Application of SIRT7 and histone acetyltransferase inhibitor in HBV inhibition and hepatitis B treatment

Abstract

The invention discloses application of SIRT7 serving as a target in screening of reagents or drugs for inhibiting HBV replication and screening of drugs for treating hepatitis B. The invention also discloses application of the histone acetyltransferase inhibitor in preparation of a reagent or a medicine for inhibiting HBV replication and in preparation of a medicine for treating hepatitis B. The research finds that histone acetylation can promote HBV expression, SIRT7 can inhibit HBV replication through deacetylation of histone H3K18Ac, and H3K18Ac can be used as an important target for HBV treatment; the histone acetyltransferase specific inhibitor inhibits H3K18 acetylation on ccc-DNA, further inhibits HBV replication, is expected to serve as a new medicine for inhibiting HBV replication, and provides a new direction and thought for treatment of HBV.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of SIRT7 and histone acetyltransferase inhibitors in inhibiting HBV and treating hepatitis B. Background Art

[0002] Hepatitis B virus (HBV) infection is a major cause of liver disease and cancer. HBV is a hepatotropic DNA virus whose 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 of which is that HBV can replicate its own DNA using a highly stable mimic minichromosome as a template (cccDNA). The consequences of HBV infection depend on the interaction between the virus and the host immune system.

[0003] Histones are alkaline proteins in eukaryotic somatic cell chromatin and prokaryotic cells, and together with DNA, they form the nucleosome structure. They are the main protein components of chromatin, acting as the spool around which DNA is wound, and play a role in gene regulation. Histones usually contain five components: H1, H2A, H2B, H3, and H4. Except for H1, the other four histones are combined as dimers (total octamers) to form the nucleosome core. DNA is then wound around the nucleosome core. Adenovirus E1A-related 300kDa protein (adenoviral E1A binding protein of 300kDa, p300) and cyclic adenosine monophosphate response element binding protein (CREB binding protein, CBP) are a pair of highly homologous histone acetyltransferases, and H3K18 can be acetylated by the CBP / p300 complex at DNA double-strand breaks.

[0004] Acetylation of proteins is a modification similar to phosphorylation. It occurs after translation and can regulate the function of proteins by changing the surface charge of proteins and regulating protein conformation or protein interaction. Histone acetyltransferase is the key enzyme of histone acetylation reaction in vivo. There are two confirmed and widely accepted views: the first is that histone acetyltransferase catalyzes the transfer of the acetyl group on acetyl coenzyme A to the ε-NH2 group on the specific lysine residue at the N-terminus of histones, neutralizing the positive charge of the lysine residue and reducing the affinity between histones and DNA; the other is that the acetylation signal changes the nucleosome and chromatin structure, achieving the result of chromatin remodeling. Both mechanisms ultimately make the structure of chromatin tend to be loose, which is more conducive to DNA replication and transcription. H3K18Ac, that is, acetylation of the 18th lysine of histone H3 (Histone H3 Lysine 18acetylation), is an important modification in epigenetics. In the nucleus of eukaryotic organisms, DNA and histones are tightly bound to form chromatin, and the N-terminal tail of histones can undergo a variety of chemical modifications, which are called histone modifications. These modifications play a key role in regulating gene expression, DNA replication, DNA repair, and cell differentiation. Acetylation is a common type of histone modification, which usually occurs on the lysine residues of the histone tail. H3K18Ac, as one of them, is closely related to the activation of gene expression. When H3K18 is acetylated, it may change the structure of chromatin, making it looser, which is conducive to the binding of transcription factors and other regulatory proteins, thereby promoting gene transcription and expression. In addition, H3K18Ac also interacts with other histone modifications to jointly regulate the expression pattern of genes. For example, it often appears in the promoter region of genes with activating modifications such as H3K4me3 (trimethylation of the 4th lysine of histone H3), indicating that these genes are in an active transcription state. In disease research, abnormal modification of H3K18ac is also related to the occurrence and development of many diseases. For example, in cancer, abnormalities in H3K18Ac may lead to abnormal expression of tumor-related genes, thereby promoting the occurrence and development of tumors. Therefore, studying the modification mechanism of H3K18Ac and its role in diseases is of great significance for understanding the pathogenesis of diseases and developing new treatment strategies.

[0005] The silent information regulator (SIRT) family of proteins consists of seven highly conserved NAD+-dependent protein 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. Summary of the invention

[0006] The purpose of the present invention is to provide a use of SIRT7 and histone acetyltransferase inhibitors in inhibiting HBV and treating hepatitis B in view of the above problems.

[0007] In order to achieve its purpose, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides the use of SIRT7 as a target in screening agents or drugs that inhibit HBV replication.

[0009] The second aspect of the present invention provides the use of SIRT7 as a target in screening drugs for treating hepatitis B.

[0010] In the above-mentioned application technology scheme, SIRT7 inhibits the replication of HBV by deacetylation of H3K18Ac.

[0011] The third aspect of the present invention provides the use of a histone acetyltransferase inhibitor 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 a histone acetyltransferase inhibitor in the preparation of a medicament for treating hepatitis B.

[0013] In the above application technology solution, the histone acetyltransferase inhibitor inhibits H3K18 acetylation.

[0014] The histone acetyltransferase inhibitor inhibits H3K18 acetylation of the ccc-DNA minichromosome.

[0015] A fifth aspect of the present invention provides a drug for inhibiting HBV replication or treating hepatitis B, wherein the active ingredient includes a histone acetyltransferase inhibitor.

[0016] In the above-mentioned drug technical solution, the histone acetyltransferase inhibitor is histone acetyltransferase inhibitor II.

[0017] The beneficial effects of the present invention are:

[0018] The present invention has found that histone acetylation can promote HBV expression, SIRT7 can inhibit HBV replication by deacetylation of histone H3K18Ac, H3K18Ac can be used as an important target for HBV treatment, and targeting H3K18Ac may play a role in treating HBV to a certain extent. The present invention has further found that histone acetyltransferase-specific inhibitors inhibit H3K18 acetylation on ccc-DNA, thereby inhibiting HBV replication, which is expected to be a new drug to inhibit HBV viral replication, providing a new direction and idea for the treatment of HBV. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figure 2 shows that in 2215 cells, SIRT7 was knocked down. (A) Western blotting was used to analyze the SIRT7 protein expression level, and real-time fluorescence quantitative PCR was used to detect the SIRT7 mRNA level. (B) ELISA was used to detect the secretion levels of HBeAg and HBsAg in the supernatant of SIRT7 knockdown cells, and quantitative RT-PCR was used to analyze the expression levels of cccDNA, HBS, PreC RNA, and pg RNA. Flag SIRT7 was transfected by mass gradient. (C) Western blotting was used to analyze the SIRT7 protein expression level, and real-time fluorescence quantitative PCR was used to detect the SIRT7 mRNA level. (D) ELISA was used to detect the secretion levels of HBeAg and HBsAg in the cell supernatant, and quantitative RT-PCR was used to analyze the expression levels of cccDNA, HBS, PreC RNA, and pg RNA. *, p<0.05; **, p<0.01; ns indicates no statistical difference.

[0020] Figure 2 (A) After knocking down or overexpressing SIRT7 in human hepatoma cell line 2215, total H3K18 acetylation level and ccc-DNA minichromosome H3K18 acetylation level were detected by Western blot and ChIP;

[0021] *, p<0.05; **, p<0.01.

[0022] Figure 3The results show that in human hepatoma cell line 2215, (A) after the application of histone acetyltransferase specific inhibitor, the total H3K18 acetylation level and the ccc-DNA minichromosome H3K18 acetylation level were detected by Western blot and ChIP; (B) the secretion level of HBeAg and HBsAg in the cell supernatant was detected by ELISA, and the expression levels of cccDNA, HBS, PreC RNA and pg RNA were detected by quantitative RT-PCR; *, p<0.05; **, p<0.01. 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] The siRNA-SIRT7 sequence and its negative control were synthesized by Shanghai Jima Pharmaceutical Co., Ltd.

[0031] Table 1. siRNA sequence information

[0032]

[0033] 1.3 Plasmid

[0034] SIRT7 overexpression plasmid Flag SIRT7 was customized by Beijing Zhongyuan Heju Biotechnology Co., Ltd. (Beijing, China). pcDNA 3.1 is a control empty vector. Plasmids HA-P300 and HA-CBP encoding P300 and CBP were customized by Beijing Zhongyuan Heju Biotechnology Co., Ltd. (Beijing, China).

[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] Gene name Upstream / Downstream Sequence (5'-3') SEQ ID NO H-GAPDH Upstream 5'-GCATCCTGGGCTACACTGAG-3' SEQ ID NO.7 H-GAPDH Downstream 5′-GTCAAAGGTGGAGGAGTGGG-3′ SEQ ID NO.8 SIRT7 Upstream 5'-AGTCTGTACCTCCTGCGTTC-3' SEQ ID NO.9 SIRT7 Downstream 5'-GGACCCTAGACACAGGATGG-3' SEQ ID NO.10 cccDNA Upstream 5'-TGCACTTCGCTTCACCT-3' SEQ ID NO.11 cccDNA Downstream 5'-AGGGGCATTTGGTGGTC-3' SEQ ID NO.12 pgRNA Upstream 5'-CACCTCTGCCTAATCATC-3' SEQ ID NO.13 pgRNA Downstream 5'-GGAAAGAAGTCAGAAGGCAA-3' SEQ ID NO.14 PreCRNA Upstream 5'-GGTCTGCGCACCAGCACC-3' SEQ ID NO.15 PreCRNA Downstream 5'-GGAAAGAAGTCAGAAGGCAA-3' SEQ ID NO.16 HBS Upstream 5'-GTTGCCCGTTTGTCCTCTAA-3' SEQ ID NO.17 HBS Downstream 5'-CAGCAACAGGAGGGATTCAT-3' SEQ ID NO.18 ChIP-cccDNA Upstream 5'-CTCCCCGTCTGTGCCTTCT-3' SEQ ID NO.19 ChIP-cccDNA Downstream 5'-GCCCCAAAGCCACCCAAG-3' SEQ ID NO.20

[0039] 1.5 Main reagents and sources

[0040]

[0041]

[0042] Molecular formula of histone acetyltransferase inhibitor II: C 20 H 16 Br2O3

[0043] 2 Experimental methods

[0044] 2.1 Cell culture

[0045] 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.

[0046] 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.

[0047] 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 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 1ml of 10% 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.

[0048] 2.2 Transfection

[0049] siRNA transfection: The cells were plated in a 6-well plate one day before transfection to ensure that the cell confluence was 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:

[0050] ①400μl opti-MEM medium + 14μl Lipo2000, mix gently and let stand at room temperature for 5 minutes;

[0051] ②200μl opti-MEM medium + 5μl siRNA to be transfected, 200μl opti-MEM + 5μl control siNC; mix gently and let stand at room temperature for 5 minutes;

[0052] ③ Add ① into the two EP tubes of ② respectively, mix gently, and let stand for 20 minutes;

[0053] 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);

[0054] ⑤ Gently add the mixed solution in ④ to the corresponding wells (experimental well, control well), and place the culture dish in a constant temperature incubator at 37°C and 5% CO2 to continue culturing;

[0055] 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.

[0056] DNA transfection: The cells were plated in a 6-well plate one day before transfection to ensure that the cell confluence was about 70-80% on the second day of transfection. Cell transfection can be performed 12-24 hours after cell plating. The final concentration of transfection DNA is 10nM. The transfection method is as follows:

[0057] ①500μl opti-MEM medium + 2μl Lipo8000 + 1ug DNA, mix gently;

[0058] ② Gently add the mixed solution in ① to the corresponding wells (experimental well, control well), and place the culture dish in a constant temperature incubator at 37°C and 5% CO2 to continue culturing.

[0059] 2.3Histone Acetyltransferase Inhibitor II Treatment

[0060] Dosing: The cells were plated in a 12-well plate one day before drug addition to ensure that the cell confluence was about 50-60% when drug addition was performed the next day. The cells could be treated with drugs 12-24 hours after cell plating.

[0061] ① Prepare the drug into a stock solution with a final concentration of 1mM.

[0062] ② Calculate the dilution multiple from the original solution concentration to the cell treatment concentration.

[0063] ③Replace the medium of the cells plated the day before.

[0064] ④According to the calculation results, the volume of drug solution that needs to be added to each well is obtained.

[0065] ⑤ Place the culture dish in a constant temperature incubator at 37°C and 5% CO2 and continue culturing.

[0066] ⑥After 48h, collect the cell suspension for subsequent experiments.

[0067] 2.4 Western blotting analysis

[0068] ① Collect cells

[0069] 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 800rpm at room temperature for 3 minutes, discard the supernatant, resuspend and wash again with 1×PBS.

[0070] ②Protein extraction

[0071] Prepare cell lysis buffer, 1ml lysis buffer = 920μl RIPA strong + 40μl PMSF + 10μl A + 10μl B + 10μl P + 10μl V (protease inhibitor), add appropriate amount of pre-cooled cell lysis buffer (prepared and used immediately) to the cell pellet collected in ①, repeatedly resuspend and blow the cell suspension with a pipette tip on ice, and lyse the cells for 10 minutes. 13.2×103g, centrifuge at 4℃ for 5min, and absorb the supernatant. The supernatant is the total cell protein lysate. Absorb 5μl of supernatant and place it in 95μl ddH2O for protein concentration measurement, add 1 / 4 volume of 5× buffer to the remaining supernatant, shake and mix, place in a metal bath at 97℃ for 10 minutes to denature the protein, immediately place on ice to cool for 2-3 minutes, and then place in a -20℃ refrigerator for standby use.

[0072] ③BCA method to measure protein concentration

[0073] The total protein concentration of cells extracted in ② was determined using the BCA kit.

[0074] ④SDS-PAGE gel electrophoresis

[0075] A. Preparation of protein samples

[0076] Generally, the amount of protein loaded in each lane is 30 μg. According to the protein concentration measured in the previous step, the protein concentration of each tube is adjusted to be consistent and the volume of protein loaded in each lane is calculated.

[0077] B. Protein electrophoresis

[0078] Prepare 5% concentrated gel and 10% separation gel according to the above reagent formula, put the gel rack on the rack of the inner tank of the electrophoresis tank, buckle it according to the groove to prevent leakage during electrophoresis, add 1× electrophoresis solution to the inner tank of the electrophoresis tank until the sample well inside the gel is immersed, and then add the adjusted sample to the sample well. Avoid bubbles when loading, as bubbles will flush the sample out of the sample well. Sample overflow will cause the total amount of protein sample to be inconsistent, affecting the analysis of experimental results. Pour 1× electrophoresis solution into the outer tank of the electrophoresis tank (the electrophoresis solution in the outer tank can be recycled and reused 2-3 times), run the concentrated gel at 70V constant voltage electrophoresis, and when bromophenol blue shows that the protein runs to the lower separation gel, adjust the voltage to 110V, run the separation gel at constant voltage electrophoresis until bromophenol blue appears at the bottom of the gel, turn off the power supply, and stop electrophoresis. Remove the glass plate from the electrophoresis tank, carefully take out the gel containing the protein sample and put it into 1× transfer solution to prepare for transfer.

[0079] C. Transfer

[0080] Before transfer, cut the 0.22μm PVDF membrane to a suitable size for the gel, soak it in methanol, and activate it on a shaker for 5 minutes. Soak the sponge pad, thick filter paper, and thin filter paper in the pre-cooled 1× transfer solution, and put them into the transfer clip in the order of transfer clip white board-sponge pad-1 layer of thick filter paper-2 layers of thin filter paper-a PVDF membrane-protein gel-2 layers of thin filter paper-1 layer of thick filter paper-sponge pad-transfer clip black board. When placing, pay attention to using the transfer solution to expel bubbles between the layers, and then lock the transfer clip to prevent bubbles from affecting the transfer process. Then insert the transfer clip into the red and black box of the transfer, and pay attention to the white board of the transfer clip facing the positive pole of the electrotransfer tank, and the black board of the transfer clip facing the negative pole of the electrotransfer tank. Then put the electrotransfer tank into the electrotransfer box, and finally fill the electrotransfer box with pre-cooled 1× electrotransfer solution so that the electrotransfer solution submerges the gel position in the transfer tank. Put the entire transfer tank into the ice-water mixture and transfer the membrane at a constant current of 200mA for 2 hours.

[0081] D. Closed

[0082] According to the above reagent formula, 5% skim milk was prepared with 1×TBST. After the transfer, the PVDF membrane was taken out from the transfer folder with tweezers and soaked in 1×TBST. According to the size of the required protein and the protein marker, the PVDF membrane was cut, and then the cut membrane was placed in the pre-prepared 5% skim milk (about 10 ml per membrane), placed on a shaker, blocked at room temperature for 60 minutes, and then washed with 1×TBST on a shaker 4 times, 5 minutes each time.

[0083] E. Incubation with primary antibody

[0084] The ratio of primary antibody was prepared according to the recommended ratio on the antibody manual. The antibody diluent used was 1×TBST solution containing 5% BSA, anti-SIRT7 (Cell Signaling Technology, 1:1000); GAPDH antibody was prepared according to (Cell Signaling Technology, 1:3000). The washed membrane was soaked in the prepared primary antibody, incubated overnight at 4°C on a shaker, and the primary antibody was recovered the next day and stored in a 4°C refrigerator, and then the membrane was washed 4 times with 1×TBST, 5 minutes each time.

[0085] F. Incubation with secondary antibody

[0086] Select the corresponding secondary antibody according to the species corresponding to the primary antibody, prepare it with 5% skim milk in TBST, and dilute the secondary antibody (1:10000) according to the antibody instructions of the secondary antibody. After washing the membrane, discard the 1×TBST, put the PVDF membrane into the diluted secondary antibody, and incubate it on a low-speed shaker at room temperature for 60 minutes. After incubation, discard the secondary antibody, wash the membrane 4 times with 1×TBST, 5 minutes each time, and wash the membrane twice with 1×TBS, 3 minutes each time.

[0087] G. Development

[0088] Prepare an appropriate amount of ECL supersensitive developing substrate (brown bottle and white bottle 1:1) in a dark place, evenly apply the developing substrate to the PVDF membrane, and after 1-2 minutes, sandwich it between the transparent film of the developing plate, drive out the bubbles between the transparent film and the PVDF membrane, cover the developing plate, and enter the darkroom for development. In the darkroom, sandwich an appropriate amount (usually 3 sheets) of Kodak film between the developing plate, take out the film at an appropriate time (usually 10 seconds), and soak it in the developer. After the target band is clearly displayed, immediately transfer the film to the fixing solution and soak it evenly for about 10 seconds. Wash the film with tap water, and after drying, the film can be scanned and analyzed.

[0089] 2.5 Real-time fluorescence quantitative PCR

[0090] The target samples were quantified using the SYBR Premix Ex Taq (Perfect Real-Time) quantification kit, and the cDNA obtained by reverse transcription was subjected to quantitative PCR according to the following system and amplification procedure:

[0091] Reaction system: cDNA template 4μl, 2×SYBER 10μl, Dye ROX 10.4μl, primer 0.4μl, ddH2O 5.2μl.

[0092] Amplification program: pre-denaturation at 95°C for 30 s; 95°C for 5 s, 60°C for 31 s, 40 cycles.

[0093] 2.6 Chromatin immunoprecipitation (ChIP)

[0094] Day 1

[0095] (I) Formaldehyde cross-linking and ultrasonic disruption of cells

[0096] ① A 10cm culture dish full of cells, ensure that there is 9ml of original culture medium in the cell culture dish, add 243μl of 37% formaldehyde to the culture medium, so that the final concentration of formaldehyde is 1%. Incubate at 37℃ for 10 minutes (the length of cross-linking time with formaldehyde and the degree of cross-linking will affect the effect of ultrasonic fragmentation. The higher the degree of cross-linking, the less likely it is for ultrasonic fragmentation to break the gene into fragments).

[0097] ②Terminate cross-linking: Add 450 μl of 2.5 M glycine to a final concentration of 0.125 M, mix well and let stand at room temperature for 5 minutes.

[0098] ③ Aspirate the culture medium and wash the cells twice with pre-cooled 1× PBS (Note: Add PBS when it adheres to the wall). Collect the cells into a 1.5ml centrifuge tube with a cell scraper. Centrifuge at 4℃ at 1000g for 5 minutes.

[0099] ④Discard the supernatant and add SDS lysis buffer containing protease inhibitors so that each 100 μl SDS lysis buffer contains

[0100] 1×106 cells were lysed on ice for 10 minutes and resuspended by pipetting every minute.

[0101] ⑤ Ultrasonic fragmentation: First clean the probe of the ultrasonic fragmentation instrument with 75% alcohol, and then clean it with ddH2O for 3 times. Note: Ultrasonic fragmentation of different samples requires ultrasonic optimization experiments. Adjust the power and time according to the size of the ultrasonic fragment to make the fragmented DNA length between 200-300bp.

[0102] ⑥ Centrifuge at 13000rpm for 5 minutes at 4℃ and transfer the supernatant to a new 1.5ml centrifuge tube.

[0103] (II) Impurity Removal and Antibody Incubation

[0104] ① Collect 20 μl of sample as input and store at -20℃.

[0105] (Input can be used as a control to calculate the efficiency of ChIP and is indispensable for ChIP experiments.)

[0106] ②Take 2 tubes of samples, 90μl in each tube, pipette 2ml ChIP dilution buffer, add 20μl PMSF, 20μl A, 20μl B, 20μl P, 20μl V, 20μl protease inhibitors, mix well, take 810μl of each and add to the 2 samples, gently invert to mix, and incubate at 4℃ for 1 hour.

[0107] ③ After incubation for 1 hour, centrifuge at 500g for 5 minutes at 4°C. Take 740μl of supernatant from each tube, add 2μl of antibody to the supernatant, add IgG to one tube, and add primary antibody anti-MCM5 (Proteintech, 1:200) to the other tube, so that the groups are supernatant + IgG, supernatant + primary antibody. Slowly invert to mix, and incubate at 4°C overnight.

[0108] the next day

[0109] (I) Precipitation and washing of immune complexes

[0110] ① Prepare beads, take 50μl beads and add 1ml 1×PBS, centrifuge at 500g at 4℃ for 5 minutes, wash 1-2 times. Discard the supernatant, add 1ml ChIP dilution buffer + 50μl PMSF to the precipitate, centrifuge at 500g at 4℃ for 5 minutes, wash 2 times.

[0111] ② Centrifuge the samples that were incubated overnight the day before at 500g for 1 minute at 4℃. Add the supernatant to the prepared beads and mix them by inversion at 4℃ for 1 hour. Centrifuge at 500g for 5 minutes at 4℃. Discard the supernatant as much as possible, wash the beads once with ChIPdilutionbuffer, centrifuge at 500g for 5 minutes at 4℃, and discard the supernatant.

[0112] ③ Wash the precipitated complex according to the following steps:

[0113] Add 1 ml of Low salt immune complex wash buffer to the beads, mix by inversion on ice, centrifuge at 500 g for 5 minutes at 4°C and discard the supernatant.

[0114] Add 1 ml of high salt immune complex wash buffer to the beads, mix by inversion on ice, centrifuge at 500 g for 5 minutes at 4°C, and discard the supernatant.

[0115] Add 1 ml of LiCl immune complex wash buffer to the beads, mix by inversion on ice, centrifuge at 500 g for 5 minutes at 4°C, and discard the supernatant.

[0116] Add 1 ml 1×TE buffer to the beads, mix by inverting on ice, centrifuge at 500 g for 5 minutes at 4°C, and discard the supernatant. Add 1 ml 1×TE buffer to wash once, mix by inverting on ice, centrifuge at 500 g for 5 minutes at 4°C, and discard the supernatant. Discard the supernatant as much as possible.

[0117] ④ When washing with TE buffer, prepare the elution buffer (1% SDS, 0.1M sodium bicarbonate). The elution buffer should be prepared and used immediately. Add 250μl of elution buffer to each tube, mix with a vertical mixer at room temperature for 15 minutes, centrifuge at 500g for 5 minutes at room temperature, collect the supernatant into a new centrifuge tube, add 250μl of elution buffer and repeat elution 3 times, and collect all the supernatant into a new centrifuge tube.

[0118] (II) Reversal of cross-linking

[0119] ① Add 30 μl 5M NaCl, 15 μl 0.5M EDTA, 20 μl 1M Tris-HCl (pH 6.5) and 1.5 μl 20 mg / ml proteinase K to each tube.

[0120] ② Seal the centrifuge tube with sealing film and reverse the crosslinking in a 65℃ water bath for at least 4 hours, preferably overnight.

[0121] Three days

[0122] (I) DNA sample recovery

[0123] ①After the reverse cross-linking is completed, tear off the sealing film, wipe the surface of the centrifuge tube clean, and place it at room temperature for 10-20

[0124] minute.

[0125] ② Add 1 / 10 volume of sodium acetate (3M, pH 5.2) and an equal volume of DNA extraction buffer (phenol: chloroform: isoamyl alcohol = 25:24:1), gently invert up and down to mix, and then centrifuge at 13000g for 10 minutes at room temperature.

[0126] ③ Take the supernatant, add 1 μl of glycogen and an equal volume of isopropanol, gently invert upside down to mix, and then place in a -80℃ refrigerator to precipitate for 4 hours.

[0127] ④After precipitation is completed, centrifuge at 13000g for 20 minutes in a 4℃ high-speed centrifuge and discard the supernatant.

[0128] ⑤ Add 1 ml of 75% ethanol to wash the DNA, centrifuge at 13000g, 4℃ for 10 minutes, discard the supernatant, and repeat the wash twice.

[0129] ⑥ Place it at the vent inside the clean bench and ventilate and dry for 3 minutes. Add 40μl sterile ddH2O to each tube to dissolve the DNA. Seal it with sealing film to prevent moisture evaporation and store it in a -20℃ refrigerator for later use.

[0130] (II) PCR quantitative analysis

[0131] 2.7 Enzyme-linked immunosorbent assay

[0132] Both HBeAg and HBsAg are transcribed from HBV cccDNA as a template, and can reflect the presence and transcriptional activity of cccDNA to a certain extent. Currently, HBeAg and HBsAg are used clinically to reflect the infection and replication of HBV. In order to detect HBsAg and HBeAg secreted by cells, a human hepatitis B surface antigen / e antigen detection kit is used. The principle of this type of kit is a double antibody sandwich enzyme-linked immunosorbent assay. In the detection well, the company has pre-coated antibodies that will capture HBsAg / HBeAg. When used for detection, the sample and the detection antibody labeled with horseradish peroxidase (HRP) are added to the well in turn, and after incubation and washing, the substrate 3',3',5,5'-tetramethylbenzidine (TMB) is used for color development. TMB turns blue under the catalysis of HRP and finally turns yellow under the action of acid. The depth of the final substrate color is proportional to the concentration of the substance to be tested in the sample. The detection steps are as follows:

[0133] ① Collect the sample to be tested. Collect the cell supernatant in a sterile 15ml centrifuge tube, centrifuge at room temperature, 1000rpm for 20 minutes, carefully aspirate the supernatant, and store at 4℃ for later use. (If not tested on the same day, store in a -20℃ refrigerator for later use)

[0134] ② Balance. Take the test kit out of the refrigerator and equilibrate it at room temperature for 20 minutes.

[0135] ③ Add samples. Set up blank control wells, standard wells and sample wells. Nothing is added to the blank wells, different concentrations of standard products are added to the standard wells, and the supernatant of the cells to be tested is added to the sample wells. Add 50 μl to each well.

[0136] ④ Incubate with antibodies. Add 100 μl of HRP-labeled detection antibody to each well except the blank well, seal the wells with a sealing film, and incubate in a 37°C constant temperature incubator for 60 minutes.

[0137] ⑤ Washing. Discard the solution and wash with 1× washing solution. Fill each well with about 350 μl washing solution, let it stand for 1 minute to wash, flick off the washing solution with a plate, fill it with washing solution again, let it stand to wash, flick off the washing solution with a plate, and repeat 5 times.

[0138] ⑥ Color development. Add 50 μl of substrate A and B to each well, and incubate at 37°C in the dark for 15 minutes.

[0139] ⑦ Stop. Add 50 μl of stop solution to each well to stop color development.

[0140] ⑧ Detection: Detect the absorbance value of each well at a wavelength of 450 nm using a multifunctional microplate reader within 15 minutes.

[0141] 2.8 Statistics

[0142] 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.

[0143] 3 Experimental results

[0144] 3.1 SIRT7 can regulate HBV replication

[0145] First, SIRT7 is a specific deacetylase. In order to study the effect of SIRT7 on HBV replication, we knocked down SIRT7 in human hepatoma cell 2215 cells. The results of ELISA experiments showed that the secretion levels of HBeAg and HBsAg in the cell supernatant were significantly decreased compared with those in the non-knockdown group. The expression levels of cccDNA, HBS, PreC RNA, and pg RNA in quantitative RT-PCR analysis were also reduced ( Figure 1 B). In order to further verify that SIRT7 is closely related to HBV replication, we overexpressed SIRT7 plasmids. The experimental results showed that the replication ability of HBV does not gradually increase with the dose of SIRT7 overexpression. Within a certain range of transfection doses, the replication ability of HBV increases with the increase of SIRT7 transfection dose. When a certain critical value is exceeded, the replication ability of HBV decreases with the increase of SIRT7 transfection dose. When a low-dose SIRT7 overexpression plasmid is transfected, the secretion levels of HBeAg and HBsAg in the cell supernatant increase, and the expression levels of cccDNA, HBS, PreC RNA, and pgRNA in quantitative RT-PCR analysis also increase, and the replication ability of HBV is improved; when a high-dose SIRT7 overexpression plasmid is transfected, the replication ability of HBV decreases. From the above experimental results, it can be concluded that SIRT7 and HBV replication are not linearly related ( Figure 1 D). Therefore, it is necessary for us to find the key targets of SIRT7 in the process of HBV replication.

[0146] 3.2H3K18Ac is a key target of SIRT7 in regulating HBV replication

[0147] Previous studies by our research group have found that SIRT7 is a deacetylase, and the acetylation modification of histones in the DNA double helix affects DNA replication and transcription. Therefore, we speculate that SIRT7 may inhibit the replication of HBVDNA by deacetylation of histones. To confirm our hypothesis, we knocked down SIRT7 in human liver cancer cell 2215 cells, and the results of ChIP experiments showed that the acetylation level of H3K18 in ccc-DNA minichromosomes increased, while overexpression of SIRT7 significantly decreased the acetylation level of H3K18 in ccc-DNA minichromosomes ( Figure 2 A). This indicates that SIRT7 is closely related to H3K18 acetylation. In order to confirm that H3H18Ac plays a key role in SIRT7 regulation of HBV DNA replication, we transfected plasmids HA-P300 and HA-CBP targeting H3K18 acetylation. Experimental results ( Figure 2 B) It was concluded that after overexpression of a plasmid specifically targeting H3K18 acetylation, the secretion levels of HBeAg and HBsAg in the cell supernatant increased, and the expression levels of cccDNA, HBS, PreC RNA, and pg RNA in quantitative RT-PCR analysis also increased. From this, we can conclude that H3K18Ac can be used as a key target in the process of SIRT7 on HBV replication.

[0148] 3.3HA-P300 histone acetylation-specific inhibitor can inhibit H3K18 acetylation on ccc-DNA and inhibit HBV replication

[0149] The results in the previous section show that H3K18 acetylation level plays a key role in HBV replication, so we hope to find a specific drug that can target H3K18 acetylation. Finally, it was found that after applying the histone acetyltransferase specific inhibitor Histone Acetyltransferase Inhibitor II (compound 2c) to human liver cancer cell 2215 cells, the H3K18 acetylation level on ccc-DNA was inhibited to a certain extent ( Figure 3 A). This indicates that this drug may be used to inhibit HBV replication. Next, we treated liver cancer cell 2215 cells with a histone acetyltransferase-specific inhibitor and found that the secretion levels of HBeAg and HBsAg and the expression levels of cccDNA, HBS, PreCRNA, and pg RNA were also inhibited by the decrease in the expression level of H3K18Ac ( Figure 3 B). Therefore, we believe that histone acetyltransferase inhibitor II (compound 2c) has the potential to be used as a drug to inhibit HBV viral replication and provide a new direction and idea for the treatment of HBV.

[0150] 4 Analysis

[0151] SIRT7 is primarily localized to the nucleolus and has emerged as an important regulator of cellular homeostasis, transcription, ribosome biogenesis, chromatin structure, and cell proliferation. SIRT7 was first described as a β-NAD+-dependent deacetylase that removes the Nε-acetyl-lysine residue at position 18 on the histone H3 protein on chromatin. SIRT7-mediated H3K18 deacetylation is restricted by the binding of this sirtuin protein to the Myb binding protein 1a (Mybbp1a) at its N and C termini. Another SIRT7 target is PAF53, an RNA polymerase I complex subunit that, after deacetylation, recruits RNA polymerase I to rDNA promoters. Other known SIRT7 substrates are p53, nucleophilin (NPM1), and GA binding protein β1 (GABP-β1), a key regulator of mitochondrial function. In addition, SIRT7 inhibits the transcriptional activity of HIF-1α and HIF-2α, thereby regulating the cellular response to oxygen. Subsequently, SIRT7 was found to be a key regulator of pre-rRNA processing and maturation through the deacetylation of the non-histone protein U3-55k, a component of the U3 small nucleolar RNP (snoRNP) complex. SIRT7 also catalyzes the deacetylation of cyclin-dependent kinase 9 (CDK9), leading to its activation. SIRT7 additionally interacts with mammalian mTOR targets, forming a binary complex with TFIIIC2, a transcription factor that forms the POLIII complex. Thus, since NAD is a cofactor for SIRT7 activity and SIRT7 interacts with both mTOR and the POLIII complex, this SIRT establishes a link between protein synthesis and POLIII function. SIRT7 also represses the expression of ribosomal proteins and reduces endoplasmic reticulum stress. Finally, SIRT7 promotes genomic stability because it is recruited to DNA double-strand breaks (DSBs) in a PARP1-dependent manner, resulting in amberization of H3K122, leading to chromatin condensation and DSB repair.

[0152] SIRT7 exhibits pro-oncogenic properties by deacetylation of H3K18Ac and inhibition of transcription of several genes involved in contact inhibition and anchorage-independent growth of hepatocellular carcinoma, gastric cancer, and colorectal cancer. In addition, SIRT7 reduces the protein expression and stability of HIF-1α and HIF-2α, thus affecting the occurrence and development of various inflammatory conditions and tumors.

[0153] P300 and CBP (CREB-binding protein) are both common histone acetyltransferases in mammals. H3K18 can be acetylated by the CBP / p300 complex at DNA double-strand breaks. In 1994, Eckner R et al. proposed that p300 interacts with adenovirus protein E1A to activate DNA transcription. In 1993, JC Chrivia et al. reported that CBP specifically binds to CREB protein phosphorylated by protein kinase A, thereby promoting DNA transcription. The genes of P300 associated with E1A and CBP associated with CREB are different, but they have high homology in structure and function and belong to the conservative family of coactivators.

[0154] Through this study, we found that SIRT7 inhibits HBV replication by removing histone acetylation. SIRT7 is a NAD-dependent + Histone H3K18Ac deacetylase. Although SIRT7 is mainly considered to be a nucleolar protein, and some studies have found that SIRT7 is enriched in the nucleolus and activates RNA polymerase I, previous studies have reported that SIRT7 is detected not only in the nucleoplasm and chromatin-related components, but also in the cytoplasmic components. HBV cccDNA exists in the nucleus of infected cells as a free plasmid-like molecule, thereby producing progeny viruses. Based on these results, we speculate that SIRT7 is likely to have an effect on HBV cccDNA and its associated histones, which are also localized in the nucleus. It has been reported that acetylation of histones bound to cccDNA significantly increases HBV replication. Therefore, we speculate that SIRT7 can inhibit HBV replication by deacetylation of histone H3K18Ac. Subsequently, we found through experimental studies that overexpression of SIRT7 can indeed hinder HBV replication. After inhibiting the expression of SIRT7, the acetylation level of H3K18 increased, ultimately promoting HBV replication. The present study showed that H3K18Ac can be used as an important target for HBV treatment, and targeting H3K18Ac may play a role in treating HBV to a certain extent.

Claims

1. Application of SIRT7 as a target in screening agents or drugs that inhibit HBV replication.

2. Application of SIRT7 as a target in screening drugs for the treatment of hepatitis B.

3. The use according to claim 1 or 2, characterized in that: SIRT7 inhibits HBV replication by deacetylation of H3K18Ac.

4. Use of histone acetyltransferase inhibitors in the preparation of reagents or drugs for inhibiting HBV replication.

5. Application of histone acetyltransferase inhibitors in the preparation of drugs for the treatment of hepatitis B.

6. The use according to claim 4 or 5, characterized in that: The histone acetyltransferase inhibitor inhibits H3K18 acetylation.

7. The use according to claim 6, characterized in that: The histone acetyltransferase inhibitor inhibits H3K18 acetylation of the ccc-DNA minichromosome.

8. A drug for inhibiting HBV replication or treating hepatitis B, characterized in that: Its active ingredients include histone acetyltransferase inhibitors.

9. The use according to claim 4 or 5 or the medicine according to claim 8, characterized in that: The histone acetyltransferase inhibitor is histone acetyltransferase inhibitor II.

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