CN107177598B - Oligonucleotide molecule for inhibiting mRNA expression of BIRC5 target gene and its composition set - Google Patents

Abstract

The invention discloses an oligonucleotide molecule for inhibiting mRNA expression of a BIRC5 target gene and a complete set of composition thereof. The invention provides siRNA, which consists of a sense strand consisting of 19-27 nucleotides and an antisense strand reverse-complementary to the sense strand; 5-9 consecutive nucleotides from the 5 ' end and 5-9 consecutive nucleotides from the 3 ' end of the sense strand are both 2 ' -ribo-modified nucleotides. The mixture of siRNA molecules in the invention can affect the expression of target genes in at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% of cells, and the inhibition rate is at least 45%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%.

Description

Oligonucleotide molecule for inhibiting mRNA expression of BIRC5 target gene and its composition set

The application is a divisional application of an invention patent application with the name of 'an oligonucleotide molecule for inhibiting the expression of mRNA of a target gene and a complete set composition thereof', which is 2016-08-18 on the filing date and 201610687850.2 on the filing date.

Technical Field

The invention relates to the field of molecular biology, in particular to an oligonucleotide molecule for inhibiting the expression of target gene mRNA and a complete set of composition thereof.

Background

Since the first discovery of RNAi phenomenon in nematodes (Caenorhabditis elegans) by Andrewfire and Craig Mello et al in 1998, research on the mechanistic principle, gene function and clinical application of RNAi has been advanced after Tuschl and Phil Sharp et al have demonstrated the existence of RNAi in mammals in 2001. RNAi plays a key role not only in defense against viral infection, anti-transposon jumping and other various body protection mechanisms (Huntvagner et al, 2001; Tuschl, 2001; Waterhouse et al, 2001; Zamore 2001), but also its related products are promising candidates.

Elbashir et al,2001, found that siRNA inhibited silencing of specific genes in mammalian cells, and studies showed that siRNA could specifically bind to and degrade target mRNA that is complementary to the sequence. The long-fragment double-stranded RNA is cleaved by Dicer enzyme into short-fragment RNA of 21-23 bases in length, wherein the strand bound to the target mRNA in both strands is called the antisense strand or guide strand, and the other strand is called the sense strand or passenger strand. Researches find that siRNA chemically synthesized in vitro also plays the role of RNAi after entering cells, and effectively reduces the immune response caused by long-chain RNA.

However, since the effectiveness of siRNA is affected by various factors such as sequence specificity, target cell specificity, target spot, etc., siRNA obtained based on the existing design principle cannot reach effective silencing effect; generally, more than 50% of siRNA has the effect of silencing target mRNA, and only 25% of siRNA has more than 75% silencing effect, so that the siRNA designed and synthesized subsequently needs experimental verification, screening or optimization, and is time-consuming and labor-consuming; based on this, a general, efficient and fast RNAi technology and product are in urgent need of development.

Disclosure of Invention

It is an object of the present invention to provide an siRNA that inhibits or reduces the expression of BIRC5 target gene.

The siRNA provided by the invention consists of a sense strand and an antisense strand which is reverse complementary (complete reverse complementary) to the sense strand;

the sense strand consists of 19-27 nucleotides, and the sense strand is 2 ' -O-ribose modified from 5-9 consecutive nucleotides from the 5 ' terminus and from 5-9 consecutive nucleotides from the 3 ' terminus.

The antisense strand is reverse complementary to a segment on the target gene, which is BIRC 5;

the base composition sequence of the sense strand is selected from SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO. 13;

the base composition sequence of the antisense chain is selected from SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO. 14.

The siRNA is reversely and complementarily combined with a target sequence on a target gene through an antisense strand thereof;

in some embodiments of the invention, the siRNA molecule includes at least one non-natural nucleotide, such as a chemically modified nucleotide, preferably a chemical modification in the sense strand or sense region. In some embodiments, the 2 '-O-ribose modification of ribose is specifically a 2' -O-methyl modification, a 2 '-O-fluoro modification, a 2' -MOE modification. Sense strand modifications of the invention can serve as: (1) enhancing the stability of siRNA molecules; (2) reducing off-target of siRNA molecules; (3) improving the specificity of siRNA molecules; (4) reducing immune activation reaction, etc.

In the above siRNA, the sense strand consists of 24, 25 or 26 nucleotides;

or, the sense strand is 2 ' -O-ribose modified from 6 or 7 or 8 consecutive nucleotides from the 5 ' terminus and 6 or 7 or 8 consecutive nucleotides from the 3 ' terminus.

In the siRNA, the 2 ' -O-ribose modification is a 2 ' -O-methyl (2 ' -O-Me) modification, a 2 ' -O-fluoro modification, or a 2 ' -MOE modification.

Another objective of the invention is to provide a siRNA set for inhibiting or reducing the expression of a target gene.

The invention provides a set of siRNA, comprising at least 5 siRNAs as described above.

Each siRNA corresponds to a different target sequence of the same target gene.

In the above siRNA kit, the siRNA kit comprises 5,6,7,8,9 or 10 siRNAs.

In the above siRNA set, the amount of each siRNA molecule in the siRNA set can be random, and the amount ratio of any 2 siRNA molecules is 1:1-1: 5; preferably, the amount of the substance of the individual siRNA molecules is equal to 1: 1.

Another object of the present invention is to provide another substance 1) or 2) as follows.

The invention provides 1) or 2) substance:

1) an agent that inhibits or reduces expression of a target gene, which is a or B:

a comprises the siRNA and transfection reagent;

b comprises the siRNA and transfection reagent set;

2) a kit for inhibiting or reducing expression of a target gene comprising an siRNA as described above or a set of sirnas as described above or said agent.

In the above substance, the total concentration of all siRNA molecules in the siRNA kit in the reagent is 2-100 nM; each siRNA molecule is present in the agent at a concentration of 10-20 nM;

the use of the above siRNA or the above siRNA set or the above substance in inhibiting or reducing the expression of a target gene is also within the scope of the present invention;

or the above siRNA set or the above substance in inhibiting or reducing the expression of a target gene in a cell is also within the scope of the present invention;

or the siRNA set or the substance in the product for inhibiting or reducing the expression of the target gene is also the protection scope of the invention;

or the siRNA set or the substance in the siRNA is used for preparing the product for inhibiting or reducing the expression of the target gene in the cell;

or the siRNA set or the substance in the product for preventing or relieving or treating diseases caused by the expression of the target gene is also the protection scope of the invention.

The method provided by the invention comprises the following steps: 2 ' -O-ribose modification is carried out on a sense strand of siRNA for inhibiting or reducing the expression of a target gene, wherein 5-9 continuous nucleotides from the 5 ' end and 5-9 continuous nucleotides from the 3 ' end are both subjected to modification;

the siRNA consists of a sense strand and an antisense strand reverse-complementary to the sense strand;

the sense strand consists of 19-27 nucleotides;

the antisense strand is reverse complementary to a segment on the target gene.

Or the 2 '-O-ribose modification is specifically a 2' -O-methyl modification, a 2 '-O-fluoro modification, or a 2' -MOE modification.

A fourth object of the invention is to provide a product.

The product provided by the invention comprises the siRNA or the siRNA set or the substance;

and/or the product has at least one function of 1) to 3) as follows:

1) inhibiting or reducing expression of a target gene;

2) inhibiting or reducing expression of a target gene in a cell;

3) preventing or alleviating or treating a disease caused by expression of a target gene.

In the above, the target gene is a tumor-, cancer-, cardiovascular-disease-, inflammation-, infectious-or rare-disease-related gene;

or, the tumor, cancer, cardiovascular disease, inflammation, infectious disease or rare disease-associated gene is specifically BIRC 5;

the cells are vertebrate cells, mammalian cells, primate cells, and human cells;

or, cancer cells, tumor cells, inflammatory cells, blood cells, leukocytes, brain cells, liver cells, lung cells, kidney cells, breast cells, cervical cells, endothelial cells, nerve cells, glial cells, or the like, wherein the target gene is expressed aberrantly (at a higher level than in normal subjects) or is genetically deficient (e.g., chromosomal abnormalities or genetic mutations);

or, the cell is in particular a HeLa, 293T, A549 or HUVEC cell;

or, the product is in particular a medicament.

The target gene may be a tumor-or cancer-associated gene, preferably BIRC5 gene.

The siRNA corresponding to the above target gene is as follows:

the target gene is BIRC5, and the corresponding siRNA sets are at least 6 of RB-BIR-D1, RB-BIR-D2, RB-BIR-D3, RB-BIR-D4, RB-BIR-D5, RB-BIR-D6 and RB-BIR-D7. The invention also includes a method of reducing expression of a target gene in a cell, said method comprising using a mixture as described above, the method comprising a) obtaining a mixture of siRNA molecules or a mixture thereof, said mixture of siRNA molecules comprising at least 5,6,7,8,9,10 sirnas; b) the mixture of siRNA molecules is delivered into the cell.

The present invention is a method for inhibiting expression of a target gene by introducing an siRNA molecule or a mixture thereof into a cell in a mixture of siRNA molecules (siRNA set); introduction may be direct introduction or indirect introduction, and in addition to the use of transfection reagents, other known means of delivering siRNA molecules, e.g., into cells, such as injection, transfection with vectors (which may be plasmids or viruses), electroporation, lipofection, and the like, may be used.

"complementary" refers to the nuclear base pairing ability.

The length of a nucleotide or RNA molecule chain can also be expressed in terms of bases or base lengths in the present invention.

The tolerance mismatch of the siRNA molecules of the invention is at least 1-5 nucleotides, preferably at the single or double stranded end, and the number of bases tolerated is influenced by the length of the complementary region. "mismatch" refers to the nuclear base can not be paired.

The mixture of siRNA molecules is preferably obtained by solid phase synthesis, and may also be synthesized by transcription or other methods.

The kit of the invention can also comprise buffer solution, markers (the markers can be dyes, radioactive labeling substances or fluorescent labeling substances, the marked positions can be at the tail ends of an antisense strand or a sense strand), transfection reagents, containers, test tubes, annealing reagents, control siRNA (including NC control and N control) and the like besides the siRNA molecules and/or the siRNA molecule mixture; the siRNA molecule mixture in the kit can be separately packaged or placed in a container after being combined; the components of the kit can be freeze-dried powder or solution.

Experiments prove that the stability of a single siRNA molecule is good, and on one hand, the resistance to nuclease in vitro or in vivo inhibition tests is improved; on the other hand, the storage and the transportation are facilitated; and simultaneously reduces off-target effect of siRNA molecules. The siRNA kit is a universal, effective and rapid RNAi tool, and has the advantages that: (1) the inhibition rate of more than 60 percent can be ensured, the inhibition rate of more than 75 percent of siRNA molecule mixture can reach more than 75 percent, the inhibition rate of more than 50 percent of siRNA molecule mixture can reach more than 85 percent, and the silencing probability and the silencing efficiency are integrally improved compared with a single siRNA molecule; in the prior art, the siRNA generally designed has a probability of inhibiting the expression of a target gene only by 50%, and the inhibition rate of the siRNA can reach more than 75% only by 25%. (2) No special design is needed, the siRNA does not need to be screened and optimized subsequently, or the effect of the siRNA is verified experimentally, so that the time and the labor are saved. (3) Solves the problem of inconsistent siRNA effects in different cell lines and different transfection reagents, can effectively silence target genes in a plurality of cell lines, and is not influenced by the transfection reagents. (4) Enhances the gene silencing effect of a single siRNA molecule and plays a role in synergy. (5) The off-target effect of the siRNA molecule is reduced.

The mixture of siRNA molecules of the present invention can affect the expression of the target gene in at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% of the cells with an inhibition rate of at least 45%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. The siRNA molecule mixture of the invention can ensure at least 60 percent of target gene inhibition effect when being treated by different transfection reagents in different cell lines. "inhibit" or similar expression in accordance with the present invention can refer to a decrease in the expression, activity or index of RNA or protein levels or related biochemical indicators relative to a negative control.

Drawings

FIG. 1 shows the results of siRNA in vitro stability assay.

Detailed Description

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

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of siRNA

All designed single siRNAs can target all transcripts of a target gene, and the design method refers to Elbashir et al 2002; paddison et al.2002; reynoldset al.2004; the method of Ui-Tei et al 2004 et al (Elbashir, S.M., Harborth, J., Weber, K., and Tuschl, T.2002.analysis of genetic in colloidal cells using colloidal interfering RNAs, methods 26: 199. 213; Paddis, P.J., Caudy, A.A., Bernstein, E.J., Hannon, G.J., and Conklin, D.S.2002.short hairpin RNAs (shRNAs) index sequence-specific colloidal cells genes&Dev.16: 948-; reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W.S., and Khvorova, A.2004.rational siRNA design for RNAi interface reference. Nat.Biotechnol.22: 326-) -330; Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki, H., Juni, A., Ueda, R., and Saigo, K.2004.guidelines for the selection of high efficiency siRNA sequences for mammalia and chip RNAi reference.nucleic Acids Res.32: 936-); to ensure the specificity of siRNA, BLAST (Basic Local Alignment Search Too,http://www.ncbi.nlm.gov) Analysis of sequence identity (identity), and selection of the sequence with the least identity to the other sequences.

The siRNA consists of an SS sense strand consisting of 25 nucleotides and an antisense strand AS reverse-complementary to the SS sense strand, and is blunt-ended;

each siRNA binds complementary in reverse direction to a target sequence on the target gene through its antisense strand;

the AS strand and the SS strand are completely reverse-complementary, the AS strand is completely reverse-complementary to a target sequence on a target gene, and 7 consecutive nucleotides from the 5 ' end and 7 consecutive nucleotides from the 3 ' end of the SS are both modified by 2 ' -O-Me.

The target genes are shown in Table 1, and the specific sequences of siRNAs corresponding to the target genes are shown in Table 2.

Table 1 target genes

Table 2 shows the sequence table of siRNA molecules

In the above tables, mA, mU, mC and mG are respectively A after 2 '-O-Me modification, U after 2' -O-Me modification, C after 2 '-O-Me modification and G after 2' -O-Me modification.

Example 2 cellular level inhibition assay of siRNA

The sirnas corresponding to BIRC5 target genes shown in table 2 prepared in example 1 were transfected into HeLa cells (from ATCC) separately as follows:

1. LF2K transfection

100 μ L LF2K transfection system: 1 μ L of LF2K (Invitrogen, 11668019), 5 μ L of siRNA (final concentration 100nM) and 94 μ L of Opti-MEM cell culture medium (Thermo Fisher Scientific, 31985070).

The above siRNAs are siRNAs corresponding to the TP53, BIRC5, CTNNB1, COPS5, STAT3, VEGFA and KRAS target genes prepared in example 1, respectively.

HeLa cells were cultured on a cell culture plate, and then 100. mu.L of the above transfection system was added to each well for transfection for 48 hours to obtain cells transfected with siRNA corresponding to different target genes.

2. Inhibition rate of RT-PCR detection

After 48h of transfection, cells transfected with siRNA corresponding to different target genes were collected, RNA was extracted by Trizol method, and the Reverse Transcription mix Reverse Transcription kit was used for Reverse Transcription (C10170, acute Bo Biotechnology Co., Ltd., Guangzhou) to obtain cDNA transfected with siRNA corresponding to different target genes. The primer pair of the target gene corresponding to siRNA shown in Table 3 was used for RT-PCR amplification using cDNA as template, human housekeeping gene actin as reference gene (Forward:5-TCAAGATCATTGCTCCTCCTGAG-3(SEQ ID NO. 15); Reverse:5-ACATCTGC

TGGAAGGTGGACA-3SEQ ID NO.16)), and Real-time fluorescent quantitative PCR reaction was performed using Real-time PCR kit SYBR Premix (2 ×) (BIO-RAD 750000131). The Ct error for 9 replicates of one sample (3 replicates per individual sample at transfection and 3 replicates per replicate at qPCR) was ± 0.5, and then relative quantification was performed using CFX 2.1. SPSS19.0 data statistics software data analysis, the data in the table are the mean values, and the P values are all less than 0.05.

NC negative control group: the transfected siRNA is irrelevant non-specific siRNA,

5'UUCUCCGAACGUGUCACGU dTdT 3'(SEQ ID NO.17)

5'ACGUGACACGUUCGGAGAA dTdT 3'；(SEQ ID NO.18)

n blank control group: normal cells, no siRNA transfection.

The Real-Time PCR inhibition rate calculation mode comprises the following steps:

inhibition rate-NC negative control mRNA relative expression level-mRNA relative expression level of siRNA group

NC negative control group mRNA relative expression level

The relative expression level of the NC negative control mRNA was 1.

Table 3 shows the primer sequences

As a result, as shown in Table 4, it can be seen that the designed siRNA all act to suppress the expression of the target gene against BIRC5 target gene.

Table 4 shows comparison of inhibition rates of individual siRNA in HeLa cells

D1-D7 in the above table are 7 siRNAs against the BIRC5 target gene, respectively.

Example 3 preparation of siRNA set

1. Principle of design

The siRNA set consists of 5-10 siRNA molecules;

each siRNA consists of an SS sense strand consisting of 25 nucleotides and an antisense strand AS reverse-complementary to the SS sense strand, and is blunt-ended; each siRNA binds complementarily to a target sequence on a target gene through its antisense strand;

the AS strand and the SS strand are completely reverse-complementary, the AS strand is completely reverse-complementary to a target sequence on a target gene, and 7 consecutive nucleotides from the 5 ' end and 7 consecutive nucleotides from the 3 ' end of the SS are both modified by 2 ' -O-Me.

The target genes are shown in Table 1, and the siRNAs corresponding to the target genes are shown in Table 2.

The set of siRNAs for the target gene may include 5,6,7 or 10 siRNAs, and the grouping is shown in Table 5.

TABLE 5 composition of siRNA set of target genes

The siRNAs in the above-mentioned siRNA set RM-2(7 siRNA mixtures), RM-3(6 siRNA mixtures) were mixed according to the grouping requirements shown in Table 5, and the siRNAs were mixed in an equimolar ratio.

Example 4 study of the inhibition of target Gene expression by Whole set of siRNA

The following examples were conducted by taking the siRNA set RM-2(7 siRNA cocktail) in Table 5 as an example:

first, the whole set of siRNA group RM-2 and single siRNA molecule are compared with the inhibition rate of target genes in HeLa cells

HeLa cells were transfected with the siRNA set RM-2 corresponding to the BIRC5 target gene shown in Table 5 in example 3, respectively, in the same manner as in example 2, wherein the total concentration of all siRNA molecules in the RM-2 mixture was 100nM, and the amounts of the substances, each siRNA molecule being equal, were mixed.

Control was transfected with a single siRNA molecule at a concentration of 100 nM.

The results of the inhibition rate of the complete siRNA group RM-2 and the inhibition rate of single siRNA are shown in Table 6, and it can be seen that the inhibition effect of the complete siRNA group RM-2 is greater than 90% for 7 target genes; and compared with 7 single siRNA molecules with the same concentration, the complete siRNA group RM-2 has the synergistic effect.

TABLE 6 comparison of RM-2 to Single siRNA in HeLa cells

Gene	RM-2	D1	D2	D3	D4	D5	D6	D7
									BIRC5	93	90	92	91	73	19	17	50

D1-D7 in the above table are 7 siRNAs corresponding to the BIRC5 target genes, and RM-2 is a mixed sample of 7 siRNAs corresponding to each target gene, respectively.

Secondly, comparing the inhibition effect of the siRNA RM-2 in the middle-formed set of different cell lines

4 different cell lines (human embryonic kidney cell 293T, cervical cancer cell HeLa, non-small cell lung cancer cell A549 and human umbilical vein endothelial cell HUVEC (both from ATCC) were transfected by the set of siRNA RM-2 corresponding to the BIRC5 target gene shown in Table 5 in example 3, and transfected 24h after inoculation and culture, cells were observed, and transfection was started in a good state.

1. Transfection

(1)50 μ L of riboFECT transfection system, 5 μ L of riboFECTTMCP Reagent (C10511-05, Izod, C.), 5 μ L of siRNA set RM-2 prepared in example 3 (total concentration of all siRNAs was 100nM) and 40 μ L of riboFECTTMCP Buffer (C10511-05, Izod, C.).

(2)100 μ L LF2K transfection system: mu.L of LF2K (Invitrogen, 11668019), 5. mu.L of the set RM-2 siRNA set prepared in example 3 (total final concentration of all siRNAs is 100nM) and 94. mu.L of Opti-MEM cell culture medium (ThermoFisher Scientific, 31985070).

The 50. mu.L of the L ibofect transfection system or the 100. mu.L of the LF2K transfection system was added to each well of 4 different cell line culture plates, cells were harvested after transfection for 48 hours, RNA was extracted by Trizol method, and the Reverse transcription kit was used for Reverse transcription (C10170, Liebo Biotech, Guangzhou) to obtain cDNA.

2. Inhibition rate of RT-PCR detection

The procedure was as in example 2.

The results are shown in Table 7, and compared with NC control, the RM-2 mixture can achieve more than 60% of inhibition effect in different cell lines by using different transfection reagents aiming at different target genes.

TABLE 7 comparison of RM-2 inhibition in different cell lines (%)

Thirdly, comparing the inhibition results of the mixture of RM-2 mixture and siRNA molecules of general structure in HeLa cells

Two target genes, namely SOD1 and EIF4E, are selected, and the inhibition effect of the corresponding RM-2 mixture and the mixture of siRNA molecules with the common structure in HeLa cells is compared.

The nucleotide sequence of the general structural siRNA of each of the above genes was different from RM-2 corresponding to each of the genes prepared in example 3 in that each of the nucleotides in the general structural siRNA was not modified with 2 ' -O-Me, and was 19bp in length (6 nucleotides at the 5 ' -end of the sense strand and 6 nucleotides at the 3 ' -end of the antisense strand were removed, respectively), and 2 dTdT overhangs were present at the ends, and the rest were identical.

The procedure was the same as the one described above, except that HeLa cells (M) were transfected with the siRNA set RM-2 corresponding to SOD1 and EIF4E prepared in example 3.

Control was performed with a mixture of siRNA molecules transfected with SOD1 and EIF4E corresponding to the general structure (S). The transfection reagent was LF 2K.

In the above transfection, the total concentration of siRNA was 100 nM.

As shown in Table 8, the mixture of RM-2 structures showed better inhibitory effect than the mixture of siRNA molecules of the general structure against 2 target genes.

TABLE 8 comparison of RM-2 modification results in HeLa cells

Four, comparison of inhibition effects of different groups of siRNA sets transfected HeLa cells

2 target genes SOD1 and MYC are selected to compare the inhibition effect of different siRNA sets in HeLa cells.

The procedure was the same as the one described above, except that HeLa cells were transfected with the siRNA set RM-2, RM-3 and RM-6 corresponding to SOD1, MYC prepared in example 3, respectively. The transfection reagent was LF 2K.

The results are shown in Table 9 below,

TABLE 9 siRNAs mixtures with different siRNA numbers

The results show that the mixtures RM-1, RM-3, RM-2 and RM-6 can all play a role in efficiently inhibiting a plurality of genes.

Example 5 in vitro stability assay

Each siRNA RB-KRA-D1, RB-TP5-D6 and RB-EIF-D3 was diluted to 5. mu.M with RNase-free water, and an equal volume of fresh rat serum (a product of bioscience, Inc., Yuanmu, Shanghai) was added, and samples were taken after incubation at 37 ℃ for 6 hours to electrophoretically observe the integrity of different siRNAs.

The results are shown in FIG. 1, where it is seen that siRNA is stable in serum, which is expected to have better efficacy in vivo.

The results of the stability determination experiments for other siRNAs are the same, and the detailed diagram is omitted.

The technical features of the embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the combinations should be considered as the scope of the description in the present specification.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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Claims (10)

1. An siRNA for inhibiting or reducing the expression of a BIRC5 target gene, which consists of a sense strand and an antisense strand reverse-complementary thereto;

the sense strand consists of 19-27 nucleotides and is 2 ' -O-ribose modified from 5-9 consecutive nucleotides from the 5 ' terminus and 5-9 consecutive nucleotides from the 3 ' terminus;

the antisense strand is reverse complementary to a segment on the target gene, and the antisense strand is unmodified;

the base composition sequence of the sense strand is selected from SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO. 13;

the base composition sequence of the antisense chain is selected from SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO. 14.

2. siRNA according to claim 1, characterized in that:

the sense strand consists of 24, 25, or 26 nucleotides;

or, the sense strand is 2 ' -O-ribose modified from 6 or 7 or 8 consecutive nucleotides from the 5 ' terminus and 6 or 7 or 8 consecutive nucleotides from the 3 ' terminus.

3. siRNA according to claim 2, characterized in that:

the 2 '-O-ribose modification is a 2' -O-methyl modification, a 2 '-O-fluoro modification, or a 2' -MOE modification.

4. A set of sirnas that inhibits or reduces expression of BIRC5 target gene, comprising at least 5 sirnas: the siRNA of any one of claims 1 to 3, wherein the sense strand has a base sequence of SEQ ID NO.9 or SEQ ID NO.11, the antisense strand has a base sequence of SEQ ID NO.10 or SEQ ID NO.12, and the sense strand has 2 ' -O-ribose modifications of 5 to 9 consecutive nucleotides from the 5 ' terminus and 5 to 9 consecutive nucleotides from the 3 ' terminus.

5. A siRNA set according to claim 4 wherein: the siRNA set consists of 5,6,7,8,9 or 10 siRNAs.

6. A siRNA set according to claim 5 wherein: the quantity ratio of any 2 siRNA substances in the siRNA set is 1:1-1: 5.

7. A siRNA set according to claim 5 wherein: the quantity ratio of any 2 siRNA substances in the siRNA set is 1: 1.

8. A kit for inhibiting or reducing the expression of BIRC5 target gene, comprising the siRNA of any one of claims 1 to 3 or the siRNA set of any one of claims 4 to 7.

9. Use of the siRNA of any one of claims 1 to 3 or the siRNA set of any one of claims 4 to 7 for the preparation of a medicament for preventing or alleviating or treating a disease caused by the expression of BIRC5 target gene.

10. A product comprising an siRNA of any one of claims 1 to 3 or a set of sirnas of any one of claims 4 to 7;

and/or the product has at least one function of 1) to 3) as follows:

1) inhibiting or reducing expression of a target gene;

2) inhibiting or reducing expression of a target gene in a cell;

3) preventing or alleviating or treating a disease caused by expression of a target gene.