Application of siRNA in preparation of TILRR expression inhibitor

Abstract

The invention relates to application of siRNA in preparation of a TILRR expression inhibitor. A target sequence interfered by the siRNA comprises a nucleic acid sequence as shown in SEQ ID NO. 1 and / or SEQ ID NO. 2, and the nucleic acid sequence of the siRNA comprises a sequence as shown in SEQ ID NO. 3 and / or SEQ ID NO. 4. According to the invention, the TILRR is taken as a treatment target, the expression of the TILRR is reduced by using siRNA, the response of cells to inflammation stimulation is reduced, an inflammation signal channel is not completely blocked, and side effects brought by blocking innate immune receptor treatment in different inflammation-related diseases are reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of siRNA in the preparation of a TILRR expression inhibitor. Background Art

[0002] The TIR (Toll-like receptor and Interleukin-1 receptor) family contains two groups of transmembrane proteins that share functional and structural characteristics, including the IL-1 receptor (IL-1R) subfamily and the Toll-like receptor (TLR) subfamily. The hallmark of the TIR family is the cytoplasmic TIR domain, which is essential for signal transduction. This domain can serve as a scaffold for a series of protein interactions, activating signaling modules composed of MyD88, interleukin-1 receptor-associated kinase (IRAK) family members and Tollip to activate signaling pathways such as NF-κB, leading to inflammatory responses. TLR4 is the main receptor for lipopolysaccharide (LPS) and some DAMP molecules. It interacts with LPS / DAMP molecules. IL-1R1 is a receptor for IL-1β. Activation of TLR4 and IL-1R1 receptors will induce cells to release pro-inflammatory factors such as TNF-α, IL-6, and IL-1β, which play a core role in inflammatory responses. The signal transduction mechanisms of TLR4 and IL-1R1 are important targets for studying inflammatory diseases, and they play key roles in infection, autoimmune diseases and chronic inflammation.

[0003] However, direct targeting of inflammatory factors or receptors has certain side effects. For example, IL-1R1, as a receptor for the inflammatory factor IL-1β, plays a "double-edged sword" role in injury repair. Knocking out IL-1R1 can reduce the pathological progression of atherosclerotic plaques, but it also enhances the instability of atherosclerotic plaques. In renal injury, knocking out IL-1R1 in proximal tubular cells can shorten the disease progression of renal injury induced by aristolochic acid (AA), while specifically knocking out IL-1R1 in endothelial cells shows an aggravation of the disease progression of AA-induced renal injury. Further analysis of its mechanism revealed that specific knockout of IL-1R1 in proximal tubules and endothelial cells did not affect the inflammatory response after AA-induced renal injury, and that cell damage and death caused by activation of IL-1R1 in the proximal tubules were related to lipid metabolism. Therefore, although directly using innate immune receptors as targets for the treatment of inflammatory-related diseases has certain effects, there is a risk of side effects.

[0004] Toll receptor / IL-1 receptor regulator (TILRR, Frem1 isoform 2) interacts with IL-1R1 and can increase the recruitment of MYD88 by the intracellular TIR domain of IL-1R1, thereby increasing the downstream NF-κB signaling flux and promoting the expression of related pro-inflammatory factors. Knocking out TILRR or blocking the interaction between TILRR and IL-1R1 can not only reduce the pathological process of atherosclerotic plaques, but also increase the stability of plaques. Further studies have found that overexpression of TILRR can increase the response of cells to LPS stimulation. Therefore, using TILRR as a therapeutic target can reduce the inflammatory response and reduce the risk of side effects caused by blocking innate immune receptors.

[0005] siRNA (small interfering RNA) is a double-stranded RNA molecule composed of 20-25 nucleotides. It mainly plays a role in the RNA interference (RNAi) pathway, inhibiting or shutting down the expression of specific genes by specifically degrading homologous mRNA. siRNA is incorporated into the RNA-induced silencing complex (RISC). In RISC, the siRNA double strands are unwound, the reverse strand is degraded, and the guide strand remains in RISC. The guide strand complementary to the target mRNA guides the RISC complex to the mRNA, and the nuclease (such as Argonaute protein) in the RISC complex degrades the target mRNA, preventing it from being translated into protein, thereby achieving gene silencing. Due to its high specificity and effectiveness, siRNA technology has very important application value in gene function research, disease treatment and new drug development. Targeting mRNA of specific genes through siRNA can be used to treat a variety of diseases including tumors, infectious diseases, genetic diseases, etc., so it has received widespread attention in the field of life sciences and is regarded as a direction with great development potential in the field of new drug development in the future.

[0006] In summary, how to reduce the response of cells to inflammatory stimuli without completely blocking the inflammatory signaling pathway is one of the urgent problems to be solved in this field. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides the use of siRNA in the preparation of a TILRR expression inhibitor, using siRNA to achieve TILRR gene silencing, thereby reducing the response of cells to inflammatory stimuli.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides the use of siRNA in the preparation of a TILRR expression inhibitor, wherein the target sequence interfered by the siRNA includes the nucleic acid sequence shown in SEQ ID NO.1 and / or SEQ ID NO.2.

[0010] SEQ ID NO. 1: GAAGUGGUCUCAUAUUGAAUG.

[0011] SEQ ID NO. 2: CCAGGAAUGUCAACUAAGAUG.

[0012] At present, the therapeutic effect of diseases mediated by innate immune inflammatory signals such as interleukin 1 and lipopolysaccharide is mainly through targeting inflammatory factors / innate immune receptors, but this treatment strategy will lead to complete blocking of innate immune-related signaling pathways. Although these treatment methods have certain effects, they often bring side effects. The present invention uses siRNA to reduce TILRR expression and reduce the response of cells to inflammatory stimuli without completely blocking the inflammatory signaling pathway, which can avoid the side effects caused by completely blocking the innate immune-related signaling pathway. The TILRR RNA target sequence provided by the present invention has an appropriate GC ratio, and the siRNA targeting the target sequence has a high efficiency in reducing TILRR gene expression.

[0013] Preferably, the nucleic acid sequence of the siRNA includes the sequence shown in SEQ ID NO.3 and / or SEQ ID NO.4.

[0014] SEQ ID NO. 3: CAUUCAAUAUGAGACCACUUCTT.

[0015] SEQ ID NO. 4: CAUCUUAGUUGACAUUCCUGGTT.

[0016] Preferably, the target sequence of the siRNA interference is the nucleic acid sequence shown in SEQ ID NO.1, and the nucleic acid sequence of the siRNA is shown in SEQ ID NO.3.

[0017] Preferably, the target sequence of the siRNA interference is the nucleic acid sequence shown in SEQ ID NO.2, and the nucleic acid sequence of the siRNA is shown in SEQ ID NO.4.

[0018] In a second aspect, the present invention provides the use of a TILRR expression inhibitor in the preparation of a preparation for inhibiting the expression of pro-inflammatory factors.

[0019] Preferably, the TILRR expression inhibitor comprises any one of a nucleic acid molecule, a nucleic acid construct, a lentivirus, an antibody or a small molecule compound, or a combination of at least two thereof.

[0020] Preferably, the nucleic acid molecule comprises any one of double-stranded RNA, siRNA or shRNA, or a combination of at least two of them.

[0021] Preferably, the nucleic acid sequence of the siRNA includes the sequence shown in SEQ ID NO.3 and / or SEQ ID NO.4.

[0022] Preferably, the source of the pro-inflammatory factors includes: obtaining them by stimulating cells or organs using exogenous or endogenous inflammatory factors.

[0023] Preferably, the inflammatory factors include IL-1β and / or lipopolysaccharide.

[0024] Preferably, the pro-inflammatory factors include any one of CXCL8, TNF or CCL2, or a combination of at least two of them.

[0025] The specific TILRR expression inhibitor designed in the present invention can be effectively used to inhibit the expression of pro-inflammatory factors, can be used as a drug for inhibiting the expression of pro-inflammatory factors, and can also be used for basic research for non-disease treatment purposes or for constructing experimental models (cells / animals / organoids / microphysiological models), etc.

[0026] In a third aspect, the present invention provides a drug for inhibiting the expression of pro-inflammatory factors, wherein the drug comprises siRNA, and the nucleic acid sequence of the siRNA comprises the sequence shown in SEQ ID NO.3 and / or SEQ ID NO.4.

[0027] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0028] Preferably, the auxiliary materials include any one or a combination of at least two of a carrier, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The siRNA interference target sequence and siRNA provided by the present invention can effectively reduce the expression level of TILRR mRNA in different types of cells and organoids, thereby partially reducing the expression of pro-inflammatory factors induced by cells / organoids in response to different inflammatory stimuli, and will not completely block the innate immune signaling pathway. The operation method is simple and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the result graph of TILRR expression level after siRNA transfection of HUVEC for 48 hours in Example 3.

[0032] Figure 2 This is the result diagram of gene expression after lipopolysaccharide stimulation of HUVEC in Example 4.

[0033] Figure 3 This is the result diagram of gene expression after ASC stimulation by lipopolysaccharide in Example 4.

[0034] Figure 4 This is a graph showing the gene expression results after IL-1β stimulated kidney organoids in Example 4. DETAILED DESCRIPTION

[0035] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0036] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0037] Example 1

[0038] This embodiment provides a siRNA for preparing a TILRR expression inhibitor. According to the siRNA design principle, a siRNA sequence with a GC content of 40-50% is selected, and the siRNA sequence is compared with the human genome sequence through the blast function of the GenBank database to ensure that there is no homology. Two siRNAs, siTILRR-1 and siTILRR-2, are determined and commissioned to Guangzhou Aiki Biotechnology Co., Ltd. to synthesize by chemical method. The nucleic acid sequence of siTILRR-1 is shown in SEQ ID NO.3, and the nucleic acid sequence of the target sequence it interferes with is shown in SEQ ID NO.1, corresponding to the 1809-1829 nucleotide sites in TILRR isoform2 RNA. The nucleic acid sequence of siTILRR-2 is shown in SEQ ID NO.4, and the nucleic acid sequence of the target sequence it interferes with is shown in SEQ ID NO.2, corresponding to the 1999-2020 nucleotide sites in TILRRisoform2 RNA.

[0039] SEQ ID NO. 1: GAAGUGGUCUCAUAUUGAAUG.

[0040] SEQ ID NO. 2: CCAGGAAUGUCAACUAAGAUG.

[0041] SEQ ID NO. 3: CAUUCAAUAUGAGACCACUUCTT.

[0042] SEQ ID NO. 4: CAUCUUAGUUGACAUUCCUGGTT.

[0043] Example 2

[0044] In this example, the siRNA provided in Example 1 was transfected into primary umbilical vein endothelial cells (HUVEC), primary human astrocytes (ASC), and kidney organoids induced by human iPSCs. The cells were plated in 12-well plates at 130,000 cells / well and cultured at 37°C, 5% CO2, and 90% relative humidity for 24 hours. Cell transfection was performed using a universal DNA / siRNA transfection reagent, and the transfection steps were performed according to the product instructions. Kidney organoids were cultured in 6-well plates at 200-300 cells / well, and transfection was performed using the lipid nanoparticle (LNP) method. Encapsulated siRNA-LNP was prepared by microfluidics, and the prepared siRNA-LNP was added to the organoid culture medium at 2000ng / mL. After 48h of culture, RNA was extracted for subsequent experiments.

[0045] Example 3

[0046] In this embodiment, total RNA of cells or organoids after transfection for 48 hours was extracted, and the content of TILRR cDNA was detected by qPCR after reverse transcription. Specifically as follows: Extraction of total RNA: Total RNA of cells or organoids after siRNA transfection was extracted using the Trizol method; Reverse transcription: Reverse transcription was performed using HiScript III Reverse Transcriptase (Nuoweizan); qPCR detection: qPCR detection was performed using SsoAdvanced Universal SYBR Green Supermix (2X) (BioRad), the reaction system was 10 μL, each reaction system contained 2 μL cDNA template, the reaction program was a. 98°C 3min, b. 98°C 10s, c. 60°C 60s, bc cycle 40 times, GAPDH was used as the internal reference (NC), and the nucleic acid sequences of the amplification primers of TILRR cDNA were shown in SEQ ID NO.5 to SEQ ID NO.6 and SEQ ID NO.7 to SEQ ID NO.8, respectively.

[0047] SEQ ID NO. 5: GGACGAGATCCCTCCAAAAT.

[0048] SEQ ID NO. 6: GGCTGTTGTCATACTTCTCATGG.

[0049] SEQ ID NO. 7: AGAGCCCTGCCTGTGGTAAC.

[0050] SEQ ID NO. 8: GAAGGGGAATGCAAGAGTGTGATA.

[0051] qPCR results are as follows Figure 1 As shown, TILRR siRNA can significantly reduce the expression of TILRR in HUVEC, and the expression of TILRR in renal organoids induced by ASC and human iPSCs is also significantly inhibited.

[0052] Example 4

[0053] In this example, cells or organoids 48 hours after transfection were stimulated with 100 ng / mL IL-1β or 1 μg / mL lipopolysaccharide (LPS) for different time periods, and then total RNA was extracted. After reverse transcription, qPCR was used to detect the expression levels of mRNAs of different inflammatory factors in cells or organoids. The qPCR detection method and system refer to Example 3.

[0054] Figure 2 The expression of TILRR, pro-inflammatory factor CXCL8, pro-inflammatory factor TNF and EndoMT-related marker gene CDH2 after HUVEC was stimulated with 1μg / mL LPS for 1h or 24h. The expression levels of the above genes were significantly reduced, indicating that the expression of pro-inflammatory factors in HUVEC cells was inhibited after the expression of TILRR was reduced. The previous experiment found that the mRNA expression level of TNF increased rapidly 1h after endothelial cells responded to LPS stimulation, while the mRNA expression level of TNF had decreased several times after 3h of stimulation. The mRNA expression level of TNF 24h after stimulation was equivalent to the level before stimulation. Therefore, this experiment mainly detected the mRNA expression level of TNF after short-term inflammatory stimulation. The purpose of this experiment is to verify the effect of reducing TILRR on the induced expression of genes after cells respond to inflammatory stimulation. The previous experiment found that 6h after endothelial cells responded to LPS stimulation did not affect the expression of EndoMT marker gene CDH2, but LPS stimulation could increase the expression of endothelial cell CDH2 after 24h, so the results only showed the expression of CDH2 after 24h of LPS treatment.

[0055] Figure 3 After ASC was transfected with siTILRR-1, the expression of pro-inflammatory factors CXCL8 and CCL2 was measured after ASC was stimulated with 1 μg / mL LPS for 1 h or 3 h. The expression levels of the above genes were significantly reduced, indicating that the reduction in TILRR expression inhibited the expression of pro-inflammatory factors in ASC cells.

[0056] Figure 4 The expression of TILRR and proinflammatory factor CXCL8 in kidney organoids after stimulation with 100 ng / mL IL-1β for 48 h. The expression levels of the above genes were significantly reduced, indicating that the reduced expression of TILRR inhibited the expression of proinflammatory factors in kidney organoids induced by human iPSCs.

[0057] In summary, the siRNA interference target sequence and siRNA provided by the present invention can effectively reduce the expression level of TILRR mRNA in different types of cells and organoids, thereby reducing the expression of pro-inflammatory factors induced by cells / organoids in response to different inflammatory stimuli, and will not completely block the innate immune signaling pathway. The operation method is simple and has broad application prospects.

[0058] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. The use of siRNA in the preparation of a TILRR expression inhibitor, characterized in that: The target sequence of the siRNA interference includes the nucleic acid sequence shown in SEQ ID NO.1 and / or SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The nucleic acid sequence of the siRNA includes the sequence shown in SEQ ID NO.3 and / or SEQ ID NO.

4.

3. The use according to claim 1 or 2, characterized in that: The target sequence of the siRNA interference is the nucleic acid sequence shown in SEQ ID NO.1, and the nucleic acid sequence of the siRNA is shown in SEQ ID NO.

3.

4. The use according to any one of claims 1 to 3, characterized in that: The target sequence of the siRNA interference is the nucleic acid sequence shown in SEQ ID NO.2, and the nucleic acid sequence of the siRNA is shown in SEQ ID NO.

4.

5. Use of TILRR expression inhibitors in the preparation of preparations for inhibiting the expression of pro-inflammatory factors.

6. The use according to claim 5, characterized in that: The TILRR expression inhibitor includes any one or a combination of at least two of a nucleic acid molecule, a nucleic acid construct, a lentivirus, an antibody or a small molecule compound; Preferably, the nucleic acid molecule comprises any one or a combination of at least two of double-stranded RNA, siRNA or shRNA; Preferably, the nucleic acid sequence of the siRNA includes the sequence shown in SEQ ID NO.3 and / or SEQ ID NO.

4.

7. The use according to claim 5 or 6, characterized in that: The sources of the pro-inflammatory factors include: obtaining by stimulating cells or organs using exogenous or endogenous inflammatory factors; Preferably, the inflammatory factors include IL-1β and / or lipopolysaccharide.

8. The use according to any one of claims 5 to 7, characterized in that: The pro-inflammatory factors include any one of CXCL8, TNF or CCL2, or a combination of at least two of them.

9. A drug for inhibiting the expression of pro-inflammatory factors, characterized in that: The drug includes siRNA, and the nucleic acid sequence of the siRNA includes the sequence shown in SEQ ID NO.3 and / or SEQ ID NO.

4.

10. The drug according to claim 9, characterized in that The drug also includes pharmaceutically acceptable excipients; Preferably, the auxiliary materials include any one or a combination of at least two of a carrier, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

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