CN118750616A - Application of c-Fos gene in the preparation of products for preventing and treating influenza virus - Google Patents

Abstract

The invention belongs to the field of biological medicine, and in particular relates to an application of a c-Fos gene in preparation of influenza virus prevention and treatment products. The invention designs siRNA aiming at c-Fos gene, knocks down c-Fos gene of H1395 cell, suppresses expression of the gene by suppressing transcription of c-Fos gene, and compared with a control group, the cell knocked down c-Fos gene can significantly suppress replication level of influenza virus, which indicates that c-Fos gene is an important host factor for replication of influenza virus, and the drug for suppressing expression of c-Fos gene is designed by taking c-Fos gene as a target, thereby providing new direction for research and development of anti-influenza drug, and having good anti-influenza virus target application prospect.

Description

Application of c-Fos gene in the preparation of products for preventing and treating influenza virus

Technical Field

The invention belongs to the field of biomedicine, and specifically relates to an application of a c-Fos gene in preparing a product for preventing and treating influenza virus.

Background Art

Influenza virus infection can cause respiratory diseases in humans with respiratory inflammation as the main symptom, resulting in sporadic and seasonal epidemics. Acute complications of influenza mainly affect vulnerable groups, such as young children, the elderly, pregnant women and individuals with specific chronic diseases. This situation significantly increases the morbidity and mortality associated with influenza infection. Currently, the prevention, control and treatment of influenza mainly rely on vaccines and antiviral drugs.

Traditional influenza virus vaccines mainly target the surface glycoprotein of the virus, namely hemagglutinin (HA). Researchers have identified 18 influenza A hemagglutinin (HA) subtypes, labeled H1 to H18, and two influenza B lineages. Conventional seasonal inactivated influenza vaccines usually contain three or four components. These components can be antigens or strains. The trivalent vaccine includes two influenza A subtypes (H1N1 and H3N2) and one influenza B lineage; the quadrivalent vaccine consists of two influenza A subtypes (H1N1 and H3N2) and two influenza B lineages (Victoria and Yamagata). These seasonal vaccines mainly induce immune responses against the specific HA subtypes they contain, and their broad spectrum is limited. However, influenza viruses have the characteristics of antigenic drift and antigenic conversion, and the epidemic strains change every year. The preventive effect of influenza virus vaccines is limited and is not enough to cope with pandemics.

In terms of drug treatment, the drugs used to treat patients infected with influenza A include neuraminidase inhibitors and ion channel M2 blockers. The main neuraminidase inhibitors include oseltamivir, zanamivir and peramivir. Ion channel M2 blockers include amantadine and rimantadine. Early symptomatic use of anti-influenza virus drugs can effectively shorten the course of the disease and alleviate symptoms. However, drug resistance has become the main problem encountered in the clinical use of anti-influenza virus drugs. Studies have shown that almost 100% of influenza A H3N2 viruses are resistant to amantadine, and 15.5% of influenza A H1N1 viruses are resistant to amantadine; in the 2007-2008 influenza season, seasonal influenza A H1N1 virus oseltamivir-resistant strains first appeared in Europe, among which the proportion of oseltamivir-resistant strains in Norway was the highest, with resistant strains as high as 67%, and the proportion of oseltamivir-resistant strains in France was 47%. Therefore, clinical influenza medication has great limitations.

In order to effectively prevent and treat influenza, it is necessary to explore the important host factors for influenza virus replication, and it is particularly important to actively find new antiviral targets.

Summary of the invention

In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide an application of a c-Fos gene in the preparation of a product for preventing and treating influenza virus.

Another object of the present invention is to provide the use of the above-mentioned c-Fos gene in the preparation of products for regulating influenza virus replication.

Another object of the present invention is to provide a siRNA.

The purpose of the present invention is achieved through the following technical solutions:

A use of a c-Fos gene in preparing a product for preventing and treating influenza virus, wherein the nucleotide sequence of the c-Fos gene is shown in SEQ ID No. 1;

The expression level of the protein encoded by the c-Fos gene is positively correlated with the influenza virus replication level;

The use of the c-Fos gene in preparing a product for preventing and treating influenza virus preferably comprises the following steps:

Inhibit, silence or knock out the c-Fos gene, thereby inhibiting influenza virus replication;

The use of the c-Fos gene in preparing a product for regulating influenza virus replication, wherein the nucleotide sequence of the c-Fos gene is shown in SEQ ID No. 1;

The regulation includes inhibiting, silencing or knocking out the c-Fos gene to inhibit influenza virus replication;

An siRNA, which is at least one of siFOS-1, siFOS-2 and siFOS-3; the nucleotide sequence of which is as follows:

siFOS-1-sense:5'-UCUGCUUUGCAGACCGAGAUUTT-3';

siFOS-1-antisense:5'-AAUCUCCGGUCUGCAAAGCAGATT-3';

siFOS-2-sense:5'-GUGGAACAGUUAUCUCCAGAATT-3';

siFOS-2-antisense:5'-UUCUGGAGAUAACUGUUCCACTT-3';

siFOS-3-sense:5'-CACUGCUUACACGUCUUCCUUTT-3';

siFOS-3-antisense:5'-AAGGAAGACGUGUAAGCAGUGTT-3';

The siRNA is preferably a mixture of siFOS-1, siFOS-2 and siFOS-3;

The molar ratio of siFOS-1, siFOS-2 and siFOS-3 is preferably 1:1:1;

The use of the siRNA in preparing a product for preventing and treating influenza virus;

Use of the siRNA in preparing a product for inhibiting influenza virus replication;

The influenza virus is preferably at least one of influenza virus strain PR8, strain WSN and other influenza strains;

Compared with the prior art, the present invention has the following advantages and effects:

(1) The present invention uses RNA interference technology to design siRNA targeting the c-Fos gene, knocks down the c-Fos gene in human lung cancer cells (H1395 cells), and inhibits the expression of the gene by inhibiting the transcription of the c-Fos gene. Compared with the control group, the cells with the c-Fos gene knocked down can significantly inhibit the replication level of influenza virus. Compared with the control group, the titer of influenza virus can be reduced by about 10 times when c-Fos is knocked down (Figure 5).

(2) The present invention discovered for the first time that the c-Fos gene is an important host factor for influenza virus replication. By targeting the c-Fos gene and designing drugs that inhibit its expression, it can provide new targets and directions for the research and development of anti-influenza drugs, and has a good application prospect for anti-influenza virus targets.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an analysis diagram showing the results of influenza virus-induced upregulation of c-Fos gene in H1395 cells.

FIG. 2 is an analysis diagram showing the results of detecting the interference efficiency of siRNA on c-Fos gene in H1395 cells.

Figure 3 is a result analysis diagram of the expression levels of c-Fos and NP mRNA after H1395 cells with c-Fos knockdown were infected with influenza virus PR8 or WSN, wherein A: c-Fos mRNA expression level, B: NP mRNA expression level, siFOS-PR8: siFOS co-transfection 24h + PR8 infection 24h, siNC-PR8: siNC transfection 24h + PR8 infection 24h, siFOS-WSN: siFOS co-transfection 24h + WSN infection 24h, siNC-WSN: siNC transfection 24h + WSN infection 24h, siFOS-MOCK: no virus infection after siFOS co-transfection 24h, siNC-MOCK: no virus infection after siNC transfection 24h.

FIG4 is a western blot diagram of NP protein expression after c-Fos knockdown H1395 cells were infected with influenza virus PR8 or WSN.

Figure 5 is an analysis diagram of the virus liquid plaque detection results, wherein siFOS-PR8: siFOS co-transfection 24h + PR8 infection 24h, siNC-PR8: siNC transfection 24h + PR8 infection 24h, siFOS-WSN: siFOS co-transfection 24h + WSN infection 24h, siNC-WSN: siNC transfection 24h + WSN infection 24h.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

The H1395 cells and MDCK cells in the examples were commercially available.

The influenza virus strain PR8 in the embodiment is influenza virus A/PR/8 (H1N1); the influenza virus strain WSN is influenza virus A/WSN/1933, both of which are from Liu Shuwen Laboratory of Southern Medical University, amplified by SPF chicken embryos in the laboratory, and stored at -80°C.

All experiments related to live viruses in the examples were performed in biosafety level 2 or 3 facilities.

Example 1 Detection of c-Fos gene transcription level in H1395 cells infected with influenza virus

(1) H1395 cells were seeded in a 24-well plate at a cell density of 2 × 10 ⁵ cells/mL, 500 μL/well;

(2) 24 hours after inoculation, H1395 cells were divided into three groups: PR8 group, WSN group and control group. H1395 cells in PR8 group and WSN group were infected with influenza virus strains PR8 and WSN, respectively. PR8 virus solution diluted with 1640 culture medium (MOI=1) was added to the PR8-infected wells, and WSN virus solution diluted with 1640 culture medium (MOI=1) was added to the WSN-infected wells. An equal amount of 1640 culture medium without virus was added to the H1395 cells in the control group. Cells from the above groups were added to the wells and incubated for 2 hours before the medium was replaced (the replacement medium was: 0.05% 1 mg/mL TPCK-treated trypsin + 3% 100 mg/mL BSA + 97% 1640 culture medium + 1% double antibody, all percentages are volume percentages), 500 μL/well;

(3) 24 h after infection, cells from the PR8 group, WSN group, and control group were collected and RNA was extracted and reverse transcribed according to conventional methods. Real-time fluorescence quantitative PCR was performed. The primer sequences for detecting the c-Fos gene and the internal reference gene GAPDH were as follows:

c-Fos-F: 5’-CCGGGGATAGCCTCTCTTACT-3’;

c-Fos-R: 5’-CCAGGTCCGTGCAGAAGTC-3’;

GAPDH-F: 5’-GGAGCGAGATCCCTCCAAAAT-3’;

GAPDH-R: 5’-GGCTGTTGTCATACTTCTCATGG-3’;

The results are shown in Figure 1. After influenza virus PR8 and WSN infected H1395 cells, they could significantly upregulate the transcription level of c-Fos gene.

Example 2 Design and screening of optimal siRNA for interfering with c-Fos gene

(1) According to the c-Fos gene sequence published by NCBI (NCBI Gene: 2353), this example designed three pairs of siRNA sequences targeting the c-Fos gene. The siRNA sequences are shown in Table 1.

Nucleotide sequence of c-Fos gene:

ATGAGTGTTCTCGGGCTTCAACGCAGACTACGAGGGCTCATCCTCCCGCTGCAGCAGCGCGTCCCCGGCCGGGGATAGCCTCTCTTACTACCACTCACCCGCAGACTCCTTCTCCAGCATGGGCTCGCCTGTCAACGCGCAGGACTTCTGCACGGACCTGGCCGTCTCCAGTGCCAACTTCATTCCCACGGTCACTGCCATCTCGACCAGTCCGGACCTGCAGTGGCTGGTGCAGCCCGCCCTCGTCTCCTCCGTGGCCCCAT CGCAGACCAGAGCCCCTCACCCT TTCGGAGTCCCCGCCCCCTCCGCTGGGGCTTACTCCAGGGCTGGCGTTGTGAAGACCATGACAGGAGGCCGAGCGCAGAGCATTGGCAGGAGGGGCAAGGTGGAACAGTTATCTCCAGAAGAAGAAGAGAAAAGGAGAATCCGAAGGGAAAGGAATAAGATGGCTGCAGCCAAATGCCGCAACCGGAGGAGGGAGCTGACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGC AGACCGAGATTGCCAACCTGCTGAAGGAGAAGG AAAAACTAGAGTTCATCCTGGCAGCTCACCGACCTGCCTGCAAGATCCCTGATGACCTGGGCTTCCCAGAAGAGATGTCTGTGGCTTCCCTTGATCTGACTGGGGGCCTGCCAGAGGTTGCCACCCCGGAGTCTGAGGAGGCCTTCACCCTGCCTCTCCTCAATGACCCTGAGCCCAAGCCCTCAGTGGAACCTGTCAAGAGCATCAGCAGCATGGAGCTGAAGACCGAGCCCTTTTGATGACTTCCTGTTCCCAG CATCATCCAGGCCCAGTGGCTCTGAGACAGC CCGCTCCGTGCCAGACATGGACCTATCTGGGTCCTTCTATGCAGCAGACTGGGAGCCTCTGCACAGTGGCTCCCTGGGGATGGGGCCCATGGCCACAGAGCTGGAGCCCCTGTGCACTCCGGTGGTCACCTGTACTCCCAGCTGCACTGCTTACACGTCTTTCCTTCGTCTTCACCTACCCCGAGGCTGACTCCTTCCCCAGCTGTGCAGCTGCCCACCGCAAGGGCAGCAGCAGCAATGAGCCTTCCTCTGACTCGCTCA GCTCACCCACGCTGCTGGCCCTGTGA

Table 1 siRNA sequences

(2) The above four groups of siRNA and a mixture of siFOS-1, siFOS-2 and siFOS-3 (volume ratio of 1:1:1) were transfected into H1395 cells respectively. The specific transfection method is as follows:

① Before preparing the transfection mixed reagent, remove the culture medium in the 24-well plate containing H1395 cells, add 400 μL Opti-MEM/well, and temporarily place in a 37°C, 5% _CO2 cell culture incubator.

② Take an EP tube and add 50μL Opti-MEM, then add 2.5μL 20μM siRNA, and gently blow and mix 3-5 times to mix; gently invert and mix the transfection reagent lipofectamine 2000, take an EP tube and add 50μL Opti-MEM, then add 2μL lipofectamine 2000, and gently blow and mix 3-5 times, and let stand at room temperature for 5 minutes.

③Mix the transfection reagent and siRNA diluent, pipette gently 3-5 times to mix, and let stand at room temperature for 20 minutes to obtain the transfection complex; add the transfection complex to the 24-well plate in step ①, 100μL/well, and place it in a 37℃, 5% CO ₂ incubator for culture. After 6 hours, replace the culture medium (90% 1640 culture medium + 10% FBS, the above percentages are volume percentages), 500μL/well.

(3) 24 hours after transfection, cells from each group were collected and RNA was extracted, reverse transcribed and real-time fluorescence quantitative PCR was performed according to conventional methods. The primer sequences for detecting c-Fos gene and internal reference gene GAPDH were the same as those in Example 1.

The results are shown in Figure 2. All three groups of siRNA had a certain interference efficiency, but the highest interference efficiency was obtained by mixing and transfecting siFOS-1, siFOS-2 and siFOS-3 at a concentration of 20 μM in a 1:1:1 volume ratio (2.5 μL in total). The mRNA level of the c-Fos gene in H1395 cells transfected with siFOS1+2+3 was significantly lower than that in the MOCK control group.

Example 3 Replication level of c-Fos knockdown cells after infection with influenza virus PR8 and WSN strains

(1) Referring to Example 2, siFOS-1, siFOS-2 and siFOS-3, all at a concentration of 20 μM, were mixed at a volume ratio of 1:1:1 and transfected into H1395 cells (siFOS group), and the control group was transfected with siNC (siNC group).

(2) 24 h after transfection, the siFOS group and siNC group were infected with PR8 and WSN influenza virus strains, respectively, and a virus-free control group was added. PR8 virus solution diluted with 1640 medium (MOI=1) was added to the PR8-infected wells, and WSN virus solution diluted with 1640 medium (MOI=1) was added to the WSN-infected wells. An equal amount of virus-free 1640 medium was added to the H1395 cells in the control group. The above groups were added to the wells and incubated for 2 h before the medium was replaced (the replacement medium was: 0.05% 1 mg/mL TPCK-treated trypsin + 3% 100 mg/mL BSA + 97% 1640 medium + 1% double antibody, all percentages are volume percentages), 500 μL/well.

(3) 24 hours after infection, cells and viral supernatants from each group were collected, and a portion of the cells were lysed and subjected to western blot (protein immunoblot) detection, with the primary antibodies being mouse anti-influenza NP antibody and rabbit anti-human GAPDH antibody (both commercially available); another portion of the cells were subjected to RNA extraction, reverse transcription, and real-time fluorescence quantitative PCR detection according to conventional methods, with the primer sequences for detecting the c-Fos gene and the internal reference gene GAPDH being the same as in Example 1, and the primer sequences for detecting the viral NP gene being as shown below;

NP-F: 5’-TCAAGTGAGAGAGAGCCGGA-3’;

NP-R: 5’-TCAAAGTCGTACCCACTGGC-3’.

(4) The virus supernatant of each group collected in step (3) was tested for virus titer by virus plaque assay. The specific method is as follows:

① MDCK cells were plated at a density of 4×10 ⁵ /mL in 12-well plates, with 500 μL per well, and grown for 24 hours until the monolayer was confluent.

② Wash the cells twice with PBS, add serially diluted virus supernatant (10-fold dilution series, add 500 μL of diluted virus supernatant to each well, and 3 replicate wells for each dilution), and adsorb in a 37°C incubator for 1 hour.

③ Wash the cells twice with PBS, add 1.5 mL of semi-fixation maintenance solution (a mixture of 2×DMEM and cellulose solution in a ratio of 1:1 (V:V) containing 0.15% 1 mg/mL TPCK-treated trypsin + 5% 100 mg/mL BSA storage solution + 1% double antibody, all percentages are by volume) to each well, and culture at 37°C for 48 h.

④After culturing at 37℃ for 48h, aspirate and discard the semi-fixation maintenance solution, and wash the cells 1-2 times with PBS until the cellulose is completely washed away.

⑤ Staining: Add 500 μL tissue cell fixative to each well for 1 hour, discard the fixative and add 500 μL crystal violet, and stain for 2 hours.

⑥Discard crystal violet, wash with ddH ₂ O 5 times, observe and count the plaques.

The results of real-time fluorescence quantitative PCR are shown in Figure 3. It can be seen from the figure that the downregulation of c-Fos levels significantly inhibits the transcription level of the influenza virus NP gene (Figure 3). In addition, interference with c-Fos can significantly inhibit the protein level of the influenza virus protein NP (Figure 4).

The virus titer results are shown in Figure 5. The titer of siNC-PR8 calculated by plaque assay is 12.6×10 ⁵ PFU/mL, which is 9.7 times that of siFOS-PR8 (1.3×10 ⁵ PFU/mL); the virus titer of siNC-WSN is 17.3×10 ⁴ PFU/mL, which is 14 times that of siFOS-WSN (1.2×10 ⁴ PFU/mL). This result further proves that the c-Fos gene is a key host factor for influenza virus replication. The c-Fos gene has a good application prospect as a target for designing inhibition of influenza virus replication.

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (10)

1. A use of a c-Fos gene in preparing a product for preventing and treating influenza virus, wherein the nucleotide sequence of the c-Fos gene is as shown in SEQ ID No.1. 2. The use according to claim 1, characterized in that: The expression level of the protein encoded by the c-Fos gene is positively correlated with the influenza virus replication level. 3. The method according to claim 1, characterized in that it comprises the following steps: Inhibiting, silencing or knocking out the c-Fos gene can inhibit influenza virus replication. 4. A use of a c-Fos gene in preparing a product for regulating influenza virus replication, wherein the nucleotide sequence of the c-Fos gene is as shown in SEQ ID No.1. 5. The use according to claim 4, characterized in that: The regulation includes inhibiting, silencing or knocking out the c-Fos gene to inhibit influenza virus replication. 6. A siRNA, characterized in that the siRNA is at least one of siFOS-1, siFOS-2 and siFOS-3; and its nucleotide sequence is as follows: siFOS-1-sense:5'-UCUGCUUUGCAGACCGAGAUUTT-3'; siFOS-1-antisense:5'-AAUCUCCGGUCUGCAAAGCAGATT-3'; siFOS-2-sense:5'-GUGGAACAGUUAUCUCCAGAATT-3'; siFOS-2-antisense:5'-UUCUGGAGAUAACUGUUCCACTT-3'; siFOS-3-sense:5'-CACUGCUUACACGUCUUCCUUTT-3'; siFOS-3-antisense: 5'-AAGGAAGACGUGUAAGCAGUGTT-3'. 7. The siRNA according to claim 6, characterized in that the siRNA is a mixture of siFOS-1, siFOS-2 and siFOS-3; The molar ratio of siFOS-1, siFOS-2 and siFOS-3 is 1:1:1. 8. Use of the siRNA according to claim 6 or 7 in preparing a product for preventing and treating influenza virus. 9. Use of the siRNA according to claim 6 or 7 in preparing a product for inhibiting influenza virus replication. 10. The use according to any one of claims 1 to 5 or the use according to claim 8 or 9, characterized in that: The influenza virus is at least one of influenza virus strain PR8 and strain WSN.