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

Abstract

The invention discloses an application of a Hacl1 gene in preparation of a medicine for treating RNA virus infection related diseases and maintaining innate immune homeostasis, relates to the technical field of biological medicines, and finds that the transcriptional level of Hacl1 is remarkably up-regulated in the process that RNA viruses represented by NDV and VSV infect eukaryotic cells. After Hacl1 is knocked down by siRNA, virus proliferation is significantly inhibited, and overexpression of HACL1 has a significant promotion effect on proliferation of NDV virus in eukaryotic cells. Further detection shows that after the Hacl1 is knocked down, the transcriptional levels of IFN beta, ISG15, IFIT2 and MX1 in cells can be improved, so that the replication of viruses in the cells is inhibited. The HACL1 protein plays an important role in infectious diseases related to RNA viruses and the innate immune regulation process, a new basis for HACL1 to participate in regulation of innate immune response is provided clinically, and the HACL1 protein has a wide application prospect 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 Hacl1 gene in preparing medicines 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 with a carboxyl group at the end. 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 for 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 to generate α-hydroxy fatty acids (2OH-FA) in the microsomes, and the process in which 2OH-FA continues to oxidize and decarboxylate to generate odd-numbered carbon fatty acids is called α-oxidation of fatty acids (FAO). Figure 1 ).

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

[0004] Fatty acid oxidation is very important for the balance of cell proliferation and function, because the carbon of fatty acids replenishes TCA in large quantities for the synthesis of aspartic acid (a nucleotide precursor) and uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates), and finally for the synthesis of DNA. The reduction of fatty acid oxidation will deplete the deoxynucleoside triphosphates stored in the cell and further impair the synthesis of new nucleotides required for DNA replication. In addition, as a cellular component, lipids are essential at all stages of the viral life cycle. They enter cells as part of the virus and are used for viral replication (for example: structural remodeling of the cell membrane; modification of viral protein maturation, and production of mature particles and infectivity of enveloped viruses). Lipids can also provide an energy source for viral replication through the oxidation of fatty acids. In order to utilize the host's fatty acid metabolism, viruses take different approaches to affect the biosynthesis of fatty acids. For example, viral infection induces reprogramming of immune cell lipid metabolism, thereby promoting viral replication and immune escape. Many viruses suppress host innate immune responses and form persistent infections by manipulating host fatty acid metabolism (such as cholesterol metabolism, lipid peroxidation, and the use of fatty acids to participate in protein post-translational modification). Studies have reported that myristic acid (C14:0), as a natural long-chain saturated fatty acid, participates in a variety of intracellular physiological activities, and herpes simplex virus 1 (HSV-1) infection selectively reduces the intracellular level 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 not affected by C14:0. Unlike DNA viruses, RNA viruses are characterized by poor genome stability and easy mutation, so that they can quickly adapt to environmental changes, which also makes RNA viruses more transmissible. Diseases caused by RNA viruses such as COVID-19 seriously threaten human life and health, hindering people's normal life and socioeconomic development. More and more reports focus on RNA viruses changing host fatty acid metabolism to promote their own proliferation. Taking hepatitis C virus (HCV) as an example, enveloped RNA viruses can change lipid homeostasis and enhance viral replication and infectivity.

[0005] It is known that HACL1 is the main enzyme for α-oxidation of straight-chain fatty acids and is closely related to the endogenous production of odd-chain fatty acids. However, there is no relevant report on whether α-oxidation of straight-chain fatty acids or odd-chain fatty acids can affect RNA virus infection. The present invention explores its effect on RNA virus infection by changing the expression level of HACL1 in cells, and finds that increasing the expression level of HACL1 can promote viral replication, while knocking down Hacl1 can inhibit viral replication, which indicates that the enhancement of α-oxidation of straight-chain fatty acids or the increase in the content of odd-chain fatty acids can affect RNA virus infection. This provides key target selection for the preparation of drugs for treating diseases related to RNA virus infection and maintaining 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, provided is 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. By knocking down the Hacl1 gene, the transcription and protein level 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 a use of the above-mentioned 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 immunoregulatory response and leading to the collapse of the autoimmune system.

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

[0016] After knocking down Hacl1 by siRNA, the present invention found that the proliferation of RNA viruses represented by NDV and VSV in eukaryotic cells was inhibited, and the proliferation of NDV virus was promoted after overexpression of HACL1. By constructing an overexpression plasmid or siRNA transfection, it was found that using these molecular technologies to change the expression level of HACL1 in cells can change the infection degree of RNA viruses, suggesting that HACL1 protein plays an important role in RNA virus-related infectious diseases and the regulation of innate immune function, providing key target selection for the preparation of drugs for treating diseases related to RNA virus infection and maintaining 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 in the process of generating odd-chain fatty acids through α-oxidation of even-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 To show the effect of knocking down Hacl1 on the replication levels of NDV virus and VSV virus (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 overexpression plasmids; 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 further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here 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. Technical personnel 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, and can also be obtained from commercial sources (such as the Aureus Biocell Bank).

[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 the changes in the transcription levels of key enzymes in the process of generating odd-chain fatty acids through α-oxidation of even-chain fatty acids after infection with NDV and VSV viruses at different times. The specific steps are as follows:

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

[0039] 1.2. Virus infection: DBT1 cells were infected with vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV) fused with 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, wash with 1 mL pre-cooled PBS solution for 3 times, transfer to a 1.5 mL Eppendorf tube for the last time, centrifuge and remove the 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 SYBR Green I method according to the instructions of Taq Pro Universal SYBR qPCR Master Mix (Q511-02) (Novozymes). System: 0.4 μL of upstream and downstream primers, 10 μL of 2×SYBR Green Mix, 0.5 μg of cDNA, and ddH2O to 20 μL; put into fluorescent quantitative PCR instrument for PCR amplification. β-actin was used as the internal reference gene in the experiment, and 2 -△△CT Methods The relative transcription levels of genes were calculated.

[0050] The primers used in QPCR were synthesized by Universal Company. 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 even-chain fatty acids generating odd-chain fatty acids through α-oxidation, 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 knockdown of Hacl1 on the replication level of NDV and VSV in DBT1 cells

[0060] 2.1. Design of si-Hacl1 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 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 a culture medium containing 10% FBS.

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

[0067] 2.6. Immunoblotting: Collect the cell set and wash it 24 hours after virus infection. Resuspend the cells and centrifuge them, then wash them with phosphate buffered saline (PBS) and centrifuge them. Then add the corresponding PBS according to the amount of cell precipitation, and add 4×SDS-PAGE protein loading buffer according to the added PBS at a ratio of 1:3, and react in a metal bath at 100°C for 10 minutes. After the sample cools down, perform protein immunoblotting experiments. The voltage is initially set to 80V. After bromophenol blue migrates to the separation gel, adjust the voltage to 120V and continue electrophoresis; 30 minutes before the end of electrophoresis, activate the PVDF membrane with methanol for 15 seconds, and then soak it in 1x transfer buffer for 30 minutes with two 2.5mm thick filter papers. When the electrophoresis is over, transfer the membrane and place it in a semi-dry transfer instrument in the order of filter paper-PVDF membrane-PAGE gel-filter paper from bottom to top. Use a roller to remove bubbles and transfer at 18V for 1 hour. 30min; the PVDF membrane after transfer was blocked with blocking solution (TBST solution containing 5% skim milk powder) and incubated at room temperature for 2h; 10mL of 1xTBST was added for washing for 5min, and repeated three times; then, anti-GFP, anti-HACL1, anti-VSV-G, anti-β-actin, anti-GAPDH (the corresponding concentrations have been prepared according to the antibody instructions), incubated overnight at 4℃ on a shaker, washed with 1xTBST for 5min, repeated 6 times, goat anti-rabbit IgG (H+L)-HRP and goat anti-mouse IgG (H+L)-HRP antibodies were added, incubated at room temperature for 1h, and finally ECL chemiluminescence was performed, color development solution A and solution B were mixed in a ratio of 1:1 and evenly covered on the PVDF membrane, and developed with a chemiluminescence imager. , and data analysis was performed 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 inhibition 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 transcription variant 1NM_012260.4 was selected for human HACL1, and the sequence of HACL1 encoded by it was shown in SEQ ID No.1. The mouse HACL1 was selected NM_019975.3, and the sequence of HACL1 encoded by it was 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 restriction enzyme digested vector), HACL1 overexpression plasmid was obtained, and the processed target fragment was connected to the vector. The connection system is shown in Table 7. The connection solution was reacted at a constant temperature of 37°C for 30 minutes.

[0077] Table 8. Connection system

[0078]

[0079] 4.3 Transformation and clone selection and identification: The transformation method can refer to the DH5α competent cell transformation instructions. The transformed bacteria were spread on LB plates containing appropriate antibiotics, positive clones were screened, plasmids were extracted after shaking, and sent to General Company (Hefei) for plasmid sequencing. Snapgene software was used to compare with the target fragment sequence to verify the constructed plasmid sequence.

[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, the supernatant was replaced with a culture medium containing 10% FBS.

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

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

[0086] 6. Effect of overexpression of HACL1 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, the supernatant was replaced with a culture medium containing 10% FBS.

[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 NDV carrying GFP 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 in the HACL1 overexpression group was significantly increased, and this trend was consistent in the three cell types ( Figure 4 B), which indicates that overexpression of HACL1 promotes viral infection. Changes in HACL1 expression can affect the replication level of RNA viruses in cells, further indicating that HACL1 is closely related to innate immune response.

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

[0094] 7.1. DBT1 cells were cultured at 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, the supernatant was replaced with a culture medium containing 10% FBS.

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

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

[0099] The results are as follows Figure 5 As shown, 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-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. Application of Hacl1 gene in the preparation of drugs for treating RNA virus infection-related diseases and maintaining innate immune homeostasis, It is characterized in that By knocking down the Hacl1 gene, the transcription and protein level 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.

2. The use according to claim 1, It is characterized in 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 in SEQ ID No.

1. The NCBI accession number of the mouse Hacl1 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, It is characterized in that Knocking down the Hacl1 gene can increase the transcription levels of IFNβ, ISG15, IFIT2 and MX1 in the intracellular antiviral response.

4. The use according to claim 1, It is characterized in that The RNA virus is Newcastle disease virus NDV or vesicular stomatitis virus VSV.

5. A siRNA that knocks down the expression of the Hacl1 gene, Its characteristics are that The sequences of the upstream primer and the downstream primer of the siRNA for knocking down the expression of the Hacl1 gene are shown as SEQ ID No.11 and SEQ ID No.

12.

6. Use of the siRNA for knocking down Hacl1 gene expression as claimed in claim 4 in the preparation of drugs for treating RNA virus infection-related diseases and maintaining innate immune homeostasis.

7. The use according to claim 1 or 6, It is 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 immunoregulatory responses and leading to the collapse of the autoimmune system.

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