CN112048509B - siRNA molecule for inhibiting NLRP3 gene expression and application thereof - Google Patents
siRNA molecule for inhibiting NLRP3 gene expression and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
Abstract
The invention discloses an siRNA molecule for inhibiting NLRP3 gene expression and application thereof. The siRNA molecule provided by the invention comprises any one of the combination of the first and the second, can efficiently inhibit the expression of NLRP3 gene, improve the activity of cell mitochondria and reduce the expression of cell protein, and is used for preparing medicines for inhibiting the expression of the gene NLRP3 and preparing medicines for treating cell apoptosis.
Description
Technical Field
The invention relates to the technical field of nucleic acid pharmacy, in particular to an siRNA molecule for inhibiting NLRP3 gene expression and application thereof.
Background
Small interfering RNAs (siRNA molecules) are emerging nucleic acid drugs that have been clinically validated, a class of double-stranded short RNA molecules whose antisense strand is capable of complementary pairing with a particular mRNA strand, thereby eliciting sequence-specific inhibition of gene expression (gene silencing), i.e., RNA interference (RNAi). Currently, 2 siRNA molecule drugs are commercially available worldwide (Onpattro, givlaari). In theory, siRNA molecules can be designed, screened for any gene and expression of the gene inhibited.
Inflammatory bodies play an important role in cell apoptosis by activating cytokine maturation, secreting chemokines and responding to endogenous and exogenous danger signals. Inflammatory corpuscles are a group of cytoplasmic protein complexes that play a critical role in host defense and cellular injury. Among all inflammatory bodies, NLRP3 inflammatory bodies are the most widely studied and consist of the NOD-like receptor family, NLRP3, the aptamer ASC and pro-caspase 1. Once activated, NLRP3 recruits and cleaves pro-caspase-1, mediating maturation of caspase-1, which in turn leads to apoptosis of the cell coke. The cell apoptosis plays an important role in the occurrence and development of tumors, and the deep research of the relationship between the cell apoptosis and the tumors is beneficial to providing a new thought for the occurrence, development and new prevention and treatment of the tumors.
While the medical technology is continuously developed and advanced, pathogens are continuously evolving and upgraded, and the pathogens can survive and replicate hard in the environment of protecting organism cells without being influenced by immune response. Many environmental factors can cause cell apoptosis, further leading to cell, tissue damage and tumorigenesis. The use of small interfering RNAs (siRNA molecules) can inhibit the expression of cell coke death related genes such as NLRP3 and reverse cell coke death, thereby preventing and treating diseases. siRNA molecules have great potential in clinical application value.
Disclosure of Invention
Accordingly, the present invention is directed to an siRNA molecule for inhibiting NLRP3 gene expression and its application, so as to solve the drawbacks of the prior art.
In view of the above objects, a first aspect of the present invention provides an siRNA molecule that inhibits expression of NLRP3 gene, the siRNA molecule comprising a sense strand and an antisense strand, the sequence of the siRNA molecule comprising one of the following combinations:
combining:
(1) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 1; and
(2) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 2; or (b)
(3) A nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in (1) or (2);
or (b)
And (2) combining two:
(4) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 3; and
(5) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 4; or (b)
(6) A nucleotide sequence having at least 85% identity to the nucleotide sequence of (4) or (5);
or (b)
And (3) combining three:
(7) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 5; and
(8) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 6; or (b)
(9) A nucleotide sequence having at least 85% identity to the nucleotide sequence of (7) or (8);
or (b)
Combination four:
(10) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 7; and
(11) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 8; or (b)
(12) A nucleotide sequence having at least 85% identity to the nucleotide sequence of (10) or (11); or (b)
And (5) combining:
(13) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 9; and
(14) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 10; or (b)
(15) A nucleotide sequence having at least 85% identity to the nucleotide sequence of (13) or (14);
or (b)
And (3) combining six:
(16) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 11; and
(17) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 12; or (b)
(18) A nucleotide sequence having at least 85% identity to the nucleotide sequence of (16) or (17).
In an embodiment of the invention, the sequence of the siRNA molecule comprises one of the following combinations:
combining:
(1) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 1; and
(2) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 2;
or (b)
And (2) combining two:
(4) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 3; and
(5) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 4;
or (b)
And (3) combining three:
(7) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 5; and
(8) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 6;
or (b)
Combination four:
(10) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 7; and
(11) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 8;
or (b)
And (5) combining:
(13) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 9; and
(14) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 10;
or (b)
And (3) combining six:
(16) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 11; and
(17) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 12.
In an embodiment of the present invention, the sequence of the siRNA molecule is combination four.
In an embodiment of the invention, the sequence of the siRNA molecule has a methoxy modification and a fluoro modification, the methoxy modification being methoxy substitution of ribose 2' -hydroxy in the nucleotide; the fluoro modification is fluoro substitution of ribose 2' -hydroxy in the nucleotide.
In embodiments of the invention, the 1 st, 2 nd, 3 rd, 4 th, 6 th, 10 th, 11 th, 12 th, 13 th, 14 th, 15 th, 16 th, 17 th, 18 th and 19 th nucleotides of the sense strand of the sequence of the siRNA molecule are methoxy-modified in the 5 'end to 3' end direction; fluoro-modified nucleotides at positions 5, 7, 8 and 9; 1 st, 3 rd, 4 th, 5 th, 7 th, 10 th, 12 th, 13 th, 15 th, 17 th, 18 th and 19 th of the antisense strand of the sequence of the siRNA molecule are methoxy-modified nucleotides; positions 2, 6, 8, 9, 14 and 16 are fluoro modified nucleotides.
In an embodiment of the present invention, the sequence of the siRNA molecule further has a phosphorothioate group modification in which at least one oxygen atom of the phosphodiester bond in the phosphate group is replaced with a sulfur atom.
In embodiments of the invention, both the sense and antisense strands of the sequence of the siRNA molecule have phosphorothioate modification sites;
the modification site of the sense strand is at least one of between the 1 st nucleotide and the 2 nd nucleotide of the 5 'terminal end and between the 2 nd nucleotide and the 3 rd nucleotide of the 5' terminal end;
the modification site of the antisense strand is at least one of the following positions: between nucleotide 1 and nucleotide 2 of the 5' terminal end; between nucleotide 2 and nucleotide 3 of the 5' terminal end; between nucleotide 1 and nucleotide 2 of the 3 'terminal end and between nucleotide 2 and nucleotide 3 of the 3' terminal end.
In a second aspect, the invention provides a method of inhibiting expression of a gene, said gene being NLRP3, said method comprising transfecting a cell with an siRNA molecule having a sequence as set forth in any one of the first aspects of the invention, so as to inhibit expression of the NLRP3 gene in said cell. Methods of transfecting cells include, but are not limited to: electroporation methods, cationic polymer reagent methods, exosome delivery methods, ionizable liposome delivery methods, and the like. For example, a commercial cationic polymer transfection reagent Lipofectamine 2000 can be adopted, and a polypeptide carrier synthesized in a laboratory, a high polymer carrier, a carbon nano tube, a metal nano carrier, various organic/inorganic hybridization carriers and the like can also be adopted.
In an embodiment of the present invention, the siRNA molecule is transfected into the inside of a cell by Lipofectamine 2000 and plays a role in inhibiting gene expression.
In a third aspect, the invention provides an siRNA molecule drug for inhibiting expression of NLRP3 gene, comprising an effective amount of the siRNA molecule according to the first aspect of the invention and a pharmaceutically acceptable carrier. The medicine can be prepared into different preparations by using a conventional method, for example, physiological saline or an aqueous solvent containing glucose and other auxiliary agents can be used for preparing injection by using the conventional method. The different medicaments prepared may be administered in any convenient form, for example by different routes of local, intravenous, intramuscular, subcutaneous, intradermal, intraarticular, intrathecal injection etc. The dosage of the medicine can be adaptively adjusted according to actual conditions.
In a fourth aspect, the invention provides an application of an siRNA molecule in preparing a drug for inhibiting NLRP3 gene expression, wherein the siRNA molecule is an siRNA molecule according to any one of the embodiments of the first aspect of the invention.
In an embodiment of the invention, the drug that inhibits NLRP3 gene expression is a drug that treats exogenous-induced apoptosis, spontaneous inflammation, inflammatory injury of normal tissues, or cancer.
In an embodiment of the invention, the drug that inhibits NLRP3 gene expression is a drug that treats exogenous-induced apoptosis of cells.
From the above, the siRNA molecules for inhibiting NLRP3 gene expression agents provided by the present invention, including any one of the combinations from one to six, can inhibit expression of NLRP3 gene and related proteins with high efficiency, and increase the activity of cell mitochondria. The siRNA molecule can be used for preparing a drug for inhibiting the expression of a gene NLRP3, and the drug can be used for resisting cell apoptosis; treatment of spontaneous inflammation, inflammatory lesions of normal tissues and cancer.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the effect of multiwall carbon nanotubes on macrophage mitochondrial activity according to an embodiment of the invention;
FIG. 2 is a graph showing the effect of multiwall carbon nanotubes on macrophage signaling pathways according to an embodiment of the present invention;
FIG. 3 is a graph showing the effect of multiwall carbon nanotubes on macrophage morphology according to an embodiment of the present invention;
FIG. 4 is a graph showing the effect of multi-walled carbon nanotubes on macrophage protein expression level in accordance with an embodiment of the present invention;
FIG. 5 shows the results of mitochondrial activity after transfection of THP-1 macrophages with 6 combined NLRP3siRNA molecules according to the example of the present invention;
FIG. 6 shows the results of NLRP3 protein expression level detection after transfection of THP-1 macrophages with NLRP3siRNA molecules of combination four in the examples of the present invention;
FIG. 7 shows the results of NLRP3 protein expression level detection after transfection of THP-1 macrophages with NLRP3siRNA molecules of combination four in the examples of the present invention.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
It should be noted that unless otherwise defined, technical or scientific terms used in the embodiments of the present invention should be given the ordinary meaning as understood by one of ordinary skill in the art to which the present disclosure pertains. The terms "first," "second," and the like, as used in this disclosure, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that elements or items preceding the word are included in the element or item listed after the word and equivalents thereof, but does not exclude other elements or items. The terms "connected" or "connected," and the like, are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "upper", "lower", "left", "right", etc. are used merely to indicate relative positional relationships, which may also be changed when the absolute position of the object to be described is changed.
Embodiments of the present invention provide an siRNA molecule (Small interfering RNA) that inhibits NLRP3 expression, the siRNA molecule comprising a sense strand and an antisense strand, comprising one of the following combinations.
Combining:
(1) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 1; and
(2) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 2; or (b)
(3) A nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in (1) or (2);
or (b)
And (2) combining two:
(4) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 3; and
(5) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 4; or (b)
(6) A nucleotide sequence having at least 85% identity to the nucleotide sequence of (4) or (5);
or (b)
And (3) combining three:
(7) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 5; and
(8) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 6; or (b)
(9) A nucleotide sequence having at least 85% identity to the nucleotide sequence of (7) or (8);
or (b)
Combination four:
(10) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 7; and
(11) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 8; or (b)
(12) A nucleotide sequence having at least 85% identity to the nucleotide sequence of (10) or (11); or (b)
And (5) combining:
(13) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 9; and
(14) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 10; or (b)
(15) A nucleotide sequence having at least 85% identity to the nucleotide sequence of (13) or (14);
or (b)
And (3) combining six:
(16) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 11; and
(17) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 12; or (b)
(18) A nucleotide sequence having at least 85% identity to the nucleotide sequence of (16) or (17).
In one embodiment, the sequence of the siRNA molecule comprises one of the following combinations:
combining:
(1) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 1; and
(2) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 2;
or (b)
And (2) combining two:
(4) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 3; and
(5) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 4;
or (b)
And (3) combining three:
(7) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 5; and
(8) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 6;
or (b)
Combination four:
(10) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 7; and
(11) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 8;
or (b)
And (5) combining:
(13) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 9; and
(14) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 10;
or (b)
And (3) combining six:
(16) The nucleotide sequence of the sense strand is shown as SEQ ID NO. 11; and
(17) The nucleotide sequence of the antisense strand is shown as SEQ ID NO. 12.
Wherein, the siRNA molecule inhibits the activation of NLRP3 protein by inhibiting the expression of NLRP3 gene.
Preferably, the nucleotides of the sequence of the siRNA molecule have methoxy and fluoro modifications. Wherein the methoxy modification and the fluoro modification are both substitutions to ribose 2' -hydroxy groups in nucleotides. That is, the methoxy group is modified to methoxy group substituted ribose 2' -hydroxy group in the nucleotide; the fluoro modification is fluoro substitution of ribose 2' -hydroxy in the nucleotide.
Wherein the nucleotides at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19 of the sense strand of the nucleotide sequence are methoxy modified nucleotides, and the nucleotides at positions 5, 7, 8 and 9 of the sense strand are fluoro modified nucleotides. The nucleotide at positions 1, 3, 4, 5, 7, 10, 12, 13, 15, 17, 18 and 19 of the antisense strand of the nucleotide sequence is methoxy modified nucleotide, and the nucleotide at positions 2, 6, 8, 9, 14 and 16 of the antisense strand is fluoro modified nucleotide. Different modifications are respectively carried out on ribose 2 '-hydroxyl groups in the nucleotides of the sites of the sense strand, and different modifications on corresponding sites of ribose 2' -hydroxyl groups in the nucleotides of the antisense strand are matched, so that the nuclease resistance of the siRNA molecule can be improved, the stability of the siRNA molecule can be improved, meanwhile, the immunogenicity of the siRNA molecule can be reduced, and the safety of the siRNA molecule can be improved.
Further, in the sequences shown in SEQ ID No.1 to SEQ ID No.12, a phosphorothioate group modification is also included, and both the sense strand and the antisense strand have phosphorothioate group modification sites. The phosphorothioate group is modified such that at least one oxygen atom in the phosphodiester bond in the phosphate group is replaced with a sulfur atom.
TABLE 1 siRNA molecule sequences in NLRP3 expression inhibitors
Wherein the modification site of the phosphorothioate group of the sense strand is at least one of between the 1 st nucleotide and the 2 nd nucleotide of the 5 'terminal end and between the 2 nd nucleotide and the 3 rd nucleotide of the 5' terminal end.
The modification site of the antisense strand is at least one of the following positions: between nucleotide 1 and nucleotide 2 of the 5' terminal end; between nucleotide 2 and nucleotide 3 of the 5' terminal end; between nucleotide 1 and nucleotide 2 of the 3 'terminal end and between nucleotide 2 and nucleotide 3 of the 3' terminal end.
The P-S bond is adopted to replace the thio modification of the P-O bond at the corresponding sites of the sense strand and the antisense strand respectively, so that the degradation of enzyme can be greatly influenced, the nuclease resistance of siRNA molecules is improved, and the stability of the siRNA molecules is improved.
Preferably, the sequence of the siRNA molecule is shown as SEQ ID NO.5 and SEQ ID NO. 6. By using this sequence, it is possible to suppress the expression of NLRP3 gene well by substituting the 1 st nucleotide and 2 nd nucleotide and the thio modification between the 2 nd nucleotide and 3 rd nucleotide of the 5 '-terminal end of the sense strand with the ribose 2' -hydroxyl group in the methoxy-substituted nucleotide, the ribose 2 '-hydroxyl group in the fluoro-substituted nucleotide, the P-S bond with the P-O bond, and the thio modification between the 1 st nucleotide and 2 nd nucleotide and 3 rd nucleotide of the 5' -terminal end of the antisense strand with the P-S bond with the P-O bond.
The embodiment of the invention also provides a method for inhibiting the expression of a gene, wherein the gene is NLRP3 gene, and the method comprises transfecting a cell with the siRNA molecule for inhibiting the expression of the NLRP3 gene so as to inhibit the expression of the NLRP3 gene in the cell.
The embodiment of the invention also provides a medicine for inhibiting gene expression, which comprises an effective amount of the siRNA molecule for inhibiting NLRP3 gene expression and a pharmaceutically acceptable carrier.
The embodiment of the invention also provides an application of the NLRP3 expression inhibitor in preparing a drug for inhibiting the expression of the NLRP3 gene, wherein the siRNA molecule has the sequence of the siRNA molecule for inhibiting the expression of the NLRP3 gene. The drug may specifically be an siRNA molecule. In general, the NLRP3 gene-inhibiting drugs described above are generally used in the form of pharmaceutical compositions in the present invention. The pharmaceutical composition can improve or treat related diseases by targeted inhibition of expression of NLRP3 gene. The drug for inhibiting the expression of the gene NLRP3 comprises the NLRP3siRNA molecule and a pharmaceutically acceptable carrier.
It should be noted that a pharmaceutically acceptable carrier refers to one or more solid or liquid fillers, diluents or encapsulating substances, which pharmaceutically acceptable carrier is suitable for applying the siRNA molecules of the invention to an individual. Such pharmaceutically acceptable carriers include various solutions, diluents, solvents, dispersions, liposomes, emulsions, coatings, antibacterial, antifungal agents, and the like, and other carriers suitable for use with the siRNA molecules of the invention. The injectable carrier comprises water, physiological saline, balanced salt solution, buffer solution, glucose solution, glycerol, etc.; carriers for oral administration include mannitol, lactose, starch, magnesium stearate, and the like. At the same time, as a biologically neutral carrier, the pharmacological component used may contain non-toxic auxiliary substances including wetting or emulsifying agents, preservatives, pH buffering agents, sodium acetate, monolaurate and the like.
Preferably, the pharmaceutically acceptable carrier is a nanoscale material, including liposomes, polymeric nanomicelles, exosomes and other nanoscale materials.
Specifically, the siRNA molecule drug expressed by the inhibition gene NLRP3 is a drug for resisting cell apoptosis, spontaneous inflammation, inflammatory injury of normal tissues or cancer.
Preferably, the siRNA molecule that inhibits expression of NLRP3 gene is a drug for treating exogenous-induced apoptosis.
Specifically, the drug against apoptosis enhances mitochondrial activity of cells by inhibiting expression of NLRP3 protein.
The technical scheme of the embodiment of the invention is further described below in conjunction with experiments.
The experimental methods in the following examples are conventional methods unless otherwise specified.
The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Test materials: THP-1 cell lines were purchased from the American type culture Collection (Manassas, va., USA). Multiwall carbon nanotubes (MWCNTs) were purchased from the tokyo xfnno materials technologies inc. 1:5000 beta-actin antibodies, 1:5000HRP goat anti-rabbit IgG from Proteintech, USA. Opti-MEM TM Low serum medium, available from Thermo-Fisher. Opti-MEM TM Low serum medium was purchased from Thermo-Fisher.
Example 1
The performance of siRNA determines the merits of the expression effect of the drug repressor gene NLRP 3. Example 1 multiple sets of siRNA molecules were designed and screened for mRNA sequences of NLRP3, as shown in table 1, with six superior combinations of siRNA molecules (combinations one to six) and one optimal combination of siRNA molecules (combination four) being finally selected.
The siRNA molecule sequences in table 1 were all synthesized by the biotechnology company, su zhou Bei Xin, and the siRNA molecules were dissolved in DEPC water and stored at 4 ℃ for use. In this example, a human NLRP3 (Gene ID: 114548) detection index is taken as an example.
Experimental example 1 cultivation and induced differentiation of THP-1 cells
1) THP-1 cell line cells were cultured using RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin.
2) 10ng/mL phorbol ester 12-myristate 13-acetate (PMA; sigma, st.Louis, MO, USA) were treated with the cultured cells for 24 hours, and THP-1 cells were differentiated into macrophages, to obtain THP-1 macrophages.
Experimental example 2 multiwall carbon nanotubes MWCNT induced pyroapoptosis of THP-1 macrophages
The purpose of the experiment is as follows: the diameter of the multiwall carbon nano tube with the best effect of inducing THP-1 macrophage coke death is screened.
Induction conditions: according to the diameters of the multiwall carbon nanotubes, three processing groups are set, named XFM4, XFM22 and XFM34, respectively, each processing group has a different diameter, and the diameters are in the order of XFM4< XFM22< XFM 34. Each treatment group was set to 6 different concentrations of 0, 4, 8, 16, 32 and 64 μg/mL. The time of the induction treatment was 24 hours.
The induction method comprises the following steps: 1) The 6 concentrations of the three treatment groups XFM4, XFM22 and XFM34 were respectively sonicated continuously for 16 minutes using a sonicator, and then suspended in 2% fbs.
2) THP-1 macrophages at 2.4X10 per well 5 Is injected into 24 well plates and is directly contacted with 6 concentrations of XFM4, XFM22 and XFM34, respectively, for 24 hours, 3 replicate wells per group.
And (3) detecting induction effect: 1) And detecting the activity of macrophage mitochondria after the induction of the multiwall carbon nanotube by using a CCK-8 reagent.
2) And detecting the influence on macrophage signal path after the induction of the multiwall carbon nanotube by using an RNA sequencing method.
UsingTotal RNA was extracted, purified by an Agilent 2100 bioanalyzer (Agilent Technologies, santa Clara, calif., USA), reverse transcribed into cDNA, constructed into a cDNA library and sequenced on an Illumina sequencing platform (HiSeqTM 2500). High quality reads obtained from sequencing were mapped to human genomes and compared. After comparison, the differentially expressed genes were identified using he DESeq functions estimate Size Factors and nbinomTest, fold changes were made>2 as a threshold for significant up-regulation, fold change<0.5 as a significantly down-regulated threshold. Functional enrichment analysis was performed based on Gene Ontology (GO), based on Kyoto Encyclopedia of Gene and Genomes (KEGG; https:the// www.kegg.jp/kegg/path.html). The SRA accession number from a read of the RNA sequence is PRJNA593906.
3) The change of macrophage morphology after induction of the multiwall carbon nanotubes was observed using a Transmission Electron Microscope (TEM).
The induced THP-1 macrophages were fixed overnight in PBS by trypsinization of cells with 2.5% glutaraldehyde, post-fixed with 1% oso4 for 3 hours, dehydrated in a series of gradient ethanol and embedded in epoxy. Samples were sectioned at 70nm using an ultra-thin microtome, placed on a copper grid-supported carbon film, stained with uranyl acetate and lead citrate, and observed for cell morphology under TEM (JEM-1230, jeol Ltd., tokyo, japan) operating at 80 kV.
4) Western blot (Western blot) method is used for detecting the change of the protein expression level of the macrophage after the induction of the multiwall carbon nanotube.
The induced THP-1 macrophages were washed with Hanks solution and total protein was extracted using RIPA lysis buffer (Roche Diagnostics) containing protease inhibitor (cocktail) and Phosphotm phosphatase inhibitor (Roche Diagnostics). The protein extract obtained was then mixed with loading buffer (50. Mu.g concentration per sample) and separated on SDS-PAGE. Thereafter, the samples were transferred to nitrocellulose membranes, blocked in skim milk for 1.5 hours at room temperature, and then incubated with primary antibodies (1:1000 casase1 antibodies; 1:1000NLRP3 antibodies) overnight at 4 ℃;1:5000 beta-actin antibodies, available from Proteintech, USA). Wash with 0.1% w/v Tween-PBS and mix with 1: after incubation with 5000HRP goat anti-rabbit IgG (Proteintech, USA) for 1.5 hours, the blots were detected by SuperECL Plus chemiluminescence (Thermo peptide, USA). The optical density of each image strip was determined by ImageJ (NIH).
In experimental example 2, the results obtained by the above techniques and methods were combined, and the diameters of the multiwall carbon nanotubes having the best induction effect were selected.
Experimental example 3NLRP 3siRNA molecule transfected THP-1 macrophage inhibits NLRP3 gene expression and reverses cell apoptosis.
The transfection method comprises the following steps: giant THP-1Phagocytes at 7X 10 per well 5 Is inoculated in a 6-well plate, and 40nM of chemically modified combination one to combination six NLRP3siRNA molecules are diluted in 0.5mL of Opti-MEM, respectively TM In low serum medium, 3 mu LLipofectamine 2000 reagent was also diluted in 0.5mL Opti-MEM TM In (2), each of the above was allowed to stand for 5 minutes, and then the siRNA molecule and Lipofectamine 2000 were thoroughly mixed to obtain a treatment group. Standing at room temperature for 10min, adding the mixed solution into a cell culture plate to transfect cells, adding 1mL of complete culture medium into each well after 4 hours, and continuously transfecting for 24 hours. The control group was a group to which no siRNA molecule was added, and 3 duplicate wells were formed in each of the treatment group and the control group.
The induction method comprises the following steps: THP-1 macrophages transfected with siRNA molecules were incubated with 32. Mu.g/mL XFM4 for 24 hours, respectively, for cell scorch induction.
The detection method comprises the following steps: after the induction, the activity of the cells was examined by CCK-8 method. UsingTotal RNA was extracted, reverse transcribed and subjected to fluorescent real-time quantitative PCR detection. Western blot (Western blot) was used to detect cellular protein expression levels. It should be noted that, the specific detection methods are the same as the detection method in the foregoing experimental example 2, and are not repeated here.
Analysis of results
1. Results of multiwall carbon nanotubes inducing pyro-apoptosis of THP-1 macrophages
1) CCK8 detection of macrophage mitochondrial Activity after Multi-walled carbon nanotube Induction
The detection results are shown in FIG. 1. It can be seen that XFM22 and XFM34 are most suitable for treatment groups of different concentrations at a concentration of 32. Mu.g/mL, and the effect on cell activity is significantly different from the effect of the treatment groups of the remaining concentrations, and the difference is more stable.
2) Detection of influence on macrophage signal pathway after induction of multiwall carbon nanotubes by RNA sequencing
The detection result is shown in FIG. 2. The values in fig. 2 are obtained by plotting the P-log (10) logarithm of the effect of the multiwall carbon nanotubes on the single signal path, so that the more obvious the effect of the multiwall carbon nanotubes is, the smaller the P-value is, and the larger the corresponding P-log (10) logarithm is. Wherein the value of the processing group XFM4 is the largest, and the influence on the signal path is the largest.
3) TEM detection of macrophage morphology change after multi-wall carbon nanotube induction
The detection results are shown in FIG. 3. Wherein, the A group is the morphology of THP-1 macrophages of the control group. Group B, group C and group D were morphology of THP-1 macrophages of three treatment groups XFM4, XFM22 and XFM34, respectively, at 32 μg/mL. It can be seen that the treatment group cells each endocytose a large number of MWCNTs (multi-walled carbon nanotube material). And Multi-pore (phenomenon of cell membrane perforation) occurs in the XFM4 treated group.
4) Western blot (Western blotting) method for detecting change of macrophage protein expression level after induction of multiwall carbon nanotube
The results of the detection are shown in Table 2 and FIG. 4. Wherein, the macrophage protein expression quantity of the control group is set as 100%, and the protein expression quantity of the treatment group is changed in percentage relative to the control group. Comparison shows that the percentage change of macrophage protein expression level relative to the control group is the largest and the difference is significant in the XFM4 treated group.
TABLE 2 macrophage NLRP3 protein expression level
| Control group (Control) | Processing group (XFM 4) | Processing (XFM 22) | Processing group (XFM 34) | |
| Pro-NLRP3 | 100±4.64 | 377.27±7.72 | 281.82±3.44 | 200±6.33 |
In view of the above, the greater the effect of the signaling pathway associated with the immune system/inflammatory response, the greater the extent of apoptosis. The phenomenon of cell membrane perforation is an important marker of cell coke death. Therefore, XFM4 in the treatment group with a concentration of 32. Mu.g/mL had the best effect of inducing apoptosis.
2. Effect of NLRP3siRNA molecules on THP-1 macrophage pyrodeath
1) RT-PCR (fluorescence quantitative real-time PCR) detection adopts NLRP3siRNA molecules from one to six combinations to transfect THP-1 macrophages, and the mRNA expression level of NLRP3 genes can reflect the NLRP3 gene inhibition efficiency. The test results are shown in Table 3. As can be seen from table 3, the NLRP3 gene expression can be greatly inhibited after transfection with the NLRP3siRNA molecules of combination one to six. Wherein, the inhibition rate of NLRP3siRNA molecules adopting the sequences shown in the combination IV to NLRP3 genes is higher than 90%, and the effect is best.
2) CCK8 detection of macrophage mitochondrial Activity after Multi-walled carbon nanotube Induction following transfection with NLRP3siRNA molecule
The test results are shown in Table 4 and FIG. 5. Wherein, the activity of mitochondria of the control group is set as 100%, and the activity percentage data of the treatment group is the percentage change relative to the control group. It can be seen that the percentage of macrophage mitochondrial activity in the XFM4 treated group intersected by the control group with a large variation and a significant difference.
TABLE 3 inhibition of intracellular NLRP3 Gene
TABLE 4 mitochondrial Activity of THP-1 cells (%)
3) Western blot (Western blotting) detection of macrophage protein expression level after multi-wall carbon nanotube induction after transfection of NLRP3siRNA molecules by combination four
TABLE 5 macrophage protein expression level after multiwall carbon nanotube induction following NLRP3siRNA molecule transfection
The detection results are shown in Table 5, FIG. 6 and FIG. 7. Wherein, the macrophage protein expression quantity of the control group is set as 100%, and the protein expression quantity of the treatment group is changed in percentage relative to the control group. It can be seen that the percentage change in macrophage protein expression level was greatest in the XFM4 treated group relative to the control group, and the difference was significant.
The results show that the 6 combined siRNA molecule sequences can inhibit the expression of NLRP3 genes and related proteins and improve the activity of mitochondria.
The foregoing describes specific embodiments of the present disclosure. Embodiments thereof are within the scope of the following claims. In some cases, the actions or steps recited in the claims can be performed in a different order than in the embodiments and still achieve desirable results. In addition, the processes depicted in the accompanying figures do not necessarily require the particular order shown, or sequential order, to achieve desirable results.
Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined within the idea of the invention, the steps may be implemented in any order and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.
Where specific details are set forth in order to describe example embodiments of the invention, it should be apparent to one skilled in the art that the invention can be practiced without, or with variation of, these specific details. Accordingly, the description is to be regarded as illustrative in nature and not as restrictive.
While the invention has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations of those embodiments will be apparent to those skilled in the art in light of the foregoing description.
The embodiments of the invention are intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Therefore, any omission, modification, equivalent replacement, improvement, etc. of the present invention should be included in the scope of the present invention.
Sequence listing
<110> university of Hunan traditional Chinese medicine
<120> an siRNA molecule for inhibiting NLRP3 gene expression and application thereof
<130> FI200480-ND
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<212> RNA
<223> Synthesis by hand
<400> 10
uucucauaaa ggucucuccu g 21
<210> 11
<211> 19
<212> RNA
<223> Synthesis by hand
<400> 11
ggauugaagu gaaagccaa 19
<210> 12
<211> 21
<212> RNA
<223> Synthesis by hand
<400> 12
uuggcuuuca cuucaaucca u 21
Claims (1)
1. An application of an siRNA molecule for inhibiting NLRP3 gene expression in vitro to inhibit NLRP3 gene expression, which is characterized in that the inhibition of NLRP3 gene expression is inhibition of the mRNA expression of NLRP3 gene of THP-1 macrophage; the siRNA molecule comprises a sense strand and an antisense strand, the sequence of the siRNA molecule comprising a combination of:
the nucleotide sequence of the sense strand is shown as SEQ ID NO. 7; and
the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 8;
wherein the sequence of the siRNA molecule has methoxy modification and fluoro modification, and the methoxy modification is ribose 2' -hydroxy in methoxy substituted nucleotide; the fluoro modification is ribose 2' -hydroxyl in fluoro substituted nucleotide;
1 st, 2 nd, 3 rd, 4 th, 6 th, 10 th, 11 th, 12 th, 13 th, 14 th, 15 th, 16 th, 17 th, 18 th and 19 th of the sense strand of the sequence of the siRNA molecule are methoxy-modified nucleotides in a5 '-end to 3' -end direction; fluoro-modified nucleotides at positions 5, 7, 8 and 9; 1 st, 3 rd, 4 th, 5 th, 7 th, 10 th, 12 th, 13 th, 15 th, 17 th, 18 th and 19 th of the antisense strand of the sequence of the siRNA molecule are methoxy-modified nucleotides; fluoro-modified nucleotides at positions 2, 6, 8, 9, 14 and 16;
the sequence of the siRNA molecule also has a phosphorothioate group modification in which at least one oxygen atom in the phosphodiester bond in the phosphate group is replaced with a sulfur atom; both the sense strand and the antisense strand of the sequence of the siRNA molecule have phosphorothioate modification sites; the phosphorothioate modification site of the sense strand is at least one of between nucleotide 1 and nucleotide 2 of the 5 'terminal end, and between nucleotide 2 and nucleotide 3 of the 5' terminal end;
the phosphorothioate modification site of the antisense strand is at least one of the following positions: between nucleotide 1 and nucleotide 2 of the 5' terminal end; between nucleotide 2 and nucleotide 3 of the 5' terminal end; between nucleotide 1 and nucleotide 2 of the 3 'terminal end and between nucleotide 2 and nucleotide 3 of the 3' terminal end.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110575436A (en) * | 2019-08-23 | 2019-12-17 | 华中科技大学同济医学院附属梨园医院 | NLRP3 inhibitor composition and application thereof |
| CN111377985A (en) * | 2018-12-29 | 2020-07-07 | 苏州瑞博生物技术有限公司 | Compounds and conjugates and methods of making and using the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111377985A (en) * | 2018-12-29 | 2020-07-07 | 苏州瑞博生物技术有限公司 | Compounds and conjugates and methods of making and using the same |
| CN110575436A (en) * | 2019-08-23 | 2019-12-17 | 华中科技大学同济医学院附属梨园医院 | NLRP3 inhibitor composition and application thereof |
Non-Patent Citations (2)
| Title |
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| 基于NLRP3炎性小体介导的三甲基锡所致中枢损伤作用机制研究;龙剑海;《中国博士学位论文全文数据库（电子期刊）医药卫生科技辑》;第53页 * |
| 幽门螺杆菌经ROS通路激活NLRP3炎症复合体诱导THP-1细胞分泌IL-1β和IL-18;李翔等;《中国免疫学杂志》;第31卷(第3期);材料与方法1.2.7部分 * |
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