CN112063624A

CN112063624A - SiRNA molecule for inhibiting caspase1 gene expression and application thereof

Info

Publication number: CN112063624A
Application number: CN202010990803.1A
Authority: CN
Inventors: 黄渊余; 曹毅; 夏新华; 翁郁华; 郭帅; 戴旭艳
Original assignee: Hunan University of Chinese Medicine
Current assignee: Hunan University of Chinese Medicine
Priority date: 2020-09-19
Filing date: 2020-09-19
Publication date: 2020-12-11

Abstract

The invention discloses a siRNA molecule for inhibiting caspase1 gene expression and application thereof. The siRNA molecule is any one of 5 sequence combinations, and comprises a double-chain structure, and nucleotides on two chains are chemically modified. The siRNA molecule provided by the invention can specifically target a caspase1 gene, efficiently inhibit the expression of the caspase1 gene and related proteins, improve the activity of mitochondria, and can be used for preparing medicines for treating cancers, inflammations and the like caused by the overexpression of the caspase1 gene.

Description

SiRNA molecule for inhibiting caspase1 gene expression and application thereof

Technical Field

The invention relates to the technical field of nucleic acid pharmacy, in particular to a siRNA molecule for inhibiting caspase1 gene expression and application thereof.

Background

Small interfering RNA (siRNA molecule) is an emerging nucleic acid drug that has been clinically validated, and is a double-stranded short RNA molecule whose antisense strand can be complementarily paired with a specific mRNA strand, thereby triggering sequence-specific gene expression inhibition (gene silencing), that is, RNA interference (RNAi). Currently, 2 kinds of siRNA molecular drugs are approved to be marketed (Onpattero, Givlaari) all over the world. In theory, siRNA molecules can be designed, screened for any gene and expression of the gene inhibited.

Caspase1, a regulator of inflammation, has been shown to be a key link in inflammatory regulation, apoptosis, and apoptosis. Studies have shown that caspase1 is widely involved in the progression of systemic diseases such as cardiovascular diseases, metabolic diseases, cancer, etc.

Apoptosis is a newly discovered mode of apoptosis that relies on caspase1 activation. Cell scorching is accompanied by membrane pore formation, fluid influx, cell swelling, cytolysis, eventually plasma membrane rupture and leakage of cell contents. In a classical cell apoptosis pathway, receptor proteins such as an endocytotic cell recognition receptor 3(NLRP3) and the like sense extracellular stimulation signals, a pro-caspase1 precursor of caspase1 and an endocytotic cell recognition receptor (NLR) and the like form a protein complex to activate caspase1, and the activated caspase is converted into an activated state by cutting GSDMD protein so as to cause cell apoptosis. The cell apoptosis plays an important role in the innate immunity and the occurrence and development of tumors, and the deep exploration of the relationship between the cell apoptosis and the tumors is beneficial to providing a new idea for the occurrence, development and new prevention and treatment of the tumors. Meanwhile, the siRNA molecule is used for inhibiting the expression level of caspase1, so that the cell apoptosis can be rapidly and directly prevented, and the diseases caused by the cell apoptosis can be prevented and treated. And the siRNA molecule only acts on mRNA of a target gene and does not influence the transcription and expression of other genes, so that the siRNA molecule mediated nucleic acid therapy has the characteristics of safety and high efficiency.

Disclosure of Invention

In view of the above, the present invention provides an siRNA molecule for inhibiting caspase1 gene expression and its application, aiming at solving one of the technical problems of the related researches to a certain extent. Based on the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an siRNA molecule for inhibiting caspase1 gene expression, wherein the siRNA molecule comprises a sense strand and an antisense strand, and the sequence of the siRNA molecule comprises one of the following combinations:

the combination is as follows:

(1) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 1; and

(2) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 2; or

(3) A nucleotide sequence with at least 85% of identity with the nucleotide sequence shown in (1) or (2);

or

Combining two:

(4) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 3; and

(5) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 4; or

(6) A nucleotide sequence with at least 85% of identity with the nucleotide sequence shown in (4) or (5);

or

Combining three components:

(7) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 5; and

(8) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 6; or

(9) A nucleotide sequence having at least 85% identity to the nucleotide sequence shown in (7) or (8);

or

And (4) combining:

(10) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 7; and

(11) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 8; or

(12) A nucleotide sequence having at least 85% identity to the nucleotide sequence shown in (10) or (11); or

And (5) combining:

(13) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 9; and

(14) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 10; or

(15) And (2) a nucleotide sequence having at least 85% identity to the nucleotide sequence shown in (13) or (14).

In the embodiment of the invention, the sequence of the siRNA molecule comprises one of the following combinations:

the combination is as follows:

(1) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 1; and

(2) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 2;

or

Combining two:

(4) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 3; and

(5) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 4;

or

Combining three components:

(7) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 5; and

(8) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 6;

or

And (4) combining:

(10) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 7; and

(11) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 8;

or

And (5) combining:

(13) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 9; and

(14) and the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 10.

In an embodiment of the invention, the sequence of the siRNA molecule is combination three.

According to an embodiment of the invention, the sequence of the siRNA molecule has methoxy modification and fluoro modification, the methoxy modification is methoxy substituted for ribose 2' -hydroxyl in nucleotide; the fluoro modification is that the fluoro replaces ribose 2' -hydroxyl in nucleotide.

According to an embodiment of the present invention, 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 are methoxy modified nucleotides in the direction from the 5 'end to the 3' end; fluoro-modified nucleotides at positions 5, 7, 8 and 9; 1 st, 3, 4, 5, 7, 10, 12, 13, 15, 17, 18 and 19 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.

According to an embodiment of the invention, the sequence of the siRNA molecule further has a phosphorothioate-based modification in which at least one oxygen atom in a phosphodiester bond in a phosphate group is replaced by a sulfur atom.

According to an embodiment of the invention, both the sense and antisense strands of the sequence of the siRNA molecule have a phosphorothioate-based modification site;

the modification site of the sense strand is at least one of between the 1 st nucleotide and the 2 nd nucleotide at the end of the 5 'end, and between the 2 nd nucleotide and the 3 rd nucleotide at the end of the 5' end;

the modification site of the antisense strand is at least one of the following positions: between the 1 st and 2 nd nucleotides at the end of the 5' terminus; between the 2 nd and 3 rd nucleotides at the 5' terminal end; between the 1 st and 2 nd nucleotides at the 3 'terminal end and between the 2 nd and 3 rd nucleotides at the 3' terminal end.

In embodiments of the invention, the siRNA molecules are capable of inhibiting expression of the mRNA and protein levels of the caspase1 gene.

In a second aspect, the present invention provides a method of inhibiting the expression of a gene which is caspase1, the method comprising transfecting a cell with an siRNA molecule having a sequence as described in any one of the first aspect of the invention so as to inhibit the expression of the caspase1 gene in said cell. Methods of transfecting cells include, but are not limited to: methods of electroporation, cationic polymer agent methods, exosome delivery methods, ionizable liposome delivery methods, and the like. For example, a commercial cationic polymer transfection reagent Lipofectamine 2000 can be used, and a polypeptide carrier synthesized in a laboratory, a high molecular polymer carrier, a carbon nanotube, a metal nano-carrier, various organic/inorganic hybrid carriers and the like can also be used.

In the present example, the siRNA molecule was transfected into the interior of the cell by Lipofectamine 2000 and exerted the effect of inhibiting gene expression.

In a third aspect, the present invention provides an siRNA molecule medicament for inhibiting caspase1 gene expression, comprising an effective amount of an siRNA molecule according to the first aspect of the present invention and a pharmaceutically acceptable carrier. The medicine can be prepared into different preparations by using a conventional method, for example, normal saline or an aqueous solvent containing glucose and other auxiliary agents can be prepared into injections by using a conventional method. The different prepared drugs can be administered in any convenient form, for example, by different routes such as topical, intravenous, intramuscular, subcutaneous, intradermal, intraarticular, intrathecal injection, etc. The dosage of the medicine can be adjusted according to the actual situation.

In a fourth aspect, the present invention provides an application of an siRNA molecule in preparation of a drug for inhibiting caspase1 gene expression, wherein the siRNA molecule is the siRNA molecule described in any one embodiment of the first aspect of the present invention.

In the embodiment of the invention, the medicine for inhibiting caspase1 gene expression is a medicine for treating exogenous induced cell apoptosis, spontaneous inflammation, inflammatory injury of normal tissues or cancer.

In the embodiment of the invention, the medicine for inhibiting caspase1 gene expression is an anti-cell apoptosis medicine.

As can be seen from the above, the siRNA molecule for inhibiting caspase1 gene expression provided by the invention comprises any one of sequence combinations from one to five, can effectively inhibit the expression of caspase1 gene and related protein, and improves the activity of cell mitochondria. The siRNA molecule can be used for preparing a medicament for inhibiting the expression of gene caspase1, and the medicament can be used for resisting cell apoptosis; treatment of spontaneous inflammation, inflammatory injury of normal tissues and cancer.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph of the effect of multi-walled carbon nanotubes on macrophage mitochondrial activity in an embodiment of the invention;

FIG. 2 is a graph illustrating the effect of multi-walled carbon nanotubes on macrophage signaling pathways in an embodiment of the present invention;

FIG. 3 is a graph showing the effect of multi-walled carbon nanotubes on macrophage morphology in accordance with an embodiment of the present invention;

FIG. 4 is a graph showing the effect of multi-walled carbon nanotubes on macrophage protein expression levels in an embodiment of the present invention;

FIG. 5 shows the results of mitochondrial activity measurements after THP-1 macrophages were transfected with 5 combined caspase1 siRNA molecules according to the present invention;

FIG. 6 shows the results of detecting caspase protein expression levels after THP-1 macrophages are transfected with caspase1 siRNA molecules combining with the third embodiment of the present invention;

FIG. 7 shows the result of detecting the expression level of NLRP3 protein after THP-1 macrophage transfection by caspase1 siRNA molecule combined with three in the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings.

It is to be noted that technical terms or scientific terms used in the embodiments of the present invention should have the ordinary meanings as understood by those having ordinary skill in the art to which the present disclosure belongs, unless otherwise defined. The use of "first," "second," and similar terms in this disclosure is not intended to indicate any order, quantity, or importance, but rather is used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that the element or item listed before the word covers the element or item listed after the word and its equivalents, but does not exclude other elements or items. The terms "connected" or "coupled" and the like are not restricted to physical or mechanical connections, but may include electrical connections, whether direct or indirect.

The embodiment of the invention provides an siRNA molecule (Small interfering RNA) for inhibiting caspase1 gene expression, which comprises a sense strand and an antisense strand, and comprises one of the following combinations.

The combination is as follows:

(1) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 1; and

(2) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 2; or

or

Combining two:

(4) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 3; and

(5) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 4; or

or

Combining three components:

(7) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 5; and

(8) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 6; or

or

And (4) combining:

(10) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 7; and

And (5) combining:

(13) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 9; and

In one embodiment, the sequence of the siRNA molecule comprises one of the following combinations:

the combination is as follows:

(1) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 1; and

(2) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 2;

or

Combining two:

(4) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 3; and

(5) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 4;

or

Combining three components:

(7) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 5; and

(8) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 6;

or

And (4) combining:

(10) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 7; and

(11) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 8;

or

And (5) combining:

(13) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 9; and

Wherein, the siRNA molecule inhibits the activation of GSDMD protein by inhibiting the expression of caspase1 gene.

TABLE 1 caspase1 siRNA molecular sequences

In the sequences shown in SEQ ID NO.1 to SEQ ID NO.10, the siRNA molecules are all double-stranded structures including a sense strand and an antisense strand. The sense strand consists of 19 nucleotides and the antisense strand consists of 21 nucleotides. Positions 1-19 of the nucleotide sequence of the antisense strand are completely complementary to the nucleotide sequence of the sense strand of the same siRNA molecule in the direction from the 5 'end to the 3' end.

Preferably, in the sequences shown in SEQ ID NO.1 to SEQ ID NO.10, each of the nucleotides of the sense and antisense strands is a modified nucleotide. The stability of the caspase1 expression inhibitor can be improved and the immunogenicity can be reduced by modifying the nucleotide, so that the caspase1 expression inhibitor can be directly used for preparing a medicine for inhibiting the expression of the gene caspase 1.

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 of ribose 2' -hydroxyl in the nucleotide. That is, the methoxy modification is to replace the ribose 2' -hydroxyl group in the nucleotide with a methoxy group; the fluoro modification is that the fluoro replaces ribose 2' -hydroxyl in nucleotide.

Wherein, the nucleotides at 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 positions of the nucleotide sequence sense strand are methoxy modified nucleotides, and the nucleotides at the 5 th, 7 th, 8 th and 9 th positions of the sense strand are fluoro modified nucleotides. The nucleotide at the 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 positions of the antisense strand of the nucleotide sequence is a methoxy modified nucleotide, and the nucleotide at the 2 nd, 6 th, 8 th, 9 th, 14 th and 16 th positions of the antisense strand of the nucleotide sequence is a fluoro modified nucleotide. Through respectively carrying out different modifications on the ribose 2 '-hydroxyl in the nucleotide of the site of the sense strand and matching with different modifications on the corresponding site of the ribose 2' -hydroxyl in the nucleotide of the antisense strand, the resistance of the siRNA molecule to nuclease can be improved, the stability of the siRNA molecule can be improved, and meanwhile, the immunogenicity and the safety of the siRNA molecule can be reduced.

Further, in the sequences shown in SEQ ID NO.1 to SEQ ID NO.10, modification of phosphorothioate group is also included, and both the sense strand and the antisense strand have a phosphorothioate group modification site. The phosphorothioate group is modified such that at least one oxygen atom in a phosphodiester bond in the phosphate group is substituted with a sulfur atom.

Wherein the modification site of the phosphorothioate group of the sense strand is at least one of between the 1 st and 2 nd nucleotides at the 5 'terminal end, and between the 2 nd and 3 rd nucleotides at the 5' terminal end.

The degradation effect of enzyme can be greatly influenced by adopting P-S bond to replace the sulfo-modification of P-O bond at the corresponding sites of the sense strand and the antisense strand, the nuclease resistance of the siRNA molecule is improved, and the stability of the siRNA molecule is improved.

Preferably, the sequence of the siRNA molecule is shown as SEQ ID NO.5 and 6. With this sequence, the substitution of the methoxy group at the specific site mentioned above for the ribose 2' -hydroxy group in the nucleotide, the substitution of the ribose 2' -hydroxy group in the fluorine-substituted nucleotide, the substitution of the P-S bond for the thio modification between the 1 st and 2 nd nucleotides and the 2 nd and 3 rd nucleotides at the 5' -terminal end of the sense strand by the P-O bond, P-S bonds replace P-O bonds to perform thio modification on the 1 st nucleotide and the 2 nd nucleotide at the end part of the 5 'end of the antisense strand and between the 2 nd nucleotide and the 3 rd nucleotide, and P-S bonds replace P-O bonds to perform thio modification on the 1 st nucleotide and the 2 nd nucleotide and between the 2 nd nucleotide and the 3 rd nucleotide at the end part of the 3' end of the antisense strand, so that the good inhibition effect on caspase1 genes can be achieved.

The embodiment of the invention also provides a method for inhibiting the expression of a gene, wherein the gene is a caspase1 gene, and the method comprises the step of transfecting a cell by using the siRNA molecule for inhibiting the expression of the caspase1 gene to inhibit the expression of the caspase1 gene in the cell.

The embodiment of the invention also provides a medicament for inhibiting gene expression, which comprises an effective amount of the siRNA molecule for inhibiting caspase1 gene expression and a pharmaceutically acceptable carrier.

The embodiment of the invention also provides application of the siRNA molecule for inhibiting caspase1 gene expression in preparation of drugs for inhibiting caspase1 gene expression, wherein the siRNA molecule has the sequence of the siRNA molecule for inhibiting caspase1 gene expression as described in any one of the above. The drug may specifically be an siRNA molecule. Generally, the above caspase1 gene inhibiting drugs are generally used in the form of pharmaceutical compositions in the present invention. The pharmaceutical composition can improve or treat related diseases by inhibiting the expression of caspase1 gene. The drug for inhibiting caspase1 gene expression can be an siRNA molecular drug, and comprises the caspase1 siRNA 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 are suitable for applying the siRNA molecules of the present invention to an individual. The pharmaceutically acceptable carriers include various solutions, diluents, solvents, dispersants, liposomes, emulsions, sugar coatings, antibacterial agents, antifungal agents, and the like, and other carriers suitable for use with the siRNA molecules of the present invention. The carrier for injection 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 biologically neutral carriers, the pharmacological components employed 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 molecular drug for inhibiting caspase1 gene expression is a drug for resisting cell apoptosis, spontaneous inflammation, inflammatory injury of normal tissues or cancer.

Preferably, the siRNA molecular drug for inhibiting caspase1 gene expression is a drug for resisting cell apoptosis.

Specifically, the drug for resisting cell apoptosis enhances the mitochondrial activity of cells by inhibiting the expression of caspase1 related proteins, such as pro-caspase1 protein and NLRP 3.

The technical solution of the present invention will be further described with reference to the following embodiments.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Test materials: THP-1 cell lines were purchased from the American type culture Collection (Manassas, VA, USA). Multi-walled carbon nanotubes (MWCNTs) were purchased from Nanjing XFINANO materials technology, Inc. 1: 5000 β -actin antibody, 1: 5000HRP goat anti-rabbit IgG, purchased from Proteintetech, USA. Opti-MEM^TMLow serum medium, purchased from Thermo-Fisher. Opti-MEM^TMLow serum media was purchased from Thermo-Fisher.

Example 1

The performance of siRNA molecule determines the quality of the expression effect of drug inhibition gene caspase 1. Example 1 multiple groups of siRNA molecules were designed and screened against the mRNA sequence of caspase1, and as shown in table 1, five groups of excellent siRNA molecule combinations (combination one to five) and one group of optimal siRNA molecule combinations (combination three) were finally screened.

Since Table 2 is identical to Table 1, the siRNA molecule sequences in Table 1 of Table 2 are deleted and synthesized by Bexin Biotech, Suzhou, and the siRNA molecules are dissolved in DEPC water and stored at 4 ℃ for use. In this example, the detection index of human caspase1 Gene (Gene ID: 834, updated on 21-Jul-2020) was used as an example.

Meanwhile, in order to verify the gene inhibition and cell apoptosis reversal effects of the siRNA molecules, the following experimental examples were performed.

Experimental example 1 culture and induced differentiation of THP-1 cells

1) THP-1 cell line cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.

2) Phorbol ester 12-myristate 13-acetate (PMA; sigma, St.Louis, MO, USA) for 24 hours, and differentiating THP-1 cells into macrophages to obtain THP-1 macrophages.

Experimental example 2 Multi-walled carbon nanotube MWCNT induces THP-1 macrophage apoptosis

Purpose of the experiment: and (3) screening the diameter of the multi-wall carbon nano tube with the best effect of inducing THP-1 macrophage scorching.

Induction conditions are as follows: the diameters of the multi-walled carbon nanotubes were arranged into three treatment groups, named XFM4, XFM22 and XFM34, respectively, and the diameters of each treatment group were different in the order XFM4< XFM22< XFM 34. Each treatment group was set with 6 different concentrations of 0, 4, 8, 16, 32 and 64. mu.g/mL. The induction treatment time was 24 hours.

The induction method comprises the following steps: the 6 concentrations of the three treatment groups XFM4, XFM22 and XFM34 were suspended in 2% FBS after continuous sonication for 16 minutes using a sonicator, respectively.

THP-1 macrophages were plated at 2.4X 10 per well⁵The cell density of (a) was injected into 24-well plates separately and contacted directly with 6 concentrations of XFM4, XFM22 and XFM34, respectively, for 24 hours, with 3 duplicate wells per group.

Experimental example 3 caspase1 siRNA molecule transfection THP-1 macrophage inhibited caspase1 gene expression, reversed cell apoptosis.

The transfection method comprises the following steps: THP-1 macrophages were plated at 7X 10 per well⁵The cell density of (1) was seeded in 6-well plates, and caspase1 siRNA molecules of combination one to five, chemically modified at 40nM, were diluted in 0.5mL of Opti-MEM, respectively^TMIn low serum medium, 3. mu.L of the LLIPOFECTAMINE 2000 reagent was also diluted in 0.5mL of Opti-MEM^TMAfter each standing for 5min, the siRNA molecules and Lipofectamine 2000 were mixed well to prepare a treatment group. Standing at room temperature for 10min, finally adding the mixed solution into a cell culture plate for cell transfection, adding 1mL complete culture medium into each hole after 4 hours, and continuing transfection for 24 hours. The control group was a group to which no siRNA molecule was added, and the treatment group and the control group were each 3 duplicate wells.

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 for induction of apoptosis.

The detection method comprises the following steps: after the induction is finished, the activity of the cells is detected by using a CCK-8 method. Use of

Extracting total RNA, reverse transcription and carrying out fluorescence real-time quantitative PCR detection. Western blot (Western blot) was used to detect the cellular protein expression levels. It should be noted that the specific detection methods are respectively the same as the detection method in experimental example 2, and are not described herein again.

Analysis of results

1. Results of THP-1 macrophage apoptosis induced by multiwalled carbon nanotube

1) CCK-8 method for detecting activity of macrophage mitochondria after multi-walled carbon nanotube induction

The results of the detection are shown in FIG. 1. It can be seen that the concentration of XFM4, XFM22 and XFM34 is most suitable for the treatment groups with different concentrations, and the influence of the concentration on the activity of cells is remarkably different from that of the treatment groups with the rest concentrations, and the differences are stable.

2) RNA sequencing detection of influence of multi-walled carbon nanotubes on macrophage signal path

The detection results are shown in FIG. 2. The figure 2 plots the P-log (10) logarithm of the effect of the multi-walled carbon nanotubes on a single signal path, so that the more pronounced the effect of the multi-walled carbon nanotubes, the smaller the P-log, and the greater the corresponding value obtained after the log (10) logarithm. The maximum value of the processing group XFM4 is the greatest, and the influence on the signal path is the greatest.

3) TEM observation of macrophage morphology change after multi-walled carbon nanotube induction

The results of the detection are shown in FIG. 3. Wherein, the group A is the form of the THP-1 macrophage of the control group. The morphology of THP-1 macrophages from three treatment groups, XFM4, XFM22 and XFM34, were 32 μ g/mL for group B, group C and group D, respectively. As can be seen, the treated cells all endocytosed large amounts of MWCNTs (multi-walled carbon nanotube material). And Multi-spots (phenomenon of cell membrane perforation) appeared in the XFM4 treatment group.

4) Western blot method for detecting change of macrophage protein expression level after induction of multi-wall carbon nano-tube

The results of the measurements are shown in table 2 and fig. 4. Wherein the macrophage protein expression level of the control group is set as 100%, and the protein expression level of the treatment group is percentage change relative to the control group. Compared with the XFM4 treated group, the percent change of the macrophage protein expression quantity relative to the control group is the largest and the difference is obvious.

Taken together, the signal pathway is associated with the immune system/inflammatory response, with greater effect on the signal pathway representing greater degree of cellular apoptosis. The phenomenon of cell membrane perforation is an important marker of cell scorching. Therefore, treatment group XFM4 at a concentration of 32. mu.g/mL had the best effect on induction of apoptosis.

TABLE 2 macrophage caspase 1-related protein expression levels

	Control group (Control)	Treatment group (XFM4)	Treatment (XFM22)	Treatment group (XFM34)
					Cleaved caspase 1	100±2.46	385.55±3.85	177.97±5.47	142±4.63

2. Effect of caspase1 siRNA molecules on THP-1 macrophage apoptosis

1) The RT-PCR (fluorescent quantitative real-time PCR) method detects the mRNA expression level of the pro-caspase1 gene after the THP-1 macrophage is transfected by the caspase1 siRNA molecules combined from one to five, and can reflect the inhibition efficiency of the caspase1 gene.

The results are shown in Table 3. As can be seen from Table 3, the expression of pro-caspase 1mRNA was greatly inhibited after transfection with caspase1 siRNA molecules combining one to five. Wherein, the inhibition rate of the caspase1 siRNA molecule with the sequence shown in the three combinations on the pro-caspase1 gene is higher than 80%, and the effect is the best.

TABLE 3 THP-1 cells pro-caspase 1mRNA inhibitory Effect

2) The CCK-8 method detects the activity of macrophage mitochondria after caspase1 siRNA molecule transfection and multi-wall carbon nano-tube induction.

The results are shown in Table 4 and FIG. 5. Wherein the activity of mitochondria of the control group is set as 100%, and the data of the activity percentage of the treatment group is the percentage change relative to the control group. It can be seen that the percent mitochondrial activity of macrophages in the XFM 4-treated group was significantly increased compared to the control group, and the cellular activity was highest in the combination of 3-sequence siRNA molecules.

3) And detecting the expression level of macrophage protein after combined three-caspase 1 siRNA molecule transfection and multi-wall carbon nano tube induction by a Western blot method.

The results are shown in table 5, fig. 6 and fig. 7. Wherein the macrophage protein expression level of the control group is set as 100%, and the protein expression level of the treatment group is percentage change relative to the control group. It can be seen that the percent of macrophage protein expression in the XFM 4-treated group relative to the control group varied greatly and differed significantly.

TABLE 4 THP-1 cell mitochondrial Activity (%)

TABLE 5 THP-1 cell protein expression levels following caspase1 siRNA molecule transfection, MWCNT induction

The results show that the 5 combined siRNA molecular sequences can inhibit the expression of caspase1 genes and related proteins and improve the activity of mitochondria.

The foregoing description has been directed to specific embodiments of this disclosure. Embodiments thereof are within the scope of the following claims. In some cases, the actions or steps recited in the claims may 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 understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, 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 will 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 instead of restrictive.

While the present invention has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations of these embodiments will be apparent to those of ordinary skill in the art in light of the foregoing description.

The embodiments of the invention are intended to embrace all such alternatives, modifications and variances that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.

Sequence listing

<110> Hunan university of traditional Chinese medicine

<120> siRNA molecule for inhibiting caspase1 gene expression and application thereof

<130> FI200479-ND

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 19

<212> RNA

<223> Artificial sequence

<400> 1

gacauauuc cuagaagaa 19

<210> 2

<211> 21

<212> RNA

<223> Artificial sequence

<400> 2

uucuucuagg aauacuguaa a 21

<210> 3

<211> 19

<212> RNA

<223> Artificial sequence

<400> 3

guucaugucu caugguauu 19

<210> 4

<211> 21

<212> RNA

<223> Artificial sequence

<400> 4

aauaccauga gacaugaaca c 21

<210> 5

<211> 19

<212> RNA

<223> Artificial Synthesis

<400> 5

gaagagaucc uucuguaaa 19

<210> 6

<211> 21

<212> RNA

<223> Artificial Synthesis

<400> 6

uuuacagaag caucucuuca c 21

<210> 7

<211> 19

<212> RNA

<223> Artificial Synthesis

<400> 7

gaaagaguga cuuugacaa 19

<210> 8

<211> 21

<212> RNA

<223> Artificial Synthesis

<400> 8

uugucaaagu cacucuuuca g 21

<210> 9

<211> 19

<212> RNA

<223> Artificial Synthesis

<400> 9

gagauauacu acaacucaa 19

<210> 10

<211> 21

<212> RNA

<223> Artificial Synthesis

<400> 10

uugaguugua guauaucugg g 21

Claims

1. An siRNA molecule for inhibiting caspase1 gene expression, wherein the siRNA molecule comprises a sense strand and an antisense strand, and the sequence of the siRNA molecule comprises one of the following combinations:

the combination is as follows:

(1) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 1; and

(2) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 2; or

or

Combining two:

(4) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 3; and

(5) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 4; or

or

Combining three components:

(7) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 5; and

(8) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 6; or

or

And (4) combining:

(10) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 7; and

(12) A nucleotide sequence having at least 85% identity to the nucleotide sequence shown in (10) or (11);

or

And (5) combining:

(13) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 9; and

2. An siRNA molecule according to claim 1, wherein the sequence of said siRNA molecule comprises one of the following combinations:

the combination is as follows:

(1) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 1; and

(2) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 2;

or

Combining two:

(4) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 3; and

(5) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 4;

or

Combining three components:

(7) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 5; and

(8) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 6;

or

And (4) combining:

(10) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 7; and

(11) the nucleotide sequence of the antisense chain is shown as SEQ ID NO. 8;

or

And (5) combining:

(13) the nucleotide sequence of the sense chain is shown as SEQ ID NO. 9; and

3. An siRNA molecule according to claim 1, wherein the sequence of said siRNA molecule is a combination of three.

4. An siRNA molecule according to claim 1, wherein the sequence of said siRNA molecule has methoxy modification and fluoro modification, said methoxy modification being a methoxy substituted for ribose 2' -hydroxyl in nucleotide; the fluoro modification is that the fluoro replaces ribose 2' -hydroxyl in nucleotide.

5. An siRNA molecule according to claim 4, characterized in that 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 of the sense strand of the sequence of said siRNA molecule are methoxy modified nucleotides in the 5 'end to 3' end direction; fluoro-modified nucleotides at positions 5, 7, 8 and 9; 1 st, 3, 4, 5, 7, 10, 12, 13, 15, 17, 18 and 19 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.

6. An siRNA molecule according to claim 1, wherein the sequence of said siRNA molecule further has a phosphorothioate modification in which at least one oxygen atom in a phosphodiester bond in a phosphate group is replaced by a sulfur atom.

7. An siRNA molecule according to claim 6, characterized in that both the sense and antisense strands of the sequence of said siRNA molecule have a phosphorothioate-based modification site;

the phosphorothioate-based modification site of the sense strand is at least one of between the 1 st and 2 nd nucleotides at the 5 'terminal end, and between the 2 nd and 3 rd nucleotides at the 5' terminal end;

the phosphorothioate-based modification site of the antisense strand is at least one of the following positions: between the 1 st and 2 nd nucleotides at the end of the 5' terminus; between the 2 nd and 3 rd nucleotides at the 5' terminal end; between the 1 st and 2 nd nucleotides at the 3 'terminal end and between the 2 nd and 3 rd nucleotides at the 3' terminal end.

8. A method of inhibiting the expression of a gene wherein the gene being inhibited is a caspase1 gene, said method comprising transfecting a cell with an siRNA molecule of any one of claims 1 to 7 to inhibit the expression of said gene in said cell.

9. A medicament for inhibiting caspase1 gene expression, comprising an effective amount of the siRNA molecule of any one of claims 1-7 and a pharmaceutically acceptable carrier.

10. An application of siRNA molecule for inhibiting caspase1 gene expression in preparation of drugs for inhibiting caspase1 gene expression, wherein the siRNA molecule is the siRNA molecule in any one of claims 1-7.