CN112972651B

CN112972651B - Application of IFITMs in preparation of EBV (Epstein Barr virus) epithelial infection inhibitor and epithelial tumor prevention and treatment drugs

Info

Publication number: CN112972651B
Application number: CN202110197479.2A
Authority: CN
Inventors: 李欣; 杨英桂; 丁腾腾
Original assignee: Shenzhen Hospital of Southern Medical University
Current assignee: Shenzhen Hospital of Southern Medical University
Priority date: 2021-02-22
Filing date: 2021-02-22
Publication date: 2024-04-02
Anticipated expiration: 2041-02-22
Also published as: CN112972651A

Abstract

The invention discloses application of IFITMs in preparation of an EBV epithelial infection inhibitor and an epithelial tumor prevention and treatment drug, wherein the IFITMs comprise IFITM1, IFITM2 and IFITM3. The invention confirms that IFITM1 plays an inhibitory role in the EBV infection process for the first time by constructing an interferon inducible transmembrane protein over-expression and knock-down cell line, further reveals the function of IFITM1 in EBV positive nasopharyngeal carcinoma and the relation between the IFITM1 and LMP1, and also indicates that IFITM1 can reduce the EBV infection epithelial cells in the EBV exposure state, can reduce the growth rate of tumors, and can be used for developing medicines for preventing and treating the EBV infection and nasopharyngeal carcinoma.

Description

Application of IFITMs in preparation of EBV (Epstein Barr virus) epithelial infection inhibitor and epithelial tumor prevention and treatment drugs

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to application of IFITMs in preparation of an EBV epithelial infection inhibitor and an epithelial tumor prevention and treatment drug.

Background

EBV (EBV) belongs to one of the members of the gamma herpesvirus family, also known as human herpesvirus type 4. The EBV infection rate is extremely high, and investigation shows that more than 95% of people are infected with the EBV in young, and the EBV infection population is widely distributed, and the infants to the elderly can cause diseases. EBV reaches B lymphocytes mainly by infecting the epithelial cells of the oropharynx, following vascular trafficking in the epithelial tissue, and then enters a latent infection phase in the B lymphocytes. EBV infection can induce various diseases such as infectious mononucleosis, haemophilus cell syndrome, oral hairy leukoplakia, chronic active EBV infection and other non-neoplastic diseases, and in addition, the existing research shows that EBV infection can also induce the occurrence of various malignant tumors: such as B/T/NK cell lymphoma in hematological tumors, burkitt's lymphoma, hodgkin's lymphoma, etc.; nasopharyngeal carcinoma, gastric cancer, hepatocellular carcinoma, breast cancer, etc. in nonhematological tumor. From the eyes, nasopharynx, to the skin, hematopoietic system, stomach, liver, lung, breast, etc., multiple organs of the whole body can be involved. Thus, EBV infection is extremely threatening to human life health.

IFITMs (interferon-induced transmembrane protein, interferon-inducible transmembrane proteins) are ubiquitously expressed in eukaryotic and prokaryotic animals. The IFITMs can be divided into three categories according to their function: the first class includes mainly human and mouse IFITM1, IFITM2 and IFITM3 and mouse IFITM6 and IFITM7 molecules, the first three proteins originally designated CD225 (or 9-27), 1-8D and 1-8U, respectively. CD22, 1-8D and 1-8U are also known as fragilis2, fragilis3 and fragilis on mice. All three are known as immune-related IFITMs because they have strong antiviral activity. IFITMs have been found to inhibit infection by human pathogenic viruses such as hepatitis virus (HCV), human Immunodeficiency Virus (HIV), ZIKV, etc., but their role in EBV infection and its associated diseases has not been known.

Disclosure of Invention

The invention aims to provide an application of interferon inducible transmembrane protein or an over-expression cell thereof in preparing an EBV infection inhibitor;

another object of the present invention is to provide an application of interferon-inducible transmembrane proteins or overexpressed cells thereof in the preparation of a medicament for preventing and treating cancer;

another object of the invention is to provide the use of interferon-inducible transmembrane proteins or overexpressed cells thereof in the preparation of inhibitors of latent membrane protein LMP 1;

it is another object of the present invention to provide the use of an interferon inducible transmembrane protein or overexpressed cells thereof in the preparation of an EBV receptor or a promoter antagonist;

another object of the present invention is to provide a medicament;

it is another object of the present invention to provide a cell line.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided:

the application of interferon inducible transmembrane proteins or overexpression cells thereof in the preparation of various EBV infection inhibitors;

wherein the interferon inducible transmembrane proteins include IFITM1, IFITM2, and IFITM3.

Further, the amino acid sequence of the IFITM1 protein is:

MHKEEHEVAVLGPPPSTILPRSTVINIHSETSVPDHVVWSLFNTLFLNWCCLGFIAFAYSVKSRDRKMVGDVTGAQAYASTAKCLNIWALILGILMTIGFILLLVFGSVTVYHIMLQIIQEKRGY(SEQ ID NO.1)。

further, the amino acid sequence of the IFITM2 protein is:

MNHIVQTFSPVNSGQPPNYEMLKEEQEVAMLGVPHNPAPPMSTVIHIRSETSVPDHVVWSLFNTLFMNTCCLGFIAFAYSVKSRDRKMVGDVTGAQAYASTAKCLNIWALILGIFMTILLIIIPVLVVQAQR(SEQ ID NO.2)。

further, the amino acid sequence of the IFITM3 protein is:

MNHTVQTFFSPVNSGQPPNYEMLKEEHEVAVLGAPHNPAPPTSTVIHIRSETSVPDHVVWSLFNTLFMNPCCLGFIAFAYSVKSRDRKMVGDVTGAQAYASTAKCLNIWALILGILMTILLIVIPVLIFQAYG(SEQ ID NO.3)。

still further, the interferon inducible transmembrane protein further includes: the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 has the functions of inhibiting the fusion of EB virus and host cell, the entry of EB virus into host cell or the replication of EB virus in host cell after one or more amino acids and/or terminal modification are substituted, deleted and/or added.

Further, the interferon-inducible transmembrane proteins are IFITM1, IFITM2, and IFITM3.

In the embodiment of the invention, the interferon inducible transmembrane protein is IFITM1.

EBV primarily infects B cells and epithelial cells. The inventors found that the mechanism of entry of EBV into epithelial cells is different from B cells. Whether the IFITMs are used as barriers for preventing and treating EBV-related diseases, especially malignant epithelial tumors, can bring great benefit to human health by researching whether the IFITMs are used as EBV receptor-related proteins or competitive binding proteins to influence the EBV infection, especially the infection process in epithelial cells and the mechanism of the IFITMs.

Further, the EBV infection inhibitor acts on epithelial cells.

Further, the above-mentioned epithelial cells include normal epithelial cells (NP 69 cells, NP460 cells) and epithelial tumor cells (HK 1 cells).

The invention should include other epithelial cells that may be infected by EBV, depending on the actual use requirements.

In a second aspect of the invention, there is provided:

use of interferon-inducible transmembrane proteins or overexpressed cells thereof in the preparation of a medicament for the prevention and treatment of cancer; wherein the interferon inducible transmembrane proteins include IFITM1, IFITM2, and IFITM3.

Further, such cancers include nasopharyngeal carcinoma and other epithelial tumors associated with EBV.

The embodiment of the invention proves that the IFITM1 recombinant protein can reduce the growth speed of tumors, so that a novel prevention and treatment means for nasopharyngeal carcinoma can be developed based on the IFITM1.

Further, the dosage forms of the above-mentioned cancer prevention and treatment drugs include, but are not limited to, tablets, capsules, injections, granules, solutions and powders; of course, the person skilled in the art can reasonably adopt any dosage form to prepare the cancer prevention and treatment medicament according to the actual use requirement.

In a third aspect of the invention, there is provided:

use of an interferon-inducible transmembrane protein or an overexpressed cell thereof in the preparation of a latent membrane protein LMP1 inhibitor;

The inventor finds that IFITM1 can inhibit proliferation of nasopharyngeal carcinoma and delay tumor progression by reducing expression of key oncoprotein LMP1 in latency after EBV infects nasopharyngeal epithelial cells and develops into nasopharyngeal carcinoma.

Further, dosage forms of the latent membrane protein LMP1 inhibitors include, but are not limited to, tablets, capsules, injections, granules, solutions and powders; of course, the person skilled in the art can reasonably adopt any formulation to prepare the latent membrane protein LMP1 inhibitor according to the actual use requirement.

In a fourth aspect of the invention, there is provided:

use of an interferon-inducible transmembrane protein or an overexpressed cell thereof in the preparation of an EBV receptor or a promoter antagonist; wherein the interferon inducible transmembrane proteins include IFITM1, IFITM2, and IFITM3.

Further, the EBV receptors or promoters include MYH9, EGFR, ITGAV and EphA2.

Further, the EBV receptor or promoting factor is EphA2.

EphA2 is a transmembrane tyrosine kinase receptor, which belongs to one of the superfamily of tyrosine kinase receptor (receptor tyrosine kinases, RTKs). EphA2 is a major host-derived receptor for EBV entry into epithelial cells by binding to EBV envelope glycoproteins (gH/gL, gB) and promoting EBV infection. IFITMs may differ in their role in antiviral effects in different host cells, and thus IFITM1 may co-affect the viral infection process by interacting with host proteins.

The examples in this invention show that, on the basis that IFITM1 can inhibit the EBV-infected epithelium, the mechanism of action of IFITM1 on EphA2 is: IFITM1 inhibits or impairs binding of the latter to EBV glycoproteins (gH/gL, gB) by binding to EphA2, thereby reducing the ability of EBV to infect epithelial cells.

In a fifth aspect of the invention, there is provided:

a medicament comprising an interferon inducible transmembrane protein or overexpressing cells thereof; wherein the interferon-inducible transmembrane proteins include IFITM1, IFITM2, and IFITM3, and the medicament is for preventing EBV infection.

Further, the medicine also contains other pharmaceutically acceptable auxiliary materials.

Further, the dosage forms of the above drugs include, but are not limited to, tablets, capsules, injections, granules, solutions, powders, and exosome preparations, AAV preparations.

In a sixth aspect of the invention, there is provided:

a cell line that expresses one or more of IFITM1, IFITM2, or IFITM3.

The invention constructs an over-expression IFITM1 cell line, and the constructed cells can effectively and stably express the IFITM1.

The beneficial effects of the invention are as follows:

1. the invention confirms that IFITM1 plays an inhibitory role in the EBV infection process for the first time by constructing an interferon inducible transmembrane protein over-expression and knock-down cell line, and the inhibitory effect exists in normal epithelium (NP 69, HEK 293) and tumor cells (HK 1). Meanwhile, the invention describes that the IFITM2 and the IFITM3 possibly play an inhibitory role in the EBV infection process, and provides a theoretical basis for developing anti-EBV infection medicines.

2. The invention also discloses the function of the IFITM1 in the EBV positive nasopharyngeal carcinoma and the relation between the IFITM1 and the LMP1, and proves that the IFITM1 can inhibit proliferation of the nasopharyngeal carcinoma and delay tumor progression by reducing the expression of the key oncoprotein LMP1 in the latency period after the EBV infects nasopharyngeal epithelial cells and develops into the nasopharyngeal carcinoma.

3. The invention also points out that IFITM1 can reduce the EBV infection of epithelial cells under the EBV exposure state, and a means for effectively preventing the EBV infection can be developed based on the IFITM1; the IFITM1 recombinant protein can reduce the growth rate of tumors, and a novel prevention and treatment means for nasopharyngeal carcinoma can be developed based on the IFITM1.

Drawings

FIG. 1 is a heat map plotted against the difference in the expression of EBV-infected epithelial cell receptors or promoters and IFITMs;

FIG. 2 shows IFITM1 expression of NP69 (A), HEK293 (B), and HK1 (C) stably overexpressing IFITM1 cell lines;

FIG. 3 shows IFITM1 expression of NP69 (A), HEK293 (B), and HK1 (C) cell lines after knocking down IFITM1;

FIG. 4 is a flow chart (A, B and C) and representative fluorescence photographs (D) of EBV-GFP infection efficiencies of NP69, HEK293, and HK1 cell lines stably overexpressing IFITM1;

FIG. 5 is a flow chart of EBV-GFP infection efficiency for NP69, HEK293, and HK1 cell lines of IFITM1 (A, B and C) and representative fluorescence photographs (D);

FIG. 6 is a fluorescent co-localization experiment (6A) and bi-directional co-immunoprecipitation (6B) of IFITM1 with EphA 2;

FIG. 7 is a flow chart (A) and representative fluorescence photograph (B) of the efficiency of EBV infection of an over-expressed IFITM1 cell line by over-expressed EphA2 in a HEK293 cell line;

FIG. 8 is a comparison of in vitro and in vivo experiments in which IFITM1 inhibited EBV+ tumor proliferation, wherein A is a comparison of in vitro inhibition of tumor proliferation by IFITM1; b is a tumor volume change photo in an in vivo experiment; c is a tumor volume increase curve in vivo experiments;

FIG. 9 is an immunoblot map of the effect of IFIMT1 over-expression (A) and knock-down (B) on LMP1 expression.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to the following specific embodiments. It should be understood that the detailed description is presented herein for purposes of illustration only and is not intended to limit the invention.

The experimental materials and reagents used, unless otherwise specified, are those conventionally available commercially.

Detection of IFITMs expression in epithelial cells

In this example, B cell line (Daudi), nasopharyngeal carcinoma cell line (HK 1), nasopharyngeal carcinoma EBV positive cell line (HK 1-EBV and C666-1), human embryonic kidney 239 cell (HEK 293), immortalized normal human nasopharyngeal epithelial cell line (NP 69), immortalized normal human nasopharyngeal epithelial cell line (NP 460) and EBV positive nasopharyngeal epithelial cell line (NP 460-EBV) were selected as test subjects, and IFITMs expression in each test subject was examined.

(1) Cell culture:

the B cell line (Daudi), the nasopharyngeal carcinoma cell line (HK 1) and the nasopharyngeal carcinoma EBV positive cell line (HK 1-EBV and C666-1) were cultured with RPMI1640 medium containing 10% fetal bovine serum.

Human embryonic kidney 239 cells (HEK 293) were cultured in DMEM medium containing 10% fetal bovine serum.

Immortalized normal human nasopharyngeal epithelial cell line (NP 69) was cultured using serum-free Defined keratinocyte-SFM medium (DKSFM) supplemented with epithelial growth factors during the culture.

Immortalized normal human nasopharyngeal epithelial cell lines (NP 460) and EBV positive nasopharyngeal epithelial cell lines (NP 460-EBV) were each cultured in mixed medium (DKSFM medium and serum-free mixed 1:1)Culture medium), and respectively supplementing the epithelial growth factors according to the respective growth conditions.

All the cells were cultured in the presence of penicillin and streptomycin in an amount of 1% and at 37℃and 5% carbon dioxide.

(2) RNA extraction and quality identification:

RNA extraction adopts phenol-chloroform extraction method, nanodrop ^TM The spectrophotometer detects the concentration of RNA, agarose gel electrophoresis detects the purity of RNA, and the Agilent 2100 bioanalyzer detects the integrity of RNA.

(3) Transcriptome sequencing library building:

transcriptome sequencing library constructionUltraTM RNA Library Prep Kit kit, methods of use refer to kit instructions. And (5) performing transcriptome sequencing after the library is established to be qualified.

(4) Differential expression analysis of IFITMs

NP69, NP460, HK1, HEK293 cells were used as the EBV (-) epithelial cell group (EBV. N), and NP460-EBV, HK1-EBV, C666-1 were used as the EBV (+) epithelial cell group (EBV. P). Each group was screened separately for differentially expressed genes (3 technical replicates per group) from the B cell line group (b_cells).

After calculating the average value of the gene expression amounts of each group, taking Log10, and drawing a heat map (figure 1) of the EBV infection related receptor or promoter such as IFITMs (IFITM 1, IFITM2 and IFITM 3) and MYH9 and EGFR, ITGAV, ephA, it is found that the IFITM1, the IFITM2 and the IFITM3 not only have lower expression level in B cells, but also basically have reverse expression trend in EBV.N and EBV.P with the EBV infection related receptor or promoter, and the IFITM1, the IFITM2 and the IFITM3 possibly play a specific inhibitory role in the process of infecting epithelial cells by the EBV. Of these, IFITM1 is most pronounced in ebv.n and ebv.p. It was demonstrated that IFITM1, IFITM2, IFITM3 (especially IFTM 1) may be associated with the inhibition of EBV-infected epithelial cells.

Construction of IFITM1 overexpressing epithelial cell lines

3 cells were selected for the construction of the overexpression cell line of IFITM1. Among them, NP69, HEK293, HK1 were selected as the test subjects. The reason is that HEK293 is an epithelial cell, which is a common model cell in the research of virus infection; NP69 is an immortalized normal human nasopharyngeal epithelial cell line, used to illustrate whether IFITM1 plays a protective role on normal epithelial cells before EBV has been contacted; HK1 is a human nasopharyngeal carcinoma cell line, a nasopharyngeal carcinoma epithelial type tumor, whose occurrence and progression are highly correlated with EBV infection. This example demonstrates by HK1 whether IFITM1 protects tumor cells after malignant transformation of epithelial cells has occurred.

(1) Cell culture:

selecting HEK293T cells for slow virus construction and amplification, and culturing the HEK293T cells by the same method. NP69, HEK293, HK1 culture methods were as in the examples above.

(2) Construction of IFITM1 over-expression plasmid and lentiviral packaging:

the IFITM1 sequence is obtained from normal nasopharyngeal cells through PCR amplification, the obtained IFITM1 sequence is cloned into a lentiviral pCW57.RFP-P2A-MCS plasmid (purchased from AddGene), and after the plasmid is confirmed by sequencing, HEK293T cells are transfected together with lentiviral packaging plasmids VSVG and psPAX2 (purchased from AddGene), and IFITM1 high-expression viruses in the cells and a control virus of unclonable IFITM1 are collected and concentrated for cell infection or stored at the temperature of-80 ℃ for standby.

(3) IFITM1 over-expressing lentivirus infected cells:

the NP69, HEK293 and HK1 cells cultured in the above steps were respectively infected after optimizing the infection efficiency of MSCV-IFITM1-ires-hCD2 obtained in the above steps and control virus MSCV-ires-hCD 2. The intensity and proportion of fluorescence expression were observed 72 hours after infection, and uninfected cells were further knocked out with 700 μg/mL puromycin (or infected cells were sorted by flow cytometry). When the fluorescence ratio is above 90%, the over-expression effect is detected by adopting a fluorescence quantitative Polymerase Chain Reaction (PCR).

Results:

the stable over-expression cell lines of IFITM1 were all effective in expressing IFITM1, compared to the expression of IFITM1 in control virus-infected cells, indicating successful construction of the stable over-expression cell line of IFITM1 (see fig. 2).

Construction of IFITM1 knock-down epithelial cell lines

NP69, HEK293, HK1 cells were selected for IFITM1 knock-down cell line construction for the same reasons as above.

(1) Cell culture:

(2) Construction of IFITM1 knockdown plasmid and lentiviral packaging:

short hairpin RNAs (shRNA) 870-hushift 1 (sequence: 5'-TGTCTACAGTGTCATTCAAT-3' (SEQ ID No. 4)) and 642-hushift 1 (sequence: 5'-TGTGACAGTCTACCATATTA-3' (SEQ ID No. 5)) were designed to target IFITM1. shRNA was cloned into the Tet-pLKO-puro vector (purchased from AddGene) followed by co-transfection of HEK293T cells with psPAX2 and pMD2 (purchased from AddGene), and after 72 hours the cell supernatant containing assembled lentivirus was collected and concentrated for cell infection or stored at-80 ℃ for later use.

(3) IFITM1 knockdown lentivirus infected cells:

after optimizing infection efficiency of 642-shift 1-containing virus, 870-shift 1-containing virus and control lentivirus sh-LacZ, NP69, HEK293 and HK1 cells are infected respectively; changing the liquid after 24 hours, continuously culturing for 1-2 days, adding 1 mug/mL puro for screening for 2 days, and then screening for 2-3 days by using 3 mug/mL puro. The resulting infected cells were stored in-80 for later use or subsequently induced with 2.5. Mu.g/mL doxycycline (Dox) for 48 hours for IFITM1 knockdown efficacy detection.

Results:

the significantly reduced IFITM1 expression levels of the 642-shift containing virus and 870-shift containing virus infected cells compared to the IFITM1 expression levels of the control lentiviral sh-LacZ infected cells, indicating successful IFITM1 knockdown cell line construction (fig. 3).

Overexpression of IFITM1 inhibits EBV infection epithelial cell validation

(1) Preparation of EBV-GFP:

EBV-GFP specifically refers to EBV carrying a GFP fluorescent marker sequence, prepared by a method referred to Zhang H, et al Nat microbiol.2018Feb;3 (2):1-8.

(2) Infection of overexpressing IFITM1 cells:

the over-expressed IFITM1 cells constructed in the above examples (NP 69, HEK293, HK1 cells) were inoculated in 6cm dishes at the appropriate density and incubated overnight. When the cells grew to a confluence of about 50%, the fresh medium was changed and incubated for 1 hour. The EBV-GFP prepared by the above steps was treated to obtain a corresponding multiplicity of infection (MOI=10 ³ ～2.5×10 ³ ) The virus infection was performed by adding, discarding the original medium after 24 hours, washing with PBS 2 times to wash out unbound virus particles, changing fresh medium and adding 0.8% (v/v) goat anti-human IgG antibody for 6 hours at 37℃to wash out residual antibody, observing the fluorescence of cells (GFP+) under a fluorescence microscope, and detecting the ratio of GFP+ cells by flow cytometry.

As shown in FIG. 4, the infection efficiency of EBV-GFP was reduced by about 50% compared to the control group after the IFITM1 was overexpressed by the epithelial cells, indicating that the IFITM1 was overexpressed effectively inhibiting the EBV infection.

Knock down of IFITM1 inhibits EBV infection epithelial cell verification

The preparation method of EBV-GFP and the method of infecting cells are the same as in the above examples.

As shown in FIG. 5, the infection efficiency of EBV-GFP was increased by 63% -119% compared to the control group after knocking down IFITM1 by the epithelial cells, indicating that IFITM1 can effectively inhibit the infection of EBV.

Effect of IFITM1 on the mechanism of EBV infection

Effect of ifitm1 on epithelial EBV infection receptor EphA2.

(1) Cell culture:

in this example, NP69, HEK293, and HK1 cells were selected as the subjects, and the cell culture method was as described in the above examples.

(2) The above-mentioned cultured NP69, HEK293, HK1 cells were cultured in 35cm confocal dishes, and when grown to 50-60%, PBS was washed once, and then 4% paraformaldehyde was added to fix the cells for 10 minutes, followed by washing off the fixative, and 0.2% Triton X-100 was added to permeabilize for 10 minutes.

(3) Blocking with 5% goat serum at room temperature for 1 hr followed by addition of primary antibodies to IFITM1 and EphA2 for overnight incubation, washing off the primary antibody the next day, adding fluorescent secondary antibody (Alexa488 goat anti-mouse IgG H&L and Alexa->647 goat anti-rabbit IgG H&L) incubated for 1 hour at room temperature in the dark and then stained with nuclear dye DAPI for 10 minutes. The staining was observed using a CQ1 carousel confocal high content quantitative analysis system.

Results:

after staining, IFITM1 will fluoresce green and EphA2 will fluoresce red, and the overlap will appear yellow (i.e. indicating co-localization of IFITM1 and EphA 2). As can be seen from fig. 6A (only NP69 results are shown), IFITM1 and EphA2 present a distinct co-localization at the cell membrane site.

Interaction of IFITM1 with EphA2

The cell culture methods in the above examples were used to prepare NP69, HEK293, and HK1 cells, and protein lysates of NP69, HEK293, and HK1 cells were prepared.

1 mg of NP69, HEK293, HK1 mill groove protein lysates prepared in the above steps were taken, respectively, and IFITM1 was added thereto, followed by gentle mixing at 4℃overnight with an equivalent amount of non-specifically immunized homologous antibody (IgG) as a control. According to the instructions of the co-immunoprecipitation kit (Abs 955), 5. Mu.L of Protein A and 5. Mu.L of Protein G were added and gently mixed at 4℃for 3 hours to obtain an immunocomplex precipitate.

Washing immune complex precipitate, adding a loading buffer solution, heating and denaturing for 5 minutes at 95 ℃, then carrying out instantaneous centrifugation for 1 minute at 14000g, and taking a proper amount (10-30 mu L) for Western immunoblotting detection.

The results are shown in FIG. 6B (only NP69 results are shown), and Western blot detection results show that IFITM1 is able to specifically precipitate EphA2 compared to the non-specific immune IgG control, and vice versa. Indicating that there is an interaction between the two.

IFITM1 partial reversion of the promotion of EBV infection by highly expressed EphA2

EphA2 lentiviruses were constructed by the IFITM1 lentivirus construction method in the above examples using the commercially available EphA2 overexpressing plasmid (EphA 2 overexpressing plasmid in this example is from the university of middle mountain Zeng Musheng subject group).

NP69, HEK293 and HK1 cells were infected with the prepared EphA2 lentivirus, respectively, and the effect of the overexpression of EphA2 on the EBV infection efficiency was examined.

The results are shown in FIG. 7, and the results show that the over-expression of EphA2 can significantly improve the EBV infection efficiency, thereby reversely verifying the conclusion that IFITM1 can inhibit the EBV infection.

In summary, based on the finding that IFITM1 can inhibit the epithelium of EBV infection in the above embodiment, the mechanism is further disclosed in this embodiment: IFITM1 inhibits or impairs binding of the latter to EBV glycoproteins (gH/gL, gB) by binding to EphA2, thereby reducing the ability of EBV to infect epithelial cells.

Inhibition of EBV positive epithelial tumor proliferation by IFITM1

(1) Cell culture:

c666-1 cells are selected as an experimental object, and the C666-1 cells are an epithelial nasopharyngeal carcinoma cell line which is naturally positive to EBV. The cultivation method is as described in the examples above.

Meanwhile, an IFITM1 over-expression C666-1 cell line (C666-1-IFITM 1-OE) was constructed as described in the above examples.

(2) IFITM1 inhibition tumor proliferation assay:

ifitm1 inhibition of tumor proliferation in vitro experiments:

the IFITM1 tumor proliferation inhibition in vitro experiment adopts an MTT proliferation method, and comprises the following specific steps:

control cells (C666-1-vector) and C666-1-IFITM1-OE overexpressing IFITM1 in logarithmic growth phase were counted separately and inoculated into well plates with 1000 cells (100. Mu.L) per well. Cell proliferation assays (1/10 volume of MTT was added, supernatant was discarded after 4h, and absorbance values were measured after shaking for 10 min with dimethyl sulfoxide (DMSO)) were performed on days 1-7, respectively, and proliferation curves were drawn.

Ifitm1 inhibition of tumor proliferation in vivo experiments:

the in vivo experiment of inhibiting tumor proliferation by IFITM1 adopts a nude mouse tumorigenesis method, which comprises the following specific steps:

taking the digestion count of C666-1-vector and C666-1-IFITM1-OE in logarithmic growth phase, washing with pre-cooled PBS, and washing with 5×10 ⁶ The amount/volume (about 200. Mu.L) was inoculated subcutaneously into the posterior portion of the armpit of the nude mice. After about 2 weeks of rearing, tumor volumes were measured every 3 days and volume increase curves were plotted.

Results:

as shown in FIG. 8, the overexpression of IFITM1 was effective in inhibiting tumor proliferation in both MTT cell proliferation experiments (in vitro) and nude mouse tumorigenesis experiments (in vitro).

Downregulating effect of IFITM1 on EBV latent membrane protein LMP1

(1) Cell culture:

the C666-1 cells were selected as subjects, and the culture methods were as described in the above examples.

Meanwhile, an IFITM1 over-expression C666-1 cell strain and an IFITM1 knockdown C666-1 cell strain are constructed, and the construction method is as shown in the embodiment.

(2) And detecting the IFITM1 expression level in the IFITM1 over-expression C666-1 cell strain and the IFITM1 knock-down C666-1 cell strain by adopting an immunoblotting method.

The immunoblotting detection procedure is shown in the examples above.

As shown in fig. 9, the overexpression of IFITM1 significantly reduced the expression of LMP1, and knock-down of IFITM1 significantly upregulated the expression of LMP 1.

In conclusion, after EBV infects nasopharyngeal epithelial cells and develops into nasopharyngeal carcinoma, IFITM1 can inhibit proliferation of nasopharyngeal carcinoma and delay tumor progression by reducing expression of key oncoprotein LMP1 in latency.

Preventive effect of IFITM1 on EBV-infected nasopharyngeal epithelial cells

Nude mice were randomly divided into two groups of 6, each of which were intraperitoneally injected with the IFITM1 recombinant protein (experimental group) and physiological saline (control group), respectively, once every 3 days. After 3 injections, HEK293, NP69 and HK1 cells overexpressing EphA2 were inoculated subcutaneously into two groups of nude mice, respectively, and then EBV-GFP carrying a fluorescent marker (preparation method as in the above example) was inoculated into the same site, and every 12 hours after the inoculation, photographed with a small animal imaging device, and the amount of fluorescent accumulation at the inoculated site was recorded. Nude mice were sacrificed after 7 days of continuous observation, and the inoculated tissues were removed to prepare cell homogenate upflow cells for EBV infection efficiency analysis and comparison.

As a result, it was found that the amount of fluorescence accumulation and infection efficiency at the inoculation site of nude mice injected with the recombinant protein IFITM1 intraperitoneally were significantly lower than those of the physiological saline group. It was demonstrated that IFITM1 confers on cells the ability to resist EBV infection.

Treatment of nasopharyngeal carcinoma by IFITM1

Nude mice were randomly divided into two groups of 6 cells each, and simultaneously inoculated subcutaneously with C666-1 cells. On day 14 after inoculation, physiological saline (control group) and IFITM1 recombinant protein (experimental group) were intraperitoneally injected every 3 days, respectively, after which tumors were measured every 3 days

As a result, it was found that the intraperitoneal injection of the IFITM1 recombinant protein significantly retarded the growth rate of subcutaneous tumors in nude mice compared with the physiological saline group.

Taken together, the above examples demonstrate that IFITM1 reduces the EBV infection of epithelial cells in the EBV exposed state, and therefore, a means for effectively preventing EBV infection can be developed based on IFITM1. However, the above examples also demonstrate that the recombinant protein of IFITM1 can reduce the growth rate of tumors, and thus new treatments for nasopharyngeal carcinoma or other epithelial tumors associated with EBV can also be developed based on IFITM1.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Li Xin

Yang Yinggui

Ding Tengteng

<120> application of IFITMs in preparation of EBV epithelial infection inhibitor and epithelial tumor prevention and treatment drug

<130>

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 125

<212> PRT

<213> IFITM1

<400> 1

Met His Lys Glu Glu His Glu Val Ala Val Leu Gly Pro Pro Pro Ser

1 5 10 15

Thr Ile Leu Pro Arg Ser Thr Val Ile Asn Ile His Ser Glu Thr Ser

20 25 30

Val Pro Asp His Val Val Trp Ser Leu Phe Asn Thr Leu Phe Leu Asn

35 40 45

Trp Cys Cys Leu Gly Phe Ile Ala Phe Ala Tyr Ser Val Lys Ser Arg

50 55 60

Asp Arg Lys Met Val Gly Asp Val Thr Gly Ala Gln Ala Tyr Ala Ser

65 70 75 80

Thr Ala Lys Cys Leu Asn Ile Trp Ala Leu Ile Leu Gly Ile Leu Met

85 90 95

Thr Ile Gly Phe Ile Leu Leu Leu Val Phe Gly Ser Val Thr Val Tyr

100 105 110

His Ile Met Leu Gln Ile Ile Gln Glu Lys Arg Gly Tyr

115 120 125

<210> 2

<211> 132

<212> PRT

<213> IFITM2

<400> 2

Met Asn His Ile Val Gln Thr Phe Ser Pro Val Asn Ser Gly Gln Pro

1 5 10 15

Pro Asn Tyr Glu Met Leu Lys Glu Glu Gln Glu Val Ala Met Leu Gly

20 25 30

Val Pro His Asn Pro Ala Pro Pro Met Ser Thr Val Ile His Ile Arg

35 40 45

Ser Glu Thr Ser Val Pro Asp His Val Val Trp Ser Leu Phe Asn Thr

50 55 60

Leu Phe Met Asn Thr Cys Cys Leu Gly Phe Ile Ala Phe Ala Tyr Ser

65 70 75 80

Val Lys Ser Arg Asp Arg Lys Met Val Gly Asp Val Thr Gly Ala Gln

85 90 95

Ala Tyr Ala Ser Thr Ala Lys Cys Leu Asn Ile Trp Ala Leu Ile Leu

100 105 110

Gly Ile Phe Met Thr Ile Leu Leu Ile Ile Ile Pro Val Leu Val Val

115 120 125

Gln Ala Gln Arg

130

<210> 3

<211> 133

<212> PRT

<213> IFITM3

<400> 3

Met Asn His Thr Val Gln Thr Phe Phe Ser Pro Val Asn Ser Gly Gln

1 5 10 15

Pro Pro Asn Tyr Glu Met Leu Lys Glu Glu His Glu Val Ala Val Leu

20 25 30

Gly Ala Pro His Asn Pro Ala Pro Pro Thr Ser Thr Val Ile His Ile

35 40 45

Arg Ser Glu Thr Ser Val Pro Asp His Val Val Trp Ser Leu Phe Asn

50 55 60

Thr Leu Phe Met Asn Pro Cys Cys Leu Gly Phe Ile Ala Phe Ala Tyr

65 70 75 80

Ser Val Lys Ser Arg Asp Arg Lys Met Val Gly Asp Val Thr Gly Ala

85 90 95

Gln Ala Tyr Ala Ser Thr Ala Lys Cys Leu Asn Ile Trp Ala Leu Ile

100 105 110

Leu Gly Ile Leu Met Thr Ile Leu Leu Ile Val Ile Pro Val Leu Ile

115 120 125

Phe Gln Ala Tyr Gly

130

<210> 4

<211> 20

<212> DNA

<213> artificial sequence

<400> 4

tgtctacagt gtcattcaat 20

<210> 5

<211> 20

<212> DNA

<213> artificial sequence

<400> 5

tgtgacagtc taccatatta 20

Claims

1. The application of the interferon-induced transmembrane protein over-expressed epithelial cells in preparing an epithelial tumor cell EBV infection inhibitor;

wherein the interferon inducible transmembrane protein is IFITM1;

such tumors include nasopharyngeal carcinoma and other EBV-associated epithelial tumors;

the interferon-inducible transmembrane protein overexpressing cells antagonize latent membrane proteins LMP1, EBV receptors or promoters;

the EBV receptors or promoters include MYH9, EGFR, ITGAV and EphA2.