Method for inhibiting expression of E6 / E7 gene in double-stranded DNA virus

Abstract

The invention relates to the technical field of biological medicines, and provides a method for inhibiting E6 / E7 gene expression in a double-stranded DNA virus. According to the invention, the E6 / E7 gene of the HPV16 virus is taken as a target for the first time, a targeted down-regulation regulator is designed, and the purpose of treating double-stranded DNA infection is achieved by silencing the E6 / E7 gene. Results prove that the si RNA designed for specific sites has excellent interference ability, can effectively inhibit HPV virus E6 / E7 genes, reduces the number of HPV viruses in vivo, and provides treatment convenience for HPV infected patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for inhibiting the expression of E6 / E7 genes in double-stranded DNA viruses. Background Art

[0002] Human papillomavirus (HPV) is a spherical DNA virus that is widely present in nature. It has humans as its only host, is very resistant to drying and can be stored for a long time. Currently, there are only more than 150 known HPV subtypes, which can be divided into low-risk and high-risk types according to the severity of the disease they cause. The clinical symptoms of the disease include various types of skin warts, such as common warts, flat warts, plantar warts, etc. In addition, high-risk HPV can cause a variety of tumors, such as cervical cancer, anal cancer, penile cancer, etc. The transmission routes mainly include contact transmission, sexual transmission and mother-to-child transmission, among which sexual contact is the most common route of infection. The HPV population is generally susceptible, but people with factors such as premature sexual intercourse, disordered sex hormone levels, low immunity, multiple sexual partners, and smoking are more likely to be infected.

[0003] In terms of prevention and treatment, we should strengthen public health publicity, promote sex education, and reduce unsafe sex. Vaccination is currently the most effective means of preventing and treating HPV infection. It is also important to identify and treat HPV infection as early as possible. Antiviral drugs and surgery are common treatments. At the same time, regular screening can help locate various tumors caused by HPV at an early stage.

[0004] Small interfering RNA is also widely used as a new therapeutic agent for the treatment and prevention of various diseases. The treatment method based on small interfering RNA includes introducing synthetic small interfering RNA into the subject to stimulate RNA interference, thereby inhibiting the mRNA expression of specific genes to produce gene silencing or inhibition. Therefore, it is expected to provide an effective and side-effect-free treatment method for HPV infection. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention provides a method for inhibiting the expression of E6 / E7 genes in double-stranded DNA viruses. The present invention is the first to target the E6 / E7 genes of HPV16 viruses, design a targeted down-regulator, and achieve the purpose of treating double-stranded DNA infection by silencing the E6 / E7 genes.

[0006] Firstly, the present invention provides the use of a double-stranded DNA virus E6 / E7 gene inhibitor in the preparation of a drug for preventing or treating double-stranded DNA virus infection.

[0007] Preferably, the inhibitor down-regulates the expression of E6 / E7 gene or its protein by RNA interference.

[0008] Preferably, the RNA interference method includes siRNA, shRNA, ASO, sgRNA and the like.

[0009] Furthermore, the RNA interference method is siRNA.

[0010] Furthermore, the siRNA is a combination of one or more selected from SEQ ID NO. 5-12.

[0011] Preferably, the double-stranded DNA virus is selected from HPV16 virus.

[0012] Another object of the present invention is to provide a double-stranded DNA virus E6 / E7 gene inhibitor.

[0013] Preferably, the inhibitor down-regulates the expression of E6 / E7 gene or its protein by RNA interference.

[0014] Preferably, the RNA interference method includes siRNA, shRNA, ASO, sgRNA and the like.

[0015] Furthermore, the RNA interference method is siRNA.

[0016] Furthermore, the siRNA is a combination of one or more selected from SEQ ID NO. 5-12.

[0017] Preferably, the double-stranded DNA virus is selected from HPV16 virus.

[0018] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating double-stranded DNA virus infection, characterized in that the pharmaceutical composition comprises the above-mentioned E6 / E7 gene inhibitor.

[0019] Preferably, the double-stranded DNA virus is selected from HPV16 virus.

[0020] The advantages of the present invention are as follows: The present invention is the first to target the E6 / E7 gene of HPV16 virus, design a targeted down-regulator, and achieve the purpose of treating double-stranded DNA infection by silencing the E6 / E7 gene. The results show that the siRNA designed for a specific site in the present invention has superior interference ability, can effectively inhibit the E6 / E7 gene of HPV virus, reduce the number of HPV viruses in the body, and provide treatment convenience for HPV-infected patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 .Analysis of E6 and E7 gene expression in HPV 16 positive and negative populations;

[0022] Figure 2.Evaluation and analysis of siRNA targeting E6 gene at the cellular level;

[0023] Figure 3 .Evaluation and analysis of siRNA targeting E7 gene at the cellular level;

[0024] Figure 4 .Evaluation and analysis of mouse models targeting E6 / E7 gene siRNA. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0026] Example 1

[0027] Analysis of the differential expression of E6 / E7 genes in HPV 16-positive and HPV 16-negative populations: Peripheral blood was collected from 25 HPV16-positive and 25 HPV16-negative populations. The total RNA in the samples was extracted using the Qiagen trace sample total RNA extraction kit, and the RNA was reverse transcribed using a reverse transcription kit. The expression of E6 / E7 genes was analyzed by qPCR.

[0028] Among them, qPCR amplification primers were designed according to the HPV 16 E6 / E7 (NC_001526.4) gene sequence in the Gen bank and synthesized by Shanghai Shenggong Biotechnology Technology Service Co., Ltd. The specific primer sequences are as follows:

[0029] HPV 16 E6 gene:

[0030] The forward primer was 5′-CAGGAGCGACCCAGAAAGTT-3′ (SEQ ID NO. 1);

[0031] The reverse primer is 5'-CAGCTGGGTTTCTCTACGTGT-3' (SEQ ID NO.2),

[0032] HPV 16 E7 gene:

[0033] The forward primer was 5′-GACAGCTCAGAGGAGGAGGA-3′ (SEQ ID NO. 3);

[0034] The reverse primer was 5'-TGAGAACAGATGGGGCACAC-3' (SEQ ID NO. 4).

[0035] qPCR reaction system: 1 μL each of forward and reverse primers (10 μmol / L), 12.5 μL of SYBR Green polymerase chain reaction system, 2 μL of cDNA template, and the rest was made up to 20 μL with ddH2O.

[0036] qPCR reaction conditions: 95°C / 10 min, (95°C / 45 s, 55°C / 60 s, 72°C / 45 s)*30 cycles, 72°C / 10 min.

[0037] And adopt 2 -ΔΔCT The relative quantification method was used, and each group of experiments was repeated 3 times. The results were expressed as mean ± standard deviation. SPSS13.0 statistical software was used for statistical analysis. The difference between the two was tested by t-test, and it was considered statistically significant when P < 0.05.

[0038] The results are as follows Figure 1 As shown in the figure, compared with the HPV 16-negative population, the expression levels of HPV 16 E6 and E7 genes in the peripheral blood of the HPV 16-positive population were significantly increased, and the difference was statistically significant (P<0.05).

[0039] Example 2

[0040] 2.1 Cell model test analysis:

[0041] In order to effectively treat HPV 16 virus infection, the inventors continued to design two groups of siRNAs targeting HPV 16 E6 and E7 genes respectively. The sequence information is shown in the following table:

[0042]

[0043] A human papillomavirus type 16 full genome infectious cell model was constructed. The specific construction method refers to the detailed method steps disclosed in the CN201811172192.9 patent. The constructed HPV 16-infected positive HaCaT cell model object was cultured in DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0044] Taking a 6-well plate as an example, before transfection, the cell model was passaged. When the cell confluence was 60-80%, fresh DMEM complete medium was replaced and 1 mL was added to each well. During transfection, 10 μL siRNA stock solution (25 nM) and 5 μL PowerFect were added to each well of the cell model group. TM In vitro siRNA transfection reagent, each group of siRNA set 6 parallel replicates. After incubation at 37℃ with 5% incubator for 24h, qPCR was used to analyze the expression of E6 / E7 genes in each well.

[0045] The results are as follows Figure 2-3 As shown, compared with the blank siRNA transfection, the expression levels of E6 / E7 genes in the cell model group were significantly decreased after the two groups of siRNA treatments of the present invention, and both achieved the purpose of efficiently silencing the E6 / E7 genes, proving that the siRNA of the present invention has the potential to inhibit the proliferation of HPV 16 virus.

[0046] 2.2 Mouse model analysis:

[0047] In order to further analyze the inhibitory effect of the siRNA of the present invention in vivo, the present invention continues to use the method described in patent CN201911320627.4 to construct a mouse model overexpressing HPV E6 / E7 genes. For detailed method steps, please refer to the contents described in the patent.

[0048] A successful mouse model was constructed and randomly and evenly divided into groups. The first group was a control group, which was only given saline during the whole process; the second group was administered with the first group of HPV 16 siRNA, the third group was administered with the second group of HPV 16 siRNA, and the fourth group was administered with the control siRNA, wherein the first group of HPV 16 siRNA and the second group of HPV 16 siRNA were a mixture of siRNAs targeting HPV E6 and E7 in an equal molar ratio, and the final concentration of siRNA in each treatment group was 100 nM. Each group was delivered intravenously by lipid carrier LP171, and the infusion dosage for each group of mice was 100 μL / time / mouse, and intravenous infusion was added once every 2 days for one week. After the administration, the expression of HPV E6 / E7 genes in the mouse models of each group was detected, and the silencing efficiency of the siRNA of the present invention was analyzed.

[0049] The results are shown in Figure 4. Compared with the fourth group, the expression level of HPV E6 / E7 genes in the mouse model was significantly decreased after intravenous injection of the siRNA described in the present invention, further confirming that the siRNA of the present invention can achieve the treatment of HPV virus infection by targeted inhibition of the expression level of HPV E6 / E7 gene expression.

[0050] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. Use of double-stranded DNA virus E6 / E7 gene inhibitors in the preparation of drugs for preventing or treating double-stranded DNA virus infections.

2. The use according to claim 1, characterized in that The inhibitor down-regulates the expression of E6 / E7 gene or its protein by RNA interference.

3. The use according to claim 2, characterized in that The RNA interference methods include siRNA, shRNA, ASO, sgRNA and the like.

4. The use according to claim 3, characterized in that The RNA interference method is siRNA.

5. The use according to any one of claims 1 to 4, characterized in that The siRNA is a combination of one or more selected from SEQ ID NO. 5-12.

6. A double-stranded DNA virus E6 / E7 gene inhibitor, characterized in that: The inhibitor down-regulates the expression of E6 / E7 gene or its protein by RNA interference.

7. The inhibitor according to claim 6, characterized in that The RNA interference methods include siRNA, shRNA, ASO, sgRNA and the like.

8. The inhibitor according to claim 7, characterized in that The RNA interference method is siRNA.

9. The inhibitor according to claim 7, characterized in that The siRNA is a combination of one or more selected from SEQ ID NO. 5-12.

10. A pharmaceutical composition for preventing or treating double-stranded DNA virus infection, characterized in that: The pharmaceutical composition comprises the E6 / E7 gene inhibitor according to any one of claims 6 to 9.

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