SiRNA for inhibiting interleukin-1alpha and application thereof

Abstract

The invention relates to siRNA for inhibiting interleukin-1 alpha and application of the siRNA, and belongs to the technical field of biology. The invention provides siRNA (small interfering Ribonucleic Acid) for inhibiting interleukin-1alpha. A positive-sense strand of the siRNA comprises a nucleic acid molecule of which the nucleotide sequence is shown as any one of SEQ ID NO.1-17; the antisense strand of the siRNA comprises a nucleic acid molecule of which the nucleotide sequence is shown as any one of SEQ ID NO.18 to 34. Experiments prove that all the siRNAs have high inhibitory activity on interleukin-1alpha. The invention also provides a modified siRNA used for inhibiting interleukin-1 alpha, wherein the modification comprises methoxy modification, fluoro modification and / or phosphorothioate group connection. Experiments prove that the modified siRNA has high inhibitory activity on interleukin-1 alpha at the concentration of 0.1-10 nM.

Description

[0001] This application is a divisional application of "an siRNA for inhibiting interleukin-1α and its application, with the application date of October 25, 2024, application number: 202411499440.6, and invention title". Technical Field

[0002] The present invention relates to an siRNA for inhibiting interleukin-1α and its application, belonging to the field of biotechnology. Background Art

[0003] Interleukin-1α (IL-1α) is encoded by the IL1A gene transcription. It is a cytokine of the chemokine family and has various biological functions. Interleukin-1α is a multifunctional signaling molecule widely present in the body. Inside the cell, as a transcription factor, it can regulate cell growth and differentiation, while outside the cell, it participates in the body's inflammatory response. Both the precursor and mature forms of interleukin-1α have biological activities, but the mature form of interleukin-1α has stronger biological activity.

[0004] Research shows that interleukin-1α can drive autoinflammatory diseases such as rheumatoid arthritis and systemic lupus erythematosus. The most consistent clinical feature of such interleukin-1α-mediated autoinflammatory diseases is episodic or chronic systemic or tissue inflammation, and the common affected areas include the skin and musculoskeletal system. Therefore, interleukin-1α can be used as a therapeutic target for autoinflammatory diseases, and interleukin-1α inhibitors can effectively treat a variety of interleukin-1α-mediated autoinflammatory diseases including rheumatoid arthritis and systemic lupus erythematosus.

[0005] In addition to driving the occurrence of autoinflammatory diseases, interleukin-1α is also involved in the occurrence and development of tumors. For example, interleukin-1α has long been proven to be related to inflammation-induced carcinogenesis, and interleukin-1α has been shown to promote the growth and metastasis of tumors such as melanoma, myeloma, ovarian cancer, and breast cancer. Further research found that the pro-tumor mechanism of interleukin-1α is very complex, including the recruitment of myeloid cells and immunosuppression, promoting angiogenesis and the activation of endothelial cells, and skewing lymphoid cell differentiation. At present, many new studies have proven that blocking with interleukin-1α inhibitors in the early stage of tumor development can effectively slow down tumor growth. Therefore, interleukin-1α inhibitors can be used for the treatment of tumors.

[0006] RNA interference (RNAi) refers to a phenomenon that is highly conserved during evolution, induced by double-stranded RNA (dsRNA), and results in the efficient and specific degradation of homologous mRNA. Small interfering RNA (siRNA) is a double-stranded RNA that is 20-25 nucleotides in length and can mediate the silencing of specific genes through the RNA interference (RNAi) mechanism. In the RNAi pathway, siRNA interferes with gene expression by hybridizing with complementary mRNA molecules, which triggers mRNA degradation and thus inhibits the gene expression of specific genes. This specific way of regulating gene expression enables siRNA to be used as a targeted therapeutic drug to specifically regulate the expression of disease-related genes and thereby achieve the purpose of treating diseases. If an siRNA that can effectively inhibit the expression of interleukin-1α can be developed, it will be a more effective and targeted therapeutic drug for inflammatory diseases and cancer. Summary of the Invention

[0007] To solve the above problems, the present invention provides an siRNA for inhibiting interleukin-1α, wherein the siRNA comprises a sense strand and an antisense strand; at least a part of the sense strand and the antisense strand are reversely complementary to form a double-stranded region; the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown in any one of SEQ ID NOs. 1-17; the antisense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown in any one of SEQ ID NOs. 18-34.

[0008] In one embodiment of the present invention, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown in SEQ ID NO. 1, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown in SEQ ID NO. 18;

[0009] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown in SEQ ID NO. 2, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown in SEQ ID NO. 19;

[0010] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown in SEQ ID NO. 3, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown in SEQ ID NO. 20;

[0011] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown in SEQ ID NO. 4, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown in SEQ ID NO. 21;

[0012] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.5, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.22;

[0013] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.6, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.23;

[0014] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.7, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.24;

[0015] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.8, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.25;

[0016] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.9, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.26;

[0017] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.10, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.27;

[0018] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.11, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.28;

[0019] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.12, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.29;

[0020] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.13, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.30;

[0021] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.14, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.31;

[0022] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO. 15, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO. 32;

[0023] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO. 16, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO. 33;

[0024] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO. 17, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO. 34.

[0025] In one embodiment of the present invention, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO. 1, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO. 18;

[0026] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO. 2, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO. 19;

[0027] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO. 3, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO. 20;

[0028] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO. 4, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO. 21;

[0029] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO. 5, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO. 22;

[0030] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO. 6, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO. 23;

[0031] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO. 7, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO. 24;

[0032] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO. 8, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO. 25;

[0033] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.9, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.26;

[0034] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.10, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.27;

[0035] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.11, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.28;

[0036] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.12, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.29;

[0037] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.13, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.30;

[0038] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.14, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.31;

[0039] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.15, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.32;

[0040] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.16, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.33;

[0041] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.17, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.34.

[0042] In one embodiment of the present invention, at least one nucleotide in the sense strand or the antisense strand of the siRNA is a modified nucleotide.

[0043] In one embodiment of the present invention, all nucleotides in the sense strand and / or the antisense strand of the siRNA are modified nucleotides, and these modifications on the nucleotide groups do not significantly weaken or lose the function of the disclosed siRNA in inhibiting interleukin-1α gene expression.

[0044] In one embodiment of the present invention, the modification includes methoxy modification, fluoro modification and / or phosphorothioate linkage.

[0045] In one embodiment of the present invention, the "phosphorothioate linkage" means that at least a part of the phosphate groups in the phosphate-sugar backbone of at least one single strand among the sense strand and the antisense strand of the siRNA is a phosphate group with a modifying group.

[0046] In one embodiment of the present invention, the phosphate group with a modifying group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom.

[0047] In one embodiment of the present invention, the fluorine-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5'-end to the 3'-end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluorine-modified nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorine-modified nucleotides;

[0048] The methoxy-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5'-end to the 3'-end, at least the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, and 19th nucleotides of the sense strand are methoxy-modified nucleotides, and at least the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, and 21st nucleotides of the antisense strand are methoxy-modified nucleotides;

[0049] The nucleotides with phosphorothioate linkage are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5'-end to the 3'-end, the nucleotides between at least the 1st and 2nd, and the 2nd and 3rd nucleotides of the sense strand are linked by a phosphorothioate group, and the nucleotides between at least the 1st and 2nd, the 2nd and 3rd, the 19th and 20th, and the 20th and 21st nucleotides of the antisense strand are linked by a phosphorothioate group.

[0050] The present invention also provides a recombinant plasmid, and the recombinant plasmid expresses the above siRNA.

[0051] In one embodiment of the present invention, the vector of the recombinant plasmid includes at least one of a viral vector or a non-viral vector; the viral vector includes at least one of a flavivirus vector, a retroviral vector, a phage vector, an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, a hybrid virus vector, a baculovirus vector, a herpes simplex virus vector, or a lentiviral vector; the non-viral vector includes a plasmid vector.

[0052] In one embodiment of the present invention, the plasmid vector includes a pUC plasmid, a derivative plasmid of the pUC plasmid, a pAAV plasmid, a derivative plasmid of the pAAV plasmid, a PGEM plasmid, and / or a derivative plasmid of the PGEM plasmid.

[0053] In one embodiment of the present invention, the method for preparing the recombinant plasmid is as follows: design shRNA according to siRNA; ligate the shRNA with the linearized vector to obtain the recombinant plasmid.

[0054] The present invention also provides a host cell, wherein the genome of the host cell integrates the above siRNA; or, the host cell carries the above recombinant plasmid.

[0055] In one embodiment of the present invention, the host cell includes fungi, bacteria, plant cells, and / or animal cells.

[0056] The present invention also provides the use of the above siRNA, the above recombinant plasmid, or the above host cell in the preparation of a drug for preventing and / or treating a disease, wherein the disease is a disease related to the expression of interleukin-1α.

[0057] In one embodiment of the present invention, the diseases related to the expression of interleukin-1α include cancer and / or inflammatory diseases.

[0058] In one embodiment of the present invention, the cancer includes melanoma, myeloma, ovarian cancer, and / or breast cancer.

[0059] In one embodiment of the present invention, the inflammatory disease includes an autoinflammatory disease; the autoinflammatory disease includes rheumatoid arthritis and / or systemic lupus erythematosus.

[0060] The present invention also provides an interleukin-1α inhibitor, and the components of the inhibitor include the above siRNA, the above recombinant plasmid, and / or the above host cell.

[0061] In one embodiment of the present invention, the components of the inhibitor further include pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include carriers, diluents, binders, and / or lubricants.

[0062] The present invention also provides a drug for preventing and / or treating a disease, wherein the disease is a disease related to the expression of interleukin-1α; the components of the drug include the above siRNA, the above recombinant plasmid, and / or the above host cell.

[0063] In one embodiment of the present invention, the diseases related to the expression of interleukin-1α include cancer and / or inflammatory diseases.

[0064] In one embodiment of the present invention, the cancer includes melanoma, myeloma, ovarian cancer, and / or breast cancer.

[0065] In one embodiment of the present invention, the inflammatory disease includes autoinflammatory disease; the autoinflammatory disease includes rheumatoid arthritis and / or systemic lupus erythematosus.

[0066] In one embodiment of the present invention, the composition of the drug further comprises a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient includes a carrier, a diluent, a binder, and / or a lubricant.

[0067] The technical solution of the present invention has the following advantages:

[0068] The present invention provides an siRNA for inhibiting interleukin-1α. The sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown in any one of SEQ ID NO.1-17; the antisense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown in any one of SEQ ID NO.18-34. Experiments have shown that the siRNAs all have high inhibitory activity against interleukin-1α. Therefore, the siRNAs have great application prospects in the preparation of drugs for preventing and / or treating diseases related to the expression of interleukin-1α (such as cancer and inflammatory diseases, etc.).

[0069] Furthermore, the 7th, 8th, and 9th nucleotides of the sense strand of the siRNA are fluorinated nucleotides, the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, and 19th nucleotides are methoxylated nucleotides, and the nucleotides between the 1st and 2nd, and the 2nd and 3rd are linked by phosphorothioate groups; the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand of the siRNA are fluorinated nucleotides, the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, and 21st nucleotides are methoxylated nucleotides, and the nucleotides between the 1st and 2nd, the 2nd and 3rd, the 19th and 20th, and the 20th and 21st are linked by phosphorothioate groups. Experiments have shown that the modified siRNAs all have high inhibitory activity against interleukin-1α at a concentration of 0.1-10 nM. Therefore, the modified siRNAs have great application prospects in the preparation of drugs for preventing and / or treating diseases related to the expression of interleukin-1α (such as cancer and inflammatory diseases, etc.). Detailed implementation mode

[0070] The following embodiments are provided to better understand the present invention further. It is not limited to the best mode described, and does not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other prior arts falls within the protection scope of the present invention.

[0071] For those not specifying specific experimental steps or conditions in the following embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0072] In the following embodiments, the capital letters C, G, U, and A represent ribonucleotides; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a nucleotide modified with a methoxy group; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a nucleotide modified with a fluorine group; the lowercase letter s indicates that there is a phosphorothioate group modification between the two nucleotides adjacent to the left and right of the letter s.

[0073] Example 1: An siRNA for inhibiting interleukin-1α

[0074] This example provides an siRNA for inhibiting interleukin-1α, and the nucleotide sequence of the siRNA is designed based on the target mRNA, as shown in Table 1.

[0075] Table 1. siRNAs for inhibiting interleukin-1α and their sequences

[0076]

[0077] Example 2: An siRNA for inhibiting interleukin-1α

[0078] This embodiment provides an siRNA for inhibiting interleukin-1α. On the basis of Example 1, the nucleotides at positions 7, 8, and 9 of the sense strand of the siRNA are replaced with fluorine-modified nucleotides, and the nucleotides at positions 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are replaced with methoxy-modified nucleotides. The nucleotides between position 1 and position 2 and between position 2 and position 3 are linked by phosphorothioate groups. The nucleotides at positions 2, 6, 14, and 16 of the antisense strand of the siRNA are replaced with fluorine-modified nucleotides, and the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are replaced with methoxy-modified nucleotides. The nucleotides between position 1 and position 2, between position 2 and position 3, between position 19 and position 20, and between position 20 and position 21 are linked by phosphorothioate groups, as shown in Table 2.

[0079] Table 2. siRNA for inhibiting interleukin-1α and its sequences

[0080]

[0081] Experimental Example 1: Detection of the inhibitory activity of siRNA for inhibiting interleukin-1α

[0082] This experimental example provides an experiment for detecting the inhibitory activity of siRNA for inhibiting interleukin-1α. The experimental procedure is as follows:

[0083] Hey cells (human ovarian cancer cells, purchased from Wuhan Punosai Life Science Co., Ltd., product number: CL-0671) were inoculated into DMEM medium (purchased from Transgen Biotech, product number FI101-01) containing 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin mixture (Penicillin-Streptomycin, purchased from Gibco, product number 15140122), and cultured in a 5% (v / v) CO2, 37 °C cell culture incubator for 48 h; after the culture was completed, the Hey cells obtained by culture were digested with trypsin (purchased from GIBCO, product number 25200-072); after the digestion was completed, they were first rinsed with PBS buffer and then resuspended with DMEM medium to obtain a cell suspension with a cell concentration of 3×10 5 cells / mL;

[0084] Dilute different siRNAs separately using opti-MEM (purchased from Gibco, catalog number 31985-070) to obtain siRNA dilutions containing different siRNAs; mix 25 μL of opti-MEM with 0.25 μL of Lipofectamine RNAiMAX transfection reagent (purchased from Thermo Fisher, catalog number 11668-019) to obtain a transfection reagent dilution; after taking 25 μL of siRNA dilutions containing different siRNAs and mixing them with the transfection reagent dilution respectively, let them stand at room temperature (25 °C) for 15 min to obtain transfection solutions containing different siRNAs;

[0085] After inoculating the cell suspension into a 96-well plate at an inoculation volume of 50 μL / well, set up a transfection reagent control group (MOCK), IL1A-1M1 experimental group, IL1A-2M1 experimental group, IL1A-3M experimental group, IL1A-4M1 experimental group, IL1A-5M1 experimental group, IL1A-6M1 experimental group, IL1A-7M1 experimental group, IL1A-8M1 experimental group, IL1A-9M1 experimental group, IL1A-10M1 experimental group, IL1A-11M1 experimental group, IL1A-12M1 experimental group, IL1A-13M1 experimental group, IL1A-14M1 experimental group, IL1A-15M1 experimental group, IL1A-16M1 experimental group, and IL1A-17M1 experimental group in the 96-well plate, with 4 replicates in each group;

[0086] After setting up, add 50 μL of transfection solutions containing different siRNAs to the wells of the experimental groups (the IL1A-1M1 experimental group is given the transfection solution containing IL1A-1M1, the IL1A-2M1 experimental group is given the transfection solution containing IL1A-2M1, and so on. Among them, the final concentration of siRNA in the wells is 10 nM), add 50 μL of transfection solution without any siRNA to the wells of the transfection reagent control group (MOCK), and culture for 48 h in a 5% (v / v) CO2, 37 °C cell culture incubator for transfection; after the transfection is completed, discard the liquid in the wells, collect the cells, and extract the total RNA in the cells using a magnetic bead method cell total RNA extraction kit (Gemagene - E31008-96) to obtain an RNA extraction solution;

[0087] Configure the genomic DNA removal reaction system according to Table 3 to remove genomic DNA in the RNA extraction solution, and obtain the processed RNA extraction solution (the reaction program for removing genomic DNA is: 42 °C, 2 min); configure the cDNA synthesis reaction system according to Table 4, and reverse-transcribe the total RNA in the processed RNA extraction solution to obtain a cDNA solution (the conditions for reverse transcription are: 50 °C, 15 min; 85 °C, 2 min); after diluting the cDNA solution 5-fold with enzyme-free water, use the diluted cDNA solution as a template, use the gene encoding glyceraldehyde-3-phosphate dehydrogenase (HGAPDH gene) as an internal reference gene, configure the RT-qPCR probe method reaction system according to Table 5 (the primers used in the reaction system are shown in Table 6), and use the configured RT-qPCR probe method reaction system at Perform fluorescence quantitative PCR reaction (the amplification program for the fluorescence quantitative PCR reaction is: pre-denaturation at 95 °C for 10 min, denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s, repeat the above denaturation, annealing, and extension processes 40 times in total to obtain product W containing the amplified target gene IL1A and internal reference gene GAPDH; product W is then incubated successively at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s, and the real-time fluorescence quantitative PCR instrument collects the melting curves of the target gene IL1A and internal reference gene GAPDH in product W), and obtain the Ct values of the target gene (IL1AmRNA) and internal reference gene (GAPDH) in the test group; use the comparative Ct (ΔΔCt) method to perform relative quantitative calculation on the target gene (IL1AmRNA) in each test group, and the calculation results are shown in Table 7;

[0088] Among them, the calculation method for relative quantification is as follows:

[0089] ΔCt (test group) = Ct (test group target gene) – Ct (test group internal reference gene);

[0090] ΔCt (control group) = Ct (control group target gene) – Ct (control group internal reference gene);

[0091] ΔΔCt (test group) = ΔCt (test group) - ΔCt (average of control group);

[0092] ΔΔCt (control group) = ΔCt (control group) - ΔCt (average of control group);

[0093] When calculating, use the transfection reagent control group as a reference to normalize the expression level of IL1AmRNA in the test group, and define the expression level of IL1AmRNA in the transfection reagent control group as 100%;

[0094] Relative expression level of IL1AmRNA in the test group = 2-ΔΔCt(测试组) × 100%;

[0095] For the siRNA of the same test group, the average relative expression level of the test group IL1A mRNA at each concentration is the arithmetic mean of the relative expression levels of 3 culture wells at this concentration;

[0096] The inhibition rate of siRNA on the expression level of IL1A mRNA is calculated according to the following equation: Inhibition rate = (1 - relative expression level of test group IL1A mRNA) × 100%.

[0097] As can be seen from Table 7, compared with the transfection reagent control group (MOCK), except for IL1A - 9M1, IL1A - 1M1 to IL1A - 8M1 and ILlA - 10M1 to IL1A - 17M1 all have inhibitory activity on interleukin - 1α. Among them, IL1A - 1M1, IL1A - 2M1, IL1A - 3M1, IL1A - 4M1, IL1A - 5M1, IL1A - 6M1, IL1A - 7M1, IL1A - 10M1, IL1A - 11M1, IL1A - 12M1, IL1A - 16M1 and IL1A - 17M1 etc. have higher inhibitory activity on interleukin - 1α.

[0098] Table 3. Genome removal reaction system

[0099] Reagent Name Volume per Well (μL) 4× gDNA Removal Premix / 4× gDNA wiper Mix 4 RNA Extraction Solution 12

[0100] Table 4 cDNA synthesis reaction system

[0101] Reagent Name Volume per Well (μL) 5× Reverse Transcription Premix / 5× RT Mix 4 RNA Extraction Solution 16

[0102] Table 5. RT - qPCR probe method reaction system

[0103] Reagent Name Volume per Well (μL) 2× Fluorescent Quantitative PCR Premix by Dye Method / 2× SYBR Mix 6.5 Forward Primer (10 μM) 0.26 Reverse Primer (10 μM) 0.26 <![CDATA[Enzyme-free water / RNase-free ddH2O]]> 0.78 Template 5.2 Total Volume 13

[0104] Table 6. Primer information

[0105]

[0106] Table 7. Inhibition rates of different siRNAs (IL1A - 1M1 to IL1A - 21M1) on the expression level of IL1A mRNA

[0107] Group Relative Expression Level of IL1A mRNA Transfection Reagent Control Group 1.00 IL1A-1M1 Experimental Group 0.20 IL1A-2M1 Experimental Group 0.31 IL1A-3M1 Experimental Group 0.30 IL1A-4M1 Experimental Group 0.25 IL1A-5M1 Experimental Group 0.35 IL1A-6M1 Experimental Group 0.47 IL1A-7M1 Experimental Group 0.28 IL1A-8M1 Experimental Group 0.63 IL1A-9M1 Experimental Group 1.16 IL1A-10M1 Experimental Group 0.45 IL1A-11M1 Experimental Group 0.48 IL1A-12M1 Experimental Group 0.19 IL1A-13M1 Experimental Group 0.74 IL1A-14M1 Experimental Group 0.73 IL1A-15M1 Experimental Group 0.80 IL1A-16M1 Experimental Group 0.24 IL1A-17M1 Experimental Group 0.23

[0108] Experimental Example 2: Detection of the inhibitory activity of siRNA for inhibiting interleukin - 1α at different concentrations

[0109] This experimental example provides an experiment for detecting the inhibitory activity of siRNA for inhibiting interleukin - 1α at different concentrations. The experimental process is as follows:

[0110] On the basis of Experimental Example 1, a blank control group (BLANK) and a negative control group (NC) were added, and IL1A-1M1, IL1A-2M1, IL1A-3M1, IL1A-4M1, IL1A-5M1, IL1A-7M1, IL1A-12M1, IL1A-16M1 and IL1A-17M1 with relatively high inhibitory activities were selected as the research objects. During the experiment, by adjusting the concentration of siRNA diluted with opti-MEM, the concentration of siRNA in the wells was replaced from 10 nM to 0.1 nM and 1 nM respectively. Among them, in the blank control group (BLANK), no reagent was added to the wells as a blank control, and in the negative control group (NC), 50 μL of transfection solution containing NC (the final concentration of NC in the wells was 10 nM) was added as a negative control. The experimental results are shown in Table 8.

[0111] As can be seen from Table 8, compared with the blank control group (BLANK) and the transfection reagent control group (MOCK), IL1A-1M1, IL1A-2M1, IL1A-3M1, IL1A-4M1, IL1A-5M1, IL1A-7M1, IL1A-12M1, IL1A-16M1 and IL1A-17M1 all had high inhibitory activities against interleukin-1α at lower concentrations of 0.1 - 1 nM.

[0112] Table 8. Different siRNAs

[0113] (IL1A-1M1 / IL1A-2M1 / IL1A-3M1 / IL1A-4M1 / IL1A-5M1 / IL1A-7M1 / IL1A-12M1 / IL1A-16M1 / IL1A-17M1) Inhibition rates of IL1A mRNA expression levels at different concentrations (0.1 nM, 1 nM and 10 nM)

[0114]

[0115] Obviously, the above-mentioned embodiments are only examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or variations can be made on the basis of the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. An siRNA for inhibiting interleukin-1α, characterized in that, The siRNA contains a sense strand and an antisense strand; at least part of the sense strand and the antisense strand are reversely complementary to form a double-stranded region; the sense strand of the siRNA contains a nucleic acid molecule having a nucleotide sequence shown in any one of SEQ ID NOs. 2 to 17; the antisense strand of the siRNA contains a nucleic acid molecule having a nucleotide sequence shown in any one of SEQ ID NOs. 19 to 34.

2. The siRNA according to claim 1, wherein The sense strand of the siRNA contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 2, and the antisense strand contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 19; Alternatively, the sense strand of the siRNA contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 3, and the antisense strand contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 20; Alternatively, the sense strand of the siRNA contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 4, and the antisense strand contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 21; Alternatively, the sense strand of the siRNA contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 5, and the antisense strand contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 22; Alternatively, the sense strand of the siRNA contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 6, and the antisense strand contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 23; Alternatively, the sense strand of the siRNA contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 7, and the antisense strand contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 24; Alternatively, the sense strand of the siRNA contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 8, and the antisense strand contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 25; Alternatively, the sense strand of the siRNA contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 9, and the antisense strand contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 26; Alternatively, the sense strand of the siRNA contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 10, and the antisense strand contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 27; Alternatively, the sense strand of the siRNA contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 11, and the antisense strand contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 28; Alternatively, the sense strand of the siRNA contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 12, and the antisense strand contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 29; Alternatively, the sense strand of the siRNA contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 13, and the antisense strand contains a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 30; Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.14, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.31; Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.15, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.32; Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.16, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.33; Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.17, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.

34.

3. The siRNA according to claim 1 or 2, characterized in that, At least one nucleotide in the sense strand or the antisense strand of the siRNA is a modified nucleotide.

4. The siRNA according to claim 3, wherein The modification includes methoxy modification, fluoro modification and / or phosphorothioate linkage.

5. The siRNA according to claim 4, wherein The fluoro-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5'-end to the 3'-end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluoro-modified nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluoro-modified nucleotides; The methoxy-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5'-end to the 3'-end, at least the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, and 19th nucleotides of the sense strand are methoxy-modified nucleotides, and at least the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, and 21st nucleotides of the antisense strand are methoxy-modified nucleotides; The nucleotides with phosphorothioate linkages are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5'-end to the 3'-end, the nucleotides between at least the 1st and 2nd, and the 2nd and 3rd positions of the sense strand are linked by phosphorothioate linkages, and the nucleotides between at least the 1st and 2nd, the 2nd and 3rd, the 19th and 20th, and the 20th and 21st positions of the antisense strand are linked by phosphorothioate linkages.

6. A recombinant plasmid, characterized in that, The recombinant plasmid expresses the siRNA according to any one of claims 1 to 5.

7. A host cell, characterized in that, The genome of the host cell integrates the siRNA according to any one of claims 1 to 5; or, the host cell carries the recombinant plasmid according to claim 6.

8. Use of the siRNA according to any one of claims 1 to 5, the recombinant plasmid according to claim 6, or the host cell according to claim 7 in the preparation of a medicament for preventing and / or treating a disease, characterized in that The disease is a disease related to the expression of interleukin-1α.

9. An interleukin-1α inhibitor, characterized in that, The composition of the inhibitor comprises the siRNA according to any one of claims 1 to 5, the recombinant plasmid according to claim 6, and / or the host cell according to claim 7.

10. A drug for preventing and / or treating diseases, characterized in that, The disease is a disease related to the expression of interleukin-1α; the composition of the drug comprises the siRNA according to any one of claims 1 to 5, the recombinant plasmid according to claim 6, and / or the host cell according to claim 7.

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