune homeostasis refers to preventing RNA virus infection from causing excessive immune regulatory response and leading to the collapse of the autoimmune system.

[0015] The present invention has the following beneficial effects:

[0016] The present invention found that knocking down HACL1 using siRNA inhibited the proliferation of RNA viruses, such as NDV and VSV, in eukaryotic cells, while overexpressing HACL1 promoted NDV proliferation. By constructing an overexpression plasmid or using siRNA transfection, the authors found that altering HACL1 expression levels in cells using these molecular techniques could alter the degree of RNA virus infection. This suggests that the HACL1 protein plays an important role in RNA virus-related infectious diseases and in regulating innate immune function, providing a key target for the development of drugs to treat diseases associated with RNA virus infection and maintain innate immune homeostasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The process by which even-chain saturated fatty acids are oxidized by reducing one carbon atom to produce odd-chain saturated fatty acids.

[0018] Figure 2 Fluorescence real-time quantitative PCR was used to detect the changes in the transcription levels of key enzymes involved in the α-oxidation of even-chain fatty acids to odd-chain fatty acids after infection with NDV and VSV viruses at different times (in the figure, A corresponds to NDV virus; B corresponds to VSV virus);

[0019] Figure 3 The effect of knocking down Hacl1 on the replication levels of NDV and VSV viruses (in the figure, A corresponds to the transcription level of HACL1; B corresponds to the protein level of HACL1; C corresponds to the GFP protein expression of NDV virus; D corresponds to the expression of VSV-G protein);

[0020] Figure 4 To verify the effect of HACL1 overexpression (in the figure, A is the HACL1 expression level in DBT1 cells, VeroE6 cells and HEK-293T cells transfected with the overexpression plasmid; B is the effect of overexpression of HACL1 on the viral replication level of NDV virus);

[0021] Figure 5The transcription levels of intracellular cytokines and immune-related genes after knocking down Hacl1 and infecting with NDV or VSV virus (in the figure, A corresponds to ISG15; B corresponds to MX1; C corresponds to IFIT2; D corresponds to IFNβ). DETAILED DESCRIPTION

[0022] The present application is described in further detail below in conjunction with the accompanying drawings. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0023] 1. Materials and Reagents

[0024] The experimental methods in the following examples are all conventional biochemical methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0025] 1. Cell lines

[0026] Human embryonic kidney cells HEK-293T were preserved in our laboratory, African green monkey kidney cells Vero E6 and mouse astroglioma cells DBT1 were donated by the Military Medical Research Institute of the Academy of Military Sciences. They can also be obtained from commercial sources (such as the Auricell Biobank).

[0027] 2. Viruses

[0028] Vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV) fused with GFP gene were donated by the Military Medical Research Institute of the Academy of Military Sciences.

[0029] 3. Plasmid

[0030] The pCMV3.0-Flag plasmid was stored in our laboratory;

[0031] 4. Molecular biology reagents and antibodies

[0032] The transfection reagent Lipofectamine 8000 was purchased from Beyotime; anti-GFP, anti-HACL1, anti-VSV-G, anti-β-actin, anti-GAPDH, goat anti-rabbit IgG (H + L)-HRP, and goat anti-mouse IgG (H + L)-HRP antibodies were purchased from Proteintech;

[0033] QPCR primers and plasmid construction primers were purchased from General Biotechnology;

[0034] siRNA was purchased from Qingke Biotechnology Co., Ltd.

[0035] 2. Methods

[0036] 1. Fluorescence real-time quantitative PCR detection

[0037] Fluorescence real-time quantitative PCR was used to detect changes in the transcription levels of key enzymes involved in the α-oxidation of even-chain fatty acids to odd-chain fatty acids after infection with NDV and VSV viruses for different periods of time. The specific steps are as follows:

[0038] 1.1. DBT1 cells were cultured at a rate of 2×10 5 Cells were seeded into 6-well plates and cultured for 18-24 hours;

[0039] 1.2 Viral infection: DBT1 cells were infected with vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV) fused with the GFP gene for 0, 6, 12, or 24 h, respectively;

[0040] 1.3. Extraction of total RNA from cells: After virus infection, discard the culture medium from the cells and wash them three times with 1 mL of pre-cooled PBS solution. The final wash was transferred to a 1.5 mL Eppendorf tube and centrifuged to remove any remaining supernatant.

[0041] (1) Cell RNA extraction: According to the total RNA extraction kit of Novozyme Biotechnology Co., Ltd.

[0042] (ER501-01-V2)Instruction sheet extraction;

[0043] (2) RNA was reverse transcribed to obtain cDNA samples. The reaction system was shown in Table 1, and the reaction conditions were shown in Table 2.

[0044] Table 1. Reaction system

[0045]

[0046] Table 2. Reaction conditions

[0047]

[0048] 1.4 Real-time fluorescence quantitative PCR

[0049] The cDNA samples obtained above were amplified by PCR using the SYBR Green I method according to the instructions of Taq Pro Universal SYBR qPCR Master Mix (Q511-02) (Novozymes). The system was as follows: 0.4 μL of upstream and downstream primers, 10 μL of 2× SYBR Green Mix, 0.5 μg of cDNA, and ddH2O was added to 20 μL. PCR amplification was performed in a fluorescent quantitative PCR instrument. β-actin was used as the internal reference gene, and 2 -△△CT Methods The relative transcription levels of genes were calculated.

[0050] The primers used for QPCR were synthesized by Universal Biotechnology. The primer information is shown in Table 3 , the reaction system is shown in Table 4 , and the reaction conditions are shown in Table 5 .

[0051] Table 3. Primer information

[0052]

[0053]

[0054] Table 4. Reaction system

[0055]

[0056] Table 5. Reaction conditions

[0057]

[0058] The results showed that compared with the control group, in DBT1 cells, with the extension of NDV infection time, the transcription level of Hacl1, a key enzyme in the process of α-oxidation of even-chain fatty acids to odd-chain fatty acids, changed most significantly ( Figure 2 A). For VSV, the infection effect on DBT1 cells and the change trend of key enzymes are consistent with those of NDV ( Figure 2 B).

[0059] 2. Effect of Hacl1 knockdown on the replication levels of NDV and VSV viruses in DBT1 cells

[0060] 2.1. Design of si-Hacl1, which was synthesized by Qingke Biotechnology Co., Ltd. (Table 6).

[0061] Table 6. si-Hacl1 primer sequence information

[0062]

[0063] 2.2. DBT1 cells were cultured at a rate of 2×10 5 Cells were seeded into 6-well plates and cultured for 18-24 h.

[0064] 2.3. Transfer si-Hacl1 into a 6-well plate.

[0065] 2.4. After culturing for 4-6 hours, the supernatant was replaced with 10% FBS culture medium.

[0066] 2.5. Virus infection: 24 hours after transfection, the DBT1 cells transfected with si-Hacl1 were infected with vesicular stomatitis virus (VSV) and GFP-tagged Newcastle disease virus (NDV) and continued to be cultured.

[0067] 2.6. Immunoblotting: 24 hours after viral infection, the cells were collected and washed. The cells were resuspended and centrifuged, then washed once with phosphate buffered saline (PBS) and centrifuged. Then, the corresponding PBS was added according to the amount of cell pellet, and 4×SDS-PAGE protein loading buffer was added in a ratio of 1:3 according to the added PBS, and reacted in a metal bath at 100°C for 10 minutes. After the sample cooled, the protein immunoblotting experiment was performed. The voltage was initially set to 80V. After bromophenol blue migrated to the separation gel, the voltage was adjusted to 120V and electrophoresis was continued; 30 minutes before the end of electrophoresis, the PVDF membrane was activated with methanol for 15 seconds, and then soaked in 1x transfer buffer for 30 minutes with two 2.5mm thick filter papers. After the electrophoresis was completed, the membrane was transferred and placed in a semi-dry transfer instrument in the order of filter paper-PVDF membrane-PAGE gel-filter paper from bottom to top. A roller was used to remove bubbles and the membrane was transferred at 18V for 1 hour. The transferred PVDF membrane was blocked with blocking buffer (TBST solution containing 5% skim milk powder) and incubated at room temperature for 2 hours. The membrane was washed with 10 mL of 1xTBST for 5 minutes, repeated three times. Anti-GFP, anti-HACL1, anti-VSV-G, anti-β-actin, and anti-GAPDH (prepared according to the antibody instructions) were then added and incubated overnight at 4°C on a shaker. The membrane was washed with 1xTBST for 5 minutes, repeated six times, and goat anti-rabbit IgG (H+L)-HRP and goat anti-mouse IgG (H+L)-HRP antibodies were added and incubated at room temperature for 1 hour. Finally, ECL chemiluminescence was performed by mixing color development solutions A and B in a 1:1 ratio and evenly covering the PVDF membrane. The membrane was developed using a chemiluminescence imager. Data were analyzed using Image J software.

[0068] The results showed that si-Hacl1 significantly inhibited the transcription and protein expression of HACL1 in DBT1 cells ( Figure 3 A, 3B), compared with the control group, the GFP protein expression of NDV virus was significantly inhibited after si-Hacl1 was introduced, and the inhibition trend was consistent under different virus titers ( Figure 3 C); During VSV virus infection, si-Hacl1 can also inhibit the expression of VSV-G protein, and this inhibitory trend does not change under different virus titers ( Figure 3 D) These results indicate that knockdown of Hacl1 significantly inhibited the proliferation of NDV and VSV viruses in cells.

[0069] 3. Query the CDS sequence of HACL1

[0070] The human and mouse genes of HACL1 were searched through the website https: / / www.ncbi.nlm.nih.gov / , and the CDS sequences of human and mouse HACL1 were downloaded. Among them, the transcript variant 1NM_012260.4 of human HACL1 was selected, and the sequence of HACL1 encoded by it is shown in SEQ ID No. 1. The transcript variant NM_019975.3 of mouse HACL1 was selected, and the sequence of HACL1 encoded by it is shown in SEQ ID No. 2.

[0071] 4. Construction of HACL1 overexpression plasmid

[0072] 4.1. Design primers for cloning human and mouse HACL1.

[0073] Table 7. Primers designed for constructing overexpression plasmids

[0074]

[0075] 4.2. The primers are used to amplify the fragment PCR product, which is then recombined into the target vector pCMV by homologous recombination.

[0076] 3.0-FLAG (KpnI-EcoRI digested vector) to obtain a HACL1 overexpression plasmid. The processed target fragment was ligated to the vector. The ligation system is shown in Table 7. The ligation solution was incubated at 37°C for 30 minutes.

[0077] Table 8. Connection system

[0078]

[0079] 4.3 Transformation and Clone Identification: Transformation methods can be followed according to the DH5α competent cell transformation instructions. Plate the transformed bacteria onto LB plates containing appropriate antibiotics to screen for positive clones. After shaking, extract the plasmids and send them to General Biotechnology (Hefei) for plasmid sequencing. Verify the constructed plasmid sequence by comparing it with the target fragment sequence using Snapgene software.

[0080] 5. Verification of HACL1 overexpression effect

[0081] 5.1. DBT1 cells, Vero E6 cells, and HEK-293T cells were cultured at a rate of 2×10 5 Cells were seeded into 6-well plates and cultured for 18-24 h.

[0082] 5.2. Transfect the constructed HACL1 overexpression plasmid into a 6-well plate.

[0083] 5.3. After culturing for 4-6 hours, replace the supernatant with 10% FBS culture medium.

[0084] 5.4. Immunoblotting: 36-48 hours after transfection, collect cells, wash and perform protein immunoblotting.

[0085] DBT1 cells were transfected with mouse Hacl1 plasmid, and Vero E6 and HEK-293T cells were transfected with human Hacl1 plasmid. The results showed that compared with the control group transfected with empty vector, the expression level of HACL1 in DBT1 cells, Vero E6 cells and HEK-293T cells transfected with overexpression plasmid was significantly increased ( Figure 4 A).

[0086] 6. Effect of HACL1 overexpression on NDV viral replication

[0087] 6.1. DBT1 cells, Vero E6 cells, and HEK-293T cells were cultured at a rate of 2×10 5 Cells were seeded into 6-well plates and cultured for 18-24 h.

[0088] 6.2. Transfect the constructed HACL1 overexpression plasmid into a 6-well plate.

[0089] 6.3. After culturing for 4-6 hours, replace the supernatant with 10% FBS culture medium.

[0090] 6.4. Virus infection: 24 hours after transfection, DBT1 cells, HEK-293T cells, and Vero E6 cells transfected with HACL1 overexpression plasmid were infected with GFP-tagged Newcastle disease virus (NDV) and continued to be cultured.

[0091] 6.5 Immunoblotting: 24 h after virus infection, cells were collected, washed, and subjected to protein immunoblotting.

[0092] The results showed that compared with the empty vector group, the viral replication level of the HACL1 overexpression group was significantly increased, and this trend was consistent in the three cell types ( Figure 4 B), indicating that overexpression of HACL1 promotes viral infection. Alterations in HACL1 expression can affect the replication of RNA viruses in cells, further demonstrating that HACL1 is closely linked to the innate immune response.

[0093] 7. Expression detection of cytokines and immune-related genes

[0094] 7.1. DBT1 cells were cultured at a rate of 2×10 5 Cells were seeded into 6-well plates and cultured for 18-24 h.

[0095] 7.2. Transfer si-Hacl1 into a 6-well plate.

[0096] 7.3. After culturing for 4-6 hours, replace the supernatant with 10% FBS culture medium.

[0097] 7.4. Virus infection: 24 hours after transfection, the DBT1 cells transfected with si-Hacl1 were infected with vesicular stomatitis virus (VSV) and GFP-tagged Newcastle disease virus (NDV) and continued to be cultured.

[0098] 7.5. Wash with PBS and perform RNA extraction, reverse transcription, and RT-qPCR (same experimental steps as 1, real-time fluorescence quantitative PCR).

[0099] The results are as follows Figure 5 As shown in the results, after knocking down Hacl1 and infecting NDV or VSV virus, the transcription levels of intracellular IFNβ, ISG15, IFIT2 and MX1 were significantly increased compared with the control group, indicating that si-Hacl1 can enhance the intracellular innate immune antiviral response, thereby inhibiting intracellular viral infection and further inhibiting RNA virus replication.

[0100] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. Knockdown Hacl1 The use of gene-expressed siRNA in the preparation of drugs for treating RNA virus infection-related diseases and maintaining innate immune homeostasis is characterized in that: By knocking down Hacl1 The gene inhibits the transcription and protein expression of HACL1 in cells, thereby inhibiting the proliferation of RNA viruses in cells, clearing RNA virus infection and maintaining innate immune homeostasis. Hacl1 The sequences of the upstream primer and the downstream primer of the siRNA for gene expression are shown in SEQ ID No. 11 and SEQ ID No. 12, and the RNA virus is Newcastle disease virus NDV or vesicular stomatitis virus VSV.

2. The use according to claim 1, characterized in that described Hacl1 The gene is derived from human or mouse, the human Hacl1 The NCBI accession number of the gene is NM_012260.4, and the sequence of the encoded HACL1 is shown in SEQ ID No.

1. Hacl1 The NCBI accession number of the gene is NM_019975.3, and the sequence of the encoded HACL1 is shown in SEQ ID No.

2.

3. The use according to claim 1, characterized in that Knockdown Hacl1 The gene can increase the transcription levels of IFNβ, ISG15, IFIT2 and MX1 in the intracellular antiviral response.

4. The use according to claim 1, characterized in that The RNA virus infection-related disease refers to an immune disease caused by RNA virus infection, and maintaining innate immune homeostasis refers to preventing RNA virus infection from causing excessive immune regulatory response and leading to the collapse of the autoimmune system.

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