SaRNA for promoting expression of abcg2 gene and application thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to saRNA for promoting expression of ABCG2 gene and application thereof; saRNA targeting ABCG2 gene is designed for the first time, including human ABCG2 saRNA, which is saRNA-1', saRNA-2', mouse ABCG2 saRNA, which is saRNA-1, saRNA-2; by transfecting saRNA into cells, high expression of ABCG2 gene is realized in human and mouse renal tubular epithelial cells; and through UA efflux experiments at the cell level and in a mouse model, it is proved that improving the expression of ABCG2 gene can promote the efflux of UA in cells, and the excretion of uric acid in blood to urine; an experimental basis is provided for preparing small RNA drugs for treating hyperuricemia or gout with ABCG2 as a target.

Description

Technical fields:

[0001] This invention belongs to the field of biomedical technology and relates to a saRNA that promotes the expression of the ABCG2 gene and its application in the preparation of drugs for treating hyperuricemia or gout. Background technology:

[0002] Uric acid (UA) plays a dual role in the body. Once inside cells, UA activates NADPH oxidase, becoming a pro-oxidant and causing oxidative stress. However, in the extracellular environment, UA exerts a strong antioxidant effect, being one of the main antioxidant molecules in blood plasma, potentially contributing more than 50% to the blood's antioxidant capacity.

[0003] However, excessively high serum uric acid (SUA) levels can lead to hyperuricemia (HUA), which in turn can cause other diseases. In recent years, the incidence of HUA has been on the rise and affecting younger individuals. In the United States, asymptomatic HUA patients account for up to 20% of the general population. Oxidative stress caused by HUA leads to DNA damage, enzyme oxidation and inactivation, the production of inflammatory cytokines, and cell apoptosis, affecting multiple organs and systems, including the kidneys and small intestine. Even mild HUA can cause kidney damage, including renal fibrosis, glomerulosclerosis, interstitial fibrosis, and nephritis. HUA also increases ROS production and intestinal permeability. The occurrence of HUA is also closely related to metabolic syndrome, including hypertension, diabetes, insulin resistance, and obesity. HUA is also considered a prognostic indicator for kidney disease, cardiovascular disease, and inflammation.

[0004] Most notably, high levels of urate (SUA) can lead to gout. Globally, the incidence of gout ranges from 0.1% to 10%, and it is increasing in developed countries. When SUA concentration exceeds its saturation point, sodium urate (MSU) crystals are formed and deposited in areas such as joints. Urate stones and urate crystals can cause acute kidney injury.

[0005] Since the discovery of small activating RNA (saRNA) phenomena, numerous studies have used saRNA transfection to enhance the expression of target genes. saRNA transfection can typically increase the expression of target genes by 2-5 times, and a single transfection can maintain the increased protein expression for more than 10 days. With its well-defined targets and long-lasting effects, saRNA is a relatively ideal method for enhancing the expression of target genes. saRNA has already played a significant role in various fields, particularly in tumor research.

[0006] ABCG2 (ATP-binding cassette superfamily G member 2, also known as BCRP) is a key protein for UA transport in the body. It is expressed in various tissues, including the kidneys and small intestine, enabling the kidneys to excrete UA into the urine and the small intestine into the intestinal contents. Therefore, theoretically, promoting ABCG2 gene expression is one strategy to increase UA excretion and reduce SUA. Currently, there are no reports on saRNAs that promote ABCG2 gene expression. Summary of the Invention:

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a saRNA that promotes ABCG2 gene expression and its application in the preparation of drugs for treating hyperuricemia or gout. By designing a saRNA targeting the ABCG2 gene promoter sequence, the transcription of the ABCG2 gene is specifically promoted. The effect of high ABCG2 protein expression on blood UA concentration in HUA model mice is examined, verifying the feasibility of the ABCG2 gene as a target for treating gout, and laying the foundation for the development of new gout drugs.

[0008] To achieve the above objectives, the present invention provides saRNAs that promote the expression of the ABCG2 gene, namely saRNA-1′ and saRNA-2′ for humans and saRNA-1 and saRNA-2 for mice; the sequences of saRNA-1′ are SEQ ID NO:1 and SEQ ID NO:2; the sequences of saRNA-2′ are SEQ ID NO:3 and SEQ ID NO:4; the sequences of saRNA-1 are SEQ ID NO:5 and SEQ ID NO:6; and the sequences of saRNA-2 are SEQ ID NO:7 and SEQ ID NO:8.

[0009] The saRNA-1′ sequence is:

[0010] Chain of Justice: 5′-CAAGCAUCCACUUUCUCAGdTdT-3′(SEQ ID NO:1)

[0011] Antonym: 5′-CUGAGAAAGUGGAUGCUUGdTdT-3′(SEQ ID NO:2);

[0012] The saRNA-2′ sequence is:

[0013] Chain of Justice: 5′-AGACGAGGUACUGAUCAGCdTdT-3′(SEQ ID NO:3)

[0014] Antisense chain: 5′-GCUGAUCAGUACCUCGUCUdTdT-3′(SEQ ID NO:4);

[0015] The saRNA-1 sequence is:

[0016] Chain of Justice: 5′-UGUCUGUGUGUUCACAGCACdTdT-3′(SEQ ID NO:5)

[0017] Antonym: 5′-GUGCUGUGAACACACAGACAdTdT-3′(SEQ ID NO:6);

[0018] The saRNA-2 sequence is:

[0019] Chain of Justice: 5′-CCACAGAAACUUGGUUAAGGdTdT-3′(SEQ ID NO:7)

[0020] Antisense chain: 5′-CCUUAACCAAGUUUCUGUGGdTdT-3′(SEQ ID NO:8).

[0021] The present invention also provides the use of the above-mentioned saRNA that promotes ABCG2 gene expression in the preparation of drugs for treating hyperuricemia or gout.

[0022] The inventive concept of this application is as follows: First, nine saRNAs targeting the mouse ABCG2 gene were designed. Through RT-qPCR and Western blotting experiments, three saRNAs with high activation efficiency were screened. Finally, two saRNA-1 / 2 sequences that significantly promoted ABCG2 protein expression were selected and tested in vivo on animals. Based on this, three saRNAs targeting the human ABCG2 gene were designed, and through RT-qPCR and Western blotting experiments, the two saRNA-1′ / 2′ sequences with the highest activation efficiency were screened.

[0023] Compared with existing technologies, this invention is the first to design a saRNA targeting ABCG2. By transfecting saRNA into cells, high expression of the ABCG2 gene was achieved in both human and mouse renal tubular epithelial cells. Cell-level UA efflux experiments demonstrated that increasing ABCG2 gene expression can promote the efflux of intracellular UA. In HUA mice, tail vein injection of saRNA successfully reduced SUA in the HUA model mice and improved UA excretion capacity of the kidneys and small intestine. It also reduced serum urea nitrogen (BUN) and creatinine (Cr) levels, protecting kidney and small intestinal function. Compared with benzbromarone (BEN), saRNA has a stronger ability to promote UA excretion and a stronger protective effect on the kidneys and small intestine, while avoiding the side effects of BEN. This invention provides an experimental basis for further in-depth development of small RNA drugs targeting ABCG2. Attached image description:

[0024] Figure 1 This is a schematic diagram of the results of saRNA screening targeting the human ABCG2 gene in Example 2 of the present invention, wherein A represents the expression of ABCG2 mRNA in HK-2 cells; and B and C represent the expression of ABCG2 protein in HK-2 cells.

[0025] Figure 2 This is a schematic diagram showing the experimental results of the effect of saRNA of the human ABCG2 gene on the intracellular and extracellular UA content of HK2 cells in Example 3 of the present invention. In this diagram, A represents the intracellular UA content of each group; and B represents the UA content in the culture medium of each group.

[0026] Figure 3 This is a schematic diagram of the saRNA screening results targeting the mouse ABCG2 gene in Example 4 of the present invention, where A represents the expression of ABCG2 mRNA in TCMK-1 cells; and B represents the expression of ABCG2 protein in TCMK-1 cells.

[0027] Figure 4 This is a schematic diagram of the experimental results of the effect of saRNA co-transfection on ABCG2 protein expression in TCMK-1 cells according to Example 5 of the present invention, where A represents the expression of ABCG2 mRNA in TCMK-1 cells; B and C represent the expression of ABCG2 protein in TCMK-1 cells.

[0028] Figure 5 This is a schematic diagram of the results of the fluorescent saRNA transfection of TCMK-1 cells in Example 5 of the present invention, wherein A is saRNA-1 transfection alone (FAM); B is saRNA-2 transfection alone (CY5); and C is saRNA-1 and saRNA-2 co-transfection.

[0029] Figure 6 This is a schematic diagram showing the experimental results of the effect of saRNA of the mouse ABCG2 gene on the intracellular and extracellular UA content of TCMK-1 cells in Example 6 of the present invention. In this diagram, A represents the intracellular UA content of each group; and B represents the UA content in the culture medium of each group.

[0030] Figure 7 This is a schematic diagram of the experimental results of the effect of in vivo transfection of saRNA on uric acid in a hyperuricemia model mouse according to Example 7 of the present invention, where A is the result of mouse SUA detection; B is the result of mouse UUA detection; and C is the result of mouse intestinal UA detection.

[0031] Figure 8 This diagram illustrates the experimental results of Example 7 of the present invention regarding the effect of in vivo transfection of saRNA on blood urea nitrogen (BUN) and creatinine (Cr) in mice with hyperuricemia. In the diagram, A represents the detection results of serum BUN in each group of mice, and B represents the detection results of serum Cr in each group of mice.

[0032] Figure 9 This is a schematic diagram illustrating the experimental results of Example 7 of the present invention regarding the effect of in vivo transfection of saRNA on xanthine oxidase in the liver of hyperuricemic mice.

[0033] Figure 10 This is a schematic diagram of the experimental results of the effect of in vivo transfection of saRNA on the growth status of mice in Example 7 of the present invention.

[0034] Figure 11 This is a schematic diagram of HE staining results of the kidneys and small intestines of mice in each group according to Example 7 of the present invention. In A, the pathological changes of the kidney tissue of mice in each group are shown, with black arrows representing renal capsules and red arrows representing renal tubules; in B, the pathological changes of the small intestine tissue of mice in each group are shown, with black arrows representing small intestinal villi epithelial cells and red arrows representing small intestinal villi capillaries.

[0035] Figure 12 This is a schematic diagram showing the expression results of ABCG2 mRNA in the kidney and small intestine of mice in Example 7 of the present invention, where A represents the expression level of ABCG2 mRNA in the kidney tissue of each group of mice; and B represents the expression level of ABCG2 mRNA in the small intestine tissue of each group of mice.

[0036] Figure 13 This is a schematic diagram showing the expression results of ABCG2 protein in mouse kidney and small intestine in Example 7 of the present invention, wherein A and B are the Western blotting results of ABCG2 in mouse kidney tissue; and C and D are the Western blotting results of ABCG2 in mouse small intestine tissue.

[0037] Figure 14This diagram illustrates the immunohistochemical results of mouse kidney and small intestine tissues in Example 7 of the present invention. A and C represent the expression and localization of ABCG2 protein in mouse kidney tissue detected by immunohistochemistry (magnification × 200), and the average optical density of ABCG2 protein in mouse kidney tissue within the same field of view. B and D represent the expression and localization of ABCG2 protein in mouse small intestine tissue detected by immunohistochemistry (magnification × 200), and the average optical density of ABCG2 protein in mouse small intestine tissue within the same field of view. Detailed implementation method:

[0038] The present invention will be further described below through specific embodiments and in conjunction with the accompanying drawings.

[0039] Example 1:

[0040] This embodiment involves the design and synthesis of ABCG2 gene saRNA. The coding sequences (CDS) of the human and mouse ABCG2 genes were retrieved from the NCBI website, and the promoter sequence information of the ABCG2 gene was obtained. Based on existing saRNA design methods, mouse and human ABCG2 saRNA sequences of 19-21 bp in length were designed upstream of the transcription start site (TSS), and two dTdT vertical tails were added to the 3' end of the sequences to enhance the stability of the saRNA sequences. The saRNA sequences and negative control sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The saRNA sequence design is as follows:

[0041] Table 1 Human saRNA Sequence

[0042]

[0043]

[0044] Table 2 Mouse saRNA Sequences

[0045]

[0046] Example 2:

[0047] This embodiment involves the screening of saRNA targeting the human ABCG2 gene. The expression of ABCG2 mRNA and protein in human renal tubular epithelial cells (HK-2) was detected by RT-qPCR and Western blotting experiments.

[0048] Cell transfection and RT-PCR steps:

[0049] (1) Human renal tubular epithelial cells (HK-2) purchased from Hunan Fenghui Biotechnology were cultured in medium supplemented with 12% FBSDMEM / high glucose at 37°C and 5% CO2. When the cell confluence reached 80%-90%, the cells were passaged. Two days before transfection, the cells were cultured at 10... 6 Seed cells per well into 6-well plates; when cell confluence reaches 40-60%, perform cell transfection;

[0050] (2) Add 12.5 μl (0.25 nmol) of the human saRNA working solution from Example 1 to a centrifuge tube, and dilute to 50 μl with serum-free medium; add 5 μl of Entranster to another centrifuge tube. TM -R4000 transfection reagent, diluted with serum-free medium to 50 μl; after standing at room temperature for 5 min, mix the solutions in the two centrifuge tubes thoroughly, and after standing at room temperature for 15 min, the transfection complex is ready;

[0051] (3) Use a pipette to aspirate the culture medium from the 6-well plate and wash the cells twice with PBS; add 2500 μl of complete culture medium to the blank well (BCG), and add 2400 μl of complete culture medium + 100 μl of transfection complex to each experimental well. The transfection concentration of saRNA is 100 nmol / L; observe the cell status 6 h after transfection and change the medium as needed; if the cell status is good, continue to culture for 24 h.

[0052] (4) After the treatment was completed, the cells were collected, lysed with Trizol, RNA was extracted with isopropanol, and then precipitated with ethanol.

[0053] (5) Following the instructions for MonScript™ RTIII All in One Mix with dsDNase, the obtained total RNA was reverse transcribed into cDNA;

[0054] (6) According to MonAmp TM The SYBR Green qPCR Mix (Low ROX) kit was used for quantitative PCR detection according to the instructions. The reaction system is shown in Table 3, and the primer sequences are shown in Table 4. Each sample was tested in triplicate, with GAPDH used as an internal control.

[0055] The PCR reaction conditions are as follows:

[0056]

[0057] Table 3 qPCR reaction system

[0058]

[0059]

[0060] Table 4 qPCR primer sequences

[0061]

[0062] The total protein extraction method is as follows: (1) Prepare the total protein extraction solution, which includes RIPA lysis buffer, PMSF, phosphorylated protease inhibitor, and 50×Cocktail protease inhibitor; (2) Wash the cells twice with 1×PBS, and add 200 μl of total protein extraction solution to each well of a six-well plate; (3) Scrape off the cells attached to the bottom of the well plate and transfer them to a 1.5 ml centrifuge tube; (4) Place on ice for 30 min, and invert twice during the process; (5) Centrifuge at 12000 rpm / min for 20 min, and transfer the supernatant (to prevent aspiration of precipitate) into a new 1.5 ml centrifuge tube; (6) Determine the concentration of total protein in the supernatant by BCA method.

[0063] The experimental procedure for Western blotting is as follows:

[0064] (1) Prepare a 10% SDS-PAGE separation gel; (2) Add an appropriate amount of RIPA cell lysis buffer to adjust the total protein concentration and 5× protein loading buffer so that the total protein concentration of different samples is approximately the same; (3) Place the protein solution in boiling water at 100℃ and boil for 10 min; (4) Electrophoresis: Add 20-30 μg of total protein to the wells of the SDS-PAGE gel for each sample. First, maintain a constant voltage of 80V. After the protein enters the separating gel and the marker bands separate, adjust the constant voltage to 120V. (3) Electrophoresis ends when the protein sample reaches near the bottom of the glass plate; (4) Transfer membrane: Take a PVDF membrane of appropriate size and soak it in methanol for about 20 min; Assemble the gel, PVDF membrane, filter paper and sponge, and electrophoresis for 2 h at a constant current of 300 mA under ice water bath conditions; (5) Blocking: After electrophoresis, soak the PVDF membrane in 5% skim milk powder at 4℃ and block it on a shaker for 2 h at room temperature; (6) Incubation with primary antibody: After blocking, wash the membrane with TBST solution and then incubate it with ABCG2 primary antibody (1:1000); β-actin is selected as the internal control. (7) Incubate with primary antibody (1:1000); after incubating at room temperature on a shaker for 30 min, place the membrane in a 4℃ refrigerator and continue incubating overnight; (8) Incubate with secondary antibody: take the membrane incubated with primary antibody overnight out of the 4℃ refrigerator, wash the membrane with TBST every 10 min for a total of 3 times; add secondary antibody (1:5000) and incubate at room temperature on a shaker for 1-2 h; (9) Develop: after incubating with secondary antibody, wash the membrane with TBST every 10 min for a total of 3 times; place the membrane in the prepared chemiluminescence developing solution, and after 1-2 min, place the membrane in the gel imaging system for chemiluminescence.

[0065] from Figure 1 As can be seen from RT-qPCR and Western blotting experiments, the expression levels of ABCG2 mRNA (P<0.01) and protein (P<0.01) in the saRNA-1′ and saRNA-2′ groups in HK-2 cells were significantly higher than those in the control group (BCG), indicating that the saRNA-1′ and saRNA-2′ groups significantly promoted ABCG2 expression at both the mRNA and protein levels. saRNA-1′ and saRNA-2′ were selected for subsequent experiments.

[0066] Example 3:

[0067] This embodiment involves an experiment on the effect of human saRNA on uric acid efflux from renal tubular epithelial cells. To verify whether highly expressed ABCG2 protein can promote UA efflux from HK-2 cells, the levels of intracellular and extracellular UA in HK-2 cells were detected using a UA detection kit after transfection with human saRNA. The specific steps for renal tubular epithelial cells are as follows:

[0068] HK-2 cells were seeded in six-well plates and divided into four groups: blank (BCG), MG (UA), UA+saRNA-1′, UA+saRNA-2′, and UA+saRNA-1′+2′. After 96 hours of saRNA transfection, the complete culture medium in the six-well plates was replaced with complete culture medium containing 400 μmol / L UA, while the blank wells remained in standard complete culture medium. Cells were cultured for another 24 hours. Cells were washed three times by centrifugation with PBS, digested with trypsin, and the cells were collected. The same number of cells (approximately 200,000) were counted, centrifuged, and the pellet was collected. 30 μl of pure water was added, and the cells were lysed using a heat shock method. The supernatant was collected by centrifugation at 12000 rpm for 15 minutes. The UA content in the supernatant and the UA content in the culture medium were measured using a uric acid assay kit.

[0069] The results are as follows Figure 2 As shown. From Figure 2As shown in Figure A, the intracellular UA content was significantly higher in the model group (MG group) compared to the blank group (BCG) (P<0.001). However, compared to the model group, the UA content was significantly lower in the saRNA-1′ group (P<0.05), saRNA-2′ group (P<0.01), and saRNA-1′+2′ group (P<0.01). The saRNA-1′+2′ group had even lower UA content than the saRNA-1′ and saRNA-2′ groups. This indicates that saRNA significantly promoted the excretion of intracellular UA, and the combined application of saRNA had a stronger effect on promoting UA efflux than its use alone. The UA efflux effect is consistent with the trend of ABCG2 protein expression.

[0070] from Figure 2 As can be seen from B, the extracellular UA content, i.e. the UA content in the culture medium, was significantly higher in each experimental group than in the control group (P<0.001), because no UA was added to the culture medium in the control group; the UA content in the culture medium of each experimental group did not vary much, because the culture medium contained sufficient UA, and its content was much greater than that in the cells. Even if some intracellular UA was excreted into the culture medium from the extracellular space, it would not have a significant impact on the UA content in the culture medium.

[0071] Example 4:

[0072] This embodiment involves a screening experiment using mouse saRNA. To detect the effect of saRNA on mouse ABCG2 gene expression, mouse renal tubular epithelial cells (TCMK-1, purchased from Hunan Fenghui Biotechnology) were transfected with ABCG2 saRNA from Example 1 at a concentration of 100 nmol / L, following the same transfection procedure as in Example 2. The expression of mouse ABCG2 mRNA was detected by real-time quantitative PCR (RT-qPCR), and the expression of ABCG2 protein in TCMK-1 cells was detected by Western blotting. The specific experimental procedures for RT-qPCR and Western blotting were the same as in Example 2, except that the qPCR primer sequences are shown in Table 5.

[0073] Table 5 qPCR primer sequences

[0074]

[0075] RT-qPCR results are as follows Figure 3 As shown in Figure A, compared with the control group (BCG), the expression levels of ABCG2 mRNA were significantly increased in the saRNA-1 group (P<0.001), saRNA-2 group (P<0.01), and saRNA-3 group (P<0.05). saRNA-1, saRNA-2, and saRNA-3 were selected for subsequent experiments.

[0076] The results of the Western blotting experiment are as follows: Figure 3 As shown in Figure B, compared with the blank control group (BCG) and the negative control group (NCG), the expression levels of ABCG2 protein were significantly increased in the saRNA-1 group (P<0.05) and the saRNA-2 group (P<0.05), while the protein expression in the saRNA-3 group was not significantly increased. Therefore, saRNA-1 and saRNA-2 were selected for subsequent experiments.

[0077] Example 5:

[0078] This embodiment relates to an experiment on the effect of mouse saRNA co-transfection on ABCG2 gene expression in TCMK-1 cells. To investigate the effect of saRNA alone and in combination on ABCG2 gene expression in TCMK-1 cells, saRNA-1 and saRNA-2 were co-transfected into TCMK-1 cells. The expression levels of ABCG2 mRNA and protein were detected by RT-qPCR and Western blotting experiments. The results are as follows: Figure 4 As shown.

[0079] from Figure 4 As shown in Figure A, the saRNA groups (saRNA-1, saRNA-2, saRNA-1+2, P<0.05) significantly enhanced ABCG2 mRNA transcription. Compared with the saRNA-1 and saRNA-2 groups, the saRNA-1+2 group (P<0.001) showed higher ABCG2 mRNA expression levels, indicating that the combined use of saRNAs had a stronger ability to activate ABCG2 gene transcription. Figure 4 As shown in B and 4C, the saRNA group (P<0.05) significantly increased the expression of ABCG2 protein. Compared with the saRNA-1 and saRNA-2 groups, the saRNA-1+2 group (P<0.05) showed a higher expression level of ABCG2 protein, indicating that the combined use of saRNAs has a stronger ability to activate ABCG2 protein expression. Although saRNA-1 has a stronger ability to activate mRNA transcription, saRNA-2 showed higher protein expression than saRNA-1.

[0080] To verify the interaction during co-transfection of saRNAs, two saRNAs with different fluorescent tags were transfected into TCMK-1 cells. The results are as follows: Figure 5 As shown. From Figure 5 As can be seen, the two co-transfected saRNAs do not interfere with each other and can co-localize in the cell. The fluorescence intensity in the cell nucleus is brighter than that in the cytoplasm, indicating that the saRNA has been transported into the cell nucleus. This is consistent with the mechanism by which saRNA promotes the transcription of the target gene by specifically binding to the promoter sequence of the target gene.

[0081] Example 6:

[0082] This embodiment involves an experiment on the effect of mouse saRNA on uric acid efflux from renal tubular epithelial cells. To verify whether highly expressed ABCG2 protein can promote UA efflux from TCMK-1 cells, the levels of UA inside and outside TCMK-1 cells were detected using a UA detection kit after cell transfection with saRNA. Specific steps are as described in Example 3:

[0083] from Figure 6 It can be seen that, compared with the control group, the intracellular UA level in the model group MG (P<0.001) was significantly increased. Compared with the MG group, the intracellular UA levels in the saRNA-1 group (P<0.05), saRNA-2 group (P<0.05), and saRNA-1+2 group (P<0.01) were significantly decreased. Compared with the saRNA-1 and saRNA-2 groups, the UA level in the saRNA-1+2 group (P<0.05) was significantly lower, and this trend was consistent with the ABCG2 protein expression level. Regarding the UA level in the culture medium, the UA levels in the culture medium of each experimental group were significantly higher than those in the control group (P<0.001), because no UA was added to the culture medium in the control group. The UA content in the culture medium of each experimental group did not vary much, because the UA content in the culture medium is much higher than that in the cells, and even if some intracellular UA is excreted into the culture medium from the extracellular space, it has little effect on the UA content in the culture medium.

[0084] Example 7:

[0085] This embodiment relates to animal experiments involving in vivo transfection of mouse saRNA in mice.

[0086] 1. Establishment, treatment, and sample collection of a mouse model of hyperuricemia.

[0087] (1) Twenty-five KM mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. and acclimatized for one week. The mice were randomly divided into five groups according to their weight, with five mice per group. The groups were: blank group (BCG), model group (MG), saRNA group (saRNA-2), saRNA-1+2 group (saRAN-1+2), and benzbromarone group (BEN).

[0088] (2) The normal group mice were fed a normal diet and were given 0.5% sodium carboxymethyl cellulose by gavage every morning, with the same volume as the other groups. The model group, saRNA-1 group, saRNA-1+2 group and benzbromarone group were fed a model diet (containing 10% yeast powder + 10% fructose + 80% basic diet) and were given potassium oxonate (containing 0.5% sodium carboxymethyl cellulose to enhance the solubility of potassium oxonate) by gavage at a dose of 250 mg / kg every morning.

[0089] (3) During the establishment of the HUA mouse model, the model was established and the mice were treated simultaneously for a total of 30 days. The weight of the mice was measured before modeling, and on days 10, 20 and 30 after modeling. The status of the mice was recorded during the modeling process.

[0090] (4) Twelve days after modeling, the RNA was transfected in vivo (Entranster) TM According to the instructions for use (in vivo), saRNA was packaged into lipid nanoparticles and administered at a dose of 2 mg / (kg·3d). The saRNA-2 group and the saRNA-1+2 group were administered via tail vein injection for a total of 6 injections; the benzbromarone group was administered at a dose of 6.5 mg / (kg·d) via daily gavage.

[0091] (5) 72 hours after the last injection of saRNA, the mice were fasted and deprived of water for 10 hours; urine was collected by stimulating the bladder of the mice to urinate; blood was collected by taking blood from the eye sockets of the mice; 1 ml of physiological saline was injected from the upper end of the duodenum and collected from the lower end of the ileum to obtain the contents of the small intestine of the mice; after the mice were sacrificed, the kidneys, small intestine and liver of the mice were collected.

[0092] (6) Mouse kidneys: one kidney was fixed at room temperature (protected from light) in 4% paraformaldehyde, and the other kidney was stored at -80℃. Mouse small intestine: part of it was fixed at room temperature (protected from light) in 4% paraformaldehyde, and the other part was stored at -80℃. Liver was stored at -80℃. Blood collected was allowed to stand at 4℃ for 2 hours, and then centrifuged at 2000 rpm / min for 10 min to separate serum. Urine and small intestinal contents were centrifuged at 5000 rpm / min for 10 min.

[0093] 2. Effects of in vivo saRNA transfection on uric acid in hyperuricemia model mice

[0094] After the HUA mouse model was established, UA levels in mouse serum, urine, and intestines were detected using a UA detection kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.) to verify the effect of saRNA on UA ​​in HUA mice. The results are shown in Table 6 and 7. Figure 7As shown in the figure. Compared with the control group, the levels of SUA (serum uric acid) (P<0.001), UUA (urinary uric acid) (P<0.01), and intestinal UA (P<0.05) in the model group were significantly increased, indicating that UA production was increased and the excretion capacity of the kidneys and intestines was insufficient to maintain the normal level of SUA, thus the HUA mouse model was successfully established. Compared with the model group, the saRNA groups (saRNA-2 group and saRNA-1+2 group) showed significantly decreased SUA (P<0.01) and significantly increased UUA (P<0.01) and intestinal UA (P<0.05), indicating that saRNA can promote the excretion of UA in the blood through the kidneys and intestines by promoting the expression of ABCG2 protein. Compared with the saRNA-2 group, the saRNA-1+2 group had lower SUA concentration and higher UUA and intestinal UA concentration, indicating that the combined use of saRNA has a stronger ability to promote UA excretion than its use alone. Compared with the model group, the BEN group showed a significant decrease in SUA (P<0.05) and a significant increase in UUA (P<0.01), while the intestinal UA (P>0.05) test results showed no significant change. This indicates that BEN can increase renal UA excretion but cannot promote intestinal UA excretion.

[0095] Table 6. SUA and UUA concentrations in the intestines of mice in each group (Mean ± SD, n = 5)

[0096]

[0097] 3. Effects of in vivo saRNA transfection on blood urea nitrogen and creatinine in hyperuricemic mice

[0098] During the modeling process, changes in blood urea nitrogen (BUN) and creatinine (Cr) concentrations were simultaneously detected while detecting UA to verify the effect of HUA on mouse kidney function.

[0099] The concentrations of blood urea nitrogen (BUN) and creatinine (Cr) were measured using a blood urea nitrogen assay kit and a creatinine assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.), respectively. The results are shown in Table 7 and... Figure 8As shown in the figure. Compared with the control group, the concentration of BUN (P<0.01) in the model group mice was significantly increased, and Cr (P<0.01) also showed the same trend as BUN, indicating that the renal excretion of BUN and Cr in the model group mice was insufficient, and HUA caused damage to the renal function of the mice. Compared with the model group, the BUN content in the saRNA group was significantly decreased (P<0.05), and the Cr concentration in the saRNA group was also significantly lower than that in the model group (P<0.05). Compared with the saRNA-2 group, the BUN and Cr concentrations in the saRNA-1+2 group were even lower. This indicates that saRNA can improve the renal function of HUA model mice by promoting UA excretion, and the combined use of saRNA has a stronger ability to promote BUN and Cr excretion than its use alone. Compared with the model group, the BUN and Cr concentrations in the BEN group mice did not change significantly, indicating that BEN cannot promote the excretion of BUN and Cr.

[0100] Table 7. BUN and Cr concentrations in mice of each group (Mean ± SD, n = 5)

[0101]

[0102] 4. Effects of in vivo saRNA transfection on hepatic xanthine oxidase in hyperuricemic mice

[0103] Since UA is produced in the liver by the catalysis of xanthine oxidase (XOD), the activity of XOD in the liver of mice in each group was measured to determine whether saRNA would affect the activity of XOD in mice.

[0104] The XOD detection steps are as follows: (1) Accurately weigh 1g of mouse liver tissue, add 9ml of physiological saline (g:ml = 1:9), and homogenize thoroughly using a tissue homogenizer at 0℃ to prepare a 10% homogenate; (2) Centrifuge at 3000rpm / min for 10min in a low-temperature centrifuge; take the supernatant. Then, XOD activity is detected using a xanthine oxidase detection kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.), and the results are shown in Table 8 and Figure 9 As shown.

[0105] From Table 8 and Figure 9 As can be seen, compared with the blank group, the XOD activity in the liver of mice in the model group (P<0.05) was significantly increased, which is consistent with the increased SUA level in the model group. There was no significant difference in XOD activity in the liver of mice in the saRNA group and BEN group compared with the model group (P>0.05), indicating that saRNA and BEN had little effect on XOD activity in the liver of mice and were not statistically significant.

[0106] Table 8. XOD activity levels in mice of each group (Mean±SD, n=5)

[0107]

[0108] 5. Effects of in vivo saRNA transfection on growth status in mice

[0109] During the establishment of the HUA mouse model, the weight and growth status of the mice were recorded, and the results are shown in Table 9. Figure 10 As shown in the figure, after the experiment, the model group mice gained more body weight than other groups, and significantly more than the control group mice (P<0.05). Mice in the BEN group, after being administered BEN by gavage, exhibited lethargy, decreased appetite, rough and dull fur, and continuous weight loss, indicating that BEN damaged liver and kidney function, leading to weight loss, with the mice's weight significantly lower than the model group (P<0.001). Mice in the saRNA group showed no significant difference in weight compared to the model group (P>0.05), and exhibited good mental state, smooth and glossy fur, and normal appetite. The results indicate that saRNA did not produce significant toxic side effects in mice and is more suitable for animal experiments than BEN.

[0110] Table 9. Changes in body weight of mice in each group (Mean±SD, n=5, g)

[0111]

[0112] 6. HE staining was used to observe the histopathological changes in the kidney tissue of mice in each group.

[0113] The kidney and small intestine tissues of mice in each group were stained using hematoxylin-eosin (HE) staining to observe pathological changes in the kidneys and small intestines, in order to verify the effects of HUA on mice. The results are as follows: Figure 11 As shown. From Figure 11 As shown in Figure A, the renal tubular structure of the control group mice was normal, with neat and regular arrangement of renal tubular epithelial cells and normal renal interstitium, without swelling or exudation. Compared with the control group, the renal tissue cells of the model group mice were disordered, with indistinct boundaries, accompanied by mild fibrosis, tissue hyperplasia, and inflammatory cell infiltration. The renal capsule boundary was narrowed and accompanied by exudation, renal tubular edema and exudation, and renal interstitial capillary congestion and dilation. Compared with the model group mice, the pathological damage of the renal tissue in the saRNA-2 group and the saRNA-1+2 group mice was significantly improved. However, compared with the model group mice, the damage of the renal tissue in the BEN group mice was not improved. Figure 11B shows that, compared with the control group, the model group mice exhibited disordered arrangement of intestinal villus epithelial cells, and the intestinal villi were swollen and exudative. Compared with the model group, the small intestinal tissue damage in the saRNA-2 group and saRNA-1+2 group mice was significantly improved. Compared with the model group mice, the BEN group did not show improvement in small intestinal tissue damage; on the contrary, the arrangement of intestinal villus epithelial cells was more disordered, and the swelling and exudation were more severe, with altered blood flow. This indicates that although BEN can reduce SUA, it cannot protect against kidney and small intestinal damage caused by HUA, and may even exacerbate the damage.

[0114] 7. ABCG2 mRNA expression in mouse kidneys and small intestines

[0115] The expression of ABCG2 mRNA in mouse tissues was detected by RT-qPCR. The specific steps were as follows: 100 mg of mouse kidney or ileum tissue was taken and 1 ml of Trizol reagent was added; the tissue homogenate was homogenized three times at 4°C for 3 min each time; the homogenate was then allowed to stand at room temperature for 5 min; the resulting tissue homogenate was extracted with chloroform, precipitated with isopropanol, and purified with ethanol under low temperature conditions to obtain pure total RNA; 20 μl of ddH2O was added, and the RNA concentration was adjusted to approximately the same level after measurement; the RT-qPCR experimental procedure was the same as in Example 4, and the results were as follows. Figure 12 As shown.

[0116] from Figure 12 As shown in Figure A, the expression levels of ABCG2 mRNA in the kidney tissues of mice in the blank group, model group, and BEN group were basically the same; however, the expression levels of ABCG2 mRNA in the saRNA-2 group (P<0.01) and the saRNA-1+2 group (P<0.01) were significantly higher than those in the normal group. Figure 12 As shown in Figure B, the expression levels of ABCG2 mRNA in the small intestine of mice in each group followed the same trend as those in the kidney. The expression levels of ABCG2 mRNA in the saRNA-2 group (P<0.01) and the saRNA-1+2 group (P<0.01) were significantly higher than those in the normal group. This indicates that saRNA can promote the transcription of ABCG2 mRNA in the kidney and small intestine of mice.

[0117] 8. Expression of ABCG2 protein in mouse kidney and small intestine

[0118] The expression level of ABCG2 protein in the kidney and small intestine of mice was detected by Western blotting. The specific steps are as follows: First, extract total protein from mouse tissue: (1) Pre-cool 1 ml of RIPA lysis buffer to 4°C; add PMSF, phosphorylated protease inhibitor, and 50×Cocktail a few minutes before use to prepare protein extraction solution; (2) Weigh 0.1 g of kidney tissue, add 1 ml of pre-cooled protein extraction solution (g: ml = 1:10), and homogenize the tissue; grind thoroughly in a tissue homogenizer to prepare tissue homogenate; (3) Incubate on ice for 30 min, and invert and mix once every 10 min; (4) Centrifuge at 12000 rpm / min for 20 min; (5) Transfer the supernatant to a new 1.5 ml centrifuge tube using a pipette, and determine the concentration of total protein using BCA; (6) Add an appropriate amount of 5× protein loading buffer and RIPA cell lysis buffer to adjust the total protein concentration; boil to denature; (7) After aliquoting, store in a -80°C freezer; then perform Western blotting experiment, the specific steps are the same as in Example 2, and the results are as follows. Figure 13 As shown.

[0119] from Figure 13 As shown in A and B, the expression levels of ABCG2 protein in mouse kidney tissue were basically the same in the blank group, model group, and BEN group; however, the expression levels of ABCG2 protein were significantly increased in the saRNA-2 group (P<0.01) and the saRNA-1+2 group (P<0.001) compared with the normal group. Figure 13 As shown in C and D, the expression level of ABCG2 protein in the small intestine tissue of mice follows the same trend as that in the kidney tissue. Compared with the normal group, the expression level of ABCG2 protein in the saRNA-2 group (P<0.01) and the saRNA-1+2 group (P<0.001) was significantly increased.

[0120] 9. Immunohistochemical experiments on mouse kidney and small intestine tissues

[0121] Immunohistochemical experiments were used to further verify the expression sites and levels of ABCG2 protein. The specific steps are as follows:

[0122] (1) The dried paraffin sections were treated with xylene I, II, and III for 15 min each to perform dewaxing. Then the sections were placed in anhydrous ethanol, 85% ethanol, and 75% ethanol for 5 min respectively, and then rinsed with distilled water.

[0123] (2) Place the tissue sections in 0.01M citrate antigen retrieval solution, microwave on medium heat for 8 minutes until boiling, turn off the heat and keep warm for 8 minutes, then microwave on medium-low heat for 7 minutes to perform antigen retrieval. After natural cooling, place in PBS and wash on a shaker for 5 minutes, for a total of 3 washes;

[0124] (3) Under normal temperature conditions, the slides were treated in 3% H2O2 in the dark for 25 min to block endogenous peroxidase; the slides were placed in PBS and washed on a shaker for 5 min, for a total of 3 times.

[0125] (4) Add 3% BSA to the immunohistochemical site to ensure even coverage of the tissue; block at room temperature for 1 hour.

[0126] (5) Remove BSA, add diluted primary antibody, incubate in a humidified chamber at room temperature for 1 hour, and then incubate overnight at 4°C;

[0127] (6) Place the slides in PBS and wash on a shaker for 5 min, repeating the washing process 3 times. Add secondary antibody to the immunohistochemical sites and incubate at room temperature for 1 h.

[0128] (7) Place the slides in PBS and wash on a shaker for 5 min, repeating the washing process 3 times; add DAB for color development.

[0129] (8) Use hematoxylin to counterstain the sections for 3 minutes, and after differentiation, use reverse blue solution to counterstain the cell nuclei.

[0130] (9) The sections were placed in ethanol of different concentrations as well as n-butanol and xylene for dehydration and clearing treatment. After drying, they were mounted with neutral resin.

[0131] (10) The slides were examined using a microscope and photographed using an electron microscope. The results are as follows: Figure 14 As shown in A and 14B.

[0132] from Figure 14 As shown in Figure A, ABCG2 protein is mainly expressed in the brush border membrane of the renal tubules in kidney tissue. Analysis of the average optical density of ABCG2 protein expression in the kidney tissues of each group of mice revealed that, compared with the model group, the average optical density of ABCG2 protein expression in the saRNA-2 group (P<0.05) and the saRNA-1+2 group (P<0.01) was significantly higher than that in other groups. Figure 14 C.

[0133] from Figure 14 As shown in Figure B, ABCG2 protein is mainly expressed on the brush border of the villi of small intestinal epithelial cells, consistent with its expression in kidney tissue. Compared with the model group, the mean optical density of ABCG2 protein expression in the saRNA-2 group (P<0.05) and the saRNA-1+2 group (P<0.01) was significantly higher than that in other groups. Figure 14 D. This indicates that saRNA can promote the expression of ABCG2 protein in mouse kidney and small intestinal tissues.

Claims

1. A saRNA that promotes the expression of the human ABCG2 gene, characterized in that, saRNA is either saRNA-1′ or saRNA-2′, with the saRNA-1′ sequence being SEQ ID NO:1 and SEQ ID NO:2; and the saRNA-2′ sequence being SEQ ID NO:3 and SEQ ID NO:

4. The saRNA-1′ sequence is: Chain of Justice: 5′-CAAGCAUCCACUUUCUCAGdTdT-3′(SEQ ID NO:1) Antonym: 5′-CUGAGAAAGUGGAUGCUUGdTdT-3′(SEQ ID NO:2); The saRNA-2′ sequence is: Chain of Justice: 5′-AGACGAGGUACUGAUCAGCdTdT-3′(SEQ ID NO:3) Antisense chain: 5′-GCUGAUCAGUACCUCGUCUdTdT-3′(SEQ ID NO:4).

2. A saRNA that promotes the expression of the mouse ABCG2 gene, characterized in that, saRNA is either saRNA-1 or saRNA-2; the saRNA-1 sequence is SEQ ID NO:5 and SEQ ID NO:6; the saRNA-2 sequence is SEQ ID NO:7 and SEQ ID NO:8; The saRNA-1 sequence is: Chain of Justice: 5′-UGUCUGUGUGUUCACAGCACdTdT-3′(SEQ ID NO:5) Antonym: 5′-GUGCUGUGAACACACAGACAdTdT-3′(SEQ ID NO:6); The saRNA-2 sequence is: Chain of Justice: 5′-CCACAGAAACUUGGUUAAGGdTdT-3′(SEQ ID NO:7) Antisense chain: 5′-CCUUAACCAAGUUUCUGUGGdTdT-3′(SEQ ID NO:8).

3. The use of the saRNA that promotes the expression of the human ABCG2 gene as described in claim 1 in the preparation of a drug for treating hyperuricemia or gout.

4. The use of the saRNA that promotes the expression of the mouse ABCG2 gene as described in claim 2 in the preparation of a drug for treating hyperuricemia or gout in mice.

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