SiRNA for inhibiting Wdfy1 gene expression, siRNA delivery system and preparation method and application of siRNA delivery system

Abstract

The invention belongs to the field of biological medicine, and discloses siRNA for inhibiting Wdfy1 gene expression, an siRNA delivery system as well as a preparation method and application of the siRNA delivery system. According to the siRNA for inhibiting Wdfy1 gene expression and the siRNA delivery system RVG-9R-siRNA composed of the siRNA and the chimeric peptide RVG-9R, WDFY1 protein expression in cerebral neuronal cells can be inhibited and lowered in a targeted mode, bone-fat imbalance caused by aging is relieved, the bone mass and bone mechanical strength are improved, and the effect of inhibiting Wdfy1 protein expression in cerebral neuronal cells is achieved. The application of the pharmaceutical composition in preparation of drugs for relieving bone-fat imbalance, preventing and treating osteoporosis, and improving bone mass or / and bone mechanical strength is outstanding in application effect, and more choices are provided for treatment of diseases such as bone-fat imbalance and osteoporosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a siRNA for inhibiting Wdfy1 gene expression, a siRNA delivery system for targeted inhibition of Wdfy1 gene expression in brain neurons, and a preparation method and application thereof. Background Art

[0002] Osteoporosis (OP) is a systemic bone metabolic disease that often occurs in middle-aged and elderly people. It is characterized by decreased bone mass, destruction of bone microstructure, and increased bone brittleness. It is easy to cause fractures, resulting in a decline in the quality of life of patients and even premature death, which seriously endangers human life and health. At present, clinical OP treatment drugs mainly include bone mineralization promoters, bone formation stimulators, and bone resorption inhibitors, which have certain effects on alleviating OP and reducing the incidence of fractures, but there are problems such as long medication cycles, high treatment costs, and side effects. Therefore, in-depth exploration of the molecular mechanism of OP pathogenesis and the search for new prevention and treatment targets have become key issues that need to be urgently addressed in research related to OP prevention and treatment.

[0003] The nervous system is an important participant in the regulation of bone metabolism. Studies have found that the nervous system can regulate bone development, growth and reconstruction through a variety of pathways and participate in bone physiological and pathological processes. Clinical evidence shows that people with certain neurological diseases (such as Alzheimer's disease, Parkinson's disease, stroke, etc.) or certain mental and psychological problems (such as long-term stress, schizophrenia, etc.) are often accompanied by OP. Therefore, it is necessary to study a new drug that can effectively deal with osteoporosis by regulating the nervous system, which is of great significance to this field.

[0004] WD repeat and FYVE domain containing 1 (WDFY1), also known as FENS-1. Studies have found that this protein can act as an adaptor protein of Toll-like receptor 3 / 4 (TLR3 / 4) to recruit interferon TIR domain adaptor protein (TRIF) to activate cellular immunity and inflammatory response. Neural stem cells and cortical neurons express WDFY1. Appropriate levels of WDFY1 are beneficial to the normal differentiation and dendritic growth of neural stem cells, but excessive increase of WDFY1 can lead to reduced dendritic branching of neurons. In addition, WDFY1 is also involved in regulating endosome maturation, content transport and autophagosome formation. Its upstream molecule neuropilin 2 promotes tumor cell autophagy by inhibiting WDFY1, and upregulation of WDFY1 can lead to tumor cell death. However, there is no relevant research reporting whether this protein has a role in regulating bone metabolism. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology. Based on the confirmation that neuronal cells in the aging brain cause bone-fat imbalance and osteoporosis by delivering extracellular vesicles rich in WDFY1 to the bones, the present invention develops a siRNA for inhibiting the expression of the Wdfy1 gene, a siRNA delivery system for targeted inhibition of the expression of the Wdfy1 gene in brain neurons, and a preparation method and application thereof.

[0006] Rabies virus glycoprotein (RVG) has neural targeting and can be transmitted retrogradely through axons and between synapses without being hindered by the blood-brain barrier. Nonamer of arginine (9R) is a positively charged cell-penetrating peptide that can efficiently load nucleic acid substances and promote their penetration into cells. The present invention adds triglycine spacer (GGG) and 9R residues (RRRRRRRRR) to the carboxyl end of a 29-amino acid-long peptide chain (YTIWMPENPRPGTPCDIFTNSRGKRASNG) derived from RVG to obtain a 41-amino acid-long chimeric peptide (RVG-9R) capable of binding and loading siRNA, and the sequence is YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGRRRRRRRRR. RVG-9R was then combined with siRNA that inhibits the expression of the Wdfy1 gene to obtain RVG-9R-siWdfy1, which was injected into 16-month-old mice via the tail vein. It can downregulate the expression of WDFY 1 protein in brain neuronal cells, alleviate the bone-fat imbalance phenotype caused by aging, and increase the bone mass and bone mechanical strength of aged mice.

[0007] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0008] In a first aspect, the present invention provides a siRNA for inhibiting the expression of Wdfy1 gene, comprising a sense strand and an antisense strand:

[0009] The positive strand is 5'-GGAATTTCACGTTTCTGAA-3',

[0010] The antisense strand is 5'-TTCAGAAACGTGAAATTCC-3'.

[0011] In a second aspect, the present invention provides a siRNA delivery system for targeted inhibition of Wdfy1 gene expression in brain neurons, comprising the siRNA for inhibiting Wdfy1 gene expression and a chimeric peptide RVG-9R.

[0012] In the above-mentioned siRNA delivery system, preferably, the amino acid sequence of the chimeric peptide RVG-9R is: YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGRRRRRRRRR.

[0013] Preferably, the molar ratio of the siRNA for inhibiting Wdfy1 gene expression to the chimeric peptide RVG-9R is 1:1.

[0014] In a third aspect, the present invention provides a method for preparing a siRNA delivery system for inhibiting Wdfy1 gene expression in brain neurons, comprising the following steps: mixing the siRNA for inhibiting Wdfy1 gene expression and the chimeric peptide RVG-9R, and incubating them in a glucose solution.

[0015] In the above preparation method, preferably, the concentration of the glucose solution is 5% (w / v), the incubation temperature is 20-25° C., and the incubation time is 10-20 min.

[0016] In a fourth aspect, the present invention provides an siRNA for inhibiting the expression of the Wdfy1 gene, an siRNA delivery system, or the use of the siRNA delivery system obtained by the preparation method in down-regulating the expression of the WDFY1 protein in brain neuronal cells.

[0017] In a fifth aspect, the present invention provides a product for inhibiting the expression of the Wdfy1 gene for use in the preparation of a drug for alleviating bone-fat imbalance, preventing and treating osteoporosis, increasing bone mass and / or increasing bone mechanical strength.

[0018] In the above application, preferably, the product for inhibiting Wdfy1 gene expression comprises the siRNA for inhibiting Wdfy1 gene expression according to claim 1, the siRNA delivery system according to any one of claims 2-4, or the siRNA delivery system obtained by the preparation method according to any one of claims 5-6.

[0019] Preferably, the bone-fat imbalance and osteoporosis are bone-fat imbalance and osteoporosis caused by the accumulation of WDFY1 in bone tissue via brain-derived EVs after aging.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention discovered for the first time the important role of WDFY1 protein in bone homeostasis, defined that aging neurons are the main source of WDFY1 protein in brain-derived EVs, and that WDFY1 protein enriched in bone tissue after aging mainly comes from brain-derived extracellular vesicles (EVs), and provided a technical idea for alleviating bone-fat imbalance, preventing and treating osteoporosis, increasing bone mass and improving bone mechanical strength by inhibiting Wdfy1 gene expression, laying a solid foundation for the further development of related inhibitory agents and therapeutic drugs.

[0022] 2. The present invention provides siRNA for inhibiting the expression of Wdfy1 gene, and siRNA delivery system RVG-9R-siRNA composed of the siRNA and chimeric peptide RVG-9R, which can target and inhibit down-regulate the expression of WDFY1 protein in brain neuronal cells, alleviate the bone-fat imbalance caused by aging, and increase bone mass and bone mechanical strength.

[0023] 3. The preparation method of the present invention has simple operation, simple reaction conditions and high production efficiency.

[0024] 4. The present invention provides siRNA for inhibiting the expression of Wdfy1 gene and its delivery system RVG-9R-siRNA for down-regulating the expression of WDFY1 protein in brain neuronal cells, and for use in preparing drugs for reducing bone-fat imbalance, preventing and treating osteoporosis, increasing bone mass and / or improving bone mechanical strength. The application effect is outstanding, providing more options for the treatment of diseases such as bone-fat imbalance and osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 It is a Western blot image of the expression of WDFY1 protein in brain tissues of young and old mice;

[0027] Figure 2 This is an immunofluorescence staining image of WDFY1 protein in the neurons of the hippocampus of the mouse brain after injection of different reagents;

[0028] Figure 3 are micro-CT images of the femurs of mice after injection of different reagents;

[0029] Figure 4 The data of femoral cancellous bone, cortical bone and three-point bending test of mice after injection of different reagents;

[0030] Figure 5 is the immunohistochemical staining of OCN and perilipin in femoral sections of mice injected with different reagents;

[0031] Figure 6 It is the number of osteoblasts per unit circumference of trabecular surface and the number of adipocytes per unit area of ​​bone marrow tissue in mouse femoral slices after injection of different reagents;

[0032] Figure 7 ARS (Alizarin Red) and ORO (Oil Red O) staining images of the intervention effect of EVs in brain tissue of mice injected with different reagents on BMSCs;

[0033] Figure 8 The data of Alizarin Red-positive area and Oil Red O-positive area of ​​BMSCs intervened by EVs in brain tissue of mice after injection of different reagents;

[0034] In the above figures: RV-MAT-9R-siCon represents a siRNA-chimeric peptide that is not neurotropic and does not target the Wdfy1 gene, RV-MAT-9R-siWdfy1 represents a siRNA-chimeric peptide that is not neurotropic but targets the Wdfy1 gene, RVG-9R-siCon represents a siRNA-chimeric peptide that does not target the Wdfy1 gene, and RVG-9R-siWdfy1 represents a siRNA-chimeric peptide that targets the Wdfy1 gene. DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0036] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0037] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0038] The present invention found that WDFY1 is highly expressed in brain neurons of aging organisms and can be transported to bones through extracellular vesicles of neurons to induce OP. Based on this, the present invention prepared specific small interfering RNAs (siRNAs) targeting the Wdfy1 gene, and used the currently well-researched brain-targeted peptide, i.e., the neurotropic rabies virus glycoprotein-derived peptide (RVG) to modify Wdfy1 siRNAs, and obtained siRNAs (RVG-9R-siWdfy1) that can target and downregulate neuronal Wdfy1. It was injected into the aging organism through the blood vessels and found that it can significantly reduce the senile OP phenotype.

[0039] Example:

[0040] An siRNA for inhibiting the expression of Wdfy1 gene, which has a sense strand and an antisense strand, and the sense strand is completely complementary to the antisense strand:

[0041] The positive strand is 5'-GGAATTTCACGTTTCTGAA-3' (see SEQ ID No. 1),

[0042] The antisense strand is 5'-TTCAGAAACGTGAAATTCC-3' (see SEQ ID No. 2).

[0043] The siRNA was prepared by chemical synthesis method on behalf of Guangzhou Ruibo Biotechnology Co., Ltd.

[0044] To ensure silencing of Wdfy1 gene expression, this siRNA can target the only known transcript of human Wdfy1 gene, NM_020830.5, and all known transcripts of mouse Wdfy1 gene, including XR_001785590.2, XM_036153456.1, NM_001111279.1, XM_006496543.3, XM_030242794.1, XM_030242799.1, XM_017322387.1, NM_027057.3 and XM_030242801.1.

[0045] A siRNA delivery system for targeted inhibition of Wdfy1 gene expression in brain neurons, comprising siRNA for inhibiting Wdfy1 gene expression and a chimeric peptide RVG-9R, wherein the amino acid sequence of the chimeric peptide RVG-9R is: YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGRRRRRRRRR (see SEQ ID No. 3).

[0046] The preparation method of this delivery system includes the following steps: taking 45 nmol of the chimeric peptide RVG-9R and siRNA per 1 mL of reagent, mixing them in 1 mL of 5% (w / v) glucose solution, incubating at room temperature (25°C) for 10 minutes, and obtaining a reagent with an siRNA concentration of 45 nmol / mL. The positively charged RVG-9R has a high affinity for the negatively charged siRNA, and in this process, RVG-9R-siRNA (i.e., RVG-9R-siWdfy1) is connected. The RVG-9R was commissioned to be synthesized and purified by Shanghai Qiangyao Biotechnology Co., Ltd.

[0047] Experiment 1: The amount of WDFY1 protein in the brain tissue of aged mice increased significantly.

[0048] Young (2 months old) and old (18 months old) mice were killed, and their brain tissues were removed. After removing the cerebellum, olfactory bulb and lower brainstem, they were chopped with scissors, added with 600 μL protein lysis buffer (RIPA) and 10 μL protease inhibitor, and homogenized with a tissue homogenizer until no solid was visible to the naked eye. The tissue homogenate was placed on ice for 20 minutes, and then centrifuged at 13400×g for 30 minutes at 4°C. The supernatant was aspirated into another centrifuge tube, and a small amount was taken to determine the protein concentration using the BCA method. After each protein sample was diluted to the same concentration with loading buffer, it was heated at 95°C for 10 minutes. The WDFY1 protein in the protein sample was detected by Western blot, and the expression level of β-actin protein was used as an internal reference to compare the expression level of WDFY1 protein in the brain tissue of young and old mice.

[0049] Depend on Figure 1 It can be seen that Western blot results showed that the expression level of WDFY1 protein in the brain tissue of old mice was significantly higher than that in young mice.

[0050] Experiment 2: RVG-9R-siWdfy1 downregulated the expression of WDFY1 in neurons in the hippocampus region of the brain.

[0051] 100 μL of the above RVG-9R-siWdfy1 was injected into 16-month-old male C57BL / 6 mice by tail vein, once a week, for 8 weeks. Mice were killed within one week after the last injection, and brain tissue and bilateral femoral samples were collected. After the brain tissue samples were cut into left and right hemispheres along the sagittal plane, one side was fixed in 4% paraformaldehyde, and after paraffin sectioning, immunofluorescence staining was performed using antibodies against MAP2 and WDFY1 and corresponding secondary antibodies. MAP2-positive, WDFY1-positive, and double-positive cells in the hippocampus of the stained samples were observed and photographed under a fluorescence microscope.

[0052] Depend on Figure 2It can be seen that immunofluorescence staining showed that the injection of RVG-9R-siWdfy1 successfully downregulated the expression of WDFY1 protein in the neurons of the hippocampus of 16-month-old mice.

[0053] Experiment 3: RVG-9R-siWdfy1 increases bone mass and bone mechanical strength in mice.

[0054] One side of the femur obtained from the animal in Experiment 1 was fixed in 4% paraformaldehyde, and the connective tissue of the other side of the femur was stripped clean for the three-point bending test. The fixed femoral sample was subjected to micro-CT scanning imaging to analyze the cancellous bone trabecular volume fraction, trabecular number, trabecular thickness and cortical bone thickness. The other side of the femoral sample was placed on the test stand of the Instron 3343 mechanical instrument, and a vertical downward pressure was applied to the midpoint of the femoral sample. The maximum load-bearing capacity before the femur broke was recorded as the mechanical strength result.

[0055] Figure 3 Micro-CT data and Figure 4 The experimental results showed that injection of RVG-9R-siWdfy1 successfully increased the bone mass and bone mechanical strength of 16-month-old mice.

[0056] Experiment 4: RVG-9R-siWdfy1 increased osteoblasts and decreased adipocytes in bone tissue.

[0057] The fixed femoral samples were decalcified and paraffin-sectioned. Immunohistochemical staining of bone tissue sections was performed using anti-osteocalcin (OCN) antibodies and anti-perilipin antibodies. The number of positive cells for each of the two indicators was observed under a microscope, and the number of OCN-positive osteoblasts and perilipin-positive adipocytes in a certain tissue area was counted respectively.

[0058] Figure 5 OCN and perilipin immunohistochemical staining results and Figure 6 The data showed that injection of RVG-9R-siWdfy1 increased the number of osteoblasts in the bone tissue and reduced the number of adipocytes in the bone marrow of 16-month-old mice.

[0059] Experiment 5: RVG-9R-siWdfy1 attenuated the in vitro adipogenic and anti-osteogenic effects of EVs derived from the brain of aged mice.

[0060] The cerebellum, olfactory bulb and lower brainstem of the unfixed brain tissue on one side in Experiment 2 were removed, and the remaining tissue was minced and placed in Hibernate-A medium supplemented with 20U / mL papain, and digested at 37°C for 20 minutes. The digestion was then terminated using Hibernate-A medium supplemented with 1× protease inhibitor. The digested brain tissue was homogenized, centrifuged at 12000g, 4°C for 5 minutes, and the supernatant was collected. The supernatant was centrifuged at 300g and 2000g for 10 minutes each, and then centrifuged at 10000g for 30 minutes to remove cell debris and large particles. The supernatant from the previous step was used for Optiprep TM EVs were purified by density gradient centrifugation with 5% paraformaldehyde as the medium. The protein concentration of the obtained EVs was quantified using the BCA method. Primary bone marrow mesenchymal stem cells (BMSCs) were isolated from mouse bone marrow and cultured in vitro to induce osteogenic and adipogenic differentiation, respectively. EVs obtained in the previous step were added to the cells induced in the two differentiation directions, so that the concentration of EVs in the culture environment was 10 μg / mL. BMSCs induced for osteogenic differentiation were stained with Alizarin Red S (ARS), and the area of ​​calcified nodules with positive staining was counted. BMSCs induced for adipogenic differentiation were stained with Oil Red O (ORO), and the area of ​​lipid droplets with positive staining was counted.

[0061] Figure 7 ARS and Oil Red O staining and Figure 8 The experimental data showed that the effects of EVs derived from the brain tissue of 16-month-old mice injected with RVG-9R-siWdfy1 on promoting BMSCs adipogenesis and inhibiting BMSCs osteogenesis were reduced.

[0062] In summary, the present invention discovered for the first time the important role of WDFY1 protein in bone homeostasis and found that the amount of WDFY1 protein in the brain tissue of aged mice increased significantly. WDFY1 reached the bone tissue through EVs, causing bone-fat imbalance and reducing the bone mass and bone mechanical strength of mice.

[0063] Based on the above original discoveries, a small interfering RNA (siRNA) delivery and gene silencing technology targeting the Wdfy1 gene in brain neurons was designed, which can downregulate the expression of WDFY1 protein in aging neurons and reduce its content in brain-derived EVs, thereby alleviating bone-fat imbalance and osteoporosis caused by the enrichment of WDFY1 in bone tissue via brain-derived EVs after aging. This has laid a solid foundation for the further development of related inhibitory reagents and therapeutic drugs, and provided more options for the treatment of diseases such as bone-fat imbalance and osteoporosis, which is of great practical significance to this field.

Claims

1. A siRNA that inhibits the expression of the Wdfy1 gene, It is characterized in that Contains sense and antisense strands: The positive strand is 5'-GGAATTTCACGTTTCTGAA-3', The antisense strand is 5'-TTCAGAAACGTGAAATTCC-3'.

2. A siRNA delivery system for targeted inhibition of Wdfy1 gene expression in brain neurons, It is characterized in that The method comprises the siRNA for inhibiting Wdfy1 gene expression as claimed in claim 1 and the chimeric peptide RVG-9R.

3. The siRNA delivery system for targeted inhibition of Wdfy1 gene expression in brain neurons according to claim 2, It is characterized in that The amino acid sequence of the chimeric peptide RVG-9R is: YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGRRRRRRRRR.

4. The siRNA delivery system for targeted inhibition of Wdfy1 gene expression in brain neurons according to claim 2, It is characterized in that The molar ratio of the siRNA for inhibiting Wdfy1 gene expression to the chimeric peptide RVG-9R is 1:

1.

5. A method for preparing the siRNA delivery system for targeted inhibition of Wdfy1 gene expression in brain neurons according to any one of claims 2 to 4, It is characterized in that The method comprises the following steps: mixing the siRNA for inhibiting Wdfy1 gene expression and the chimeric peptide RVG-9R, and incubating them in a glucose solution to obtain the mixture.

6. The preparation method according to claim 5, It is characterized in that The concentration of the glucose solution is 5% w / v, the incubation temperature is 20-25° C., and the incubation time is 10-20 min.

7. Use of the siRNA for inhibiting Wdfy1 gene expression according to claim 1, the siRNA delivery system according to any one of claims 2 to 4, or the siRNA delivery system obtained by the preparation method according to any one of claims 5 to 6 in down-regulating WDFY1 protein expression in brain neuronal cells.

8. Use of a product that inhibits Wdfy1 gene expression in the preparation of drugs for alleviating bone-fat imbalance, preventing and treating osteoporosis, increasing bone mass and / or improving bone mechanical strength.

9. Use of the product for inhibiting Wdfy1 gene expression according to claim 8 in preparing a drug for preventing and treating osteoporosis, It is characterized in that The product for inhibiting Wdfy1 gene expression comprises the siRNA for inhibiting Wdfy1 gene expression according to claim 1, the siRNA delivery system according to any one of claims 2 to 4, or the siRNA delivery system obtained by the preparation method according to any one of claims 5 to 6.

10. Use of the product for inhibiting Wdfy1 gene expression according to claim 8 or 9 in preparing a drug for preventing and treating osteoporosis, It is characterized in that The bone-fat imbalance and osteoporosis are caused by the accumulation of WDFY1 in bone tissue via brain-derived EVs after aging.

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