CN108272815B - Application of Epstein-Barr virus miR-BART10-5p inhibitor - Google Patents

Abstract

The invention discloses the application of EB virus miR-BART10-5p inhibitor. The present invention uses microtubule formation experiments and chicken embryo chorioallantoic membrane experiments to find that Epstein-Barr virus miR-BART10-5p, which is highly expressed in nasopharyngeal carcinoma cells, can significantly promote nasopharyngeal carcinoma angiogenesis. The Epstein-Barr virus miR-BART10-5p inhibitor of the present invention is based on the antisense oligonucleotide of Epstein-Barr virus miR-BART10-5p, obtained through a series of modifications, and is obtained through a microtubule formation experiment, a chicken embryo chorioallantoic membrane experiment and Matrigel suppository experiments, it was confirmed that the EB virus miR-BART10-5p inhibitor can significantly inhibit the angiogenesis of nasopharyngeal carcinoma, and has the effect of treating nasopharyngeal carcinoma. Further, it can also be used to prevent and treat the recurrence of nasopharyngeal carcinoma. and transfer.

Description

Application of Epstein-Barr virus miR-BART10-5p inhibitor

technical field

The invention belongs to the field of tumor molecular biology, and more particularly, relates to the application of Epstein-Barr virus miR-BART10-5p inhibitors.

Background technique

Nasopharyngeal carcinoma (NPC) refers to a malignant tumor that occurs in the nasopharyngeal mucosa. It mostly occurs in middle-aged people and occasionally occurs in adolescents. It has a high degree of malignancy and is the disease with the highest recurrence and metastasis rates among head and neck tumors. Neck lymph node metastasis can occur and the metastasis rate is as high as 80%, which is often high in South China and Southeast Asia in China, and has obvious regional characteristics. 20-30% of patients with nasopharyngeal carcinoma still have recurrence and metastasis within 5 years after being cured, which is one of the main factors leading to clinical death. At present, the main methods of clinical treatment of nasopharyngeal carcinoma are surgery, radiotherapy and chemotherapy. Although the current treatment level has been greatly improved, the 5-year survival rate of nasopharyngeal carcinoma is still only about 50%. Therefore, it is urgent to develop more treatment methods that can further improve the survival rate of nasopharyngeal carcinoma patients with adjuvant radiotherapy and chemotherapy.

Epstein Barr virus (EBV) is a gamma herpes virus, and almost all undifferentiated and poorly differentiated nasopharyngeal carcinomas are associated with latent EB virus infection. Recent studies have found that EBV-encoded miRNAs are involved in regulating the expression of EBV-encoded genes and human host genes. For example, EBV-miR-BART2 is fully complementary to the 3'UTR of the viral DNA polymerase-encoding gene BALF5. During the lytic infection period, when the virus replicates a lot, the expression level of miR-BART2 decreases, and the decomposition of BALF5 gene is weakened, which is conducive to the viral replication cycle; with the change of the selection pressure of miR-BART2, the expression of BALF5 decreases, and the virus particles released by EBV-infected cells Decreasing, the infection enters a latent state (Nucleic Acids Res 310:1035-48, 2009). EBV-miR-BHRF1-3 is related to the expression level of IFN-induced T cell chemokine CXCL-11/I-TAC in cells. It is speculated that CXCL-11/I-TAC is the target of miR-BHRF1-3, and EBV can This acts as a means of regulation to interfere with host immune surveillance and immune clearance (Cancer Res 68:1436-42, 2008). EBV-miR-BART5 can up-regulate the expression of P53 by inhibiting the host's pre-apoptotic protein PUMAH1. If miR-BART5 is removed from EBV-infected cells, it will promote PUMA-mediated apoptosis, suggesting that EBV can inhibit apoptosis. occurs, thereby protecting the epithelial cells it infects (J Exp Med 205:2551-60, 2008; J Biol Chem 285:33358-100, 2010). miR-BART6-5p can inhibit the expression of EBNA2 virus oncogene, and the expression of this oncogene is not necessary in the transition from type I and II latent state (immune hyporesponsiveness) to type III latent state (immune hyperresponsiveness). missing, suggesting that miR-BART6 plays an important regulatory role in EBV infection and latency (J Biol Chem 285:33358-100, 2010). It can be seen that on the one hand, EB virus miRNA can act on the target genes of the virus itself to promote virus infection and latency, and on the other hand, it can also act on the target genes of the host, promote the virus to escape the immune response of host cells, and inhibit host cells. of apoptosis.

However, there are few studies on the role and mechanism of Epstein-Barr virus-encoded miR-BART10. Among them, CN105154446A and CN105154586A jointly disclosed the application method of Epstein-Barr virus-encoded microRNA BART10 and its antisense oligonucleotides. The expression level of EBV-miR-BART10 in nasopharyngeal carcinoma is positively correlated with lymph node metastasis and distant metastasis in patients with nasopharyngeal carcinoma; we compared the sequence of this patent and found that it actually involves EBV-miR-BART10-3p. Another study pointed out that EBV-miR-BART10-3p can promote the occurrence of EMT in nasopharyngeal carcinoma by targeting BTRC gene and then promote the metastasis of nasopharyngeal carcinoma (Oncotarget 39:41766-41782). However, so far, there is no report on the relationship between Epstein-Barr virus miR-BART10 and angiogenesis in nasopharyngeal carcinoma.

In 1971, Folkman proposed that tumor growth and metastasis depend on angiogenesis, and blocking angiogenesis is an effective strategy to suppress tumor growth. In the malignant growth and metastasis of primary solid tumors, tumor angiogenesis plays a key role. The growth, invasion and metastasis of tumors depend on abundant blood supply and angiogenesis. New blood vessels of tumors not only deliver nutrients and nutrients to tumor tissues. Excretion of metabolic wastes is one of the main links in the hematogenous metastasis of tumor cells. Taking tumor angiogenesis as a target and treating tumor and its metastasis by anti-angiogenesis has become a hotspot of basic and clinical research in recent years. Inhibiting tumor angiogenesis can significantly inhibit tumor growth and metastasis, which may open up a new way for the treatment and prevention of nasopharyngeal carcinoma.

In addition, in the prior art, the preparation of miRNA inhibitors mostly adopts the method of loading antisense oligonucleotides on polylysine-modified silicon nanoparticles to form nanospheres. Amine (PEI) gene vector has toxic and side effects. The transfection efficiency of PEI increases with the increase of molecular weight, but the cytotoxicity also increases. Therefore, most of the current research is on the premise of not affecting the transfection efficiency of PEI. Appropriate modification of its structure reduces its cytotoxicity.

At present, the recognized effective radical treatment for nasopharyngeal carcinoma is radiotherapy or comprehensive treatment mainly based on radiotherapy. However, while radiotherapy cures the tumor, it inevitably damages normal tissues and organs. long, more complications. Therefore, it is of great practical significance to propose a low-toxic, safe and efficient preparation for the treatment and prevention of nasopharyngeal carcinoma.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of the EB virus miR-BART10-5p inhibitor, more specifically: the EB virus miR-BART10-5p antisense oligonucleotide is used to prepare the anti-angiogenesis preparation of nasopharyngeal carcinoma.

The technical route adopted by the present invention is:

The application of Epstein-Barr virus miR-BART10-5p inhibitor in the preparation of preparations for treating nasopharyngeal carcinoma; the nucleotide sequence of the Epstein-Barr virus miR-BART10-5p is:

GCCACCUCUUUGGUUCUGUACA (SEQ ID NO: 1).

As a preference of the above application, the application of an EB virus miR-BART10-5p inhibitor in the preparation of an anti-angiogenesis preparation for treating nasopharyngeal carcinoma.

As a preference for the above application, the EB virus miR-BART10-5p inhibitor is EB virus miR-BART10-5p antisense oligonucleotide, and the EB virus miR-BART10-5p antisense oligonucleotide can interact with EB virus At least 5 oligonucleotides in the nucleotide sequence of miR-BART10-5p are complementary paired.

As a preference for the above application, the EB virus miR-BART10-5p antisense oligonucleotide is:

UGUACAGAACCAAAGAGGUGGC (SEQ ID NO: 2).

An anti-angiogenic preparation for nasopharyngeal carcinoma containing Epstein-Barr virus miR-BART10-5p inhibitor.

As a preference of the above preparation, the EB virus miR-BART10-5p inhibitor is EB virus miR-BART10-5p antisense oligonucleotide, and the EB virus miR-BART10-5p antisense oligonucleotide can interact with EB virus At least 5 oligonucleotides in the nucleotide sequence of miR-BART10-5p are complementary paired.

As a preferred option of the above preparation, the EB virus miR-BART10-5p inhibitor is chemically modified by chemically modifying the EB virus miR-BART10-5p antisense oligonucleotide, and the EB virus miR-BART10-5p antisense oligonucleotide The nucleotides can be complementary to at least 5 oligonucleotides in the nucleotide sequence of Epstein-Barr virus miR-BART10-5p.

As a preference of the above preparation, the Epstein-Barr virus miR-BART10-5p inhibitor is cholesterol-modified at the 3' end of the Epstein-Barr virus miR-BART10-5p antisense oligonucleotide sequence, and two thiols at the 5' end. The skeleton is modified, the 3' end is modified with four thio skeletons, and the whole chain is modified by 2' methoxy.

As the preference of the above preparation, the EB virus miR-BART10-5p antisense oligonucleotide is:

UGUACAGAACCAAAGAGGUGGC (SEQ ID NO: 2).

As a preference of the above formulation, the formulation can be used for preventing and treating the recurrence and metastasis of nasopharyngeal carcinoma.

The beneficial effects of the present invention are:

The present invention utilizes microtubule formation experiment and chicken embryo chorioallantoic membrane experiment to find that Epstein-Barr virus miR-BART10-5p, which is highly expressed in nasopharyngeal carcinoma cells, can obviously promote nasopharyngeal carcinoma angiogenesis.

The Epstein-Barr virus miR-BART10-5p inhibitor of the present invention is based on the antisense oligonucleotide of Epstein-Barr virus miR-BART10-5p, obtained through a series of modifications, and is obtained through microtubule formation experiments and chicken embryo chorioallantoic membrane experiments. and Matrigel suppository experiments, it was confirmed that the EB virus miR-BART10-5p inhibitor can significantly inhibit the angiogenesis of nasopharyngeal carcinoma, and has the effect of treating nasopharyngeal carcinoma. recurrence and metastasis.

The Epstein-Barr virus miR-BART10-5p inhibitor of the present invention is a single-stranded small RNA specially marked and chemically modified according to the sequence of the microRNA mature body, and is a highly efficient blocker specially used to inhibit endogenous microRNA; Compared with miRNA inhibitors, it has a higher affinity with the cell membrane, and the amount of transfection reagents used in cell transfection experiments is significantly reduced. It can be enriched in target cells and achieve efficient specific and stable interference. Systemic injection or local injection can be used. The method of administration is simple and easy to operate, and the inhibition duration is long, at least one week, and up to 5 to 6 weeks; and miRNA has no immunogenicity, which is conducive to better treatment of nasopharyngeal carcinoma and further clinical development. and application.

Description of drawings

Fig. 1: the concrete result of the microtubule formation experiment of Epstein-Barr virus miR-BART10-5p of the present invention, the control group in the figure is the result of the negative control experiment, and BART10-5p is the experimental result after the action of Epstein-Barr virus miR-BART10-5p; Figure A is a microscope observation diagram, and Figure B is a microtubule statistical data diagram;

Figure 2: The specific results of the microtubule formation experiment of the EB virus miR-BART10-5p inhibitor according to the present invention, the control group in the figure is the result of the negative control experiment, and the BART10-5p inhibitor group is the EB virus miR-BART10-5p inhibition The experimental results after the action of the agent; Figure A is a microscope observation map, and Figure B is a microtubule statistical data map;

Figure 3: The specific results of the chicken embryo chorioallantoic membrane experiment of EB virus miR-BART10-5p according to the present invention, the control group in the figure is the result of the negative control experiment, and the BART10-5p inhibitor group is the EB virus miR-BART10-5p The experimental results after the inhibitor acts; Figure A is a microscope observation picture, and Figure B is a statistical data chart of the percentage of the area occupied by blood vessels;

Figure 4: The specific results of the chicken embryo chorioallantoic membrane experiment of the EB virus miR-BART10-5p inhibitor according to the present invention, the control group in the figure is the result of the negative control experiment, and the BART10-5p inhibitor group is the EB virus miR-BART10 The experimental results after the action of -5p inhibitor; Figure A is a microscope observation picture, and Figure B is a statistical data chart of the percentage of area occupied by blood vessels;

Figure 5: The specific results of the EB virus miR-BART10-5p inhibitor Matrigel plug experiment according to the present invention, the control group in the figure is the negative control experimental result, the BART10-5p inhibitor group is the EB virus miR-BART10-5p inhibition The experimental results after the action of the agent; Figure A is the appearance of the Matrigel plug; Figure B is the statistical data of the hemoglobin content.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, but not limited to the present invention.

Unless otherwise indicated, molecular biology, microbiology, cell biology, immunology and techniques used in the present invention are within the scope of routine skill in the art.

Antisense oligonucleotide: a synthetic nucleic acid fragment complementary to a certain segment of a target gene or mRNA, which can be combined with the target gene/mRNA through the principle of base complementation, thereby blocking the expression of the gene.

The nasopharyngeal cancer cell line CNE1 used in the present invention was purchased from the central laboratory of Xiangya Medical College, Central South University, Hunan; the cell transfection vectors all used commercial liposome Lipofectamine2000 as the vector; Epstein-Barr virus miR-BART10-5p and its inhibitor Entrusted to Shanghai Gema Pharmaceutical Technology Co., Ltd. to synthesize.

Example 1. Epstein-Barr virus miR-BART10-5p, Epstein-Barr virus miR-BART10-5p antisense oligonucleotides, Epstein-Barr virus miR-BART10-5p inhibitors

(1) Epstein-Barr virus miR-BART10-5p

According to the international public sanger mirbase database, the sequence of Epstein-Barr virus miR-BART10-5p was retrieved as follows:

GCCACCUCUUUGGUUCUGUACA (SEQ ID NO: 1)

(2) Epstein-Barr virus miR-BART10-5p antisense oligonucleotide

Antisense design is carried out according to the sequence of Epstein-Barr virus miR-BART10-5p in the international public sanger mirbase database; among them, the basic principles of probe design are: 1) there are no more than two hairpin structures in the probe; The similarity between BLAST and other sequences is less than 20%; 3) The repeat of BLAST and other gene sequences is not more than 3 consecutive bases; the antisense oligonucleotide sequence is obtained as follows:

UGUACAGAACCAAAGAGGUGGC (SEQ ID NO: 2)

(3) Epstein-Barr virus miR-BART10-5p inhibitor

Its preparation method and purification process are as follows:

Based on the nucleotide sequence shown in SEQ ID NO: 2 obtained in Example 1, a standard solid-phase phosphoramidite synthesis method was used to bind Quasar-570 to the 5' end of the oligonucleotide to obtain a fluorescent label, with 5 - Ethylthiotetrazole was used as the activator, and the reaction was prolonged in 0.1M acetonitrile for 15 minutes to complete the end blocking reaction, oxidation reaction and deprotection of the oligonucleotide, and its 3' end was modified with cholesterol, 5 Two thio-skeleton modifications at the '' end, four thio-backbone modifications at the 3' end, and 2' methoxyl modification of the whole chain to obtain an inhibitor of EB virus miR-BART10-5p.

Afterwards, HPLC was used for purification. The synthetic column was eluted and the eluate was collected. Then, it was frozen, dried, deaminated, and dissolved in a 9:1 mixed solution of formamide and TBE; the sample was loaded on HPLC for purification. To contain 10% acetonitrile (solvent A) containing 20 mM NaOAC and 70% acetonitrile (solvent B) containing 20 mM NaOAC, alcohol precipitation, washing, vacuum drying, and storage at -20°C for later use.

Experimental example 1. Microtubule formation experiment

The microtubule formation assay was used to detect the effects of EB virus miR-BART10-5p and its inhibitors on the formation of HUVEC microtubules in human umbilical vein endothelial cells. The specific steps are as follows:

Take about 10 ⁵ to 2×10 ⁵ nasopharyngeal carcinoma cell line CNE1 and inoculate it in a 6-well plate in a complete medium (RPMI 1640) containing 10% fetal bovine serum without antibiotics at 37°C, 5% CO ₂ . Incubate overnight, and the next day, when the cell density reaches about 60%, start transfection. Dilute lipofectamine 2000 with Opti-men medium, and dilute Epstein-Barr virus miR-BART10-5p and its inhibitor with Opti-men medium. 5p and its inhibitor were mixed and incubated at room temperature for 20 min. After the cells in the 6-well plate were removed from the medium, they were washed twice with PBS, and 1.5 mL of Opti-men medium was added. Mix well, incubate at 37° C. in a 5% CO ₂ incubator for 6-8 h, and take the supernatant of nasopharyngeal carcinoma cell CNE1 transfected with Epstein-Barr virus miR-BART10-5p and its inhibitor for use.

Add 50 μL of diluted Matrigel gel (BD Biosciences) solution to a pre-cooled 96-well plate, place on ice for 5 minutes to make the Matrigel gel (BD Biosciences) liquid level level, and incubate at 37°C for at least 1 h to allow the gel to solidify. . The supernatant of nasopharyngeal carcinoma cell CNE1 transfected with Epstein-Barr virus miR-BART10-5p and its inhibitor was mixed with HUVEC cells and added to a 96-well plate covered with Matrigel gel (BD Biosciences), about 2 to 8 hours later. The formation of microtubules was observed under an inverted optical microscope, and the microtubules were counted and counted.

The experimental results are shown in Figure 1 and Figure 2. Figure 1 shows that compared with the control group, the number of microtubules in the EB virus miR-BART10-5p group was significantly increased, indicating that EB virus miR-BART10-5p can promote nasopharyngeal carcinoma neovascularization. Figure 2 shows that compared with the control group, the number of microtubules in the EB virus miR-BART10-5p inhibitor group was significantly reduced, indicating that the EB virus miR-BART10-5p inhibitor can significantly inhibit the formation of new blood vessels in nasopharyngeal carcinoma.

Experimental example 2. Chicken embryo chorioallantoic membrane experiment

Clean the surface of SPF-grade fertilized eggs, soak them in 1:1000 Neogel for 3 minutes, wipe them dry, put the eggs with air chamber upwards and put them into an ordinary incubator for incubation; on the 7th day of incubation, take the egg embryos and mark the air chamber and fetal position under the egg detection lamp. The fenestration site was marked at the avascular site near the fetal position. Aner iodine disinfects the top of the air chamber and the marked place, drill a small hole outside the top of the air chamber, then carefully peel off the 1x1 cm eggshell at the marked place with a small hacksaw and ophthalmic forceps, and add a drop of sterile saline to the shell. On the membrane, the rubber nipple was used to suck in the small hole at the end of the air chamber to create a negative pressure in the air chamber, the physiological saline sank, and an artificial false air chamber was formed. Remove the transparent glue and add 100ul of the supernatant of nasopharyngeal carcinoma cell CNE1 transfected with Epstein-Barr virus miR-BART10-5p and its inhibitor to the relatively avascular area on the CAM, sealed with a sterile transparent glue film, and incubated in an incubator; On the 11th day of incubation, the inoculated site and surrounding area were disinfected with iodine and ethanol, the eggshell at the closed part was torn off, the opening was enlarged with sterile tweezers, and a camera or a stereomicroscope was used to observe and take pictures.

The experimental results are shown in Figure 3 and Figure 4. Figure 3 shows that compared with the control group, the number of new blood vessels in the EB virus miR-BART10-5p group increased significantly, indicating that EB virus miR-BART10-5p can promote the new blood vessels of nasopharyngeal carcinoma. Figure 4 shows that compared with the control group, the number of new blood vessels in the EB virus miR-BART10-5p inhibitor group was significantly reduced, indicating that the EB virus miR-BART10-5p inhibitor can significantly inhibit the formation of new blood vessels in nasopharyngeal carcinoma.

Experimental example 3. Matrigel plug experiment

The low growth factor Matrigel gel (BD Biosciences) was mixed with 1 μg/mL b-FGF (PeproTech) and operated on ice. The experimental group was added with nasopharyngeal carcinoma transfected with Epstein-Barr virus miR-BART10-5p and its inhibitor. The supernatant of cell CNE1, the control group was added with PBS, and the total volume was 700 μL. The prepared Matrigel mixture was injected into the dense blood vessels in the flank of nude mice. After 7 days, the nude mice died suddenly, and the Matrigel plugs were taken out to take pictures and measure the hemoglobin content.

The experimental results are shown in Figure 5. Compared with the control group, the number of new blood vessels and the content of hemoglobin in the EB virus miR-BART10-5p inhibitor group were significantly reduced, indicating that the EB virus miR-BART10-5p inhibitor could significantly inhibit the neogenesis of nasopharyngeal carcinoma. angiogenesis.

In conclusion, the EB virus miR-BART10-5p inhibitor can significantly inhibit the angiogenesis of nasopharyngeal carcinoma, has the effect of treating nasopharyngeal carcinoma, and further, can prevent the recurrence and metastasis of nasopharyngeal carcinoma.

SEQUENCE LISTING

<110> Southern Medical University Shenzhen Hospital

<120> Application of Epstein-Barr virus miR-BART10-5p inhibitor

<130>

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 22

<212> RNA

<213> Epstein-Barr virus

<400> 1

gccaccucuu ugguucugua ca 22

<210> 2

<211> 22

<212> RNA

<213> Artificial sequences

<400> 2

uguacagaac caaagaggug gc 22

Claims (9)

1. The application of an inhibitor of EB virus miR-BART10-5p in preparing a preparation for treating nasopharyngeal carcinoma; the nucleotide sequence of the EB virus miR-BART10-5p is as follows:

   GCCACCUCUUUGGUUCUGUACA(SEQ ID NO:1)；

   the inhibitor of the EB virus miR-BART10-5p is antisense oligonucleotide of the EB virus miR-BART10-5 p.
2. 2. Use according to claim 1, characterized in that: the antisense oligonucleotide of EB virus miR-BART10-5p can be complementarily paired with at least 5 oligonucleotides in the nucleotide sequence of EB virus miR-BART10-5 p.
3. 3. Use according to claim 2, characterized in that: the antisense oligonucleotide of EB virus miR-BART10-5p is:

   UGUACAGAACCAAAGAGGUGGC(SEQ ID NO:2)。
4. 4. an anti-nasopharyngeal carcinoma angiogenesis preparation, which is characterized in that: the inhibitor containing EB virus miR-BART10-5p is antisense oligonucleotide of EB virus miR-BART10-5 p.
5. 5. The anti-nasopharyngeal cancer angiogenic formulation according to claim 4, wherein: the EB virus miR-BART10-5p antisense oligonucleotide can be complementarily paired with at least 5 oligonucleotides in the nucleotide sequence of EB virus miR-BART10-5 p.
6. 6. The anti-nasopharyngeal cancer angiogenic formulation according to claim 4, wherein: the EB virus miR-BART10-5p inhibitor is formed by chemically modifying EB virus miR-BART10-5p antisense oligonucleotide, and the EB virus miR-BART10-5p antisense oligonucleotide can be complementarily paired with at least 5 oligonucleotides in the nucleotide sequence of EB virus miR-BART10-5 p.
7. 7. The anti-nasopharyngeal cancer angiogenic formulation according to claim 6, wherein: the EB virus miR-BART10-5p inhibitor is prepared by performing cholesterol modification on the 3 'end of an EB virus miR-BART10-5p antisense oligonucleotide sequence, performing two thio-skeleton modifications on the 5' end, performing four thio-skeleton modifications on the 3 'end, and performing 2' methoxyl modification on the whole chain.
8. 8. The anti-nasopharyngeal cancer angiogenesis agent according to any one of claims 5 to 7, wherein: the EB virus miR-BART10-5p antisense oligonucleotide is:

   UGUACAGAACCAAAGAGGUGGC(SEQ ID NO:2)。
9. 9. the anti-nasopharyngeal cancer angiogenesis agent according to any one of claims 4 to 7, wherein: the preparation can be used for preventing and treating recurrence and metastasis of nasopharyngeal carcinoma.

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