SiRNA specifically inhibiting expression of LILRB4 gene and application thereof

Abstract

This invention relates to the field of biomedical technology, and discloses a siRNA that specifically inhibits LILRB4 gene expression and its applications. The siRNA contains a completely inversely complementary sense strand and an antisense strand, the nucleotide sequences of which are any pair as shown in SEQ ID NO. 1 and 2, SEQ ID NO. 3 and 4, SEQ ID NO. 5 and 6, SEQ ID NO. 7 and 8, SEQ ID NO. 9 and 10, SEQ ID NO. 11 and 12, SEQ ID NO. 13 and 14, SEQ ID NO. 15 and 16, SEQ ID NO. 17 and 18, or SEQ ID NO. 19 and 20. This invention also provides the application of this siRNA in the preparation of pharmaceuticals. The siRNA pharmaceutical composition provided by this invention can effectively silence LILRB4 gene expression, inhibit the infiltration of tumor cells into normal tissues and organs, and enhance T cell activity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an siRNA that specifically inhibits the expression of the LILRB4 gene and its applications. Background Technology

[0002] Immunotherapy is one of the most promising directions in the field of cancer treatment; however, its response rate in clinical practice is low, only 10-30%. Suppression of T cell activity in tumor tissue is a significant reason for clinical failure of immunotherapy. Studies have found that leukocyte immunoglobulin-like receptor B4 (LILRB4), a member of leukocyte Ig-like receptors (LILRs), plays a crucial role in the function of the immune system under physiological conditions through expression on various immune cells, including T cells and plasma cells. Under pathological conditions, LILRB4 influences the progression of various diseases through multiple signaling pathways, such as the transformation and invasion of tumors and leukemia.

[0003] Tumor cells that highly express LILRB4 support tumor cell infiltration into normal tissues via the ApoE / LILRB4 / SHP-2 / uPAR / Arginase-1 signaling axis, while simultaneously suppressing T cell activity. Blocking LILRB4 signaling can limit tumor development and activate T cell activity. Therefore, LILRB4 is an important target for tumor therapy.

[0004] siRNA drugs are among the most strategically promising biopharmaceutical technologies, and their approval rate has accelerated in recent years. Currently, four siRNA drugs have received FDA approval: Onpattro, Givlaari, Oxlumo, and Leqvio from Alnylam, and more are expected to enter the market in the coming years. siRNA drugs can block the expression of pathogenic proteins at the mRNA level, offering advantages such as high efficiency, good specificity, and long-lasting effects.

[0005] However, there are currently no studies on using RNA interference (RNAi) technology to inhibit LILRB4 expression, prevent tumor cells from infiltrating tissues, and enhance T cell activity. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems existing in the prior art and provide a specific siRNA that inhibits the expression of the LILRB4 gene and its application. This siRNA has high sequence stability, can effectively silence the expression of the human LILRB4 gene, block the ApoE / LILRB4 / SHP-2 / uPAR / Arginase-1 signaling pathway, prevent tumor cell infiltration and enhance T cell activity.

[0007] To achieve the above objective, a first aspect of the present invention provides an siRNA that specifically inhibits the expression of the LILRB4 gene, the siRNA containing a completely inversely complementary sense strand and an antisense strand, the nucleotide sequences of the sense strand and the antisense strand being any pair as shown in SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18 or SEQ ID NO.19 and SEQ ID NO.20.

[0008] Preferably, the nucleotide sequence of the sense strand is shown in SEQ ID NO.7, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.8.

[0009] Preferably, ribose modification is introduced onto the ribosyl group of at least one nucleotide of the sense strand and / or the antisense strand, and at least one phosphate ester group in the phosphate-sugar backbone of the sense strand and / or the antisense strand is a phosphate ester group with a modifying group.

[0010] Preferably, the ribose modification is a methoxy modification or a fluorinated modification, and the phosphate ester group with the modified group is a thiophosphate ester group formed by replacing the oxygen atom in the phosphate diester bond with a sulfur atom.

[0011] Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the sense strand are methoxylated nucleotides; the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand are methoxylated nucleotides; wherein, the fluorinated nucleotides are nucleotides formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine, and the methoxylated nucleotides are nucleotides formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group.

[0012] Preferably, the phosphate ester group having the modifying group is present at least once in the group consisting of:

[0013] Between the first and second nucleotides at the 5' end of the positive strand;

[0014] Between the second and third nucleotides at the 5' end of the positive strand;

[0015] Between the first and second nucleotides at the 3' end of the positive strand;

[0016] Between the second and third nucleotides at the 3' end of the positive strand;

[0017] Between the first and second nucleotides at the 5' end of the antisense strand;

[0018] Between the second and third nucleotides at the 5' end of the antisense strand;

[0019] Between the first and second nucleotides at the 3' end of the antisense strand; and

[0020] Between the second and third nucleotides at the 3' end of the antisense strand.

[0021] A second aspect of the present invention provides the use of the aforementioned siRNA in the preparation of a drug for inhibiting the expression of the LILRB4 gene in tumor cells.

[0022] Preferably, the tumor cells are human leukemia cells.

[0023] Preferably, the human leukemia cells are human acute myeloid leukemia cells.

[0024] Preferably, the human acute myeloid leukemia cells are human M4 or M5 subtype acute myeloid leukemia cells.

[0025] A third aspect of the present invention provides the use of the aforementioned siRNA in the preparation of a drug for blocking the ApoE / LILRB4 / SHP-2 / uPAR / Arginase-1 signaling pathway in tumor cells.

[0026] Preferably, the tumor cells are human leukemia cells.

[0027] Preferably, the human leukemia cells are human acute myeloid leukemia cells.

[0028] Preferably, the human acute myeloid leukemia cells are human M4 or M5 subtype acute myeloid leukemia cells.

[0029] A fourth aspect of the present invention provides the use of the aforementioned siRNA in the preparation of a medicament for inhibiting the infiltration of tumor cells into normal tissues.

[0030] Preferably, the tumor cells are human leukemia cells.

[0031] Preferably, the human leukemia cells are human acute myeloid leukemia cells.

[0032] Preferably, the human acute myeloid leukemia cells are human M4 or M5 subtype acute myeloid leukemia cells.

[0033] The fifth aspect of the present invention provides the use of the aforementioned siRNA in the preparation of a medicament for enhancing the activity of T cells at tumor lesions.

[0034] Preferably, the tumor is human leukemia.

[0035] Preferably, the human leukemia is human acute myeloid leukemia.

[0036] Preferably, the human acute myeloid leukemia is human M4 or M5 subtype acute myeloid leukemia.

[0037] The beneficial effects of the present invention through the above technical solution are as follows:

[0038] This invention designs and synthesizes siRNA based on the human LILRB4 encoding gene. This siRNA not only exhibits good stability but also high gene-suppressive activity, effectively inhibiting the expression of LILRB4 mRNA and protein in human acute myeloid leukemia (THP-1) cells. Furthermore, the siRNA provided by this invention can block the ApoE / LILRB4 / SHP-2 / uPAR / Arginase-1 signaling pathway, thereby preventing tumor cell infiltration into normal tissues and simultaneously enhancing T cell activity. Therefore, the siRNA of this invention has promising applications in the preparation of pharmaceutical formulations for leukemia treatment. Attached Figure Description

[0039] Figure 1 This is a graph showing the relationship between the inhibition rates of ten pairs of stabilized and modified siRNAs on the expression levels of LILRB4 mRNA in human leukemia in Example 2.

[0040] Figure 2 This is a polyacrylamide gel electrophoresis image of stabilized modified siLILRB4(4) and unmodified siRNA-4 in Example 4;

[0041] Figure 3 This is a graph showing the luminescence of Luciferase in mice treated with the PBS control group, PNP-siNC group, and PNP-siLILRB4 group in Example 7. Detailed Implementation

[0042] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0043] It should be explained that, unless otherwise specified, in the preceding and following text, uppercase letters C, G, U, and A represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; and lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by phosphate thioester groups. The term "fluorinated nucleotide" refers to a nucleotide in which the 2'-hydroxyl group on the ribose group of a nucleotide is replaced by fluorine, and the term "methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group on the ribose group of a nucleotide is replaced by a methoxy group.

[0044] In this invention, it should be noted that "reverse complementarity" has a meaning known to those skilled in the art, namely, in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases of the other strand in an inverse, complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair includes a purine and a pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence.

[0045] The first aspect of the present invention provides an siRNA that specifically inhibits the expression of the LILRB4 gene, the siRNA containing a completely inversely complementary sense strand and an antisense strand, the nucleotide sequences of the sense strand and the antisense strand being any pair as shown in SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18 or SEQ ID NO.19 and SEQ ID NO.20.

[0046] According to the present invention, siRNA uses nucleotides as basic structural units, and as is known to those skilled in the art, the nucleotides contain phosphate, ribose and bases.

[0047] According to the present invention, the nucleotide sequence length of the siRNA positive strand is 19 nucleotides, the nucleotide sequence length of the siRNA negative strand is 21 nucleotides, and the 19 nucleotides of the siRNA negative strand starting from the 5' end are completely anticomplementary to the 19 nucleotides of the corresponding siRNA positive strand; at this time, the 3' end of the siRNA negative strand is connected to 2 additional nucleotides, thereby forming a 3' overhang composed of 2 nucleotides after the siRNA negative strands are complementary and paired.

[0048] Specifically, the nucleotide sequence of the siRNA sense strand is shown in SEQ ID NO.1, and the nucleotide sequence of the siRNA antisense strand is shown in SEQ ID NO.2;

[0049] SEQ ID NO.1: 5'-GGAGAUACCGCUGUUACUA-3',

[0050] SEQ ID NO.2: 5'-UAGUAACAGCGGUAUCUCCCU-3';

[0051] Alternatively, the nucleotide sequence of the siRNA sense strand is shown in SEQ ID NO.3, and the nucleotide sequence of the siRNA antisense strand is shown in SEQ ID NO.4;

[0052] SEQ ID NO.3: 5'-GGGAGUACCGUCUGGAUAA-3',

[0053] SEQ ID NO.4: 5'-UUAUCCAGACGGUACUCCCGA-3';

[0054] Alternatively, the nucleotide sequence of the siRNA sense strand is shown in SEQ ID NO.5, and the nucleotide sequence of the siRNA antisense strand is shown in SEQ ID NO.6;

[0055] SEQ ID NO.5: 5'-GUGAAACACUCCAGACCUA-3',

[0056] SEQ ID NO.6: 5'-UAGGUCUGGAGUGUUUCACCU-3';

[0057] Alternatively, the nucleotide sequence of the siRNA sense strand is shown in SEQ ID NO.7, and the nucleotide sequence of the siRNA antisense strand is shown in SEQ ID NO.8;

[0058] SEQ ID NO.7: 5'-GAGGACAGACAGAUGGACA-3',

[0059] SEQ ID NO.8: 5'-UGUCCAUCUGUCUGUCCUCUU-3';

[0060] Alternatively, the nucleotide sequence of the siRNA sense strand is shown in SEQ ID NO.9, and the nucleotide sequence of the siRNA antisense strand is shown in SEQ ID NO.10;

[0061] SEQ ID NO.9: 5'-GAGUCCUCUUGUGACCUCA-3',

[0062] SEQ ID NO.10: 5'-UGAGGUCACAAGAGGACUCGG-3';

[0063] Alternatively, the nucleotide sequence of the siRNA sense strand is shown in SEQ ID NO.11, and the nucleotide sequence of the siRNA antisense strand is shown in SEQ ID NO.12;

[0064] SEQ ID NO.11: 5'-GAGACAGGCUGAUUUCCAA-3',

[0065] SEQ ID NO.12: 5'-UUGGAAAUCAGCCUGUCUCUG-3';

[0066] Alternatively, the nucleotide sequence of the siRNA sense strand is shown in SEQ ID NO.13, and the nucleotide sequence of the siRNA antisense strand is shown in SEQ ID NO.14;

[0067] SEQ ID NO.13: 5'-GGGUCUUGGUGGUCCCAU-3',

[0068] SEQ ID NO.14: 5'-AUGGAGACCACCAAGACCCCG-3';

[0069] Alternatively, the nucleotide sequence of the siRNA sense strand is shown in SEQ ID NO.15, and the nucleotide sequence of the siRNA antisense strand is shown in SEQ ID NO.16;

[0070] SEQ ID NO.15: 5'-CAAGGCCAGAUUCUCCAUC-3',

[0071] SEQ ID NO.16: 5'-GAUGGAGAAUCUGGCCUUGUU-3';

[0072] Alternatively, the nucleotide sequence of the siRNA sense strand is shown in SEQ ID NO.17, and the nucleotide sequence of the siRNA antisense strand is shown in SEQ ID NO.18;

[0073] SEQ ID NO.17: 5'-CAUCCCCUACUGCAUCUGA-3',

[0074] SEQ ID NO.18: 5'-UCAGAUGCAGUAGGGGAUGGG-3';

[0075] Alternatively, the nucleotide sequence of the siRNA sense strand is shown in SEQ ID NO.19, and the nucleotide sequence of the siRNA antisense strand is shown in SEQ ID NO.20;

[0076] SEQ ID NO.19: 5'-CCUGGAGCUCAUAGUCUCA-3',

[0077] SEQ ID NO. 20: 5'-UGAGACUAUGAGCUCCAGGGG-3'.

[0078] According to the present invention, the method for designing siRNA that specifically inhibits LILRB4 gene expression includes the following steps:

[0079] (1) Select sequence-conserved coding genes in the human LILRB4 genome as targets for siRNA;

[0080] (2) Generate candidate siRNAs against LILRB4 by comprehensively utilizing multiple siRNA design software;

[0081] (3) Homology analysis is performed on the sequences of the selected siRNAs to exclude non-specific inhibition of siRNAs. Finally, two or more siRNA sequences designed by the software are selected that overlap or are in close positions to screen out theoretically efficient siRNAs.

[0082] According to the present invention, more preferably, 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.8.

[0083] According to the present invention, the siRNA design process further includes stabilizing the sense and / or antisense strands of the siRNA, and screening for the optimal siRNA molecule using gene repression rate and stability as indicators.

[0084] According to the present invention, a ribose modification is introduced onto the ribosome of at least one nucleotide of the sense strand and / or the antisense strand. Preferably, a ribose modification is introduced onto the ribosome of each nucleotide of the sense strand and the antisense strand.

[0085] According to the present invention, the ribose modification is methoxy modification or fluorination modification. The inventors have found that, under this preferred embodiment, siRNA can achieve a high balance between stability and gene expression inhibition efficiency.

[0086] According to the present invention, the fluorinated nucleotide is a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine, and the methoxylated nucleotide is a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with methoxy group.

[0087] Specifically, the siRNA provided by the present invention is an siRNA with the following modifications: the nucleotides at positions 5, 7, 8, and 9 of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the sense strand are methoxylated nucleotides, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand are methoxylated nucleotides.

[0088] According to the present invention, at least one phosphate ester group in the phosphate-sugar backbone of the sense strand and / or the antisense strand is a phosphate ester group with a modifying group. Preferably, the phosphate ester group with the modifying group is a thiophosphate ester group formed by replacing the oxygen atom in the phosphodiester bond with a sulfur atom. The inventors have found that, in this preferred embodiment, thiophosphate modification can effectively stabilize the double-stranded structure of siRNA, maintaining high specificity and high affinity of base pairing.

[0089] According to the present invention, the phosphate ester group having the modifying group is present at least once in the group consisting of:

[0090] Between the first and second nucleotides at the 5' end of the positive strand;

[0091] Between the second and third nucleotides at the 5' end of the positive strand;

[0092] Between the first and second nucleotides at the 3' end of the positive strand;

[0093] Between the second and third nucleotides at the 3' end of the positive strand;

[0094] Between the first and second nucleotides at the 5' end of the antisense strand;

[0095] Between the second and third nucleotides at the 5' end of the antisense strand;

[0096] Between the first and second nucleotides at the 3' end of the antisense strand; and

[0097] Between the second and third nucleotides at the 3' end of the antisense strand.

[0098] Specifically, the phosphate groups of the modifying groups are present between the first and second nucleotides, and between the second and third nucleotides at the 5' end of the sense strand, and between the first and second nucleotides, and between the second and third nucleotides at the 5' end of the antisense strand, and between the first and second nucleotides, and between the second and third nucleotides at the 3' end.

[0099] According to the present invention, siRNA can be modified by using a nucleoside monomer with the corresponding modification to introduce a modified nucleotide group into the corresponding siRNA. The method for preparing the nucleoside monomer with the corresponding modification and the method for introducing the modified nucleotide group into the siRNA can be conventional in the art. The corresponding modified nucleoside monomers can be commercially available or prepared by known methods.

[0100] Based on the aforementioned siRNA that specifically inhibits LILRB4 gene expression, a second aspect of the present invention provides the use of the aforementioned siRNA in the preparation of a medicament for inhibiting LILRB4 gene expression in tumor cells.

[0101] According to the present invention, a method for inhibiting LILRB4 gene expression in tumor cells comprises administering an effective amount of the aforementioned siRNA and / or drug to a subject in need. The siRNA and / or drug may be administered via any suitable route known in the art, including but not limited to: oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration, pulmonary administration, nasal administration, rectal administration, and local administration. The dosage of the siRNA and / or drug may be a conventional dosage in the art and may be determined based on various parameters, particularly the subject's age, weight, and sex.

[0102] According to the present invention, preferably, the tumor cells are human leukemia cells.

[0103] According to the present invention, preferably, the human leukemia cells are human acute myeloid leukemia cells.

[0104] According to the present invention, preferably, the human acute myeloid leukemia cells are human M4 or M5 subtype acute myeloid leukemia cells.

[0105] A third aspect of the present invention provides the use of the aforementioned siRNA in the preparation of a drug for blocking the ApoE / LILRB4 / SHP-2 / uPAR / Arginase-1 signaling pathway in tumor cells.

[0106] According to the present invention, preferably, the tumor cells are human leukemia cells.

[0107] According to the present invention, preferably, the human leukemia cells are human acute myeloid leukemia cells.

[0108] According to the present invention, preferably, the human acute myeloid leukemia cells are human M4 or M5 subtype acute myeloid leukemia cells.

[0109] According to the present invention, a method for blocking the ApoE / LILRB4 / SHP-2 / uPAR / Arginase-1 signaling pathway in tumor cells includes administering an effective amount of the above-mentioned siRNA and / or drug to a subject in need.

[0110] A fourth aspect of the present invention provides the use of the aforementioned siRNA in the preparation of a medicament for inhibiting the infiltration of tumor cells into normal tissues.

[0111] According to the present invention, a method for inhibiting the infiltration of tumor cells into normal tissues includes administering an effective amount of the above-mentioned siRNA and / or drug to a subject in need.

[0112] According to the present invention, preferably, the tumor cells are human leukemia cells.

[0113] According to the present invention, preferably, the human leukemia cells are human acute myeloid leukemia cells.

[0114] According to the present invention, preferably, the human acute myeloid leukemia cells are human M4 or M5 subtype acute myeloid leukemia cells.

[0115] The fifth aspect of the present invention provides the use of the aforementioned siRNA in the preparation of a medicament for enhancing the activity of T cells at tumor lesions.

[0116] According to the present invention, a method for enhancing T cell activity at a tumor lesion site includes administering an effective amount of the above-mentioned siRNA and / or drug to a subject in need.

[0117] According to the present invention, preferably, the tumor is human leukemia.

[0118] According to the present invention, preferably, the human leukemia is human acute myeloid leukemia.

[0119] According to the present invention, preferably, the human acute myeloid leukemia is human M4 or M5 subtype acute myeloid leukemia.

[0120] Based on the aforementioned application of siRNA that specifically inhibits LILRB4 gene expression, the aforementioned siRNA that specifically inhibits LILRB4 gene expression can be used to prepare a drug for treating tumors, which is a pharmaceutical composition containing the aforementioned siRNA that specifically inhibits LILRB4 gene expression.

[0121] According to the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or other pharmaceutically acceptable excipients. The pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA delivery, such as, but not limited to, liposomes, nanovesicles, polymers, magnetic nanoparticles, carbon nanotubes, mesoporous silica, calcium phosphate nanoparticles, etc.; the excipients may be one or more of various formulations or compounds conventionally used in the art, such as pH buffers, protectants, osmotic pressure regulators, etc.

[0122] According to the present invention, there are no particular requirements for the content of siRNA and pharmaceutically acceptable carriers and excipients in the pharmaceutical composition, as long as the siRNA can exert its corresponding pharmaceutical effect. The pharmaceutical composition can be a liquid formulation, such as an injection; or it can be a lyophilized powder for injection, which is mixed with liquid excipients to form a liquid formulation for administration. The liquid formulation can be used, but is not limited to, for subcutaneous, intramuscular, or intravenous injection, and can also be administered via a spray to the lungs, or via a spray to other organs and tissues through the lungs.

[0123] The present invention will be described in detail below through embodiments.

[0124] In the following examples, human acute myeloid leukemia cells THP-1, THP-1-Luc, and human T lymphocytes were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The transfection reagent Lipofectamine 2000 was purchased from Invitrogen (catalog number 11668019), the reverse transcription kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. (catalog number R323-01), the CFDA-S cell proliferation and tracing detection kit, PCR reaction system reagents, and CFSE dye were purchased from Yisheng Biotechnology Co., Ltd. (catalog numbers 40714ES76 and 11201ES03, respectively), the BCA protein quantification kit was purchased from Beijing Kangwei Century Biotechnology Co., Ltd. (catalog number CW0014), NTG mice were purchased from Spiford (Beijing) Biotechnology Co., Ltd., and Luciferase substrate was purchased from Promega Biotechnology (Beijing) Biotechnology Co., Ltd. (catalog number E1501). All other reagents were conventional commercially available products.

[0125] In the following embodiments, unless otherwise specified, the room temperature is 25±5℃.

[0126] Example 1: Design and Synthesis of siRNA Sequences

[0127] (1) The mRNA sequence of the human LILRB4 gene was obtained from Genebank using bioinformatics methods (Genebank ID: NM_001278427.3), and the sequence-conserved coding gene was selected as the target of siRNA.

[0128] (2) Based on the siRNA design principles, candidate siRNA sequences against LILRB4 were generated using online siRNA design software such as siDESIGN Center (https: / / horizondiscovery.com / products / tools / siDESIGN-Center), siDirect (http: / / sidirect2.rnai.jp / ), DSIR (http: / / www.bioinfo.ensmp.fr / dsir / ), GenScript siRNATarget Finder (https: / / www.genscript.com / tools / sirna-target-finder), and Block-iT RNAi Designer (https: / / rnaidesigner.invitrogen.com / rnaiexpress / setOption.do?designOption=sirna);

[0129] (3) Based on the design principles of siRNA, homology analysis was performed on the sequences to exclude non-specific inhibition of siRNA. Finally, two or more siRNA sequences designed by the software that overlapped or were in close proximity were selected to obtain ten pairs of siRNA design sequences. Their sequence information is as follows:

[0130] siRNA-1 design sequence

[0131] Chain of Justice (SEQ ID NO.1): 5'-GGAGAUACCGCUGUUACUA-3',

[0132] Antisense strand (SEQ ID NO.2): 5'-UAGUAACAGCGGUAUCUCCCU-3';

[0133] siRNA-2 design sequence

[0134] Chain of Justice (SEQ ID NO.3): 5'-GGGAGUACCGUCUGGAUAA-3',

[0135] Antisense chain (SEQ ID NO.4): 5'-UUAUCCAGACGGUACUCCCGA-3';

[0136] siRNA-3 design sequence

[0137] Chain of Justice (SEQ ID NO.5): 5'-GUGAAACACUCCAGACCUA-3',

[0138] Antisense strand (SEQ ID NO.6): 5'-UAGGUCUGGAGUGUUUCACCU-3';

[0139] siRNA-4 design sequence

[0140] Chain of Justice (SEQ ID NO.7): 5'-GAGGACAGACAGAUGGACA-3',

[0141] Antisense chain (SEQ ID NO.8): 5'-UGUCCAUCUGUCUGUCCUCUU-3';

[0142] siRNA-5 design sequence

[0143] Chain of Justice (SEQ ID NO.9): 5'-GAGUCCUCUUGUGACCUCA-3'

[0144] Antisense chain (SEQ ID NO.10): 5'-UGAGGUCACAAGAGGACUCGG-3'.

[0145] siRNA-6 design sequence

[0146] Chain of Justice (SEQ ID NO.11): 5'-GAGACAGGCUGAUUUCCAA-3',

[0147] Antisense chain (SEQ ID NO.12): 5'-UUGGAAAUCAGCCUGUCUCUG-3';

[0148] siRNA-7 design sequence

[0149] Chain of Justice (SEQ ID NO.13): 5'-GGGUCUUGGUGGUCUCCAU-3',

[0150] Antisense strand (SEQ ID NO.14): 5'-AUGGAGACCACCAAGACCCCG-3';

[0151] siRNA-8 design sequence

[0152] Chain of Justice (SEQ ID NO.15): 5'-CAAGGCCAGAUUCUCCAUC-3',

[0153] Antisense strand (SEQ ID NO.16): 5'-GAUGGAGAAUCUGGCCUUGUU-3';

[0154] siRNA-9 design sequence

[0155] Chain of Justice (SEQ ID NO.17): 5'-CAUCCCCUACUGCAUCUGA-3',

[0156] Antisense strand (SEQ ID NO.18): 5'-UCAGAUGCAGUAGGGGAUGGG-3';

[0157] siRNA-10 design sequence

[0158] Chain of Justice (SEQ ID NO.19): 5'-CCUGGAGCUCAUAGUCUCA-3',

[0159] Antisense chain (SEQ ID NO.20): 5'-UGAGACUAUGAGCUCCAGGGG-3'.

[0160] The ten pairs of siRNA design sequences mentioned above were synthesized by Suzhou Beixin Biotechnology Co., Ltd. The positive and negative strands of the siRNA designs were stabilized and modified, including: nucleotides at positions 5, 7, 8, and 9 of the positive strand were fluorinated, and the remaining nucleotides in the positive strand were methoxylated; nucleotides at positions 2, 6, 8, 9, 14, and 16 of the negative strand were fluorinated, and the remaining nucleotides in the negative strand were methoxylated; phosphate groups, representing modifying groups, were present between the first and second nucleotides at the 5' end of the positive strand, between the second and third nucleotides at the 5' end of the negative strand, and between the first and second nucleotides at the 5' end of the negative strand, and between the first and second nucleotides at the 3' end of the negative strand.

[0161] The modified siRNA sequence information is shown in Table 1:

[0162] Table 1

[0163]

[0164]

[0165] Example 2: Verification of gene silencing efficiency

[0166] (1) Cell culture: Human acute myeloid leukemia cells THP-1 were inoculated into 1640 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin and cultured in a saturated humidity environment with a temperature of 37°C and a CO2 content of 5% by volume.

[0167] (2) Real-time PCR was used to investigate the mRNA expression level of LILRB4.

[0168] The human acute myeloid leukemia cells THP-1 obtained in step (1) were cultured at 2 × 10⁻⁶. 5 / wells were seeded in 6-well plates and cultured overnight in an incubator at 37°C and 5% CO2. The culture medium was replaced with opti-MEM medium, and the ten pairs of siRNAs shown in Table 1 of Example 1, namely siLILRB4(1), siLILRB4(2), siLILRB4(3), siLILRB4(4), siLILRB4(5), siLILRB4(6), siLILRB4(7), siLILRB4(8), siLILRB4(9), and siLILRB4(10), were transfected according to the instructions of the transfection reagent Lipofectamine 2000. The final concentration of siRNA was 50 nM. Finally, the cells were collected, washed three times with pre-cooled PBS, and total RNA was extracted with trizol.

[0169] Total RNA from each group of cells was reverse transcribed into cDNA using a reverse transcription kit. The specific procedure is as follows: First, the following reaction system was prepared to remove genomic DNA: 4×g DNA wiper Mix: 4.0 μL; Total RNA: 1000 ng; nuclease-free water added to 16 μL. After mixing, the mixture was incubated at 42°C for 2 min. Then, 5×qRT SuperMix II: 4 μL was added to the above mixture. After mixing, it was incubated at 50°C for 15 min and then at 85°C for 5 sec.

[0170] After reverse transcription is complete, prepare the PCR reaction system: qPCR Green Master Mix: 10 μL; PCR Forward Primer (10 μM): 0.5 μL; PCR Reverse Primer (10 μM): 0.5 μL; cDNA template: 1 μL; Nuclease-free water added to 20 μL;

[0171] The primer sequences used for PCR are as follows:

[0172] LILRB4 gene:

[0173] Upstream (SEQ ID NO.21): 5'-CCTACAGTAAACCCACCCTTTCA-3',

[0174] Downstream (SEQ ID NO.22): 5'-GATCTCAGATGCAGTAGGGGATG-3';

[0175] GAPDH gene

[0176] Upstream (SEQ ID NO.23): 5'-AGAAGGCTGGGGCTCATTTG-3',

[0177] Downstream (SEQ ID NO.24): 5'-AGGGGCCATCCACAGTCTTC-3';

[0178] The PCR cycling program is as follows: ① 95℃, 5min, 1 cycle; ② 95℃, 10s, 40 cycles; ③ 55℃, 20s, 40 cycles; ④ 72℃, 20s, 40 cycles; ⑤ 95℃, 1s, 1 cycle.

[0179] Using the GAPDH gene as an internal reference, the Ct values ​​of the LILRB4 and GAPDH genes were detected by PCR. The mock control group was used as the final control, with the expression level of LILRB4 mRNA in the control cells set at 100%. The relative percentage of LILRB4 mRNA in each group was calculated. The results are shown in [Figure number missing]. Figure 1 .

[0180] like Figure 1 As shown, all ten pairs of stabilized siRNAs (siLILRB4(1), siLILRB4(2), siLILRB4(3), siLILRB4(4), siLILRB4(5), siLILRB4(6), siLILRB4(7), siLILRB4(8), siLILRB4(9), siLILRB4(10)) can effectively silence the expression of LILRB4 mRNA to a certain extent. Among them, siLILRB4(4) has the most obvious effect in silencing the expression of LILRB4 mRNA, with a silencing efficiency of 45.3%.

[0181] Example 3: Western blot analysis of LILRB4 protein expression levels

[0182] Using the ten pairs of siRNAs shown in Table 1 of Example 1, human acute myeloid leukemia cells THP-1 were transfected according to the method shown in step (2) of Example 2. The final concentration of siRNA was 100 nM. After transfection, the cells were lysed with RIPA lysis buffer (containing 1% wt% phenylmethylsulfonyl fluoride PMSF), and the protein concentration was quantified using the BCA protein detection kit. The protein was subjected to SDS-PAGE electrophoresis, transferred to a membrane, blocked with 5% skim milk, and incubated overnight at 4°C with LILRB4 primary antibody. After the incubation, the membrane was washed with TBST buffer (2.42 g Tris, 8 g NaCl, 1 mL Tween 20 dissolved in 1 L ultrapure water), incubated with secondary antibody at room temperature, and washed again with TBST buffer. ECL luminescent solution was evenly spread on the PVDF membrane, and the membrane was imaged using a 5200 multi gel imaging system. The protein bands were quantified using ImageJ software. The quantification results are shown in Table 2.

[0183] Table 2

[0184]

[0185]

[0186] As can be seen from the data in Table 2, all ten pairs of stabilized siRNAs have an inhibitory effect on LILRB4 protein expression. Among them, siLILRB4(4) has the highest inhibition rate on LILRB4 protein expression. This result is consistent with the results of quantitative real-time PCR. Therefore, siLILRB4(4) is a more preferred siRNA that specifically inhibits LILRB4 gene expression.

[0187] Example 4: Polyacrylamide gel electrophoresis to investigate siRNA stability

[0188] The stabilized modified siLILRB4(4) and unmodified siRNA-4 shown in Table 1 of Example 1 were mixed with 10% fetal bovine serum and incubated at 37°C for 2h, 4h, 8h, 24h, 48h, and 72h. 10μL samples were then electrophoresed on a 20% polyacrylamide gel and imaged using a 5200multi Tianneng gel imaging system to further analyze siRNA stability. The results are shown in [Table 1]. Figure 2 .

[0189] like Figure 2As shown, the results of polyacrylamide gel electrophoresis showed that the stabilized siLILRB4(4) could still be detected after incubation with 10% fetal bovine serum at 37°C for 72 h, while the unmodified siRNA-4 began to degrade after incubation with serum for 2 h. This indicates that the stability of the stabilized siRNA was significantly improved. That is, the introduction of 2'-methoxy or 2'-fluorine modification on the nucleotide sugar ring of the siRNA sequence and the modification of the phosphate backbone with thiophosphate can significantly improve the resistance of siRNA to nuclease degradation.

[0190] Example 5: Western blot investigation of the effect of LILRB4 silencing on the ApoE / LILRB4 / SHP-2 / uPAR / Arginase-1 signaling pathway.

[0191] Using siLILRB4(4) as shown in Table 1 of Example 1, human acute myeloid leukemia cells THP-1 were transfected according to the method shown in step (2) of Example 2. The final concentration of siRNA was 100 nM. After transfection, nuclear proteins were extracted using a nuclear protein extraction kit. After quantifying the protein concentration using a BCA protein detection kit, Western blot was performed according to the method described in Example 3 to detect the protein levels of LILRB4 and phosphorylated SHP-2 (p-SHP-2) in the cells. β-tubulin was used as an internal control. The results were quantified using ImageJ. The quantification results are shown in Table 3.

[0192] Table 3

[0193] Group Protein inhibition rate (%) Mock 0±1.3 p-SHP-2 31±3.0 LILRB4 46±2.6

[0194] As shown in Table 3, compared with the Mock group, the lipid nanoparticle formulation loaded with siLILRB4(4) can effectively inhibit the expression levels of LILRB4 and phosphorylated SHP-2 protein in human acute myeloid leukemia THP-1 cells, indicating that silencing LILRB4 can effectively block the ApoE / LILRB4 / SHP-2 / uPAR / Arginase-1 signaling pathway and reduce the level of phosphorylated SHP-2 protein.

[0195] Example 6: Flow cytometry detection of T cell activity

[0196] Using siLILRB4(4) as shown in Table 1 of Example 1, human acute myeloid leukemia cells THP-1 were transfected according to the method shown in step (2) of Example 2. The final concentration of siRNA was 100 nM. After transfection, the cells were cultured for another 24 h to obtain THP-1 cells with silenced LILRB4 gene (THP-1). LILRB4- T cells (human T lymphocytes) were labeled with CFSE fluorescence and then treated as follows:

[0197] (1) Fluorescently labeled T cells (4×10) 4 (1) No processing is performed;

[0198] (2) THP-1 cells (10 4 (4 × 10) and fluorescently labeled T cells (4 × 10) 4 (each) is co-incubated at a ratio of 1:4;

[0199] (3) THP-1 LILRB4- (10 4 (4 × 10) and fluorescently labeled T cells (4 × 10) 4 (each) is co-incubated at a ratio of 1:4;

[0200] After 48 hours of treatment, the proliferation of T cells was detected by flow cytometry, and the quantitative results are shown in Table 4.

[0201] Table 4

[0202] Group T cell activity (%) T 100±2.3 THP-1 / T 42±1.6 <![CDATA[THP-1 LILRB4- / T]]> 79±2.7

[0203] As can be seen from the data in Table 4, the siLILRB4(4) provided by the present invention can effectively enhance the activity of T cells at the tumor lesion site.

[0204] Example 7: In vivo treatment of acute myeloid leukemia with LILRB4

[0205] (1) Preparation of lipid nanoparticle formulations loaded with negative control siRNA (PNP-siNC) and lipid nanoparticle formulations loaded with siLILRB4(4) (PNP-siLILRB4):

[0206] Four lipid components, A1-D1-5, DSPC, Chol, and DMG-PEG-2000, were dissolved in anhydrous ethanol to a final concentration of 20 mg / ml. The components were mixed in a molar ratio of 54:12:60:2.5 and injected into sodium citrate buffer at a uniform rate. The mixture was then incubated with siRNA to obtain a lipid nanoparticle formulation (PNP-siRNA).

[0207] When NC is used as siRNA, a lipid nanoparticle formulation loaded with negative control siRNA (PNP-siNC) is obtained. When siRNA is the stabilized modified siLILRB4 (4) shown in Table 1 of Example 1, a lipid nanoparticle formulation loaded with siLILRB4 (4) is obtained (PNP-siLILRB4).

[0208] The sense strand (SEQ ID NO.25) of the siRNA drug NCsiRNA is:

[0209] CCUUGAGGCAUACUUCAAAdTdT;

[0210] The antisense strand (SEQ ID NO.26) of the siRNA drug NC is as follows:

[0211] UUUGAAGUAUGCCUCAAGGdTdT;

[0212] (2) Establishment of an acute myeloid leukemia cell model: THP-1-Luc cells were suspended in PBS buffer and administered at a rate of 8 × 10⁸ cells per NTG mouse. 6 A humanized mouse leukemia model was established by tail vein injection of cells.

[0213] (3) Seven days after cell inoculation, each mouse was injected intraperitoneally with Luciferase substrate (150 mg / kg) and the disease status was detected;

[0214] (4) Mice were randomly divided into 3 groups and treated as follows: (a) PBS control group; (b) lipid nanoparticle formulation (PNP-siNC) group loaded with negative control siRNA; (c) lipid nanoparticle formulation (PNP-siLILRB4) group loaded with siLILRB4. The siRNA was administered via tail vein at a dose of 1 mg / kg. After 3 weeks of treatment (day 28), the progression of leukemia in mice was detected again by Luciferase luminescence. The results are shown in the figure. Figure 3 .

[0215] like Figure 3 As shown, compared with PBS and lipid nanoparticle formulations loaded with negative control siRNA-4 (PNP-siNC), lipid nanoparticle formulations loaded with siLILRB4 (PNP-siLILRB4) significantly inhibited leukemia cell infiltration and effectively stopped the progression of leukemia, showing good application prospects.

[0216] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention. SEQUENCE LISTING <110> Beijing Institute of Technology, Tianjin Medical University <120> siRNAs that specifically inhibit LILRB4 gene expression and their applications <130> June 2, 2022 <160> 26 <170> PatentIn version 3.3 <210> 1 <211> 19 <212> RNA <213> Artificial sequence <400> 1 ggagauaccg cuguuacua 19 <210> 2 <211> twenty one <212> RNA <213> Artificial sequence <400> 2 uaguaacagc gguaucuccc u 21 <210> 3 <211> 19 <212> RNA <213> Artificial sequence <400> 3 gggaguaccg ucuggauaa 19 <210> 4 <211> twenty one <212> RNA <213> Artificial sequence <400> 4 uuauccagac gguacucccg a 21 <210> 5 <211> 19 <212> RNA <213> Artificial sequence <400> 5 gugaaacacu ccagaccua 19 <210> 6 <211> twenty one <212> RNA <213> Artificial sequence <400> 6 uaggucugga guguuucacc u 21 <210> 7 <211> 19 <212> RNA <213> Artificial sequence <400> 7 gaggacagac agauggaca 19 <210> 8 <211> twenty one <212> RNA <213> Artificial sequence <400> 8 uguccaucug ucuguccucu u 21 <210> 9 <211> 19 <212> RNA <213> Artificial sequence <400> 9 gaguccucuu gugaccuca 19 <210> 10 <211> twenty one <212> RNA <213> Artificial sequence <400> 10 ugaggucaca agaggacucg g 21 <210> 11 <211> 19 <212> RNA <213> Artificial sequence <400> 11 gagacaggcu gauuuccaa 19 <210> 12 <211> twenty one <212> RNA <213> Artificial sequence <400> 12 uuggaaauca gccugucucu g 21 <210> 13 <211> 19 <212> RNA <213> Artificial sequence <400> 13 gggucuuggu ggucuccau 19 <210> 14 <211> twenty one <212> RNA <213> Artificial sequence <400> 14 auggagacca ccaagacccc g 21 <210> 15 <211> 19 <212> RNA <213> Artificial sequence <400> 15 caaggccaga uucuccauc 19 <210> 16 <211> twenty one <212> RNA <213> Artificial sequence <400> 16 gauggagaau cuggccuugu u 21 <210> 17 <211> 19 <212> RNA <213> Artificial sequence <400> 17 cauccccuac ugcaucuga 19 <210> 18 <211> twenty one <212> RNA <213> Artificial sequence <400> 18 ucagaugcag uaggggaugg g 21 <210> 19 <211> 19 <212> RNA <213> Artificial sequence <400> 19 ccuggagcuc auagucuca 19 <210> 20 <211> twenty one <212> RNA <213> Artificial sequence <400> 20 ugagacuaug agcuccaggg g 21 <210> twenty one <211> twenty three <212> DNA <213> Artificial sequence <400> twenty one cctacagtaa acccaccctt tca 23 <210> twenty two <211> twenty three <212> DNA <213> Artificial sequence <400> twenty two gatctcagat gcagtagggg atg 23 <210> twenty three <211> 20 <212> DNA <213> Artificial sequence <400> twenty three agaaggctgg ggctcatttg 20 <210> twenty four <211> 20 <212> DNA <213> Artificial sequence <400> twenty four aggggccatc cacagtcttc 20 <210> 25 <211> twenty one <212> DNA <213> Artificial sequence <400> 25 ccuugaggca uacuucaaat t 21 <210> 26 <211> twenty one <212> DNA <213> Artificial sequence <400> 26 uuugaaguau gccucaaggt t 21

Claims

1. The application of siRNA that specifically inhibits LILRB4 gene expression in the preparation of a drug for inhibiting LILRB4 gene expression in tumor cells; wherein the tumor cells are human acute myeloid leukemia cells; the siRNA contains a perfectly inversely complementary sense strand and an antisense strand; the nucleotide sequence of the sense strand is shown in SEQ ID NO.7, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

7. As shown in NO.8; the positive strand has phosphate groups with modifying groups between the first and second nucleotides at the 5' end, between the second and third nucleotides at the 5' end, and between the first and second nucleotides at the 5' end, between the second and third nucleotides at the 5' end, and between the first and second nucleotides at the 3' end, and between the second and third nucleotides at the 3' end; the phosphate groups with modifying groups are thiophosphate groups formed by replacing the oxygen atom in the phosphodiester bond with a sulfur atom; the nucleotides at positions 5, 7, 8, and 9 of the positive strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the positive strand are methoxylated nucleotides; the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand are methoxylated nucleotides; wherein, The fluorinated nucleotide is a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine, and the methoxylated nucleotide is a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group.

2. The application of siRNA that specifically inhibits LILRB4 gene expression in the preparation of drugs for blocking the ApoE / LILRB4 / SHP-2 / uPAR / Arginase-1 signaling pathway in tumor cells; wherein the tumor cells are human acute myeloid leukemia cells; wherein the siRNA contains a completely reverse complementary sense strand and an antisense strand; the nucleotide sequence of the sense strand is shown in SEQ ID NO.7, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

7. As shown in NO.8; the positive strand has phosphate groups with modifying groups between the first and second nucleotides at the 5' end, between the second and third nucleotides at the 5' end, and between the first and second nucleotides at the 5' end, between the second and third nucleotides at the 5' end, and between the first and second nucleotides at the 3' end, and between the second and third nucleotides at the 3' end; the phosphate groups with modifying groups are thiophosphate groups formed by replacing the oxygen atom in the phosphodiester bond with a sulfur atom; the nucleotides at positions 5, 7, 8, and 9 of the positive strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the positive strand are methoxylated nucleotides; the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand are methoxylated nucleotides; wherein, The fluorinated nucleotide is a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine, and the methoxylated nucleotide is a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group.

3. The application of siRNA that specifically inhibits LILRB4 gene expression in the preparation of a drug for inhibiting the invasion of tumor cells into normal tissues; wherein the tumor cells are human acute myeloid leukemia cells; wherein the siRNA contains a completely reverse complementary sense strand and an antisense strand; the nucleotide sequence of the sense strand is shown in SEQ ID NO.7, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

7. As shown in NO.8; the positive strand has phosphate groups with modifying groups between the first and second nucleotides at the 5' end, between the second and third nucleotides at the 5' end, and between the first and second nucleotides at the 5' end, between the second and third nucleotides at the 5' end, and between the first and second nucleotides at the 3' end, and between the second and third nucleotides at the 3' end; the phosphate groups with modifying groups are thiophosphate groups formed by replacing the oxygen atom in the phosphodiester bond with a sulfur atom; the nucleotides at positions 5, 7, 8, and 9 of the positive strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the positive strand are methoxylated nucleotides; the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand are methoxylated nucleotides; wherein, The fluorinated nucleotide is a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine, and the methoxylated nucleotide is a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group.

4. The application of siRNA that specifically inhibits LILRB4 gene expression in the preparation of a drug for enhancing T cell activity at tumor lesions; wherein the tumor is human acute myeloid leukemia; wherein the siRNA contains a completely reverse complementary sense strand and an antisense strand; the nucleotide sequence of the sense strand is shown in SEQ ID NO.7, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

7. As shown in IDNO.8; the positive strand has phosphate groups with modifying groups between the first and second nucleotides at the 5' end, between the second and third nucleotides at the 5' end, and between the first and second nucleotides at the 5' end, between the second and third nucleotides at the 5' end, and between the first and second nucleotides at the 3' end, and between the second and third nucleotides at the 3' end; the phosphate groups with modifying groups are thiophosphate groups formed by replacing the oxygen atom in the phosphodiester bond with a sulfur atom; the nucleotides at positions 5, 7, 8, and 9 of the positive strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the positive strand are methoxylated nucleotides; the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand are methoxylated nucleotides; wherein, The fluorinated nucleotide is a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine, and the methoxylated nucleotide is a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group.

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