Oligonucleotides targeting the RNA-binding protein SND1 and their applications

Abstract

This invention discloses an oligonucleotide targeting the RNA-binding protein SND1 and its applications, belonging to the field of biomedicine. The nucleotide sequence of the oligonucleotide targeting SND1 described in this invention is selected from SEQ ID NO. 1-3. This invention is the first to discover that the SND1 protein is associated with HBV replication and verifies that the oligonucleotide targeting SND1 significantly inhibits the expression of SND1 RNA and protein, while also significantly inhibiting the levels of HBV DNA, Total RNA, pgRNA, S protein, and HBV antigens S and E, ultimately improving or treating HBV. This invention is of great significance for the development of novel anti-HBV drugs, with broad prospects and enormous economic value.

Description

Technical Field

[0001] This invention relates to an oligonucleotide targeting the RNA-binding protein SND1 and its applications, belonging to the field of biomedicine. Background Technology

[0002] Hepatitis B virus (HBV) is a significant human pathogen that primarily infects the liver, causing acute or chronic hepatitis. Globally, there are approximately 350 million HBV carriers. Long-term infection can lead to serious consequences such as cirrhosis and hepatocellular carcinoma (HCC). Each year, 500,000 to 1 million people die from HCC or liver failure caused by chronic HBV (CHB). Therefore, HBV infection is one of the key infectious diseases to be controlled in the public health field and a major global health threat.

[0003] HBV is a partially double-stranded DNA virus, appearing as spherical particles of 42 nanometers in size. The HBV viral particle consists of an envelope and a nucleocapsid. The lipid bilayer of the viral particle envelope contains three types of hepatitis B virus surface proteins: large (L-HBs), medium (M-HBs), and small (S-HBs). The nucleocapsid is an icosahedron assembled from HBcAg, enclosing the Pol protein and a 3.2kb circular DNA genome. The main reason HBV is difficult to cure is the inability to completely eliminate covalently closed circular DNA (cccDNA) and the immune tolerance induced by the virus. Currently, commonly used nucleotide analogues and pegylated interferon therapies have significant limitations, failing to achieve complete clearance of hepatitis B surface antigen and complete inhibition of viral replication. Viral rebound often occurs after discontinuation of medication, making the management of HBV infection still challenging.

[0004] Human staphylococcal nuclease and tudordomain containing 1 (SND1), also known as Tudor-SN, TSN, or p100, is expressed by the SND1 gene located on chromosome 7. It contains four repeating staphylococcal nuclease domains (SNs) and one Tudor-SN domain. SND1 possesses endonuclease activity and acts as a transcriptional coactivator through interactions with nucleic acids, proteins, and protein complexes. The SND1 protein recognizes and binds to specific RNA sequences through its Tudor domain, thereby playing a role in RNA stability, splicing, and translation. It is involved in the activation and inhibition of multiple pathways, including NF-κb, TGFβ1 / Smad, Wnt / β-catenin, and JAK / STAT3. SND1 is closely associated with the development, progression, and invasive characteristics of various cancers, including colon cancer, breast cancer, and prostate cancer. Furthermore, the expression level of SND1 is significantly upregulated in patients with liver cancer caused by hepatitis B virus. Previous studies in our laboratory have shown that SND1 plays a role in HBV infection, but the specific effects are still unclear. Summary of the Invention

[0005] Objectives of the Invention: The first objective of this invention is to provide an oligonucleotide targeting the RNA-binding protein SND1 and its applications. The second objective of this invention is to provide an anti-hepatitis B virus pharmaceutical composition.

[0006] Technical solution: The oligonucleotide targeting RNA-binding protein SND1 described in this invention has a nucleotide sequence selected from SEQ ID NO.1~3.

[0007] Furthermore, the oligonucleotide also includes 2'-methoxyethoxy modification and / or nucleic acid chain backbone modification.

[0008] The application of the oligonucleotides described in this invention in the preparation of drugs that downregulate the expression level of SND1 protein.

[0009] The application of the oligonucleotides described in this invention in the preparation of drugs that inhibit the replication, expression, or reduce the infectivity of hepatitis B virus.

[0010] Furthermore, the oligonucleotides are used to reduce the expression levels of HBV S antigen, HBV E antigen, and HBV RNA.

[0011] Furthermore, the drug also includes medically acceptable excipients or adjuvants.

[0012] Furthermore, the drug also includes a pharmaceutical carrier.

[0013] Furthermore, the pharmaceutical carrier includes at least one of sugars, polyamines, amino acids, peptides, and lipids.

[0014] The anti-hepatitis B virus drug composition of the present invention comprises an oligonucleotide of the aforementioned RNA-binding protein SND1 as the active ingredient.

[0015] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention is the first to discover that the SND1 protein is associated with HBV replication, and verifies that oligonucleotides targeting the RNA-binding protein SND1 can significantly inhibit the expression of SND1 RNA and protein, while also significantly inhibiting the levels of HBV DNA, Total RNA, pgRNA, S protein, and HBV antigens S and E antigens, ultimately improving or treating HBV. This invention is of great significance for the development of novel anti-HBV drugs, and has broad prospects and enormous economic value. Attached Figure Description

[0016] Figure 1 The diagram shows the effect of SND1 RNA expression regulation after SND1-ASO transfection into HepG2.2.15 cells;

[0017] Figure 2 The figure shows the effect of SND1 protein expression regulation after SND1-ASO transfection into HepG2.2.15 cells;

[0018] Figure 3 The diagram shows the results of SND 1-ASO transfection of HepG2.2.15 cells on the regulation of expression of A: HBV E antigen, B: HBV S antigen, C: HBV pg-RNA and D: HBV Total RNA.

[0019] Figure 4 The diagram shows the results of SND 1-ASO transfection of HepG2.2.15 cells on the regulation of HBV DNA expression in A: intracellular HBV DNA and B: cell supernatant. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0021] Example 1: Detection of antisense oligonucleotide regulation of SND1 RNA and related protein expression levels

[0022] (1) Cell culture: HepG2.2.15 cells were donated by the Wuhan Institute of Virology, Chinese Academy of Sciences and kept in our laboratory. They were cultured in DMEM medium (gibco, catalog number 8123184) with 10% FBS (purchased from CelliGent, catalog number CG0430B) in a CO2 incubator at 37°C and 5% CO2 concentration.

[0023] (2) Constructing the ASO model: Connect 8×10 to each well in a 24-well plate. 4HepG 2.2.15 cells were divided into five groups: NC, SND 1-ASO 1, SND 1-ASO 2, SND 1-ASO 3, and entecavir (ETV) positive control group. The sequence of SND 1-ASO 1 was 5'-CGGATAGTATGTGAACCGTT-3' (SEQ ID NO.1); the sequence of SND 1-ASO 2 was 5'-CGATGATAGCATTAACAGGC-3' (SEQ ID NO.2); and the sequence of SND 1-ASO 3 was 5'-CCGGGTGTAAACTGCAATCG-3' (SEQ ID NO.3), all three having a 2'-methoxyethoxy modification. All nucleotide sequences of this invention were synthesized and provided by Suzhou Hongxun Biotechnology Co., Ltd. Cells were plated the night before transfection, and transfection was performed the next day when cell confluence reached 60-70%. In the NC group, 2 μL of liposome Lipo 2000 transfection reagent (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number TL201-01) and 25 μL of opti-MEM (purchased from gbico, catalog number 31985-070) were mixed and diluted. After standing for 5 minutes, 25 μL of opti-MEM was added, for a total of 51 μL. The mixture was incubated at room temperature for 20 minutes, and then the liposomes were added to the cell culture plate in the form of mixed droplets. In the ASO administration group, 1 μL of SND1-ASO and 25 μL of opti-MEM were first diluted together. Simultaneously, 2 μL of liposome Lipo 2000 transfection reagent (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number TL201-01) and 25 μL of opti-MEM (purchased from gbico, catalog number 31985-070) were diluted together. After standing for 5 minutes, the two were mixed, resulting in a total volume of 53 μL. This mixture was then incubated at room temperature for 20 minutes. Subsequently, 200 μL of opti-MEM medium was added to each well of a 24-well plate. The liposome-encapsulated SND1-ASO was then added to the cell culture plate as a droplet mixture. Simultaneously, the medium for the ETV group was replaced with 250 μL of opti-MEM for starvation. After 6 h of culture, the liquid in each culture dish was discarded, and DMEM medium containing 10% FBS was added. The ETV group received 0.03 μM of the drug per well. The samples were cultured at 37°C and 5% CO2 for 96 hours before being collected.

[0024] (3) Reverse transcription and RT-qPCR reaction: After culturing for 96 h, cells were collected and washed twice with self-prepared PBS (pH 7.4, containing NaCl, KCl, Na2HPO4 and KH2PO4, excluding Ca and Mg ions, preparation table as shown in Table 1). Cells were then pipetted with RNA-easy IsoLation Reagent (product number: R701-01) purchased from Nanjing Novizan Biotechnology Co., Ltd., and total RNA was extracted from the cells for reverse transcription. The reverse transcription reaction system (HiScript IV All-in-One Ultra RT SuperMix for qPCR purchased from Nanjing Novizan Biotechnology Co., Ltd., product number: R433-01) is shown in Table 2.

[0025] Table 1

[0026] NaCl 8.0 g <![CDATA[Na2HPO4·12H2O]]> 3.58 g <![CDATA[KH2PO4]]> 0.24 g KCl 0.2 g

[0027] Dissolve in double-distilled water, adjust pH to 7.4, bring volume to 1 L, and autoclave at 121℃ for 20 min.

[0028] Table 2

[0029] reaction system volume 4x All-in-One Ultra qRT SuperMix 15 μL RNA extract 5 μL

[0030] Reaction conditions: 37℃, 5 min; 85℃, 5 s; 4℃, Forever.

[0031] The primer sequences are as follows:

[0032] SND1-FP:5'-GTGGACAGCGTAGTTCGGGA-3'(SEQ ID NO.4);

[0033] SND1-RP:5'-CCCACGAGACATTTCCACACAC-3' (SEQ ID NO.5);

[0034] hGAPDH-FP:5'-AAATCAAGTGGGGCGATGCTG-3' (SEQ ID NO.6);

[0035] hGAPDH-RP: 5'-GCAGAGATGATGACCCTTTTG-3' (SEQ ID NO. 7).

[0036] The real-time quantitative PCR reaction system (real-time quantitative PCR kit purchased from Nanjing Novizan Biotechnology Co., Ltd.) and conditions are shown in Tables 3 and 4.

[0037] Table 3

[0038] reaction system volume cDNA 2 μL 2x Taq Pro SYBR qPCR Master Mix 5 μL Forward 0.2 μL Reverse 0.2 μL <![CDATA[dd H2O]]> 2.6 μL

[0039] Table 4

[0040] program temperature time Pre-variation 95℃ 30 s Cyclic reaction (40×) 95℃60℃95℃ 10 s 30 s 15 s Dissolution curve 95℃60℃95℃ 15 s60 s15 s

[0041] (4) Western blot analysis of proteins:

[0042] Add a mixture of protease and phosphatase inhibitors to RIPA lysis buffer at a ratio of 1:100;

[0043] Wash the cells twice with phosphate buffer pre-cooled to 4°C, add an appropriate amount of lysis buffer containing inhibitor to each well, and lyse the mixture on ice for 10 min.

[0044] After lysis, the lysis products were collected and transferred to new 1.5 mL centrifuge tubes. The tubes were incubated on ice for 30 min, vortexed every 5 min, and centrifuged at 10,000 rpm for 10 min. The supernatant was collected and transferred to new EP tubes. The protein concentration was detected by BCA method, and each sample was adjusted to the same concentration with PBS. Loading buffer was added to the above samples and the tubes were incubated in boiling water for 10 min.

[0045] The prepared protein samples can be used immediately for subsequent experiments or stored at -80°C for later use.

[0046] Prepare SDS-PAGE at the appropriate concentration based on the protein molecular weight.

[0047] The protein concentration of the sample was measured. The sample volume was 17 μL per well and 2 μL of protein marker was loaded. Electrophoresis was performed at a continuous voltage of 80V until the protein markers separated. Then the voltage was adjusted to 120V and electrophoresis was performed for 60 min.

[0048] Cut the PVDF membrane and soak it in methanol for 5 min to activate it. Take out the gel after electrophoresis and place it on the PVDF membrane. Add filter paper and sponge on both sides, press to remove air bubbles, and then start wet transfer printing.

[0049] Adjust the current to 340 mA, and the transfer time depends on the protein molecules. Generally, 1 KD is transferred for 1 min. Add ice cubes to the outside of the transfer tank to cool it down. After the PVDF membrane is transferred, take it out and soak it in 5% skim milk for 2 h.

[0050] Prepare a primary antibody solution with 1% BSA, remove the PVDF membrane, blot dry with filter paper, immerse it in the primary antibody solution, and incubate overnight at 4°C; the primary antibody solution is recovered the next day, and the PVDF membrane is washed 5 times with TBST for 6 minutes each time;

[0051] Prepare a secondary antibody solution with 1% BSA, take out the PVDF membrane, blot the water with filter paper, immerse it in the secondary antibody solution, and incubate at room temperature for 2 hours;

[0052] Remove the PVDF membrane and wash it with TBST 5 times, 10 minutes each time.

[0053] Prepare the exposure solution, use an exposure apparatus to expose the PVDF film, and save the image.

[0054] The results are as follows Figure 1 As shown, the SND1 RNA level in HepG 2.2.15 cells transfected with SND1-ASO1 and SND1-ASO2 was significantly lower than that in the control group (***p<0.001); the SND1 RNA level in HepG 2.2.15 cells transfected with SND1-ASO3 was also significantly lower than that in the control group (**p<0.01); protein expression results are as follows. Figure 2 As shown, the group transfected with SND 1-ASO 1 showed the most significant anti-HBV effect at both the protein and antigen levels.

[0055] Example 2: Detection of antisense oligonucleotide regulation of HBV

[0056] (1) Cell culture and construction of ASO model are the same as in Example 1.

[0057] (2) Detection of HBV S and E antigen levels

[0058] The HBsAg and HBeAg tests will be performed using the Shanghai Kehua ELISA kit. The specific steps are as follows.

[0059] HBsAg detection methods:

[0060] 1) Remove the kit from the refrigerator and allow it to reach room temperature before use;

[0061] 2) Dilute the washing solution 25 times with purified water;

[0062] 3) Add 75 μL of the sample to be tested and the negative and positive controls to each well, seal the plate with sealing paper and incubate at 37℃ for 60 min;

[0063] 4) Add 50 μL of enzyme conjugate to each well, shake the plate for 10 s, and then incubate at 37°C for 30 min;

[0064] 5) Remove the liquid from the wells, wash the plate 5 times with washing solution for 1 minute each time, and finally pat the reaction plate dry on absorbent paper.

[0065] 6) Immediately add 50 μL each of colorimetric reagent A and B to each well, mix and shake for 10 s, then incubate at 37°C for 30 min;

[0066] 7) Add 50 μL of stop solution to each well, shake the plate for 5 seconds, and then use an ELISA reader to detect the absorbance at a wavelength of 450 nm.

[0067] HBeAg detection methods:

[0068] 1) Remove the kit from the container and allow it to reach room temperature before use;

[0069] 2) Dilute the washing solution 25 times with purified water;

[0070] 3) Add 50 μL of the sample to be tested and the negative and positive controls to each well, then add 50 μL of enzyme conjugate, seal the plate with mounting paper and incubate at 37℃ for 30 min;

[0071] 4) Remove the liquid from the wells, wash the plate 5 times with washing solution for 1 minute each time, and finally pat the reaction plate dry on absorbent paper.

[0072] 5) Immediately add 50 μL each of colorimetric reagent A and B to each well, mix and shake for 10 s, then incubate at 37°C for 15 min;

[0073] 6) Add 50 μL of stop solution to each well, shake the plate for 5 seconds, and then use an ELISA reader to detect the absorbance at a wavelength of 450 nm.

[0074] (3) Extraction of total DNA from cells

[0075] 1) Set the centrifuge to 4°C in advance to collect or discard the supernatant, wash twice with PBS, add 800 μL of DNA lysis buffer to each well, incubate on ice for 10 min, and aspirate into 1.5 mL EP tubes;

[0076] 2) Vortex vigorously for 30 s, incubate on ice for 10 min, centrifuge at 14000 rpm for 10 min, and transfer the supernatant to a 2 mL EP tube;

[0077] 3) Add 20 μL proteinase K and 80 μL 10% SDS to each tube, vortex to mix for a few seconds, and then incubate in a water bath at 55°C for 2 hours.

[0078] 4) Add 500 μL of Tri-saturated phenol and 500 μL of chloroform to each tube, invert and mix well, centrifuge at 13000 rpm for 10 min, and transfer 800 μL of supernatant to a new 2 mL EP tube;

[0079] 5) Add 560 μL isopropanol, 80 μL NaAc, and 1 μL glycogen to each tube and incubate overnight at -20°C. Centrifuge at 14000 rpm for 15 min, discard the supernatant, and add 1 mL of 70% ethanol to each tube.

[0080] 6) Centrifuge at 14000 rpm for 1 min, discard the supernatant, invert the container onto absorbent paper, transfer it to a 40℃ oven, and let it stand for 5 min;

[0081] Add 30 μL ddH2O to each tube, incubate in a 56℃ water bath for 10 min to promote dissolution, vortex centrifuge, and store at -20℃.

[0082] (4) Extraction of HBV DNA from the supernatant

[0083] 1) Reagent preparation: The kit is a nucleic acid extraction kit - magnetic bead method produced by Xiamen Anpuli Biotechnology Co., Ltd. Take out the lysis buffer A, lysis buffer A0, washing buffer B, repeat washing buffer C and magnetic beads from the kit and place them at room temperature to allow them to fully equilibrate for later use;

[0084] 2) Take a clean centrifuge tube and prepare lysis buffer A0 (each person's preparation system: 30 μL lysis buffer + 0.2 μL magnetic beads, take 8 μL of magnetic beads and add them to lysis buffer A0 for one-time use); calculate the amount of reagents needed, shake to suspend and set aside;

[0085] 3) According to the number of samples to be tested and the number of quantitative standards, take centrifuge tubes, mark them, add 200 μL of lysis buffer to each tube, and shake to suspend the lysis buffer before use. Then add 100 μL of the sample to be tested or the standard to each tube. Add 30 μL of lysis buffer A0, mix well and let stand at room temperature for 10 minutes. Magnetic adsorption for 90 seconds, remove the supernatant. Add 200 μL of washing buffer, suspend, magnetic adsorption for 90 seconds, remove the supernatant. Add 200 μL of repeat washing buffer, suspend, magnetic adsorption for 60 seconds, remove the supernatant and set aside.

[0086] (5) The system and procedure for reverse transcription and RT-qPCR are the same as in Example 1, and the primers are as follows:

[0087] Total RNA-FP:5'-TCACCAGCACCATGCAAC-3' (SEQ ID NO.8);

[0088] Total RNA-RP:5'-AAGCCACCCAAGGCACAG-3' (SEQ ID NO.9);

[0089] pg RNA-FP:5'-CTGGGTGGGTGTTAATTTGG-3' (SEQ ID NO.10);

[0090] pg RNA-RP:5'-TAAGCTGGAGGAGTGCGAAT-3' (SEQ ID NO.11);

[0091] HBV DNA-FP: 5'-ACCAATCGCCAGTCAGGAAG-3' (SEQ ID NO. 12);

[0092] HBV DNA-R: 5'-ACCAGCAGGGAAATACAGGC-3' (SEQ ID NO. 13).

[0093] Figure 3 In this context, A and B represent the expression levels of HBV S and E antigens in HepG 2.2.15 cells after treatment with NC, SND 1-ASO, and ETV for 4 days. (The remaining text appears to be incomplete and requires further context.) Figure 3 As shown in A and B, the E antigen level in HepG 2.2.15 cells transfected with SND 1-ASO 1 was significantly lower than that in the control group (****p<0.0001), and the S antigen level was significantly lower than that in the control group (***p<0.001); the S antigen level in HepG 2.2.15 cells transfected with SND 1-ASO 2 and SND 1-ASO 3 was also significantly lower than that in the control group (**p<0.01), and the E antigen level was also lower than that in the control group (SND 1-ASO 2: *p=0.0126, SND 1-ASO 3: *p=0.0164); Figure 3 As shown in C and D, treatment of HepG 2.2.15 cells with SND 1-ASO resulted in a certain degree of decrease in both HBV Total RNA and pg RNA, and SND 1-ASO 1 showed a significant effect in reducing HBV pg RNA (*p=0.0338). Figure 4 As shown in A and B, the HBV DNA expression level in HepG 2.2.15 cells transfected with SND 1-ASO 1 was significantly lower than that in the control group (**p<0.01), and the HBV DNA expression level in the supernatant was significantly lower than that in the control group (****p<0.0001). The HBV DNA level in HepG 2.2.15 cells transfected with SND 1-ASO 2 was also significantly lower than that in the control group (*p=0.0138). The HBV DNA level in HepG 2.2.15 cells transfected with SND 1-ASO 3 was also significantly lower than that in the control group (****p<0.0001). The HBV DNA level in the supernatant of HepG 2.2.15 cells transfected with SND 1-ASO 2 and SND 1-ASO 3 was also significantly lower than that in the control group (****p<0.0001).

Claims

1. An oligonucleotide targeting the RNA binding protein SND 1, characterized in that, The nucleotide sequence of the oligonucleotide is selected from the group consisting of SEQ ID NO. 1~3.

2. The oligonucleotide of claim 1, wherein, The oligonucleotide further comprises 2'-methoxyethoxy modification and / or nucleic acid chain skeleton modification.

3. Use of the oligonucleotide according to any one of claims 1~2 in the preparation of a medicament for inhibiting replication, expression or reducing the infectivity of hepatitis B virus.

4. Use according to claim 3, characterized in that, The oligonucleotide is used to reduce the expression level of HBV S antigen, HBV E antigen and HBV RNA.

5. Use according to claim 3, characterized in that, The medicament further comprises a pharmaceutically acceptable excipient or adjuvant.

6. Use according to claim 3, characterized in that, The medicament further comprises a pharmaceutical carrier.

7. Use according to claim 6, characterized in that, The pharmaceutical carrier comprises at least one of sugar, polyamine, amino acid, peptide and lipid.

8. A pharmaceutical composition against hepatitis B virus, characterized by comprising the compound of claim 1 as an active ingredient. The active ingredient in the pharmaceutical composition is the oligonucleotide targeting RNA binding protein SND 1 according to any one of claims 1~2.

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