CN119158019B - Use of Hacl gene in preparing medicine for treating diseases related to RNA virus infection and maintaining innate immunity stable state - Google Patents

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

Use of Hacl gene in preparing medicine for treating diseases related to RNA virus infection and maintaining innate immunity stable state

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of Hacl gene in preparing medicines for treating diseases related to RNA virus infection and maintaining innate immune homeostasis.

Background

Fatty acid is a compound composed of carbon, hydrogen and oxygen, the main chain is a repeated methylene sequence, and the tail end carries carboxyl. Short chain fatty acids, medium chain fatty acids, long chain fatty acids and ultra-long chain fatty acids are classified according to their length, and even chain fatty acids and odd chain fatty acids are classified according to the number of carbon atoms. In the presence of oxygen, fatty acids can oxidize and release energy, a process known as Fatty Acid Oxidation (FAO). Beta-oxidation of fatty acids is the main pathway of fatty acid oxidation in the body and can supply a great deal of energy required by the body. There are also some special oxidized forms of fatty acids, such as long chain even fatty acids catalyzed in microsomes by monooxygenases and decarboxylases to produce alpha-hydroxy fatty acids (2 OH-FA), and the process of continuing oxidative decarboxylation of 2OH-FA to fatty acids of odd carbon atoms is known as alpha-oxidation of fatty acids (fig. 1).

The alpha oxidation of even fatty acids in peroxisomes is catalyzed by endoplasmic reticulum-related NAD (P) H-dependent monooxygenases and fatty acid alpha-hydroxylases (FA 2H) to form 2OH-FA. They are incorporated into sphingolipids, an important component of cerebrosides and sulfatides. These 2 OH-FAs, after activation to 2 OH-acyl-CoA, are cleaved by HACL1 and HACL2 to yield formyl-CoA and n-1 fatty aldehydes. The latter can be oxidized to n-1 fatty acids. Thus 2OH-FA is considered to be an intermediate in the production of odd-chain fatty acids, and peroxisomes may contribute to this process. In this process, 2OH-FA is endogenous, and HACL1 has been reported to contribute to odd-chain fatty acid synthesis in chinese hamster ovary Cells (CHO), with the understanding of HACL1 in depth, HACL1 is considered to be the primary enzyme for in vivo alpha oxidation of linear fatty acids.

Fatty acid oxidation is very important for the balance of cell proliferation and function, since the carbon of fatty acids largely supplements TCA for the synthesis of aspartic acid (a nucleotide precursor) and uridine monophosphate (a precursor of pyrimidine nucleoside triphosphate), and finally DNA. The reduction of fatty acid oxidation will deplete cell-stored deoxynucleoside triphosphates and further impair new nucleotide synthesis required for DNA replication. In addition, as a cellular component, lipids are required at various stages of the viral life cycle, which enter the cell as part of the virus for replication of the virus (e.g., structural remodeling of cell membranes; modification of viral protein maturation, and production of mature particles and infectivity of enveloped viruses), and can also provide an energy source for replication of the virus by oxidation of fatty acids. In order to utilize the fatty acid metabolism of the host, viruses take different ways to influence fatty acid biosynthesis. For example, viral infection induces reprogramming of immune cell lipid metabolism, thereby promoting viral replication and immune escape. Many viruses inhibit host innate immune responses by manipulating host fatty acid metabolism (e.g., cholesterol metabolism, lipid peroxidation, and utilization of fatty acids to participate in post-translational modifications of proteins) and form persistent infections. It has been reported that myristic acid (C14:0) is involved in a variety of intracellular physiological activities as a natural long chain saturated fatty acid, whereas herpes simplex virus 1 (HSV-1) infection selectively reduces intracellular levels of C14:0. C14:0 inhibits cGAS-mediated recognition of HSV-1 infection and interferon-stimulated DNA (ISD) -induced IFN- β secretion and mRNA levels. However, IFN- β expression induced by the RIG-I mediated recognition of the RNA virus Sendai virus (SeV) is not affected by C14:0. Unlike DNA viruses, RNA viruses are characterized by poor genomic stability and are prone to mutation, so that they can adapt rapidly to environmental changes, which also results in RNA viruses having greater transmissibility. Diseases caused by COVID-19 RNA viruses are serious threatened to human life and health, and prevent people from developing normal life and socioeconomic performance. More and more reports focus on RNA viruses altering host fatty acid metabolism to promote their own proliferation, for example, hepatitis C Virus (HCV), and enveloped RNA viruses can alter lipid homeostasis, enhancing viral replication and infectivity.

HACL1 is known to be the primary enzyme for alpha-oxidation of linear fatty acids and is closely related to endogenous production of odd-chain fatty acids, but whether alpha-oxidation of linear fatty acids or odd-chain fatty acids can affect RNA viral infection has not been reported so far. The invention researches the influence of HACL1 on RNA virus infection by changing the expression quantity of the HACL1 in cells, and discovers that increasing the expression quantity of the HACL1 can promote virus replication, and knocking down Hacl1 can inhibit virus replication, which shows that the enhancement of alpha-oxidation of linear fatty acid or the increase of the content of odd fatty acid can influence RNA virus infection. Thereby providing a key target selection for preparing the medicine for treating diseases related to RNA virus infection and maintaining innate immune homeostasis.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides application of Hacl gene in preparing medicaments for treating diseases related to RNA virus infection and maintaining innate immune homeostasis.

The invention realizes the above purpose through the following technical scheme:

as a first aspect of the present invention, there is provided the use of Hacl gene in the manufacture of a medicament for the treatment of diseases associated with RNA viral infection and for maintaining innate immune homeostasis, whereby transcription and protein level expression of HACL1 in a cell is inhibited by knocking down Hacl gene, thereby inhibiting proliferation of RNA virus in the cell, clearing RNA viral infection and maintaining innate immune homeostasis.

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

A further improvement is that knock-down of Hacl1 gene increases the transcript levels of IFN beta, ISG15, IFIT2 and MX1 in intracellular antiviral responses.

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

As a second aspect of the invention, there is also disclosed an siRNA for knocking down Hacl1 gene expression, wherein the sequences of the upstream primer and the downstream primer of the siRNA for knocking down Hacl gene expression are shown as SEQ ID No.11 and SEQ ID No. 12.

As a third aspect of the present invention, there is also disclosed an application of siRN A with knockdown Hacl gene expression as described above in the preparation of a medicament for treating diseases associated with RNA virus infection and maintaining innate immune homeostasis.

A further improvement is that the disease associated with RNA viral infection is an immune disease caused by RNA viral infection, and the maintenance of innate immune homeostasis is prevention of collapse of the autoimmune system caused by excessive immune regulatory response caused by RNA viral infection.

The invention has the following beneficial effects:

In the present invention, hacl1 was knocked down by siRNA, and it was found that proliferation of RNA viruses represented by NDV and VSV in eukaryotic cells was inhibited and proliferation of NDV viruses was promoted after HACL1 was overexpressed. Through constructing over-expression plasmid or siRNA transfection, it is found that the molecular technology can change the infection degree of RNA virus by changing the expression level of HACL1 in cells, which suggests that HACL1 protein plays an important role in the regulation process of RNA virus-related infectious diseases and innate immunity, and provides a key target selection for preparing medicines for treating RNA virus infection-related diseases and maintaining innate immunity homeostasis.

Drawings

FIG. 1 is a process for reducing one carbon atom of an even chain saturated fatty acid to an odd chain saturated fatty acid by alpha oxidation;

FIG. 2 shows the change in transcription level of key enzyme in the process of generating odd-chain fatty acid by alpha oxidation of even-chain fatty acid in the middle after different times of infection of NDV and VSV virus by fluorescence real-time quantitative PCR (in the figure, A corresponds to NDV virus; B corresponds to VSV virus);

FIG. 3 is a graph showing the effect of knockdown Hacl1 on replication levels of NDV virus and VSV virus (in the graph, A corresponds to the transcription level of HACL 1; B corresponds to the protein level of HACL 1; C corresponds to GFP protein expression of NDV virus; D corresponds to the expression of VSV-G protein);

FIG. 4 shows the verification 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 replication level of NDV virus);

FIG. 5 shows the transcription levels of intracellular cytokines and immune-related genes after knocking down Hacl1 and infecting NDV or VSV virus (in the figure, A corresponds to ISG15; B corresponds to MX1; C corresponds to IFIT2; D corresponds to IFNbeta).

Detailed Description

The present application will be described in further detail with reference to the accompanying drawings, wherein it is to be understood that the following detailed description is for the purpose of further illustrating the application only and is not to be construed as limiting the scope of the application, as various insubstantial modifications and adaptations of the application to those skilled in the art can be made in light of the foregoing disclosure.

1. Materials and reagents

The experimental methods in the following examples are conventional biochemical methods unless otherwise specified. The test materials used in the examples described below were purchased from conventional biochemical reagent stores unless otherwise specified.

1. Cell strain

Human embryonic kidney cells HEK-293T are preserved in the laboratory, african green monkey kidney cells Vero E6 and mouse astroglioma cells DBT1 are presented to the military medical institute of science and the military sciences, and can also be obtained by being sold in the market (such as AuRui Sich biological cell bank).

2. Virus (virus)

Vesicular stomatitis virus VSV and newcastle disease virus NDV fused with GFP gene are presented to military medical institute of military science.

3. Plasmid(s)

The pCMV3.0-Flag plasmid is stored for the laboratory;

4. Molecular biological reagents and antibodies

Transfection reagent Lipofectamine 8000 was purchased from Beyotime, anti-GFP, anti-HACL1, anti-VSV-G, anti-beta-actin, anti-GAPDH, goat anti-rabbit IgG (H+L) -HRP and goat anti-mouse IgG (H+L) -HRP antibodies were purchased from Proteintech;

the QPCR primer and the plasmid construction primer are purchased from general biological company;

siRNA was purchased from the department of prime biology.

2. Method of

1. Fluorescent real-time quantitative PCR detection

The fluorescence real-time quantitative PCR is used for detecting the transcriptional level change of key enzymes in the process of generating odd-chain fatty acid from even-chain fatty acid through alpha oxidation after the NDV and VSV viruses are infected for different time. The method comprises the following specific steps:

1.1, inoculating DBT1 cells into a 6-well plate according to 2X 10 ⁵ cells, and culturing for 18-24 hours;

1.2, virus infection, namely, infecting DBT1 cells for 0, 6, 12 or 24 hours respectively by vesicular stomatitis virus VSV and newcastle disease virus NDV fused with GFP genes;

1.3, extracting total RNA of cells, namely discarding a culture medium in the cells infected by viruses, washing 3 times by using 1mL of precooled PBS solution, transferring the cells into a 1.5mL Ep tube for the last time, centrifuging, and sucking out residual supernatant;

(1) Cell RNA extraction according to total RNA extraction kit of Norwegian biotechnology Co., ltd

(ER 501-01-V2) instruction extraction;

(2) cDNA samples were obtained by reverse transcription of RNA, the reaction system is shown in Table 1, and the reaction conditions are shown in Table 2.

TABLE 1 reaction System

TABLE 2 reaction conditions

1.4 Real-time fluorescent quantitative PCR

The cDNA sample obtained above was subjected to PCR amplification by SYBR Green I method according to Taq Pro Universal SYBR QP CR MASTER Mix (Q511-02) instructions (Norpran Corp.). The system comprises 0.4 mu L of each of the upstream and downstream primers, 10 mu L of 2 XSYBR Green Mix, 0.5 mu g of cDNA, 20 mu L of ddH ₂ O, and PCR amplification in a fluorescent quantitative PCR instrument, wherein the experiment uses beta-actin as an internal reference gene, and the relative transcription level of the gene is calculated by using a 2 ^-△△CT method.

Primers used in QPCR were synthesized by general purpose companies, 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.

TABLE 3 primer information

TABLE 4 reaction System

TABLE 5 reaction conditions

The results show that transcript levels of Hacl1 in key enzymes in the production of odd-chain fatty acids by alpha oxidation in cells were most pronounced in DBT1 cells as NDV infection time was prolonged compared to control (fig. 2A). For VSV, the infection effect on DBT1 cells and the trend of change in key enzymes were consistent with NDV (fig. 2B).

2. Effect of knockdown Hacl1 on NDV and VSV viral replication levels in DBT1 cells

2.1, Designing si-Hacl1, synthesized by the Optimago company (Table 6).

TABLE 6 si-Hacl primer sequence information

2.2 DBT1 cells were seeded in 6-well plates according to 2X 10 ⁵ cells and cultured for 18-24h.

2.3 Transfer si-Hacl1 into 6 well plates.

2.4, After 4-6 hours of culture, the supernatant was changed to 10% FBS medium for culture.

2.5 Viral infection DBT1 cells transfected with si-Hacl1 from vesicular stomatitis virus VSV and GFP tagged newcastle disease virus ND V were cultured continuously after 24h transfection.

2.6 Immunoblotting cell sets were collected and washed 24h after virus infection. Cells were centrifuged after resuspension, washed once with phosphate buffer PBS, centrifuged after which corresponding PBS was added according to the amount of cell pellet, 4 x SDS-PAGE protein loading buffer was added according to the added PBS at a ratio of 1:3, and reacted in a metal bath at 100 ℃ for 10min, after which the samples were cooled, western blot experiments were performed. The method comprises the steps of setting the voltage to 80V initially, adjusting the voltage to 120V after bromophenol blue migrates to a separating gel, continuing electrophoresis, activating a PVDF membrane with methanol for 15s 30min before electrophoresis is finished, then immersing the PVDF membrane and two pieces of filter paper with the thickness of 2.5mm in a 1x membrane transfer buffer solution for 30min, transferring the membrane after electrophoresis is finished, placing the membrane in a semi-dry membrane transfer instrument from bottom to top according to the sequence of the filter paper-PVDF membrane-PAGE gel-filter paper, removing bubbles by a roller for 1H 30min, sealing the PVDF membrane after membrane transfer by a sealing solution (TBST solution containing 5% of skimmed milk powder), incubating for 2H at room temperature, adding 10mL of 1xTBST, repeating for 5min, then adding anti-GFP, anti-HACL1, anti-VSV-G, anti-beta-actin and anti-GAPDH (prepared according to the antibody specification), incubating the sequence of the filter paper-PVDF membrane-PAGE gel-filter paper at 4 ℃ overnight, repeating for 6 times, adding goat anti-HRP IgG (H+L) -HRP and performing imaging by a chemical mixing solution at the ratio of 1:1. And data analysis was performed by Image J software.

The results show that si-Hacl1 significantly inhibited transcription and protein level expression of HACL1 in DBT1 cells (FIGS. 3A, 3B), while GFP protein expression of NDV virus was significantly inhibited after transfer of si-Hacl1 compared to the control group, the inhibition trend was consistent under different virus titers (FIG. 3C), and si-Hacl1 also inhibited VSV-G protein expression during VSV virus infection, and this inhibition trend was unchanged under different virus titers (FIG. 3D). These results indicate that NDV and VSV virus proliferation in cells is significantly inhibited after Hacl a knockdown.

3. Query CDS sequence of HACL1

Inquiring human source and mouse source genes of HACL1 through https:// www.ncbi.nlm.nih.gov/website retrieval, and downloading CDS sequences of the human source and the mouse source of the HACL1, wherein the human source HACL1 selects transcription variant 1NM_012360.4, the sequence of the encoded HACL1 is shown as SEQ ID No.1, the mouse source HACL1 selects NM_019975.3, and the sequence of the encoded HACL1 is shown as SEQ ID No. 2.

4. Construction of HACL1 overexpression plasmid

4.1, Designing primers required for cloning human and murine HACL 1.

TABLE 7 construction of primers designed for over-expression plasmids

4.2, Amplifying the primer to obtain a fragment PCR product, and recombining the fragment PCR product to a target vector pCMV by a homologous recombination method

3.0-FLAG (KpnI-EcoRI cleavage vector) to give HACL1 overexpression plasmid, and ligating the treated target fragment to the vector. The connection system is shown in Table 7. The ligation solution was reacted for 30min at 37℃under constant temperature.

TABLE 8 connection System

4.3, Transformation and selected clone identification, wherein the transformation method can be referred to a DH5 alpha competent cell transformation instruction book. The transformed bacteria are coated on LB plate containing proper antibiotics, positive clone is selected, plasmid is extracted after shaking, plasmid sequence is carried out by common company (fertilizer combination), snapgene software is used for comparing with target fragment sequence, and constructed plasmid sequence is verified.

5. Verification of HACL1 overexpression Effect

5.1 DBT1 cells, vero E6 cells, HEK-293T cells were seeded in 6-well plates according to 2X 10 ⁵ cells and cultured for 18-24h.

5.2 Transfection of the constructed HACL1 overexpression plasmid into 6-well plates.

After culturing for 4-6 hours, the supernatant was changed to 10% FBS medium for culturing.

5.4, Immunoblotting, namely, transfecting for 36-48 hours, collecting cells, cleaning and carrying out western blotting experiments.

DBT1 cells were transfected with murine Hacl plasmid, vero E6 and HEK-293T cells were transfected with human Hacl plasmid. The results showed that the HACL1 expression levels were significantly increased in DBT1 cells, vero E6 cells and HEK-293T cells transfected with the over-expression plasmid compared to the control group transfected with the empty vector (fig. 4A).

6. Effect of over-expression of HACL1 on replication level of NDV Virus

6.1 DBT1 cells, vero E6 cells, HEK-293T cells were seeded in 6-well plates according to 2X 10 ⁵ cells and cultured for 18-24h.

6.2, The constructed HACL1 overexpression plasmid was transfected into 6-well plates.

After culturing for 4-6 hours, the supernatant was changed to 10% FBS medium for culturing.

6.4, Virus infection, namely, after transfection for 24 hours, the NDV of the newcastle disease virus with GFP label is infected with DBT1 cells, HEK-293T cells and Vero E6 cells transformed with HACL1 overexpression plasmids to continue culture.

6.5 Immunoblotting cells were collected 24h after virus infection, washed and subjected to western immunoblotting experiments.

As a result, it was found that the virus replication level of HACL1 over-expressed group was significantly increased compared to the empty group, and this trend was consistent among 3 cells (fig. 4B), which suggests that over-expression of HACL1 has a promoting effect on virus infection. The change in the expression level of HACL1 can affect the replication level of RNA virus in cells further suggests that HACL1 is closely related to the innate immune response.

7. Detection of cytokine and immune related gene expression

7.1 DBT1 cells were seeded in 6-well plates according to 2X 10 ⁵ cells and cultured for 18-24h.

7.2 Transfer si-Hacl1 into 6 well plates.

After culturing for 4-6 hours, the supernatant was changed to 10% FBS medium for culturing.

7.4 Viral infection DBT1 cells transfected with si-Hacl1 were continued to be cultured 24h after transfection with vesicular stomatitis virus VSV and GFP tagged newcastle disease virus ND V.

7.5, After washing with PBS and RNA extraction, reverse transcription and RT-qPCR experiments (experimental procedure 1, fluorescent real-time quantitative PCR detection).

As shown in FIG. 5, after Hacl is knocked down and the NDV or VSV virus is infected, the transcription levels of intracellular IFNbeta, ISG15, IFIT2 and MX1 are significantly increased compared with the control group, which indicates that si-Hacl1 can promote intracellular innate immune and antiviral responses, thereby inhibiting intracellular viral infection and thus RNA viral replication.

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.

Claims (4)

1. Use of siRNA for knocking down Hacl1 gene expression in the preparation of a drug for treating RNA virus infection-related diseases and maintaining innate immune homeostasis, characterized in that 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, eliminating RNA virus infection and maintaining innate immune homeostasis. The sequences of the upstream primer and downstream primer of the siRNA for knocking down Hacl1 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 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. 3. The use according to claim 1, 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, characterized in that the RNA virus infection-related disease refers to an immune disease caused by RNA virus infection, and the maintenance of innate immune homeostasis refers to preventing RNA virus infection from causing excessive immunoregulatory response and leading to the collapse of the autoimmune system.