CN111926012A

CN111926012A - SiRNA molecule for silencing Caspase-2mRNA expression

Info

Publication number: CN111926012A
Application number: CN202010822661.8A
Authority: CN
Inventors: 季爱民; 岑柏宏; 黎权辉; 陈佳扬
Original assignee: Southern Medical University Zhujiang Hospital
Current assignee: Wuhan Zezhi Biological Pharmaceutical Co ltd
Priority date: 2020-08-14
Filing date: 2020-08-14
Publication date: 2020-11-13

Abstract

The invention discloses a group of siRNA molecules for silencing Caspase-2mRNA expression. The siRNA molecule is siCASP2-1, siCASP2-2 or siCASP 2-3; the antisense strand consists of 19-29 nucleotides, the sense strand consists of 15-21 nucleotides, and a double-stranded structure can be formed by 15-19 nucleotides between the sense strand and the antisense strand. The siRNA provided by the invention can be modified on the 2' -ribose of all nucleotides or part of the nucleotides to prevent nucleosidase degradation. The siRNA molecule or the molecular structure with homology of more than 60 percent is used independently or jointly, the silencing efficiency of the target gene Caspase-2mRNA expression reaches more than 60 percent, and the siRNA molecule can treat diseases caused by Caspase-2 mutation, expression or activation and the like.

Description

SiRNA molecule for silencing Caspase-2mRNA expression

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a siRNA sequence expressed by a silent gene or protein and application thereof.

Background

Caspases, caspases specific for caspases, are a family of cysteine proteases that have been highly conserved evolutionarily. Caspase-2 (or Caspase 2) is one of the most conserved Caspase family members, with a precursor structure similar to the initial Caspase and cleavage properties similar to the effector Caspase. A great deal of evidence suggests that Caspase-2 plays a key and unique role in apoptosis regulation. Caspase family members act as "initiation molecules" or "executive molecules" to activate apoptotic pathways to cell death. Caspase-2 is the most structurally conserved member of the family, both the apoptosis "promoter" and the "effector" of the apoptotic process. (science.2002; 297: 1352-1354.Nature Cell biol.2006; 8: 72-77. Science; 2004; 304: 843-846.) Caspase-2 causes initiation and onset of apoptosis by activating the PIDDosome pathway or the unrelated death receptor (TRAIL or FAS) pathway. Since Caspase-2 is involved in multiple stages of the apoptosis process, such as DNA damage, heat shock, stimulation of death receptors, cytoskeletal damage, endoplasmic reticulum emergency response, oxidation emergency response and the like, Caspase-2 becomes an important therapeutic target for diseases caused by cytopenia due to pathological damage. The study finds that the Caspase-2 deficient neurons can antagonize apoptosis induced by beta-amyloid peptide (beta-amyloid), and Caspase-2 in hippocampal neurons caused by cerebral ischemia is activated to cause neuronal apoptosis. (J Neurosci.2000; 20: 1386-1392. PNAS.2008; 105: 16368-16373.) furthermore, Caspase-2 is expressed at a high level in ischemic retinal optic nerve cells (RGCs) and activated, with the result that optic nerve apoptosis occurs and blindness occurs. And the silent expression of Caspase-2 inhibits the apoptosis activity of Caspase-2, can effectively delay the progress of optic neuropathy, achieves the purpose of inhibiting the progress of optic neuropathy (NAION) caused by non-cerebral infarction anterior ischemia, glaucoma row optic neuropathy and the like, and achieves good effect. (Cell Death and disease.2011; 2: E173; DOI: 10.1038/CDDIS.2011.54.nucleic Acid therapeutics 2014; 24 (4): 258-266. DOI: 10.1089/NAT.2014.0489.)

Additional studies found that Caspase-2 expression levels were positively correlated with the severity of non-alcoholic fatty liver disease (NAFLD). Caspase-2 was found to be closely associated with symptoms of nonalcoholic steatohepatitis (NASH), including lipid droplet accumulation, liver injury, inflammation, and scarring, by knocking out the Caspase-2 gene in mice that were subjected to hepatic endoplasmic reticulum stress and high-fat diet and treating these mice with specific Caspase-2 inhibitors; in addition, Caspase-2 expression levels were also increased in liver samples from patients with benign NAFLD or aggressive NASH. Researchers have also found that Caspase-2 controls the activation of SREBP1 and SREBP2 by cleaving a protease called site-1 protease (S1P), while SREBP1 and SREBP2 are the major regulatory molecules of lipogenesis (lipogenesis) (the process by which nutrients such as carbohydrates are converted to fatty acids, triglycerides and cholesterol in the liver). Caspase-2 is therefore considered to be a key driver of the initiation of NASH. Thus, inhibition of Caspase-2 expression may provide an effective means to arrest the progression of NASH disease, and may even reverse the early symptoms of the disease. (cell.2018; 175 (1): 133-145.)

Small interfering RNA (siRNA) can specifically silence the expression level of target mRNA, and the theory is awarded to Nobel biomedical science in 2006, so that the siRNA molecule has the application prospect of treating diseases caused by gene overexpression and gene mutation by specifically silencing the expression of genes causing or promoting the occurrence or development of the diseases (science.2016; 352 (6292): 1417-1420; Nat Chem biol.2006; 2 (12): 689-700.).

In the double-stranded structure of siRNA, the single strand capable of forming an RNA Interference Silencing Complex (RISC) with Argonaute protein (AGO protein) is called Antisense strand (Antisense strand) or Guide strand (Guide strand), and the RISC complex formed by the strand is capable of binding to target mRNA, cleaving and silencing the target mRNA. While the other non-functional single strand is called the sense strand (sense strand) or the Passenger strand (Passenger strand). Using the characteristics of siRNA molecules, multiple siRNA sequences can be designed to silence target gene expression (Nature Reviews genetics.2015; 16 (9): 543-552.).

In the invention, an RNAi principle is adopted to design and screen an siRNA sequence for specifically silencing Caspase-2mRNA expression, and the obtained siRNA is used for inhibiting the expression of Caspase-2mRNA and the apoptosis-promoting function thereof, thereby realizing the application prospect of treating diseases caused by Caspase-2 expression activation or mutation.

Disclosure of Invention

The purpose of the invention is as follows:

provides siRNA sequence capable of efficiently silencing and suitable for silencing Caspase-2mRNA expression used in vivo and application thereof.

The technical scheme is as follows:

in the invention, through designing and synthesizing the siRNA sequence expressed by candidate silent Caspase-2mRNA, the nucleotide sequences contained in the target sequence, the sense strand and the antisense strand are respectively any one of the following sequences:

siCASP2-1：

target sequence: CTGTTGTTGAGCGAATTGC the flow of the air in the air conditioner,

sense strand: 5 '-CUGUUGUUGAGCGAAUGUdTdT-3',

antisense strand: 5 '-ACAAAUUCGCUCAACAACAGdTdT-3'.

siCASP2-2：

Target sequence: GTTGTTGAGCGAATTGTTA the flow of the air in the air conditioner,

sense strand: 5 '-GUUGUUGAGCGAAUGUUADTT-3',

antisense strand: 5 '-UAACAUUCGCUCAACAACdTdT-3'.

siCASP2-3：

Target sequence: CCTATTGCCAGGAATGTTT the flow of the air in the air conditioner,

sense strand: 5 '-CCUAUUGCCAGGAAAUGUUdTdT-3',

antisense strand: 5 '-AAACAUUCCCUGGCAAUAGGdTdT-3'.

siCASP2-4：

Target sequence: GCAGTTTCAGCCAGAATGT the flow of the air in the air conditioner,

sense strand: 5 '-GCAGUUUCAGCCAGAAUGUdTdT-3',

antisense strand: 5 '-ACAUUCUGGCCUGAAACUGCdTdT-3'.

siCASP2-5：

Target sequence: GATTGCTTTTATTACATTA the flow of the air in the air conditioner,

sense strand: 5 '-GAUUGCUUUACAUUACAUUDTdT-3',

antisense strand: 5 '-UAAUGUAAUAAAAGCAAUCdTdT-3'.

siASP2-6：

Target sequence: CTACAGAACAAACCAAAAA

Sense strand: 5 '-CUACAGAAACAAACCAAAdTdT-3',

antisense strand: 5 '-UUUUGGUUUGUUGUUGUGUAGdTdT-3'.

siCASP2-7：

Target sequence: CTTTCTCTTTCAGTATTAA the flow of the air in the air conditioner,

sense strand: 5 '-CUUUCUUCAGUGAUUAADTdT-3',

antisense strand: 5 '-UUAUACUGAAAGAGAAGdTdT-3'.

Wherein A is adenine ribonucleotide; g is guanine ribonucleotide; c is cytosine ribonucleotide; u is uracil ribonucleotide; dT is thymine deoxynucleotide. The nucleotides are connected by phosphodiester bonds or phosphorothioate bonds.

The siRNA sequence is obtained by the following process:

1) designing siRNA molecules;

the target sequences designed for use were derived from Homo sapiens Caspase-2(CASP2), transcript variant 1, mRNA, NCBI Reference Sequence: NM _ 032982.4. siRNA sequences for screening are designed by reference to Elbashir (methods.2002; 26: 199-213.), Ryynoldset (Nat. Biotechnol.2004; 22: 326-330.), and Ui-Tei (Nucleic Acids Res.2004; 32: 936-948), and the like. To avoid off-target effects, BLAST (http:// www.ncbi.nlm.gov) was used to analyze the identity of the designed sequence and to select the sequence with the least identity to the other sequences.

2) Adopting an RT-qPCR method to screen siRNA molecules with high silencing efficiency on target gene Caspase-2 mRNA:

using the above 7 sequences and the control sequence, RT-qPCR was used to detect the level of Caspase-2mRNA expression in human Hela cells after transfection of siRNA with lipofectamine 2000, which revealed that: the silencing efficiency of siCASP2-1, siCASP2-2 and siCASP2-6 is good (namely the expression level is low), and the silencing efficiency is more than 70 percent. The silencing efficiency of siCASP2-3 is better, the silencing efficiency of siCASP2-7 and siCASP2-5 is still better, and the silencing efficiency of siCASP2-4 is not good enough.

In the invention, an siRNA molecule can be selected, wherein a sense strand of the siRNA molecule has more than 60% of homology with a sense strand sequence in the siRNA molecule, or double strands of the siRNA molecule have more than 60% of homology; or has more than 60% homology with the antisense strand sequence in the siRNA molecule, or has more than 60% homology with double strands; the sense strand and the antisense strand form a double-stranded siRNA structure, and similar silencing efficiency can be obtained. In the sequence, 15-19 nucleotide sequences are complementary between the sense strand and the antisense strand, so that better silencing efficiency can be obtained.

3) Modifying the structure of the siRNA and judging the silencing efficiency of the siRNA to the target gene.

Adopts methoxy structure modification, fluorine structure modification or thiophosphate structure modification. The silencing efficiency of the modified siRNA molecules was tested separately.

In the siRNA, ribose or deoxyribose in all nucleotides or part of nucleotides is subjected to 2 '-methoxy modification (represented by m) or 2' -fluoro modification (represented by y), so that degradation can be prevented, the action time in vivo can be prolonged, and the silencing efficiency is not changed greatly; the internucleotide backbone can also be modified by phosphorothioate (expressed by s), the modified structure increases the lipid solubility of siRNA, is suitable for in vivo use, not only can prolong the action time in vivo and antagonize the degradation of nucleosidase in vivo, but also can smoothly enter cells, and basically does not influence the silencing efficiency of the original sequence on target gene Caspase-2mRNA, which is detailed in the attached table 1.

In the invention, siCASP2 with gene silencing efficiency of more than 70 percent can be obtained by adopting an siRNA design rule, combining computer aided design software and comparing experimental screening through RT-qPCR and can be used for further biological function research. Wherein, the silencing efficiency of the siCASP2-24 is higher than that of the siCASP2-23 by more than 10 percent and is close to that of the siCASP2-2, the silencing efficiency is basically not reduced, the siCASP2-24 smoothly enters human cells, the action time in vivo is prolonged, and diseases caused by the expression and activation of Caspase-2mRNA, such as optic nerve degenerative disease and nonalcoholic fatty liver, are obviously inhibited, so the effect of treating the diseases is better.

Has the advantages that:

the siRNA molecules obtained by the invention are used singly or jointly, the silencing efficiency of the target gene Caspase-2mRNA expression reaches more than 60 percent, even more than 70 percent, and the expression and activation level of the Caspase-2mRNA and diseases caused by the expression and activation level can be obviously inhibited. Moreover, some of the modified genes can prevent degradation or successfully enter cells in vivo to treat diseases caused by Caspase-2mRNA overexpression or activation.

Drawings

FIG. 1 shows the results of detection of Caspase-2mRNA expression levels in human Hela cells after lipofectamine 2000 transfection of candidate siRNA by RT-qPCR.

In fig. 1, the ordinate chinese meaning: relative Caspase-2mRNA expression levels. Here is shown how much Caspase-2mRNA expression remains in the cells after siRNA transfection; lower expression levels indicate more efficient silencing of Caspase-2mRNA expression by the added siRNA molecules.

Detailed Description

Example 1: siRNA molecules were designed.

The target sequences designed for use were derived from Homo sapiens Caspase-2(CASP2), transcript variant 1, mRNA, NCBI Reference Sequence: NM _ 032982.4. siRNA sequences for screening are designed by reference to Elbashir (methods.2002; 26: 199-213.), Ryynoldset (Nat. Biotechnol.2004; 22: 326-330.), and Ui-Tei (Nucleic Acids Res.2004; 32: 936-948), and the like. To avoid off-target effects, BLAST (http:// www.ncbi.nlm.gov) was used to analyze the identity of the designed sequence and to select the sequence with the least identity to the other sequences. The designed sequences were synthesized by commercial companies for screening purposes. Using a control sequence: siCASP2 was the positive control siRNA sequence and siNC was the negative control siRNA sequence.

Example 2: the RT-qPCR method screens the silencing efficiency of the siCASP2 on the target gene Caspase-2 mRNA.

After a professional organization is entrusted to synthesize the siRNA molecular structure, the human Hela cells are diluted and passaged by a culture medium DMEM, and the culture medium contains 10% of bovine serum. Lipo2000 transfects siRNA with a final concentration of 100nM for each group; in addition, the administration was carried out in OPTI-MEM medium, and the control group was set as a blank control group (untreated group); culture with transfection reagentAfter the cells are cultured for 6 hours, the culture medium containing 10% FBS is replaced to continue culturing for 48 hours, the cells are collected, the total RNA of the cells is extracted, cDNA is synthesized by reverse transcription, qPCR (quantitative polymerase chain reaction) is carried out to detect the silencing efficiency, the beta-actin gene of human is used as an internal reference, and a Real-time PCR kit TB is utilized

Premix Ex Taq^TM(Tli RNaseH Plus) real-time fluorescent quantitative PCR reaction was performed. The experiment was repeated with 3 duplicate wells for each sample, with the Ct error controlled to ± 0.5, and comparative analysis was performed with GAPDH mRNA expression levels to calculate the silencing efficiency of siCASP 2. According to the test results, the silencing efficiency of the designed siCASP2 on the target gene Caspase-2mRNA is more than 50%; wherein the silencing efficiencies of the siCASP2-1 (silencing efficiency 74.25%), the siCASP2-2 (silencing efficiency 78.25%), the siCASP2-3 (silencing efficiency 66%), the siCASP2-5 (silencing efficiency 62.5%), the siCASP2-6 (silencing efficiency 75%), the siCASP2-7 (silencing efficiency 64.27%) and the like on the target gene Caspase-2mRNA are all more than 60%; wherein the silencing efficiency of the siCASP2-1, the siCASP2-2 and the siCASP2-6 to the mRNA of the target gene Caspase-2 is more than 70 percent. Specific results can be seen in fig. 1.

Example 3: modified siRNA structure and silencing efficiency of modified siRNA structure to target gene.

On the basis of example 2, siRNA sequences with the greatest silencing efficiency were structurally modified to antagonize nucleosidase-induced degradation when used internally. The designed modified structure is shown in table 1; after all the modified structures were synthesized by commercial companies, the silencing efficiency of the modified structures for the target gene was determined according to the method of example 2. The specific scheme is as follows: the human Hela cells were diluted with DMEM medium containing 10% bovine serum for passaging. Lipo2000 transfects siRNA with a final concentration of 100nM for each group; in addition, the administration was carried out in OPTI-MEM medium, and the control group was set as a blank control group (untreated group); after culturing cells for 6 hours by using transfection reagent, recovering the whole culture medium to culture for 48 hours, collecting the cells, extracting total RNA of the cells, synthesizing cDNA by reverse transcription, carrying out qPCR (quantitative polymerase chain reaction) to detect the silencing efficiency, taking a human beta-actin gene as an internal reference gene, and utilizing a Real-time PCR kit TB

Premix Ex Taq^TM(Tli RNaseH Plus) real-time fluorescent quantitative PCR reaction was performed. And carrying out real-time fluorescent quantitative PCR reaction. Experiments were repeated with 3 replicate wells per sample, with Ct error controlled at ± 0.5, using GAPDH mRNA expression levels for comparative analysis, and Caspase-2mRNA silencing efficiency was calculated for different siCASP 2. The final silencing efficiency and its modified structure sequence are shown in table 1 below. The results show that the modification mode adopted by the invention does not influence the silencing efficiency of the original sequence on the target gene Caspase-2mRNA, and is suitable for in vivo use.

TABLE 1 different siCASP2-2 structures, modifications, and their Gene silencing efficiency

Remarking: m is methoxy structure modification; f is fluorine structure modification; s is a phosphorothioate structural modification.

Claims

1. A siRNA sequence for silencing Caspase-2mRNA expression is a nucleotide sequence of a double-strand structural molecule consisting of a sense strand and an antisense strand, and is characterized in that: the target sequence, the sense strand and the antisense strand of the siRNA sequence are respectively one of the following structures:

siCASP2-1：

sense strand: 5 '-CUGUUGUUGAGCGAAUGUdTdT-3',

antisense strand: 5 '-ACAAAUUCGCUCAACAACAGdTdT-3';

siCASP2-2：

sense strand: 5 '-GUUGUUGAGCGAAUGUUADTT-3',

antisense strand: 5 '-uaacaauucgcucaacdtt-3';

siCASP2-3：

sense strand: 5 '-CCUAUUGCCAGGAAAUGUUdTdT-3',

antisense strand: 5 '-AAACAUUCCCUGGCAAUAGGdTdT-3';

siCASP2-6：

target sequence: CTACAGAACAAACCAAAAA the flow of the air in the air conditioner,

sense strand: 5 '-CUACAGAAACAAACCAAAdTdT-3',

antisense strand: 5 '-UUUUGGUUUGUUGUUGUGUAGdTdT-3'.

2. The Caspase-2mRNA expressed siRNA sequence according to claim 1, characterized in that: the sequence of the sense strand of the siRNA molecule has more than 60 percent of homology with the sequence of the sense strand of the siRNA molecule, or the sequence of the antisense strand of the siRNA molecule has more than 60 percent of homology with the sequence of the antisense strand of the siRNA molecule.

3. The Caspase-2mRNA expressed siRNA sequence according to claim 1 or 2, characterized in that: 15-19 nucleotide sequences are in a complementary double-stranded structure between the sense strand and the antisense strand, and the target sequence, the sense strand and the antisense strand are respectively one of the following structures:

siCASP2-2：

sense strand: 5 '-GUUGUUGAGCGAAUGUUADTT-3',

antisense strand: 5 '-uaacaauucgcucaacdtt-3';

siCASP2-21：

target sequence: GTTGAGCGAATTGTTA the flow of the air in the air conditioner,

sense strand: 5'-GUUGAGCGAAUUGUUA-3' the flow of the air in the air conditioner,

antisense strand: 5 '-AGAUGUUCUAACAAAUUCGCUCAACAACdTdT-3'.

4. The siRNA sequence for silencing Caspase-2mRNA expression according to claim 1 or 2, characterized in that: the ribose in the nucleotide can be modified by 2 '-methoxyl or 2' -fluorine, and the target sequence, the sense strand and the antisense strand have the following structures:

siCASP2-22：

target sequence: GTTGAGCGAATTGTTA the flow of the air in the air conditioner,

sense strand: 5 '- (mG) (mU) (mU) GAGCGAAUUG (mU) (mU) (mA) -3',

antisense strand: 5 '- (mU) (yA) (mA) CAAUUCGCUCAA (mC) (yA) (mA) (mC) dTdT-3'.

5. The siRNA sequence for silencing Caspase-2mRNA expression according to claims 1, 2 or 3, wherein said siRNA sequence comprises: adjacent two nucleotides in the sense strand or the antisense strand are modified by phosphorothioate(s) instead of phosphodiester linkage, and the target sequence, the sense strand and the antisense strand have the following structures:

siCASP2-23：

target sequence: GTTGAGCGAATTGTTA the flow of the air in the air conditioner,

sense strand: 5 '- (mG) (mU) (mU) GAGCGAAUUG (mU) (mU) (mA) -3',

antisense strand: 5 '- (mU) (yA) (mA) CAAUUCGCUCAA (mC) (yA) (mA) (mC) sdTsdT-3';

siCASP2-24：

target sequence: GTTGAGCGAATTGTTA the flow of the air in the air conditioner,

sense strand: 5 '- (mG) s (mU) (mU) GAGCGAAUUG (mU) (mU) s (mA) -3',

antisense strand: 5 '- (mU) s (yA) (mA) CAAUUCGCUCAA (mC) (yA) (mA) (mC) sdTsdT-3'.

6. The siRNA sequence for silencing Caspase-2mRNA expression according to any one of claims 1-5, wherein: can be used for treating diseases caused by Caspase-2mRNA overexpression or mutation or Caspase activation.