A siRNA inhibitor composition and its application

Abstract

This invention belongs to the field of biomedical technology, specifically relating to an siRNA inhibitor composition and its applications. The siRNA inhibitor composition provided by this invention contains at least one of siRNA-1, siRNA-2, and siRNA-3, the nucleotide sequences of which are shown in SEQ ID No. 5-10. After transfection of MC3T3-E1 cells and mBMSCs cells with this siRNA inhibitor composition and subsequent osteogenic induction differentiation, the Rhau gene is significantly knocked down, and osteogenic activity is significantly inhibited. The siRNA inhibitor composition provided by this invention can be used to construct osteoporosis cell models and can be applied to the screening of osteoporosis treatment drugs and Rhau agonists.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an siRNA inhibitor composition and its application. Background Technology

[0002] Osteoporosis is a systemic skeletal disease characterized by decreased bone mass and bone microstructure, leading to increased bone fragility, decreased strength, and a significantly increased risk of fractures. Current clinical treatments for bone metabolic diseases primarily rely on drugs that inhibit bone resorption (such as bisphosphonates and estrogen), but simply inhibiting bone resorption cannot enhance the body's own bone formation capacity. Only parathyroid hormone analogs and SOST monoclonal antibodies are clinically used to promote bone formation, but these drugs suffer from limitations such as a limited variety and significant side effects. Specifically, continuous use of parathyroid hormone analogs can activate bone resorption and increase the risk of osteosarcoma, while continuous use of SOST monoclonal antibodies for more than one year significantly reduces their effectiveness and poses a potential risk of cardiovascular disease; therefore, both drugs have strict therapeutic windows. Furthermore, existing bone formation promoters are very expensive, and subcutaneous injection leads to poor patient adherence. Therefore, there is a need to develop new drugs that regulate bone metabolism.

[0003] Currently, while research on bone metabolism pathways has become increasingly comprehensive, very few targets have been successfully translated into practical applications. In response to this situation, researchers have drawn inspiration from the central dogma, attempting to find regulatory targets and methods at the nucleic acid level. Helicases are a class of enzymes that catalyze the unwinding of DNA or RNA double strands, playing a central role in key processes such as DNA replication, repair, recombination, transcription, and translation in organisms. In the field of bone metabolism, researchers have discovered that the helicase DDX5 plays a crucial role in the pathogenesis of osteoarthritis and have achieved the goal of treating osteoarthritis by regulating DDX5 levels.

[0004] Rhau, a member of the DExD / H-box family, possesses unique nucleic acid binding capabilities and ATP-dependent helicase activity, playing a crucial role in transcriptional and post-transcriptional regulation of developmental processes such as spermatogenesis and cardiac development. Currently, research on the function and application of Rhau in bone metabolism is limited. To clarify the impact of Rhau on osteogenic / osteoclast differentiation and elucidate the specific molecular mechanisms by which Rhau regulates bone metabolism, siRNA knockdown of the target gene Rhau is the preferred method for exploring its function and mechanism. Summary of the Invention

[0005] In order to overcome the shortcomings and disadvantages of the prior art, the primary objective of the present invention is to provide an siRNA inhibitor composition that can significantly knock down the Rhau gene.

[0006] Another object of the present invention is to provide the application of the above-described siRNA inhibitor composition.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A siRNA inhibitor composition comprising at least one of siRNA-1, siRNA-2, and siRNA-3, the nucleotide sequence of which is shown below:

[0009] siRNA-1-Sense: 5'-GGGUCAUGUAAAUAGACAACA-3';

[0010] siRNA-1-Anti-Sense:5'-UUGUCUAUUUACAUGACCCUG-3';

[0011] siRNA-2-Sense: 5'-GGUGUUCGGAAAAUAGUAAUU-3';

[0012] siRNA-2-Anti-Sense:5'-UUACUAUUUUCCGAACACCUU-3';

[0013] siRNA-3-Sense: 5'-GGAUAAGCAAGAAGAAUUAAC-3';

[0014] siRNA-3-Anti-Sense: 5'-UAAUUCUUCUUGCUUAUCCAA-3'.

[0015] The siRNA inhibitor composition is preferably a mixture of siRNA-2 and siRNA-3.

[0016] The preferred molar ratio of siRNA-2 to siRNA-3 is 1:1.

[0017] The siRNA inhibitor composition may also include an RNA molecule obtained by chemically modifying the siRNA molecule; or a recombinant vector encoding the siRNA sequence molecule in vivo.

[0018] The recombinant vector can be one of plasmids, adenoviruses, lentiviruses, or adeno-associated viruses.

[0019] The siRNA inhibitor composition preferably further comprises siRNA transfection-related reagents.

[0020] The siRNA transfection reagent can be a Jet-Prime™ transfection reagent.

[0021] The use of the siRNA inhibitor composition in the preparation of products for screening Rhau promoters or drugs for the prevention and treatment of bone metabolic diseases.

[0022] The bone metabolic diseases mentioned refer to various bone loss disorders caused by osteoporosis, rickets, osteogenesis imperfecta, and other bone metabolic diseases.

[0023] The application of the siRNA inhibitor composition in the construction of osteoporosis cell models.

[0024] An osteoporosis cell model was prepared by the following method:

[0025] The siRNA in the above siRNA inhibitor composition was transfected into cells using conventional methods.

[0026] The applications or methods described in this invention do not have the purpose of treating or diagnosing diseases.

[0027] The present invention has the following advantages and effects compared with the prior art:

[0028] (1) This invention provides an siRNA inhibitor composition for knocking down the Rhau gene. The siRNA inhibitor composition contains at least one of siRNA-1, siRNA-2, and siRNA-3. Transfecting MC3T3-E1 cells with this siRNA inhibitor composition showed that siRNA-1 had the highest efficiency in knocking down the Rhau gene, followed by siRNA-2 and siRNA-3. When two siRNAs were transfected in combination, the 1:1 mixture of siRNA-2 and siRNA-3 showed the most significant Rhau gene knockdown effect. Further transfection of MC3T3-E1 and mBMSCs cells with a mixture of siRNA-2 and siRNA-3 and induction of osteogenic differentiation showed that osteogenic activity was significantly inhibited after the Rhau gene was knocked down.

[0029] (2) Based on the discovery of the present invention that the expression level of Rhau gene is significantly downregulated during the pathogenesis of osteoporosis, the siRNA inhibitor composition provided by the present invention can be used to construct osteoporosis cell models and can be used for screening osteoporosis treatment drugs and Rhau agonists. Attached Figure Description

[0030] Figure 1 This is a graph showing the results of qPCR detection of the relative expression levels of the Rhau gene in osteoblasts and osteoclasts at different differentiation days.

[0031] Figure 2 This is a graph showing the changes in the expression levels of osteogenic-related genes in MC3T3-E1 and mBMSCs cells that overexpress Rhau.

[0032] Figure 3 This is a diagram showing the changes in osteogenic differentiation activity of MC3T3-E1 and mBMSCs cells overexpressing Rhau.

[0033] Figure 4 This is a graph showing the changes in Rhau gene expression levels after siRNA transfection into MC3T3-E1 cells.

[0034] Figure 5 This is a diagram showing the changes in osteogenic differentiation activity after siRNA-2 and siRNA-3 were co-transfected into MC3T3-E1 and mBMSCs cells.

[0035] Figure 6 This is a graph showing the changes in the expression levels of key genes for osteoclast differentiation in RAW264.7 cells that overexpress Rhau.

[0036] Figure 7 This is a graph showing the changes in osteoclast differentiation activity of RAW264.7 cells overexpressing Rhau.

[0037] Figure 8 This figure shows the changes in luciferase activity after co-transfection of MC3T3-E1 cells with Rhau overexpression recombinant plasmid, pRL-SV40 and Col1a1-wt or Rhou-wt plasmid.

[0038] Figure 9 This is a graph showing the changes in Rhau gene expression levels between the control group and the osteoporosis group.

[0039] Figure 10 This is a graph showing the changes in bone mass and bone metabolism parameters in mice after treatment with recombinant AAV virus.

[0040] Figure 11 This is a graph showing the changes in bone formation rate after treatment with recombinant AAV virus.

[0041] Figure 12 This is a graph showing the changes in osteoclast activity in mice after treatment with recombinant AAV virus. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0043] 1. Cells involved in the examples: Mouse pre-osteoblasts (MC3T3-E1) were purchased from the American Technology and Materials Center (ATCC), catalog number: CRL-2594; Mouse bone marrow mesenchymal stem cells (mBMSCs) were purchased from Cyagen Biosciences, catalog number: MUBMX-01001; Human bone marrow mesenchymal stem cells (hBMSCs) were purchased from Cyagen Biosciences, catalog number: HUXMA-01001; Mouse mononuclear macrophage leukemia cells (RAW 264.7) were purchased from Pronos, catalog number: CL-0190.

[0044] 2. The cell culture and passage procedures in this embodiment are as follows: When the cells reach a confluence of over 90%, wash them once with sterile PBS, then add trypsin containing EDTA for digestion at room temperature for 2 min. Terminate the digestion reaction by adding 2 mL of complete culture medium. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells in 5 mL of fresh complete culture medium, adjust the density to 30%, and passage. Maintain the culture by passage every 48 h as usual.

[0045] Example 1

[0046] I. Experimental Methods

[0047] 1. Construction of an osteogenic differentiation model

[0048] MC3T3-E1, mBMSCs, and hBMSCs were cultured to their optimal state using standard methods, followed by routine digestion and passage. The cells were then cultured overnight to form a monolayer. The growth medium was completely aspirated, and osteogenic differentiation induction medium was added. The medium was changed every 3 days. The osteogenic differentiation induction medium consisted of DMEM high-glucose medium + 10... -7 M dexamethasone + 10 mM β-glycerophosphate sodium + 50 μM ascorbic acid + 10% (v / v) FBS + 1% (w / v) dual antibody.

[0049] 2. Construction of an osteoclast differentiation model

[0050] RAW 264.7 cell lines with good activity were seeded into plates and cultured overnight. The culture medium was then discarded. The osteoclast differentiation medium was replaced every two days. The osteoclast differentiation medium was formulated as follows: DMEM medium + 10% (v / v) FBS + 1% (w / v) penicillin-antibody + 75 ng / mL RNAKL.

[0051] 3. Changes in Rhau gene expression levels during osteogenic and osteoclast differentiation.

[0052] (1) MC3T3-E1, mBMSCs and hBMSCs cells were collected on day 0, day 3.5 and day 7 of differentiation; RAW 264.7 cells were collected on day 0, day 1.5 and day 3 of differentiation;

[0053] (2) Total RNA extraction from cells: Total RNA extraction was performed using TRNzol Universal Total RNA Extraction Reagent (DP424) from Tiangen Biotech (Beijing) Co., Ltd. The specific steps are as follows: Add 1 mL of Trizol lysis buffer to each well of a 6-well plate, mix thoroughly by pipetting, and let stand at room temperature for 5 min; transfer the lysed cells to a 1.5 mL centrifuge tube, add 200 μL of chloroform, vortex to mix, let stand at room temperature for 3 min, and centrifuge at 12000g for 15 min at 4℃; carefully aspirate the upper aqueous phase to a new centrifuge tube, add 500 μL of isopropanol, mix, let stand at room temperature for 10 min, and centrifuge at 12000g for 10 min at 4℃; discard the supernatant and retain the RNA precipitate, add 1 mL of 75% ethanol (prepared with DEPC water) to gently suspend the precipitate, and centrifuge at 7500g for 5 min at 4℃. After discarding the ethanol, dry at room temperature for 10-15 min. Finally, dissolve the RNA precipitate with 30-40 μL of DEPC water. The sample can be used immediately or stored at -80℃.

[0054] (3) Total RNA reverse transcription to cDNA: Add 500 ng of total RNA to a 200 μL EP tube, then add 2 μL of 5×Evo M-MLV RT Master Mix (Aikerui, AG11706), and make up to 10 μL of reaction system with DEPC water. After thorough mixing, place the tube in a PCR instrument and run the program of 37℃ for 15 min (reverse transcription), 85℃ for 5 seconds (enzyme inactivation), and 4℃ storage. The obtained cDNA product can be used for qPCR detection or stored at -20℃ for later use after diluting 5 times with DEPC water.

[0055] (4) Quantitative Real-Time PCR (qPCR) Amplification: Before starting the qPCR experiment, the following reagents need to be prepared in advance: cDNA template (diluted with DEPC water to a working concentration of 12.5 ng / μL), upstream and downstream detection primers for the gene to be tested (mixed at 5 μM concentrations), DEPC water, a 96-well PCR plate, and SYBR® Premix Ex Taq™ II quantitative PCR reagent. Plan the sample loading positions of the 96-well plate reasonably according to the number of samples and the number of genes to be tested. The reaction system is 10 μL per well: 5 μL 2×SYBR Premix, 1 μL primer mixture (final concentration 0.25 μM), 2 μL cDNA template (final volume 25 ng), and 2 μL DEPC water. After loading the samples, seal the plate with sealing film, centrifuge at 500×g for 2 min, and place it in a Roche LightCycler 480 II system to run the following program: 95℃ pre-denaturation for 30 seconds; 95℃ for 5 seconds, 60℃ for 30 seconds (reading), 40 cycles; melting curve analysis. Data were processed using LightCycler SW1.5 software, with three technical replicates. Outliers with CT value differences >0.5 were removed. GAPDH was used as an internal reference, and the relative gene expression level was calculated using the ΔΔCt method. The amplification specificity was verified by single peak of melting curve.

[0056] Table 1. qPCR-related primers

[0057]

[0058] II. Test Results

[0059] Rhau expression levels were assessed by qPCR using the primers in Table 1. The results showed that Rhau expression levels were significantly increased during osteogenic differentiation, while Rhau expression levels were significantly downregulated during osteoclast differentiation. Figure 1 ).

[0060] Example 2

[0061] I. Experimental Methods

[0062] 1. Rhau gene overexpression in osteoblasts

[0063] (1) Preparation and acquisition of overexpression recombinant plasmids

[0064] ① Using cDNA from MC3T3-E1 cells in Example 1 as a template, primer pairs with Kpn I and Xho I restriction sites were designed and PCR amplified to obtain the gene sequence of the mouse Rhau protein coding region. The amplified product and plasmid pcDNA3.1-3xflag-C (YouBio, Cat#VT9221) were double-digested with Kpn I and Xho I, respectively, and the gel was recovered using a gel recovery / DNA purification kit (Novozymes Biotechnology, catalog number: DC301-01). The digested DNA product and plasmid were ligated using T4 DNA Ligase (2011A) (TAKARA), and routinely transformed into DH5α. Single-clone identification yielded a strain loaded with the mouse Rhau vector. Subsequently, the strain was inoculated and cultured to expand its scale. Plasmid extraction was performed using an endotoxin-free plasmid mini-extraction kit (Tiangen, catalog number: DP118) to obtain the Rhau overexpression recombinant plasmid pcDNA3.1-Rhau (mouse).

[0065] ② Using the cDNA of hBMSCs cells in Example 1 as a template, primer pairs with hind III and Kpn I restriction sites were designed and PCR amplified to obtain the human Rhau protein coding region gene sequence; the amplification product and plasmid p3xFLAG-CMV-10 (YouBio, Cat#VT1070) were double-digested with hindIII and Kpn I, respectively, and the Rhau overexpression recombinant plasmid p3xFLAG-CMV-10-Rhau (human) was prepared according to the method in step ①.

[0066] Mouse Rhau protein coding region gene sequence:

[0067]

[0068] Amino acid sequence of the coding region of mouse Rhau protein:

[0069]

[0070] Human Rhau protein coding region gene sequence:

[0071]

[0072] The amino acid sequence of the coding region of human Rhau protein:

[0073]

[0074] (2) Cell transfection

[0075] Transfection was performed using Jet-Prime™ transfection reagent (Polyplus, Cat#101000051), and the specific method was as follows:

[0076] ① In a 24-well plate, use 2×10 5 MC3T3-E1, mBMSCs, or hBMSCs cells were seeded at a density of cells / mL and cultured in complete medium containing 10% (v / v) serum to a cell density of 60-70%.

[0077] ② The complex was prepared using Jet-Prime™ transfection reagent (Polyplus, Cat#101000051) in the following proportions: 500 ng of overexpression recombinant plasmid pcDNA3.1-Rhau (mouse) or p3xFLAG-CMV-10-Rhau (human) was diluted in 50 μL of Jet-Prime™ Buffer and centrifuged thoroughly; then 0.5 μL of Jet-Prime™ transfection reagent was added, centrifuged thoroughly again, and incubated at room temperature for 10 min to obtain the transfection complex. Plasmid pcDNA3.1-3xflag-C was used as a control (empty vector).

[0078] ③ Transfection: Replace the culture medium in the cell culture wells from step ① with 2 mL of fresh culture medium (containing serum) without antibiotics. Add the transfection complex prepared in step ② evenly to the cell culture wells in multiple portions and incubate at 37℃ and 5% CO2 for 6 h. The experimental transfection efficiency can be optimized by adjusting the plasmid dosage and reagent volume. The transfection complex must be used within 6 h to prevent degradation. The experiment is repeated 3 times to ensure data reproducibility.

[0079] (3) Six hours after transfection, the cells were induced to differentiate using osteogenic induction medium as described in Example 1. On day 3 of osteogenic differentiation, the expression levels of Rhau gene and osteogenic differentiation-related genes (Osx, Col1a1, Runx2, Ocn) were detected by qPCR as described in Example 1. On day 7 of osteogenic differentiation, alkaline phosphatase (ALP) activity was detected. On day 21 of osteogenic differentiation, mineralized nodules were stained (Alizarin Red staining, ARS). The specific methods are as follows:

[0080] ① Alkaline phosphatase activity staining: Alkaline phosphatase activity was detected on day 7 of osteogenic differentiation. First, the cells to be tested were removed from the incubator, the culture medium was carefully aspirated, and the cells were washed three times (3 min each) with PBS buffer. Then, the cells were fixed with 4% (w / w) paraformaldehyde at room temperature for 20 min. After removing the fixative, the cells were washed three more times with PBS (3 min each). The staining working solution was prepared according to the Beyotime BCIP / NBT alkaline phosphatase chromogenic kit instructions (ratio: 3 mL chromogenic buffer + 10 μL BCIP solution + 20 μL NBT solution). After washing, 500 μL of staining working solution was added to each well, and the cells were incubated at room temperature in the dark for 5-30 min until the desired color development was achieved. After staining, the staining solution was removed, and the cells were washed three times (3 min each) with triple-distilled water. Finally, the cells were observed under a microscope and photographed. The air-dried culture plates can be stored long-term.

[0081] ② Mineralized nodule staining: Mineralized nodule staining was performed on day 21 of osteogenic differentiation. First, the cell culture plate was removed from the incubator, the culture medium was carefully aspirated, and the cells were washed three times (3 min each) with PBS buffer. Then, the cells were fixed with 4% (w / w) paraformaldehyde at room temperature for 30 min. After removing the fixative, the cells were washed three more times with PBS (3 min each). 500 μL of Alizarin Red staining solution was added to each well, and the cells were incubated at room temperature in the dark for 30 min. After staining, the staining solution was removed, and the cells were thoroughly washed five times (3 min each) with triple-distilled water. Finally, the cells were observed and photographed under a microscope. The air-dried culture plates can be stored long-term.

[0082] 2. siRNA knockdown of mouse Rhau gene expression levels in MC3T3-E1 and mBMSCs cells

[0083] (1) Transfection with different siRNAs

[0084] Based on the Rhau gene sequence published by NCBI, three pairs of siRNA sequences targeting the mouse Rhau gene were designed. The siRNA sequences are shown in Table 2. Using Jet-Prime™ transfection reagent (Polyplus, Cat#101000051), siRNA-1, siRNA-2, siRNA-3, and mixtures of siRNA-1+siRNA-2, siRNA-1+siRNA-3, and siRNA-2+siRNA-3 from Table 2 were transfected into MC3T3-E1 or mBMSCs cells, respectively. The specific methods are as follows:

[0085] ① Seed MC3T3-E1 or mBMSCs cells in 6-well plates and culture them in complete medium containing 10% (v / v) serum to a cell density of 60-70%.

[0086] ② Prepare a 20 μM stock solution of siRNA. Add 200 μL of Jet-Prime™ Buffer to a 1.5 mL centrifuge tube, then add 5 μL of the siRNA stock solution to the buffer (for a mixture, the total volume of the two siRNAs is 5 μL, with a volume ratio of 1:1). Vortex thoroughly. Add 4 μL of Jet-Prime™ transfection reagent, vortex thoroughly again, and incubate at room temperature for 10 min to obtain the transfection complex.

[0087] ③ Transfection: During transfection, replace the culture medium in the cell culture wells of step ① with 2 mL of fresh culture medium without antibiotics (containing serum), and slowly add the transfection complex prepared in step ② into the six-well plate. Incubate at 37℃ and 5% CO2 for 6 h. The final concentration of siRNA in the culture medium is 50 nM.

[0088] (2) Six hours after transfection, the cells were induced to differentiate using osteogenic induction solution as in Example 1. On the third day of osteogenic differentiation, the expression level of mouse Rhau gene and other genes was detected by qPCR as in Example 1. On the seventh day of osteogenic differentiation, alkaline phosphatase activity was detected. On the twenty-first day of osteogenic differentiation, mineralized nodules were stained.

[0089] Table 2 siRNA sequences

[0090]

[0091] II. Test Results

[0092] 1. Changes in the expression levels of osteogenic-related genes and osteogenic differentiation activity after overexpression of the Rhau gene.

[0093] After successfully transfecting MC3T3-E1 and mBMSCs cells with the overexpression recombinant plasmid pcDNA3.1-Rhau (mouse), osteogenic differentiation was induced. qPCR results showed that Rhau overexpression significantly increased Rhau gene expression in MC3T3-E1 and mBMSCs cells under osteogenic differentiation-induced conditions, and the expression of osteogenic differentiation-related genes (Osx, Col1a1, Runx2, Ocn) was significantly upregulated. Figure 2 ).

[0094] Overexpression of recombinant plasmids pcDNA3.1-Rhau (mouse) and p3xFLAG-CMV-10-Rhau (human) was successfully transfected into MC3T3-E1, mBMSCs, and hBMSCs cells, and osteogenic differentiation was induced. Osteoblast differentiation activity was quantified using ALP and ARS. The results showed that osteogenic differentiation activity was significantly enhanced after Rhau overexpression. Figure 3 ).

[0095] 2. Changes in osteoblast osteogenic differentiation activity after siRNA knockdown of the Rhau gene.

[0096] In this embodiment, MC3T3-E1 cells were transfected with different siRNAs. The culture medium was changed 6 hours after transfection to induce osteogenic differentiation. On day 3 of differentiation, the Rhau gene expression level was detected by qPCR to evaluate the knockdown effect. Among the different siRNAs transfected alone, siRNA-1 showed the highest Rhau knockdown efficiency; however, when two siRNAs were transfected in combination, a 1:1 ratio of siRNA-2 to siRNA-3 resulted in the most significant Rhau knockdown effect. Figure 4 ).

[0097] MC3T3-E1 and mBMSCs cells were further transfected with a 1:1 mixture of siRNA-2 and siRNA-3 using Jet-Prime transfection reagent. The culture medium was changed after 6 hours to induce osteogenic differentiation. Alkaline phosphatase staining was performed on day 7 of differentiation, and Alizarin Red staining was performed on day 21. The results showed that osteogenic activity was significantly inhibited after Rhau knockdown. Figure 5 ).

[0098] Example 3

[0099] I. Experimental Methods

[0100] 1. Overexpression of the mouse Rhau gene in osteoclasts

[0101] (1) Preparation and acquisition of mouse Rhau gene overexpression recombinant plasmid: Same as in Example 2.

[0102] (2) Cell transfection: Mouse mononuclear macrophage RAW 264.7 cells were used as the target cells and transfected using Jet-prime™ transfection reagent (Polyplus, Cat#101000051). The specific transfection method was the same as in Example 2.

[0103] (3) Construction of osteoclast differentiation model: The culture medium was changed 6 h after transfection, and the osteoclast differentiation model was constructed according to Example 1.

[0104] 2. qPCR detection: Cell samples from day 4 of differentiation were used to perform qPCR detection on key osteoclast differentiation genes (Ctsk, Trap, Nfatc1 genes) and Rhau gene. The specific method is as described in Example 1.

[0105] 3. Tartrate acid phosphatase (TRAP) staining: Cell samples from day 4 of differentiation were subjected to TRAP staining. Specifically, the culture medium was discarded, and the cells were washed twice with PBS and the PBS was discarded. Then, the cells were fixed with 4% (w / w) paraformaldehyde at room temperature for 15 min. Subsequently, TRAP staining was performed according to the Solarbio anti-tartrate acid phosphatase staining kit (Cat#G1480) instructions. After the experiment, images were taken and statistical analysis was performed using ImageJ software.

[0106] II. Test Results

[0107] pcDNA3.1-Rhau (mouse) was overexpressed in RAW264.7 cells to construct an osteoclast differentiation model. Cell samples were taken on day 4 of differentiation, and qPCR was used to detect key genes in osteoclast differentiation. The results showed that Rhau overexpression significantly inhibited osteoclast differentiation activity (Ctsk, Trap, Nfatc1). Figure 6 Cell samples from day 4 of differentiation were stained with TRAP and their area was quantified using ImageJ software. The results showed that overexpression of Rhau significantly inhibited osteoclast activity. Figure 7 ).

[0108] Example 4: Rhau regulates the secondary structure of Col1a1 and Rhou promoter nucleic acid G-quadruplexes to regulate osteogenic and osteoclast differentiation activities.

[0109] I. Experimental Methods

[0110] 1. The nucleotide sequences of the Col1a1 promoter and the Rhou promoter were artificially synthesized. Kpn I and EcoRI restriction sites were introduced at the 5' and 3' ends of the Col1a1 promoter nucleotide sequence, and Kpn I and Nhe I restriction sites were introduced at the 5' and 3' ends of the Rhou promoter nucleotide sequence. The nucleotide sequences were then inserted into pGL3-Basic (Ubibio, Cat#VT1554) through the corresponding restriction sites to construct the Col1a1-wt and Rhou-wt recombinant plasmids.

[0111] The nucleotide sequence of the Col1a1 promoter:

[0112]

[0113] The nucleotide sequence of the Rhou promoter:

[0114]

[0115] 2. The Rhau overexpression recombinant plasmid / blank control (empty vector) was co-transfected with Col1a1-wt and pRL-SV40 (Ubibio, Cat#VT1571) in a mass ratio of 1:1:0.5 into MC3T3-E1 cells. The specific transfection method is as described in Example 2.

[0116] 3. The Rhau overexpression recombinant plasmid / blank control (empty vector) was co-transfected with Rhou-wt and pRL-SV40 (Ubibio, Cat#VT1571) in a mass ratio of 1:1:0.5 into RAW264.7 cells. The specific transfection method is as described in Example 2.

[0117] 4. The effect of Rhau overexpression on the activities of Col1a1 and Rhou promoters was evaluated by luciferase assay, using the Dual Luciferase Reporter Gene Assay Kit (Abbkine, catalog number KTA8010).

[0118] Experimental results show that Rhau can upregulate Col1a1 promoter activity and downregulate Rhou promoter activity through interactions with Col1a1 G-quadruplex and Rhou G-quadruplex. Figure 8 ).

[0119] Example 5

[0120] I. Experimental Methods

[0121] 1. Eight-week-old female C57BL / 6J mice (purchased from Guangdong Provincial Experimental Animal Center) were randomly divided into two groups (at least 3 mice in each group): a control group and an osteoporosis group. The osteoporosis group underwent bilateral ovariectomy, and the ovariectomy (OVX) model was established according to the standard method described in previous literature. The control group received no treatment.

[0122] 2. Mouse bone marrow mesenchymal stem cells (mBMSCs) were isolated and cultured using the MesenCult™ Expansion Kit (Mouse) (stemcell product number: 5513). The specific method is as follows:

[0123] (1) Sacrifice the mice: One month after the operation, the mice were euthanized according to the conventional method. After euthanasia, the mice were immersed in 75% alcohol for 5 minutes.

[0124] (2) Bone tissue acquisition: Dissect the femur and tibia of the hind limb, and thoroughly remove the surrounding muscle tissue. Place the bone in a culture dish containing buffer (buffer formula: PBS or HBSS + 2% FBS) for 30 s. Note that only the red, bone marrow-containing part of the tibia should be retained; the femur can be dissected upwards from the white ligament of the knee joint.

[0125] (3) Bone tissue cleaning: Rinse the detached bone tissue 1-2 times with serum-free culture medium MEM-α.

[0126] (4) Bone marrow rinsing: Prepare two sterile 6 cm dishes for each mouse, and add 5 mL of MesenCult complete culture medium to one of them. Cut open both ends of the bone, draw up 1 mL of complete culture medium with a syringe, and rinse the bone marrow repeatedly along the bone wall until the bone tissue turns white. Note that you should rinse from the distal end first, and then rinse from the proximal end in the opposite direction to ensure that the stem cells in the distal femur and proximal tibia are completely washed out.

[0127] (5) Cell dispersion: Gently blow the flushed bone marrow with a 1 mL pipette to disperse the cells fully.

[0128] (6) Remove impurities: Transfer the cell suspension to a new 6 cm culture dish, taking care to avoid aspirating precipitated bone fragments and other tissues.

[0129] (7) Add selection reagent: Adjust the volume of MesenCult complete medium to 5 mL, and add MesenPure while gently shaking.

[0130] (8) Preliminary culture: Place the petri dish in a 37℃ incubator and let it stand for 3 days.

[0131] (9) Medium change treatment: After 3 days, discard the culture medium, gently shake to remove non-adherent cells, wash once with PBS, and then add 5 mL of MesenCult complete medium to continue culturing.

[0132] (10) Passage culture: Usually, the stem cells can grow to fill the culture dish in about 5 days, and then passage culture is carried out. The passage is recorded as the first generation.

[0133] 3. RNA was extracted from first-generation mBMSC cells in the control group and osteoporosis group and reverse transcribed. The expression level of Rhau gene was detected according to Example 1.

[0134] II. Test Results

[0135] Compared with primary mBMSCs from healthy organisms (control group), Rhau expression levels were significantly downregulated in osteoporotic mice at the onset of osteoporosis. Figure 9This result could provide a method for diagnosing osteoporosis by detecting Rhau gene expression levels using qPCR, fluorescent probes, diagnostic kits, etc.

[0136] Example 6: Application of Rhau-loaded adeno-associated virus in the treatment of osteoporotic mice

[0137] I. Experimental Methods

[0138] 1. Preparation and Acquisition of Recombinant Adeno-Associated Virus

[0139] (1) Referring to Example 2, the gene sequence of the mouse Rhau protein coding region was inserted into the PAAV2 / 9 plasmid (Fenghui Biotechnology, catalog number: ZT836) through the Pme I and Nco I restriction sites to obtain the recombinant plasmid PAAV2 / 9-Rhau.

[0140] (2) In a φ100-mm cell culture dish containing 10% FBS DMEM medium, seed 4.0 × 10⁻⁶ cells. 6 A number of AAVpro293T cells (Clontech, catalog number 632273) were cultured.

[0141] (3) Mix 1 μg of recombinant plasmid PAAV2 / 9-Rhau, 1 μg of PAAV-RC9 plasmid (helper plasmid, Miaoling Company, catalog number: P2846) and 1 μg of pHelper plasmid (adenovirus Helper plasmid, Miaoling Company, catalog number: P0243), and transfect the above plasmid into AAVpro 293T cells at a mass-to-volume ratio of 1:2 using transfection reagent Lipo293™ (Beyotime, catalog number: C0521). 24 h after transfection, replace the medium with fresh DMEM containing 2% FBS and culture for 3 days.

[0142] (4) Then, the recombinant AAV (AAV-Rhau) virus solution was extracted, purified, and concentrated according to the method of Beyotime's AAV Purification Kit Mini for All Serotypes (catalog number C2906S). The viral titer was quantified using YEASEN's AAV9 Titration ELISA Kit (catalog number: 99306ES96), and the viral titer needed to reach 1.34E+13vg / mL or higher.

[0143] 2. Eight-week-old female C57BL / 6J mice (purchased from Guangdong Provincial Experimental Animal Center) were randomly divided into three groups (n=6 per group): sham-operated group, osteoporosis-blank treatment group, and osteoporosis-Rhau treatment group. The ovariectomy (OVX) model was established according to the standard method described in previous literature. Except for the sham-operated group, all other groups underwent bilateral ovariectomy; the sham-operated group received only sham surgery. One week post-operation, the Rhau treatment group received bilateral intra-articular injections (10 μL of recombinant AAV virus solution per side), while the blank treatment group received AAV virus transfected with an empty vector. The experimental period was two months.

[0144] 3. After the experiment, changes in bone mass and bone metabolism parameters in mice were assessed using μCT.

[0145] 4. Twenty-four days before the end of the experiment, mice were injected intraperitoneally with dimethylphenol orange (Aladdin, #X101229-5G) at a dose of 10 mg / kg. Three days before the end of the experiment, mice were injected with calcein (Sigma, #C08955G) at a dose of 20 mg / kg. After the experimental results were obtained, mouse femurs were harvested. The femur specimens were fixed in 4% (w / w) paraformaldehyde solution for 24 h, and 20 μm thick sections were cut using a hard tissue sectioning and grinding system (EXAKT). Images were acquired using an inverted microscope (Nikon, DS-Ri1). Mineral deposition rate (MAR) and bone formation rate / bone surface area (BFR / BS) were analyzed using Bioquant Osteo software (v.7.20.10, Bioquant Nashville).

[0146] 5. Mouse femurs were fixed overnight with 4% (w / w) paraformaldehyde, then embedded and sectioned. The sections were subjected to TRAP immunohistochemistry and quantified using Image J.

[0147] II. Test Results

[0148] 1. Changes in bone mass and bone metabolism parameters in mice

[0149] After the experiment, changes in bone mass and bone metabolism parameters in mice were assessed using μCT. The results showed that bone mass and bone metabolism parameters in mice treated with Rhau-loaded recombinant AAV virus significantly recovered. Figure 10 ).

[0150] 2. Fluorescence double-labeling assay for assessing bone formation rate

[0151] The results showed that treatment with Rhau-loaded recombinant AAV virus significantly increased bone formation rate in mice, indicating that Rhau-loaded recombinant AAV virus can treat osteoporosis. Figure 11 ).

[0152] 3. TRAP staining to assess osteoclast activity in Rhau-treated mice

[0153] Mouse femoral bones were fixed overnight in 4% paraformaldehyde, then embedded and sectioned. TRAP immunohistochemistry was performed on the sections, and quantification was conducted using Image J. The results showed that Rhau treatment significantly reduced the number and proportion of osteoclasts in the mouse femoral bones, indicating a significant decrease in bone resorption activity. Figure 12 ).

[0154] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A siRNA inhibitor composition, characterized in that, The siRNA is a mixture of siRNA-2 and siRNA-3, and its nucleotide sequence is shown below: siRNA-2-Sense: 5'-GGUGUUCGGAAAAUAGUAAUU-3'; siRNA-2-Anti-Sense:5'-UUACUAUUUUCCGAACACCUU-3'; siRNA-3-Sense: 5'-GGAUAAGCAAGAAGAAUUAAC-3'; siRNA-3-Anti-Sense: 5'-UAAUUCUUCUUGCUUAUCCAA-3'.

2. The siRNA inhibitor composition according to claim 1, characterized in that: The molar ratio of siRNA-2 to siRNA-3 is 1:

1.

3. The siRNA inhibitor composition according to claim 1, characterized in that: The siRNA inhibitor composition further comprises an RNA molecule obtained by chemically modifying the siRNA molecule; or a recombinant vector encoding the siRNA sequence molecule in vivo; The recombinant vector is one of plasmid, adenovirus, lentivirus, or adeno-associated virus.

4. The siRNA inhibitor composition according to claim 1, characterized in that: The siRNA inhibitor composition further comprises siRNA transfection-related reagents.

5. The use of the siRNA inhibitor composition according to any one of claims 1-4 in the preparation of a product for screening drugs to prevent and treat osteoporosis.

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