Preparation and application of multi-target small nucleic acid drug based on small activating RNA technology

Abstract

The application discloses a kind of preparation and application of multi-target small nucleic acid drug based on small activation RNA technology, and is related to the field of biotechnology.The preparation method of the multi-target small nucleic acid drug includes the following steps: (1) selecting at least two anti-disease genes according to target disease;(2) intercepting the sequence of 1000 base pairs of the promoter region upstream of the coding region of the anti-disease gene;(3) according to the promoter sequence, avoid CpG island and locate the segment of species conservation, and cut a sequence containing 19 base length as a sense strand;(4) design an antisense strand according to the sense strand, and the 19 base of the antisense strand and the sense strand are completely complementary;(5) add two deoxythymine or uracil to the 3' end of each chain.The multi-target small nucleic acid drug has good therapeutic effect on heart failure, and has strong cancer cell killing ability and inhibition ability of cancer cell migration, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the preparation and application of a multi-target small nucleic acid drug based on small activating RNA technology. Background Technology

[0002] RNA activation (RNAa) is a phenomenon and mechanism in which small double-stranded RNAs (dsRNAs) target (recognize and bind) gene promoter regions, inducing or upregulating gene expression at the transcriptional level. Small dsRNAs with activating functions are called small activating RNAs (saRNAs). Research has discovered and confirmed RNA activation in human cells, naming it RNAa / saRNA. RNA activation is a conserved phenomenon from humans and mammals such as mice and rats to plants and nematodes.

[0003] saRNAs can be obtained through artificial design and chemical synthesis, or they can be naturally occurring endogenous RNAs such as miRNAs. The RNA activation mediated by the former is called exogenous RNA activation, while that mediated by the latter is endogenous RNA activation. The saRNAs mediating exogenous RNAa are structurally similar to the siRNAs mediating RNAi, both being 21-nucleotide double-stranded RNAs (dsRNAs), with two nucleotide overhangs at their respective 3' ends. Like RNAi, RNAa requires the participation of AGO proteins, particularly AGO2, which processes and activates the saRNA molecule and mediates the recognition and binding of saRNA to its target sites on its promoter. Unlike RNAi (which mainly occurs in the cytoplasm), the RNAa mechanism occurs in the nucleus. The saRNA mediating RNAa targets the promoter DNA sequence rather than the mRNA sequence. Furthermore, RNAa has a unique time-dependent effect, with its effects (gene expression upregulation) showing a lag of approximately 48 hours and a long duration of effect (10-14 days).

[0004] Studies have reported that naturally occurring miRNAs can also target promoter sequences to induce RNAa, discovering miR-373 target sites in the promoters of human CDH1 and CSDC2 genes. Introducing miR-373 mimics into human tumor cells can activate the expression of its target genes. Further research confirmed endogenous miRNA-mediated RNAa, revealing its important role in the proliferation of both normal and tumor cells. Studies have also reported that miRNAs targeting enhancer regions can induce RNAa. Research found that miR-551b-3p promotes ovarian cancer cell proliferation, survival, and tumor growth by targeting and activating STAT3. Currently, the exact number of genes regulated by endogenous RNA activation mechanisms remains unclear, but studies have found extensive binding sites for miRNAs and AGO proteins in the promoter regions of the human genome, suggesting that miRNAs may regulate a large number of genes at the transcriptional or epigenetic level.

[0005] RNAa is a simple and readily applicable method for upregulating gene expression, with broad potential applications in the biomedical field. These include its use as a tool for studying gene function and reprogramming cells, as well as its role in treating diseases such as cancer, cardiovascular disease, and erectile dysfunction. Currently, saRNA drugs for treating liver cancer have entered clinical trials.

[0006] Heart failure is a common heart disease and a common outcome of the later stages of all different types of heart disease. In severe cases, it can lead to malignant arrhythmias and sudden death. Currently, there are no effective single-drug treatments on the market, especially for diastolic heart failure. Imbalance or disorder of myocardial calcium ion homeostasis, mitochondrial dysfunction, and cardiomyocyte death are common characteristics and markers of various types of cardiovascular diseases, and also one of their pathogenesis mechanisms. The maintenance of myocardial calcium ion homeostasis depends on the fine regulation of a regulatory network composed of a series of calcium-regulating proteins, ion channels, and enzymes. SERCA2a (sarcoplasmic / endoplasmic reticulum calcium ATPase, encoded by gene ATP2A2) is the most crucial protein for maintaining myocardial calcium ion homeostasis. It is responsible for re-uploading calcium ions released into the cytoplasm during myocardial cell contraction and storing them in the sarcoplasmic / endoplasmic reticulum calcium pool. This ensures sufficient calcium ions are released from the sarcoplasm / endoplasmic reticulum during the next contraction, thus guaranteeing normal cardiac contractile function. Most importantly, SERCA2a prevents calcium accumulation in the myocardial cell cytoplasm, maintains normal cardiac diastolic function, and prevents apoptosis caused by calcium overload. SERCA2a expression and activity control cardiac contraction and relaxation, affecting cardiac function. The ATP2A2 / SERCA2a isoform is mainly expressed in cardiomyocytes and skeletal muscle cells. SERCA2a expression and activity are significantly reduced in failing hearts and in all other types of heart disease.

[0007] Adult myocardial regeneration capacity is weak and further impaired in heart failure, with a sharp increase in apoptosis and other types of cell death leading to a significant weakening of myocardial contractile function and even loss of pumping ability. Studies have found that CAND1 (cullin-Associated and Neddylation Dissociated 1) overexpression effectively prevents myocardial hypertrophy and heart failure. This protective effect is achieved by preventing and inhibiting apoptosis while simultaneously enhancing cardiomyocyte proliferation and promoting cardiac regeneration. The increased cardiomyocyte proliferation mediated by CAND1 overexpression significantly contributes to the repair and improvement of cardiac function in adults.

[0008] p53, also known as tumor protein p53, cell tumor antigen p53, or transformation-associated protein 53 (TRP53), is a regulatory protein that is frequently mutated in human cancers. p53 proteins are essential in vertebrates, where they prevent cancer formation. Therefore, p53 maintains stability by preventing genomic mutations and is thus classified as a tumor suppressor gene. The TP53 gene is the most frequently mutated gene in human cancers (>50%), and studies have shown that the TP53 gene plays a crucial role in preventing cancer formation. p53 plays a role in the regulation or progression of cell cycle, apoptosis, and genomic stability through several mechanisms: (1) When DNA is damaged, it can activate DNA repair proteins. (2) The TP53 gene encodes proteins that bind to DNA and regulate gene expression to prevent genomic mutations; (3) It can halt growth by holding the cell cycle at the G1 / S regulatory point where DNA damage is recognized—if it holds the cell there long enough, the DNA repair proteins will have time to repair the damage, and the cell will be allowed to continue the cell cycle; (4) If the DNA damage proves irreparable, it can induce apoptosis (i.e., programmed cell death). Studies have shown that in cancer cells, saRNA activation of p53 expression leads to caspase-dependent apoptosis, significantly increasing the number of G1 / G0 and G2 / M cells, and decreasing the number of S cells. Importantly, cell cycle arrest is associated with the overexpression of the downstream cell cycle repressor protein p21 induced by p53 activation, as saRNA activation of p53 results in a 10-fold upregulation of p21 expression.

[0009] CCAAT / enhancer-binding protein α, or C / EBP-α or CEBPA for short, is a leucine zipper protein encoded by the human CEBPA gene. As a major regulator of liver homeostasis, various oncogenic processes (including cell cycle control, proliferation, and angiogenesis), and the hematopoietic myeloid cell lineage, it initiates and activates myeloid gene expression programs by binding to the promoters or enhancers of myeloid-related genes. CEBPA plays a crucial role in embryogenesis and is essential for homeostatic glucose metabolism, adipogenesis, and bone marrow development. Its ability to regulate lineage-specific gene expression and induce growth arrest contributes to the final differentiation of several cell types, including hepatocytes, adipocytes, and granulocytes. Loss-of-function mutations in CEBPA lead to the development of approximately 10% of acute myeloid leukemia (AML), thus establishing the tumor-suppressive role of C / EBPα. The role of CEBPA in granulocyte differentiation and its function as a tumor suppressor gene are crucial in the prognosis of AML. Furthermore, downregulation of C / EBPα expression has also been observed in various other human tumors, including pancreatic cancer cells, liver cancer, breast cancer, lung cancer, and head and neck squamous cell carcinoma. However, functional CEBPA mutations have not been found in solid tumors, suggesting that the elimination of C / EBPα function in non-hematopoietic tissues is regulated by other mechanisms, particularly the significant downregulation of C / EBPα expression levels.

[0010] In a normal, healthy body, pathogenic and anti-pathogenic factors exist in a state of equilibrium. Most human diseases are multifactorial, multi-mechanism, and multi-layered pathological processes because the body contains multiple pathogenic and anti-pathogenic factors that work synergistically, compensating for each other, or mutually restricting and antagonizing each other, thus forming a complex and delicate dynamic equilibrium. In a normal dynamic equilibrium, anti-pathogenic factors are sufficient to counteract the harmful effects of pathogenic factors, ensuring the body is protected from disease. However, when this balance is disrupted—that is, when the strength of pathogenic factors is superior to that of anti-pathogenic factors—disease may be induced and its development exacerbated. Therefore, the occurrence and development of human diseases are determined by the relative strength of pathogenic and anti-pathogenic factors both inside and outside the body. Treatment methods include eliminating pathogenic factors, enhancing anti-pathogenic factors, or both. The discovery of the RANa mechanism provides a novel opportunity and strategy for strengthening the power of anti-pathogenic factors.

[0011] On the other hand, due to the rapid development of medicine and related disciplines, as well as the accumulation of past medical experience in treating diseases with drugs, the principle of "single-target drugs," once considered the gold standard for drug development, has lost its luster and has been gradually replaced by the emerging branch of pharmacology, "multi-target pharmacology." In recent years, the proportion of multi-target drugs among newly approved drugs has been increasing year by year. "Multi-target pharmacology" focuses on rationally designing drugs based on the key factors in the occurrence and development of diseases, using the concept of "one drug, multiple targets." The resulting multi-target drugs can simultaneously act on one or more key pathogenic or anti-pathogenic factors, or both, of a disease. Compared to "single-target drugs," they have superior efficacy, higher safety, lower drug resistance, and the potential to cure diseases.

[0012] This invention utilizes both the RNAa principle and the principle of multi-target pharmacology to design multi-target saRNA small nucleic acid drugs based on the selected key anti-disease factors that determine the occurrence and development of target diseases. Summary of the Invention

[0013] The purpose of this invention is to provide a method for preparing and applying multi-target small nucleic acid drugs based on small activating RNA technology.

[0014] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0015] On the one hand, this invention provides a method for preparing a multi-target small nucleic acid drug based on small activating RNA technology, comprising the following steps:

[0016] (1) Select at least two disease-resistant genes based on the target disease;

[0017] (2) Extract the sequence of 1000 base pairs from the promoter region upstream of the coding region of the above-mentioned disease resistance gene;

[0018] (3) Based on the above promoter sequence, avoid CpG islands and conserved segments of local species, and extract a 19-base sequence as the semantic chain.

[0019] (4) Based on the obtained semantic chain, design an antisense chain that is completely complementary to the 19 bases of the semantic chain;

[0020] (5) Add two deoxythymine or uracil to the 3' end of each of the two chains.

[0021] Specifically, the target diseases mentioned in step (1) include heart disease and cancer.

[0022] Furthermore, the aforementioned heart diseases include heart failure, myocardial infarction, atherosclerotic heart disease, myocarditis, and angina pectoris.

[0023] Furthermore, the cancers mentioned include liver cancer, lung cancer, pancreatic cancer, leukemia, oral cancer, skin cancer, head and neck cancer, laryngeal cancer, esophageal cancer, lymphoma, stomach cancer, bone cancer, kidney cancer, neuroblastoma, glioblastoma, bile duct cancer, bladder cancer, endometrial cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, melanoma, myeloma, ovarian cancer, prostate cancer, basal cell carcinoma, rectal cancer, squamous cell carcinoma, thyroid cancer, and uterine cancer.

[0024] Furthermore, the target diseases mentioned in step (1) include heart failure, liver cancer, lung cancer, and pancreatic cancer.

[0025] Specifically, the disease-resistant gene mentioned in step (1) is a key disease-resistant gene that plays a decisive role in the occurrence and development of the target disease.

[0026] According to some embodiments of the present invention, such as when the target disease is heart failure, the disease-resistant genes may be selected from the ATP2A2 gene encoding the SERCA2a protein and the CAND1 gene encoding the CAND1 protein.

[0027] According to some embodiments of the present invention, such as when the target disease is cancer, the disease-resistant gene may be selected from the TP53 gene encoding the p53 protein and the CEBPA gene encoding the C / EBP-α protein.

[0028] Specifically, in step (2), the sequence of the selected disease-resistant gene mentioned above is queried on the GenBank website.

[0029] Specifically, the GC content in the semantic chain described in step (3) does not exceed 60%.

[0030] In another aspect, the present invention provides a small nucleic acid drug for treating heart disease, the small nucleic acid drug comprising a saRNA targeting CAND1 and a saRNA targeting ATP2A2, the sequence of the saRNA targeting CAND1 is shown in SEQ ID NO:1, and the sequence of the saRNA targeting ATP2A2 is shown in SEQ ID NO:2.

[0031] Specifically, the small nucleic acid drug for treating heart disease is saR-ATP2A2:saR-CAND1.

[0032] saR-ATP2A2:saR-CAND1 sequence:

[0033] 5'-UUGAAGCUGCAGAAGUGAU (SEQ ID NO: 1)-dTdT-3'---CAND1.

[0034] 3'-dTdT-CACUUCGACCUCUUCACUU (SEQ ID NO: 2)-5'---ATP2A2.

[0035] Specifically, the heart diseases mentioned include heart failure, myocardial infarction, atherosclerotic heart disease, myocarditis, and angina pectoris.

[0036] Furthermore, the aforementioned heart disease is heart failure.

[0037] In another aspect, the present invention provides a small nucleic acid drug for treating cancer, the small nucleic acid drug comprising a saRNA targeting TP53 and a saRNA targeting CEBPA, the sequence of the saRNA targeting TP53 is shown in SEQ ID NO:5, and the sequence of the saRNA targeting CEBPA is shown in SEQ ID NO:6.

[0038] Specifically, the cancers mentioned include liver cancer, lung cancer, pancreatic cancer, leukemia, oral cancer, skin cancer, head and neck cancer, laryngeal cancer, esophageal cancer, lymphoma, stomach cancer, bone cancer, kidney cancer, neuroblastoma, glioblastoma, bile duct cancer, bladder cancer, endometrial cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, melanoma, myeloma, ovarian cancer, prostate cancer, basal cell carcinoma, rectal cancer, squamous cell carcinoma, thyroid cancer, and uterine cancer.

[0039] Furthermore, the target diseases described in the text include liver cancer, lung cancer, and pancreatic cancer.

[0040] Specifically, the small nucleic acid drug for treating cancer is saR-p53:saR-CEBPA.

[0041] saR-p53:saR-CEBPA sequence:

[0042] 5'-UUAGGAAGGCUCUCUGGAA (SEQ ID NO: 5)-dTdT-3'---p53.

[0043] 3'-dTdT-CAUCCUUAACAGUGACCAG (SEQ ID NO: 6)-5'---CEBPA.

[0044] Specifically, all of the above-mentioned small nucleic acid drugs were obtained using the above-described preparation method.

[0045] In another aspect, the present invention provides the use of the above-mentioned small nucleic acid drugs in the preparation of medicaments for treating heart disease and / or cancer.

[0046] Specifically, the drug may also contain other active ingredients and pharmaceutically acceptable excipients.

[0047] Furthermore, the pharmaceutically acceptable excipients are selected from excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, and flavoring agents.

[0048] Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, mannitol, magnesium stearate, starch, calcium phosphate, ethyl cellulose, methyl cellulose, alginate, gelatin, gum arabic, glyceryl monostearate, sodium glycolate starch, guar gum, glycerol, and propylene glycol.

[0049] Specifically, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0050] Specifically, the emulsifier is selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, glyceryl oleate, and polyglycerol-3 polyricinoleate.

[0051] Specifically, the stabilizer is selected from at least one of acacia gum, agar, alginate, cellulose ether, and carboxymethyl chitosan.

[0052] Specifically, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0053] Specifically, the adhesive is selected from at least one of ethanol, starch paste, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, and polyvinylpyrrolidone.

[0054] Specifically, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric oxycyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0055] Specifically, the lubricant is selected from at least one of magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, and poloxamer.

[0056] Specifically, the pH adjuster is selected from at least one of citric acid, fumaric acid, succinic acid, tartaric acid, malic acid, and ascorbic acid.

[0057] Specifically, the cryoprotectant is selected from at least one of sucrose, glucose, mannitol, fructose, trehalose, dextrose, lactose, glycerol, methanol, ethanol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), acetamide, or formamide.

[0058] Specifically, the flavoring agent is selected from at least one of sweet orange flavoring, vanilla flavoring, strawberry flavoring, milk flavoring, banana flavoring, and cherry flavoring.

[0059] Specifically, the dosage form of the drug is drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, powder, emulsion, solution, drop pill, pill, lozenge, lyophilized powder for injection, gel, suppository or aerosol.

[0060] In another aspect, the present invention provides the application of the above-mentioned small nucleic acid drugs in the preparation of formulations that activate or upregulate the expression of target genes in cells.

[0061] Specifically, the small nucleic acid drug is directly introduced into the cells.

[0062] Specifically, the target genes include, but are not limited to, the ATP2A2 gene, the CAND1 gene, the TP53 gene, and the CEBPA gene.

[0063] The beneficial effects of this invention are as follows:

[0064] This invention provides a method for preparing multi-target small nucleic acid drugs based on small activating RNA technology. According to this method, small nucleic acid drugs targeting heart disease and cancer, namely saR-ATP2A2:saR-CAND1 and saR-p53:saR-CEBPA, were obtained. saR-ATP2A2:saR-CAND1 exhibits strong cardioprotective effects, reduces cell death, reverses cardiac function and damaging electrophysiological and structural remodeling processes in heart failure mice, and even has the ability to stimulate myocardial regeneration. saR-p53:saR-CEBPA possesses strong cancer cell killing ability and the ability to inhibit cancer cell migration, reversing and preventing tumor growth and organ metastasis. Furthermore, saR-p53:saR-CEBPA shows inhibitory effects on liver cancer, lung cancer, and pancreatic cancer, and its ability to induce tumor growth and metastasis. Attached Figure Description

[0065] Figure 1The effect of saRNA on ATP2A2 and CAND1 mRNA in AC16 cells (n=4); in the figure, Sa50 represents a drug concentration of 50 nM, Sa100 represents a drug concentration of 100 nM, and Sa200 represents a drug concentration of 200 nM; compared with the Scramble control group, *P<0.05, **P<0.01; compared with the Sa50 concentration group, #P<0.05.

[0066] Figure 2 The effect of saRNA on ATP2A2 and CAND1 proteins in AC16 cells (n=4); in the figure, Sa50 represents a drug concentration of 50 nM, Sa100 represents a drug concentration of 100 nM, and Sa200 represents a drug concentration of 200 nM; compared with the Scramble control group, **P<0.01.

[0067] Figure 3 The effect of saRNA on TP53 and CEBPA mRNA in A549 cells (n=4); in the figure, Sa50 represents a drug concentration of 50 nM, Sa100 represents a drug concentration of 100 nM, and Sa200 represents a drug concentration of 200 nM; compared with the Scramble control group, *P<0.05, **P<0.01; compared with the Sa50 concentration group, #P<0.05.

[0068] Figure 4 The effect of saRNA on p53 and C / EBPα proteins in A549 cells (n=4); in the figure, Sa50 represents a drug concentration of 50 nM, Sa100 represents a drug concentration of 100 nM, and Sa200 represents a drug concentration of 200 nM; compared with the Scramble control group, *P<0.05; compared with the Sa50 concentration group, #P<0.05.

[0069] Figure 5 The effect of saRNA on TP53 and CEBPA mRNA in HepG2 cells (n=4); in the figure, Sa50 represents a drug concentration of 50 nM, Sa100 represents a drug concentration of 100 nM, and Sa200 represents a drug concentration of 200 nM; compared with the Scramble control group, *P<0.05, **P<0.01; compared with the Sa50 concentration group, #P<0.05.

[0070] Figure 6 The effect of saRNA on p53 and C / EBPα proteins in HepG2 cells (n=4); in the figure, Sa50 represents a drug concentration of 50 nM, Sa100 represents a drug concentration of 100 nM, and Sa200 represents a drug concentration of 200 nM; compared with the Scramble control group, *P<0.05; compared with the Sa50 concentration group, #P<0.05.

[0071] Figure 7 The effect of saRNA on the viability of A549 and HepG2 cells (n=6); in the figure, Sa50 represents a drug concentration of 50 nM, Sa100 represents a drug concentration of 100 nM, and Sa200 represents a drug concentration of 200 nM; compared with the Scramble control group, *P<0.05, **P<0.01; compared with the Sa50 concentration group, #P<0.05.

[0072] Figure 8 The effect of saRNA on HepG2 cell migration (n=4); in the figure, Sa50 represents a drug concentration of 50 nM, Sa100 represents a drug concentration of 100 nM, and Sa200 represents a drug concentration of 200 nM; compared with the Scramble control group, *P<0.05.

[0073] Figure 9 The effect of saRNA on HepG2 cell apoptosis was detected by TUNEL assay (n=4); in the figure, Sa50 represents a drug concentration of 50 nM, Sa100 represents a drug concentration of 100 nM, and Sa200 represents a drug concentration of 200 nM; compared with the Scramble control group, *P<0.05, **P<0.01; compared with the Sa50 concentration group, #P<0.05.

[0074] Figure 10 The effect of saRNA on HepG2 cell apoptosis was detected by flow cytometry (n=4); in the figure, Sa50 represents a drug concentration of 50 nM, Sa100 represents a drug concentration of 100 nM, and Sa200 represents a drug concentration of 200 nM; compared with the Scramble control group, *P<0.05, **P<0.01; compared with the Sa50 concentration group, #P<0.05. Detailed Implementation

[0075] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0076] The design steps of this invention are as follows:

[0077] (1) Select two key anti-disease genes that play a decisive role in the occurrence and development of the target disease, such as the ATP2A2 gene encoding SERCA2a protein and the CAND1 gene encoding CAND1 protein for heart failure; and the TP53 gene encoding p53 protein and the CEBPA gene encoding C / EBP-α protein for cancer.

[0078] (2) Search for the selected gene sequence on the GenBank website and extract the sequence of 1000 base pairs of the promoter region upstream of the coding region of the gene.

[0079] (3) Based on the promoter sequence obtained above, avoid the CpG island, locate the species-conserved (especially highly conserved sequences in humans and mice) segment, and extract a 19-base-length sequence as the sense strand (SS), in which the GC content should not exceed 60%.

[0080] (4) Based on the semantic chain obtained above, design an antisense chain (antisense or AS). The antisense chain must be completely complementary to the 19 bases of the semantic chain.

[0081] (5) Add two deoxythymine (dT) or uracil (U) to the 3' end of each of the two chains.

[0082] Example 1 saR-ATP2A2:saR-CAND1

[0083] Based on the above-described multi-target saRNA design steps, this invention designs the following double-stranded oligonucleotide fragment (dsRNA), namely saR-ATP2A2:saR-CAND1. One strand can recognize and bind to the promoter region of the ATP2A2 gene, inducing the transcription of this gene; the other strand can recognize and bind to the promoter region of the CAND1 gene, inducing the transcription of the CAND1 gene.

[0084] saR-ATP2A2:saR-CAND1 sequence:

[0085] 5'-UUGAAGCUGCAGAAGUGAU (SEQ ID NO: 1)-dTdT-3'---CAND1.

[0086] 3'-dTdT-CACUUCGACCUCUUCACUU (SEQ ID NO: 2)-5'---ATP2A2.

[0087] control group saRNA sequence:

[0088] 5'-CUUACGCUGAGUACUUCGA (SEQ ID NO: 3)-dTdT-3'.

[0089] 3'-dTdT-GAAUGCGACUCAUGAAGCU (SEQ ID NO: 4)-5'.

[0090] Experimental Example 1: Effects of saR-ATP2A2:saR-CAND1 on Chronic Heart Failure

[0091] 1. Experimental Methods:

[0092] The saR-ATP2A2:saR-CAND1 was transfected into the AC16 human cardiomyocyte line using lipofectamine. The specific procedure was as follows: 10 μL of lipofectamine was diluted in 100 μL of serum-free DMEM medium, then mixed with 1 μg of saR-ATP2A2:saR-CAND1, and incubated at room temperature for 15 min. The mixture was then added to the AC16 human cardiomyocyte line, and qRT-PCR was performed 48 hours later.

[0093] Real-time quantitative PCR (qRT-PCR):

[0094] AC16 human cardiomyocytes were lysed and transfected. Total RNA was extracted using RNAzol reagent, and RNA yield was quantified using Qubit. RNA was reverse transcribed using Oligodt primers and reverse transcriptase. cDNA samples were analyzed using pre-designed real-time PCR primers to detect ATP2A2 and CAND1 mRNA levels.

[0095] Western blot:

[0096] Prepare polyacrylamide gels according to the instructions of the 12.5% ​​ExpressCast PAGE color gel rapid kit. The separating gel concentration is 12.5%, the sample loading volume is 20 μL, the stacking gel electrophoresis conditions are 80V for 30 min, and the separating gel electrophoresis conditions are 120V for 60 min.

[0097] Electrotransfer: Using polyvinylidene fluoride film, the semi-dry transfer method was used to transfer the film at 25V for 10 minutes.

[0098] Blocking: Block with 5% skim milk for 1 hour.

[0099] Primary antibody incubation: Dilute the primary antibody 1:2000 with 5% skim milk and incubate overnight at 4°C. Wash three times with TBST for 10 min each time.

[0100] Secondary antibody incubation: Dilute the secondary antibody 1:1000 with 5% skim milk and incubate at room temperature for 2 hours. Wash 3 times with TBST, 10 minutes each time.

[0101] Development: Developer A and developer B were prepared in a 1:1 ratio. The PVDF film was developed using the ChemiDocMP Imaging System, and the developed image was then analyzed for grayscale using ImageJ.

[0102] Intracellular calcium levels were detected using a reagent kit purchased from Nanjing Jiancheng Biotechnology Institute.

[0103] Methods for detecting apoptosis:

[0104] 48 h after transfection, the culture medium was aspirated and the cells were digested. The cells were centrifuged at 1000 rpm for 1 min, and then 400 μl of binding buffer was added. 2.5 μl of V-FITC dye was added to the cells and gently pipetted to mix. The cells were then incubated at 4 °C in the dark for 15 min. After that, 5 μl of PI dye was added and gently pipetted to mix. The cells were then incubated at 4 °C for 5 min. Apoptosis was detected by flow cytometry.

[0105] 2. Experimental Results:

[0106] The levels of ATP2A2 and CAND1 transcripts (mRNA) were measured by qRT-PCR, and both were found to be significantly upregulated. Figure 1 Western blot experiments confirmed that the proteins SERCA2a and CAND1, encoded by ATP2A2 and CAND1 respectively, were also significantly upregulated. Figure 2 Under hypoxic conditions, AC16 cells experience intracellular calcium overload, leading to apoptosis. After transfection with saR-ATP2A2:saR-CAND1 for 48 h, the intracellular calcium overload was reversed, and apoptosis was inhibited.

[0107] The above experimental results were further confirmed in primary cultured mouse cells.

[0108] 3. Experimental methods:

[0109] Lipid nanoparticles treated with cardiomyocyte-specific targeting peptide PCM (WLSEAGPVVTVRALRGTGSW, SEQ ID NO:9) were used to encapsulate saR-ATP2A2:saR-CAND1 (LNP-PCM / saR-ATP2A2:saR-CAND1) and injected into mice via tail vein.

[0110] The experimental procedures for qRT-PCR and Western blot are the same as above.

[0111] Detection method:

[0112] Echocardiography was performed using a high-resolution Vevo 770 imaging system platform. M-Mode ultrasound was performed using a 30MHz probe to measure ejection fraction, left ventricular fractional shortening, end-diastolic diameter, end-systolic diameter, and anterior and posterior wall thickness at end-diastole. Each mouse was examined twice on each section, with values ​​collected for five consecutive cardiac cycles each time, and the average value was taken.

[0113] 4. Experimental Results:

[0114] 48 hours later, qRT-PCR was used to measure the mRNA levels of ATP2A2 and CAND1 transcripts, and both were found to be significantly upregulated (more than +3-fold). Western blot experiments confirmed that the proteins SERCA2a and CAND1 encoded by ATP2A2 and CAND1, respectively, were also significantly upregulated.

[0115] Using mouse aortic arch coarctation (TAC) as a classic surgical model of hypertrophic cardiomyopathy and a model of decompensated transformation into chronic heart failure (similar to human hypertension or aortic valve stenosis leading to heart failure), cardiac systolic and diastolic function were examined. It was found that cardiac function indicators such as ejection fraction and left ventricular fractional shortening rate were significantly decreased in heart failure.

[0116] Indicators reflecting ventricular anatomical size, including left ventricular end-diastolic diameter and left ventricular end-systolic diameter, showed a significant increase, while the thickness of the left ventricular anterior and posterior walls at end-diastolic and end-systolic was significantly decreased; cardiomyocyte apoptosis and pyroptosis were significantly increased, while autophagy was reduced; and the arrhythmia induction rate was significantly increased.

[0117] After 48 hours of treatment with LNP-PCM / saR-ATP2A2:saR-CAND1, the aforementioned heart failure, cardiac function decline, poor structural remodeling, cell death, and impaired autophagy were significantly reversed and improved. Of particular note was the activation of the regenerative capacity of some cardiomyocytes, which is one of the mechanisms by which the reduced thickness of the anterior and posterior walls of the left ventricle was partially restored.

[0118] In all the above experiments, the control group saRNA did not produce any significant effect compared with the negative control (Mock-control) group. All statistical data were obtained by comparing the saR-ATP2A2:saR-CAND1 group with the control group saRNA and the negative control (Mock-control) group respectively.

[0119] As can be seen from the above, saR-ATP2A2:saR-CAND1 has a strong cardioprotective effect, reduces cell death, can reverse cardiac function and damaging electrophysiological and structural remodeling processes in heart failure mice, and even has the ability to stimulate myocardial regeneration. Therefore, it has the potential to become a small nucleotide drug for the treatment of chronic heart failure.

[0120] Example 2 saR-p53:saR-CEBPA

[0121] To further verify the feasibility of multi-target saRNA as a small nucleic acid drug, a double-stranded oligonucleotide sequence was designed based on the methods described in the multi-target saRNA design steps above: saR-p53:saR-CEBPA. One strand can recognize and bind to the promoter region of the p53 gene, inducing the transcription of this gene; the other strand can recognize and bind to the promoter region of the CEBPA gene, inducing the transcription of the CEBPA gene.

[0122] saR-p53:saR-CEBPA sequence:

[0123] 5'-UUAGGAAGGCUCUCUGGAA (SEQ ID NO: 5)-dTdT-3'---p53.

[0124] 3'-dTdT-CAUCCUUAACAGUGACCAG (SEQ ID NO: 6)-5'---CEBPA.

[0125] control group saRNA sequence:

[0126] 5'-CUUACGCUGAGUACUUCGA (SEQ ID NO:7)-dTdT-3'.

[0127] 3'-dTdT-GAAUGCGACUCAUGAAGCU (SEQ ID NO: 8)-5'.

[0128] 1. Experimental Methods:

[0129] The saR-p53:saR-CEBPA was transfected into the human hepatocellular carcinoma cell line HEPG2 and the human non-small cell lung cancer cell line A549 using liposomes, with the specific operation being the same as in Example 1.

[0130] The methods for detecting qRT-PCR, Western blot, and apoptosis are the same as in Example 1.

[0131] Methods for detecting cell viability: 48 h after transfection, CCK-8 reagent was added, and the cells were incubated at 37℃ in the dark for 2 h. The absorbance value at 450 nm was detected using an ELISA reader, and the results were statistically analyzed to determine the effect of saR-p53:saR-CEBPA on cell viability.

[0132] Apoptosis was detected using the TUNEL assay kit (Vazyme).

[0133] The method for detecting cell migration ability includes the following steps:

[0134] Cell migration ability was analyzed by seeding and transfecting A549 cells in 24-well plates. After 48 hours, the cells were scraped and exposed to macrosaccharides (5 mM) under normoxic (21% O2) conditions. Cells were then serum starved, and mitomycin C (10 μg / mL, Roche) was added to inhibit cell proliferation. Migration was calculated using the area of ​​cell-free regions at 0 and 24 hours.

[0135] 2. Experimental Results:

[0136] Using lipofectamine, saR-p53:saR-CEBPA was transfected into the human hepatocellular carcinoma cell line HEPG2 and the human non-small cell lung cancer cell line A549, respectively. After 48 hours, qRT-PCR was used to measure the levels of TP53 and CEBPA transcripts (mRNA), and both were found to be significantly upregulated. Figure 3 , Figure 5 Western blot experiments confirmed that the proteins p53 and C / EBPα, encoded by TP53 and CEBPA respectively, were also significantly upregulated. Figure 4 , Figure 6 ).

[0137] Cellular level experiments revealed that, compared with the scramble control group, saR-p53:saR-CEBPA induced a visually and statistically significant decrease in cell viability in three different cancer cell lines (CCK-8 assay). Figure 7 ), induces massive apoptosis (TUNEL staining) Figure 9 , Figure 10 Inhibits cancer cell proliferation (Edu assay), inhibits cancer cell migration (Transwell assay) Figure 8 It inhibits the ability of cancer cells to infiltrate (healing assay) and inhibits the ability of cancer cell colonies to form (colony formation assay).

[0138] 3. Experimental methods:

[0139] In a nude mouse tumor-bearing model, we implanted EPG2, PANC-1, and A549 cells into the backs of nude mice to induce tumor formation.

[0140] 4. Experimental Results:

[0141] Compared with the control group, direct injection of saR-p53:saR-CEBPA into tumor tissue significantly reduced tumor size, weight, and volume, and inhibited tumor growth. EPG2, PANC-1, and A549 cells transfected with saR-p53:saR-CEBPA for 60 hours almost completely lost their tumorigenicity in nude mice.

[0142] When EPG2, PANC-1, and A549 cells were injected into nude mice via tail vein, significant cross-tissue and organ metastasis was observed in each cell type. After tail vein injection of saR-p53:saR-CEBPA, cancer cell metastasis was significantly reduced or even disappeared.

[0143] Pre-transfecting EPG2, PANC-1, and A549 cells with saR-p53:saR-CEBPA for 60 h, and then injecting them into mice via tail vein, revealed that the tissue and organ metastasis of these cells was significantly reduced or even disappeared.

[0144] In all the above experiments, the control group saRNA did not produce any significant effect compared with the Mock-control group. All statistical data were obtained by comparing the saR-p53:saR-CEBPA group with the control group saRNA and the Mock-control group respectively.

[0145] Conclusion: saR-p53:saR-CEBPA possesses extremely strong cancer cell killing and cancer cell migration inhibition capabilities, reversing and preventing tumor growth and organ metastasis. In particular, we verified the inhibitory effect of saR-p53:saR-CEBPA on three types of cancer cells—liver cancer, lung cancer, and pancreatic cancer—and its ability to induce tumor growth and metastasis, indicating that saR-p53:saR-CEBPA has the potential to be developed into a broad-spectrum anticancer small nucleotide drug.

[0146] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A small nucleic acid drug for treating cancer, characterized in that, The small nucleic acid drug is a double-stranded oligonucleotide, and the double-stranded oligonucleotide is saR-p53:saR-CEBPA. The sequence of saR-p53:saR-CEBPA is: 5'-UUAGGAAGGCUCUCUGGAA-dTdT-3'; 3'-dTdT-CAUCCUUAACAGUGACCAG-5'.

2. The small nucleic acid drug of claim 1, wherein The cancer includes liver cancer, lung cancer and pancreatic cancer.

3. Use of a small nucleic acid drug according to any one of claims 1 to 2 for the manufacture of a medicament for the treatment of cancer, characterized in that, The cancer includes liver cancer, lung cancer and pancreatic cancer.

4. Use according to claim 3, characterized in that, The drug further comprises other active ingredients and pharmaceutically acceptable adjuvants.

5. Use according to claim 3, characterized in that, The dosage form of the drug is eye drops, tincture, powder, tablet, capsule, granule, ointment, powder, emulsion, pill, gel, suppository or aerosol.

6. Use according to claim 3, characterized in that, The dosage form of the drug is a freeze-dried powder injection.

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