Application of micro chromosome maintenance defect protein MCM5 in inhibition of HBV (hepatitis B virus) and treatment of hepatitis B

Abstract

The invention discloses application of MCM5 as a target in screening of reagents or drugs for inhibiting HBV replication or screening of drugs for treating hepatitis B, and further discloses application of an MCM5 expression inhibitor in preparation of reagents or drugs for inhibiting HBV replication or preparation of drugs for treating hepatitis B. According to the invention, a new target MCM5 influencing HBV replication is found, and a new thought is provided for prevention and treatment of chronic hepatitis B.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of minichromosome maintenance defect protein MCM5 in inhibiting HBV and treating hepatitis B. Background Art

[0002] Hepatitis B virus (HBV) infection is the main cause of liver disease and cancer. my country is an endemic area of ​​HBV. HBV is a hepatotropic DNA virus. Its double-stranded genomic DNA can take two forms: relaxed circular DNA (usually present in about 90% of virions) or double-stranded linear DNA (present in about 10% of virions). The HBV genome contains four overlapping open reading frames, which encode seven viral proteins, including: HBV core antigen (HBcAg), e antigen (HBeAg), surface antigen (HBsAg, in three forms: large, medium and small), polymerase (HBVpol) and x protein (HBx). Chronic HBV infection is usually lifelong (with few exceptions) because the virus has developed a variety of persistence mechanisms to evade immune surveillance. The most important one found so far is that HBV can replicate its own DNA using a highly stable mimic mini-chromosome as a template (cccDNA). The consequences of HBV infection depend on the interaction between the virus and the host immune system.

[0003] Silent information regulator (SIRT) family proteins are composed of seven highly conserved NAD + SIRTs are composed of a family of histone deacetylases that regulate a large number of cellular pathways and prevent the progression of aging and age-related diseases. SIRTs belong to a class of histone deacetylases that catalyze the removal of acetyl groups from lysine residues of histones. Silent information regulator 7 (SIRT7) is a specific histone deacetylase that has emerged as an important regulator of cellular homeostasis, transcription, ribosome biogenesis, chromatin structure, and cell proliferation. Many studies in recent years have found that SIRT7 plays an important regulatory role in the occurrence and development of hepatocellular carcinoma. However, the mechanism of action of SIRT7 in HBV replication is still not well understood.

[0004] The minichromosome maintenance (MCM) protein family is a highly conserved protein family. Studies have shown that the six members of the MCM protein family (MCM2-7) form a complex with DNA helicase activity, the minichromosome maintenance complex, which is the core of the replicative helicase in eukaryotic cells and can control DNA replication. The initiation of eukaryotic DNA replication goes through two different stages: in the first stage, the minichromosome maintenance complex assembles into a head-to-head double hexamer (MCM2-7 complex), which surrounds the double-stranded replication initiation DNA in the G1 phase; then the second stage converts it into an active CMG (Cdc45-MCM-gins) helicase in the S phase. In this complex, some people used cryo-electron microscopy to find that the (MCM2-7) helicase complex is assembled into an inactive hexamer by two single hexagons, which are arranged in a tilted and twisted manner through the interaction of the staggered amino-terminal domains, forming a twisted central channel, with MCM2 and MCM5 on both sides of the channel. There are few studies on the interaction between MCM5 (Minichromosome Maintenance complex component 5) and HBV. Summary of the invention

[0005] The purpose of the present invention is to provide a use of a minichromosome maintenance defect protein MCM5 in inhibiting HBV and treating hepatitis B in view of the above problems.

[0006] In order to achieve its purpose, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides the use of minichromosome maintenance defect protein MCM5 as a target in screening agents or drugs that inhibit HBV replication.

[0008] The second aspect of the present invention provides the use of minichromosome maintenance defect protein MCM5 as a target in screening drugs for treating hepatitis B.

[0009] The application, the reagent or drug inhibits the expression of MCM5.

[0010] Among them, the expression of MCM5 was reduced, inhibiting the replication of HBV.

[0011] The third aspect of the present invention provides the use of an inhibitor of expression of the minichromosome maintenance defect protein MCM5 in the preparation of an agent or a drug for inhibiting HBV replication.

[0012] The fourth aspect of the present invention provides the use of an inhibitor of expression of the minichromosome maintenance defect protein MCM5 in the preparation of a drug for treating hepatitis B.

[0013] The expression inhibitors include nucleic acid molecules, proteins, and compounds.

[0014] The nucleic acid molecules include siRNA and shRNA.

[0015] The siRNA includes siMCM5-1 and siMCM5-2;

[0016] The positive strand sequence of the siMCM5-1 is: 5'-GUCGUCUGUAUUGACGAGUTT-3',

[0017] The antisense sequence of siMCM5-1 is: 5′-ACUCGUCAAUACAGACGACTT-3′;

[0018] The positive strand sequence of the siMCM5-2 is: 5'-CCAAAUGUCUAUGAGGUCATT-3',

[0019] The antisense sequence of siMCM5-2 is: 5'-UGACCUCAUAGACAUUUGGTT-3'.

[0020] The beneficial effects of the present invention are as follows: The present invention has confirmed that SIRT7 can bidirectionally regulate the replication of HBV through MCM5. When SIRT7 is knocked down, the interaction between MCM5 and cccDNA is weakened, and the expression of MCM5 is reduced, thereby inhibiting the replication of HBV; when SIRT7 is overexpressed, low-dose SIRT7 promotes the expression of MCM5 and thus promotes the replication of HBV, and high-dose SIRT7 can promote the degradation of MCM5, reduce the expression level of MCM5, and thus inhibit the replication of HBV. The present invention has discovered a new target MCM5 that affects HBV replication, providing a new idea for the prevention and treatment of chronic hepatitis B. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown are: (A) Detection of interaction between SIRT7 and MCM5 by Co-IP; (B) Detection of interaction between MCM5 and cccDNA by ChIP; (C) Knockdown of SIRT7 and detection of interaction between MCM5 and cccDNA; *, p<0.05; **, p<0.01.

[0022] Figure 2The results show that: in 2215 cells, (A) knockdown of MCM5 or overexpression of MCM5, western blotting analysis of MCM5 protein expression level, and real-time fluorescence quantitative PCR detection of MCM5 mRNA level; (B) knockdown of MCM5 or overexpression of MCM5, ELISA detection of HBeAg and HBsAg secretion levels in cell supernatant, and quantitative RT-PCR analysis of cccDNA, HBS, PreC RNA, and pgRNA expression levels; (C) after knockdown or overexpression of SIRT7, western blotting detection of MCM5 protein expression level; (D) knockdown of SIRT7 reduced MCM5, then restored MCM5 expression, and western blotting analysis of MCM5 protein expression level; ELISA detection of HBeAg and HBsAg secretion levels in cell supernatant, and quantitative RT-PCR analysis of cccDNA, HBS, PreC RNA, and pgRNA expression levels; (E) after overexpression of SIRT7 (1.5ug) and then knockdown of MCM5, western blotting analysis of MCM5 protein expression level The expression level of MCM5 protein was analyzed by blotting; the secretion levels of HBeAg and HBsAg in the cell supernatant were detected by ELISA, and the expression levels of cccDNA, HBS, PreC RNA, and pg RNA were analyzed by quantitative RT-PCR; *, p<0.05; **, p<0.01; ns, no statistical difference. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0024] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0025] Example 1

[0026] 1 Experimental Materials

[0027] 1.1 Cells

[0028] HepG2.2.15 (2215) is a human hepatoma cell line expressing HBV virus and was purchased from Shanghai Xinyu Biotechnology Co., Ltd.

[0029] 1.2siRNA

[0030] siRNA-SIRT7, siRNA-MCM5 sequences and their negative controls were synthesized by Shanghai Jima Pharmaceutical Co., Ltd.

[0031] Table 1. siRNA sequence information

[0032]

[0033] 1.3 Plasmid

[0034] SIRT7 overexpression plasmid FlagSIRT7 was customized by Beijing Zhongyuan Heju Biotechnology Co., Ltd. (Beijing, China). MCM5 overexpression plasmid PRP[EXP]EGFP / Puro-CAQ>MCM5ORF000045 (hereinafter referred to as pMCM5) was customized by Guangzhou Saiye Biotechnology Co., Ltd. pcDNA3.1 is a control empty vector.

[0035] 1.4 Primers

[0036] The primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and the sequence information is as follows:

[0037] Table 2. Primer sequence information

[0038]

[0039]

[0040] 2 Main reagents and their sources

[0041] Table 3. Main reagent information

[0042]

[0043]

[0044] 3 Experimental methods

[0045] 3.1 Cell culture

[0046] Culture: Human hepatoma cell line 2215 cells were purchased from Shanghai Xinyu Biotechnology Co., Ltd. The culture medium was DMEM medium containing 10% fetal bovine serum and cultured in a constant temperature incubator containing 5% CO2 and 37°C.

[0047] Cryopreservation: Mix DMEM serum-free medium, FBS and DMSO in a volume ratio of 6:3:1 to prepare cell freezing solution. Digest the cells to be frozen with trypsin for 5 minutes, add 3 times the volume of trypsin to neutralize the DMEM complete medium, collect the cell suspension into a 1.5ml centrifuge tube, centrifuge at 1000g for 5 minutes, discard the culture medium supernatant, add an appropriate amount of cell freezing solution to resuspend the cells, transfer them into the cryopreservation tube, and mark the cell name, name of the cryopreservation person and the cryopreservation date on the cryopreservation tube with an antifreeze pen. In order to allow the cells to gradually cool down and freeze, put them in a cell freezing box and then put them in a -80℃ refrigerator. After 24 hours, the cell cryopreservation tube can be taken out of the cryopreservation box and placed in liquid nitrogen for long-term storage.

[0048] Recovery: Take a new 15ml centrifuge tube, add 5ml of DMEM culture medium containing 10% fetal bovine serum, put the cryopreserved tube of frozen cells into a 37℃ constant temperature water bath to thaw it within 3 minutes, and immediately transfer the cell suspension in the cryopreserved tube to the 15ml centrifuge tube prepared above, gently blow and mix with the gun tip, centrifuge at 500rcf for 5min at room temperature, discard the supernatant of the culture medium (since the DMSO in the cryopreserved solution is toxic to cells, the discarded solution must be discarded completely), resuspend with 1ml10% fetal bovine serum DMEM culture medium, transfer the cell suspension to a new cell culture dish, add the corresponding volume of the above complete culture medium, shake it up and down to mix, and culture it in a constant temperature cell culture incubator.

[0049] 3.2 Transfection

[0050] siRNA transfection: The cells are plated in a 6-well plate one day before transfection. The plate density is lower than the general transfection DNA density to ensure that the cell confluence is about 50-60% on the second day of transfection. Cell transfection can be performed 12-24 hours after cell plating. The final concentration of transfection RNA is 10nM. The transfection method is as follows:

[0051] ①400μl Lopti-MEM medium + 14μl Lipo2000, mix gently and let stand at room temperature for 5 minutes;

[0052] ②200μlopti-MEM medium + 5μl siRNA to be transfected, 200μlopti-MEM + 5μl control siNC; mix gently and let stand at room temperature for 5 minutes;

[0053] ③ Add ① into the two EP tubes of ② respectively, mix gently, and let stand for 20 minutes;

[0054] ④ Change the cell medium, replace the complete culture medium with Opti-MEM. (Do not change the medium in advance, wait 2 minutes before the end of the static time to change the medium);

[0055] ⑤ Gently add the mixture in ④ to the corresponding wells (siRNA well, control well), and place the culture dish in a constant temperature incubator at 37°C and 5% CO2 to continue culturing;

[0056] ⑥After 4-6 hours, change the medium, replace the opti-MEM medium with normal DMEM complete medium, and continue culturing in a constant temperature incubator for 48 hours in preparation for subsequent experiments.

[0057] DNA Transfection: The transfection procedure for DNA is similar to that for siRNA, except that the plating density is 70-80%.

[0058] 3.3 Western blotting analysis

[0059] ① Collect cells

[0060] Discard the culture medium, add an appropriate amount of 1×PBS to wash and discard the liquid. Add an appropriate amount of trypsin to digest for 5 minutes, neutralize with complete culture medium, collect cells into a 1.5ml centrifuge tube. Centrifuge at room temperature at 1000g for 5 minutes, discard the supernatant, resuspend and wash again with 1×PBS.

[0061] ②Protein extraction

[0062] ③BCA method to measure protein concentration

[0063] 3.4 Real-time fluorescence quantitative PCR

[0064] The qPCR reaction system is as follows:

[0065]

[0066] The qPCR reaction procedure is as follows:

[0067]

[0068]

[0069] 3.5 Co-immunoprecipitation (Co-IP)

[0070] 3.6 Chromatin immunoprecipitation (ChIP)

[0071] 3.7 Enzyme-linked immunosorbent assay

[0072] 3.8 Statistics

[0073] Each experiment was repeated at least three times independently. Continuous variables were expressed as mean ± standard error (SEM). Student's t test was used, and P < 0.05 was considered statistically significant.

[0074] 4 Results

[0075] 4.1 SIRT7 can interact with MCM5, which can bind to cccDNA. Knockdown of SIRT7 inhibits the interaction between MCM5 and cccDNA

[0076] In order to study the reasons why SIRT7 affects HBV replication, we conducted a study. MCM5 is an important component of the minichromosome maintenance complex MCM2-7, which has DNA helicase activity, and studies have shown that MCM7 can promote hepatitis B virus DNA replication. Therefore, we speculate that the expression of MCM5 may also affect HBV DNA replication. Through IP experimental analysis, it was found that SIRT7 can interact with MCM5 ( Figure 1A), MCM5 can bind to cccDNA, and knockdown of SIRT7 can inhibit the interaction between MCM5 and cccDNA ( Figure 1 B-1C). 4.2 MCM5 can bind to cccDNA to promote HBV replication, and SIRT7 affects HBV replication by affecting MCM5 expression

[0077] Since SIRT7 can affect the interaction between MCM5 and cccDNA, we believe that SIRT7 can affect HBV replication through MCM5. Knockdown or overexpression of MCM5 found that knockdown of MCM5 inhibited HBV replication, while overexpression of MCM5 promoted HBV replication ( Figure 2 A-2B). At the same time, we found that in 2215 cells, after knocking down SIRT7, the expression of MCM5 decreased by western blotting. When SIRT7 was overexpressed, low doses promoted the expression of MCM5, while high doses inhibited the expression of MCM5 ( Figure 2 C). After knocking down SIRT7, restoring MCM5 expression, HBV expression was restored ( Figure 2 D), after overexpression of SIRT7 increased MCM5, knocking down MCM5 reduced HBV replication levels ( Figure 2 E).

[0078] 5 Analysis

[0079] The present invention found that SIRT7 can interact with MCM5, and different doses of SIRT7 have different effects on MCM5 protein expression.

[0080] SIRT7 regulates HBV replication through MCM5: knocking down SIRT7 weakens the interaction between MCM5 and cccDNA, reducing MCM5 expression and inhibiting HBV replication; when SIRT7 is overexpressed, low-dose SIRT7 promotes MCM5 expression and thus promotes HBV replication. High-dose overexpression of SIRT7 promotes MCM5 protein degradation, reduces MCM5 protein levels, and inhibits HBV replication. By clarifying the relationship between SIRT7 and HBV, we discovered a new target, MCM5, that affects HBV replication, providing new ideas for the prevention and treatment of chronic hepatitis B.

Claims

1. Application of minichromosome maintenance defect protein MCM5 as a target in screening agents or drugs that inhibit HBV replication.

2. Application of minichromosome maintenance defect protein MCM5 as a target in screening drugs for the treatment of hepatitis B.

3. The use according to claim 1 or 2, characterized in that: The agent or drug inhibits the expression of MCM5.

4. The use according to claim 3, characterized in that: The expression of MCM5 is reduced, inhibiting the replication of HBV.

5. Use of an inhibitor of the expression of the minichromosome maintenance defect protein MCM5 in the preparation of reagents or drugs for inhibiting HBV replication.

6. Application of the minichromosome maintenance defect protein MCM5 expression inhibitor in the preparation of drugs for the treatment of hepatitis B.

7. The use according to claim 5 or 6, characterized in that: The expression inhibitors include nucleic acid molecules, proteins, and compounds.

8. The use according to claim 7, characterized in that: The nucleic acid molecules include siRNA and shRNA.

9. The use according to claim 7, characterized in that: The siRNA includes siMCM5-1 and siMCM5-2; the sense strand sequence of the siMCM5-1 is: 5'-GUCGUCUGUAUUGACGAGUTT-3', The antisense sequence of siMCM5-1 is: 5′-ACUCGUCAAUACAGACGACTT-3′; The sense strand sequence of the siMCM5-2 is: 5'-CCAAAUGUCUAUGAGGUCATT-3', The antisense sequence of siMCM5-2 is: 5'-UGACCUCAUAGACAUUUGGTT-3'.

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