Preparation and application of multi-target small nucleic acid medicine based on small activation RNA (Ribonucleic Acid) technology

Abstract

The invention discloses preparation and application of a multi-target small nucleic acid drug based on a small activation RNA technology, and relates to the technical field of biology. The preparation method of the multi-target small nucleic acid medicine comprises the following steps: (1) selecting at least two disease-resistant genes according to a target disease; (2) intercepting sequences of 1000 base pairs in a promoter region on the upstream of a disease-resistant gene coding region; (3) according to the promoter sequence, avoiding a CpG island and positioning a conservative segment of the species, and intercepting a segment of sequence containing 19 base lengths as a significance chain; (4) designing a section of antisense chain according to the meaning chain, wherein 19 basic groups of the antisense chain and the meaning chain are completely complementary; 5, two deoxidized thymine or uracil are added to the 3'ends of the two chains, the multi-target-point small nucleic acid medicine has a good treatment effect on heart failure, the cancer cell killing capacity and the cancer cell migration inhibiting capacity are high, and wide application prospects are achieved.

Description

Technical Field

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

[0002] RNA activation (RNAa) is a phenomenon and mechanism in which small double-stranded RNA (dsRNA) targets (recognizes and binds to) the gene promoter region to induce or up-regulate gene expression at the transcriptional level. The small dsRNA with activation function is called small activating RNA (saRNA). It has been found and confirmed that the RNA activation phenomenon occurs in human cells, and it is named RNAa / saRNA. RNA activation is a conserved phenomenon from mammals such as humans, rats and mice to plants and nematodes.

[0003] saRNA can be obtained through artificial design and chemical synthesis, or it can be an endogenous RNA such as miRNA that exists naturally; the former-mediated RNA activation is called exogenous RNA activation, and the latter is endogenous RNA activation. The saRNA that mediates exogenous RNAa is similar in structure to the siRNA that mediates RNAi. Both are double-stranded RNAs (dsRNAs) containing 21 nucleotides, and both strands have a 2-nucleotide overhang at their respective 3' ends. Like RNAi, RNAa also requires the participation of AGO proteins, especially AGO2, whose role is to process and activate saRNA molecules and mediate the recognition and binding of saRNA to its target site on the promoter. Different from RNAi (which mainly occurs in the cytoplasm), the RNAa mechanism occurs in the nucleus. The saRNA that mediates RNAa targets the promoter DNA sequence rather than the mRNA sequence. In addition, RNAa has special timeliness, manifested as a lag of about 48 hours in the appearance of its effect (up-regulation of gene expression) and the long-term nature of the effect (10 - 14 days).

[0004] Studies have reported that naturally occurring miRNAs can also target promoter sequences to induce RNAa. It was found that there are target sites for miR-373 in the promoters of human CDH1 and CSDC2 genes. Introduction of miR-373 mimics into human tumor cells can activate the expression of its target genes. Subsequently, endogenous miRNA-mediated RNAa was further confirmed, and it was found that this mechanism plays an important role in the proliferation of normal and tumor cells. Studies have reported that miRNAs targeting enhancer regions can also induce RNAa. It was found that miR-551b-3p promotes the proliferation, survival, and tumor growth of ovarian cancer cells by targeting and activating STAT3. Currently, it is not clear how many genes are regulated by the endogenous RNA activation mechanism, but studies have found that miRNAs and AGO proteins have extensive binding sites 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 easy method for upregulating gene expression and has extensive potential uses in the biomedical field, including as a tool for studying gene functions and reprogramming cells, and as a means of treating diseases, including tumors, cardiovascular diseases, erectile dysfunction, etc. Currently, saRNA drugs for treating liver cancer have entered clinical trials.

[0006] Heart failure is a common heart disease and the common outcome of the development of all different types of heart diseases in the late stage. Severe cases can lead to malignant arrhythmia and sudden death. Currently, there is no effective single therapeutic drug on the market, especially the lack of drugs for treating diastolic heart failure. Imbalance or disorder of myocardial cell calcium ion homeostasis, mitochondrial function failure, and myocardial cell death are the common characteristics and signs of various types of cardiovascular diseases and one of their pathogenesis. The maintenance of myocardial calcium ion homeostasis depends on the fine regulation of a regulatory network composed of a series of calcium regulation-related proteins, ion channels, and enzymes. SERCA2a (sarco / endoplasmic reticulum calcium ATPase, encoded by the gene ATP2A2) is the most critical protein for maintaining myocardial cell calcium ion homeostasis. It is responsible for reuptaking the calcium ions released into the cytoplasm during myocardial cell contraction into the sarcoplasmic / endoplasmic reticulum calcium pool for storage, so that there are sufficient calcium ions released from the sarcoplasmic / endoplasmic reticulum during the next contraction to ensure the normal contraction function of the heart. Most importantly, SERCA2a prevents calcium accumulation in the cytoplasm of myocardial cells, maintains normal heart diastolic function, and prevents apoptosis caused by calcium overload. The expression and activity of SERCA2a control the contraction and relaxation of the heart and affect heart function. The ATP2A2 / SERCA2a subtype is mainly expressed in myocardial cells and skeletal muscle cells. The expression and activity of SERCA2a are significantly reduced in failing hearts and all other types of heart diseases.

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

[0008] p53, also known as tumor protein p53, cellular tumor antigen p53, or transformation-related protein 53 (TRP53), is a regulatory protein that is frequently mutated in human cancers. The p53 protein is crucial in vertebrates, where it prevents the formation of cancer. 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 the 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 arrest growth by keeping the cell cycle at the G1 / S regulatory checkpoint for DNA damage recognition - if it holds the cell here long enough, 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 to be irreparable, it can trigger apoptosis (i.e., programmed cell death). Studies have shown that in cancer cells, saRNA activation of p53 expression can lead to caspase-dependent apoptosis, along with a significant increase in the number of G1 / G0 and G2 / M cells and a decrease in S cells. Importantly, cell cycle arrest is associated with the overexpression of the downstream cell cycle inhibitor protein p21 induced by p53 activation, as saRNA activation of p53 leads to a 10-fold upregulation of p21 expression.

[0009] CCAAT / enhancer-binding protein alpha, abbreviated as C / EBP-α or CEBPA, is a leucine zipper protein encoded by the human CEBPA gene. As a major regulator of liver homeostasis, various carcinogenic processes (including cell cycle control, proliferation, and angiogenesis), and the hematopoietic myeloid cell lineage, it initiates and activates the myeloid gene expression program by binding to the promoters or enhancers of myeloid-related genes. CEBPA plays a crucial role during 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 terminal 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, thereby establishing the tumor-suppressive role of C / EBPα. The role of CEBPA in granulocyte differentiation and its role as a tumor suppressor gene are crucial for the prognosis of acute myeloid leukemia. In addition, downregulation of C / EBPα expression also occurs in a variety of 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, indicating that the elimination of C / EBPα function in non-hematopoietic tissues is regulated by other mechanisms, particularly a substantial downregulation of C / EBPα expression levels.

[0010] In a normal and healthy organism, pathogenic factors and disease-resistant factors inside and outside the body are in a state of balance. Most of the human diseases belong to multi-factor, multi-mechanism, and multi-level pathological processes because there are multiple pathogenic factors and disease-resistant factors in the body, which cooperate with each other, compensate each other, or restrict and oppose each other, thus forming a complex and delicate dynamic balance state. In the normal dynamic balance state, the disease-resistant factors are sufficient to offset the harmful effects of pathogenic factors and ensure that the body is free from diseases. However, when this balance is broken, that is, when the power of pathogenic factors is dominant relative to the power of disease-resistant factors, it is possible to induce the occurrence of diseases and exacerbate the development of diseases. It can be seen that the occurrence and development of human diseases are determined by the relative power of pathogenic factors and disease-resistant factors inside and outside the body. The methods for treating diseases are to eliminate pathogenic factors, or enhance disease-resistant factors, or both. The discovery of the RANa mechanism provides a brand-new opportunity and strategy to strengthen the power of disease-resistant factors.

[0011] On the other hand, due to the rapid development of medicine and related disciplines and the accumulation of medical practice experience in the treatment of diseases with drugs in the past, it is found that the principle of "single-target drugs", which was once regarded as the gold standard for drug development, has declined and has been gradually replaced by "multi-target pharmacology", a branch of contemporary emerging pharmacology. Among the new drugs approved in recent years, the proportion of multi-target drugs has increased year by year. "Multi-target pharmacology" is to rationally design drugs based on the key factors of the occurrence and development of diseases and the concept of "one drug, multiple targets". The multi-target drugs developed in this way can act on more than one key pathogenic factor or anti-disease factor or both of a disease at the same time. Compared with "single-target drugs", they have better efficacy, higher safety, lower drug resistance, and have the potential to cure diseases.

[0012] The present invention simultaneously utilizes the RNAa principle and the multi-target pharmacology principle, and designs a multi-target saRNA small nucleic acid drug based on the selected key disease resistance factors that determine the occurrence and development of the target disease. Summary of the invention

[0013] The purpose of the present invention is to provide a preparation and application of multi-target small nucleic acid drugs based on small activating RNA technology.

[0014] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: In one aspect, the present invention provides a method for preparing a multi-target small nucleic acid drug based on small activating RNA technology, comprising the following steps: (1) Select at least two disease resistance genes based on the target disease; (2) extracting a 1000 base pair sequence of the promoter region upstream of the coding region of the above-mentioned disease resistance gene; (3) Based on the above promoter sequence, avoid CpG islands and locate species-conserved segments, and extract a 19-base sequence as the sense strand; (4) Based on the obtained sense strand, an antisense strand is designed, which is completely complementary to the 19 bases of the sense strand; (5) Add two deoxythymine or uracil residues to the 3' end of each chain.

[0015] Specifically, the target diseases described in step (1) include heart disease and cancer.

[0016] Furthermore, the heart diseases include heart failure, myocardial infarction, filled atherosclerotic heart disease, myocarditis, and angina pectoris.

[0017] Further, the cancers include liver cancer, lung cancer, pancreatic cancer, leukemia, oral cancer, skin cancer, head and neck cancer, laryngeal cancer, esophageal cancer, lymphoma, gastric cancer, bone cancer, kidney cancer, neuroblastoma, glioblastoma, cholangiocarcinoma, 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, uterine cancer.

[0018] Still further, the target diseases in step (1) include heart failure, liver cancer, lung cancer, and pancreatic cancer.

[0019] Specifically, the disease-resistant gene in step (1) is a key disease-resistant gene that plays a decisive role in the occurrence and development of the target disease.

[0020] According to some embodiments of the present invention, if the target disease is heart failure, the disease-resistant genes can be the ATP2A2 gene encoding the SERCA2a protein and the CAND1 gene encoding the CAND1 protein.

[0021] According to some embodiments of the present invention, if the target disease is cancer, the disease-resistant genes can be the TP53 gene encoding the p53 protein and the CEBPA encoding the C / EBP-α protein.

[0022] Specifically, in step (2), the sequences of the selected disease-resistant genes are queried on the GenBank website.

[0023] Specifically, the GC content in the sense strand in step (3) does not exceed 60%.

[0024] On the other hand, the present invention provides a small nucleic acid drug for treating heart diseases. The small nucleic acid drug includes saRNA targeting CAND1 and saRNA targeting ATP2A2. The sequence of the saRNA targeting CAND1 is as shown in SEQ ID NO:1, and the sequence of the saRNA targeting ATP2A2 is as shown in SEQ ID NO:2.

[0025] Specifically, the small nucleic acid drug for treating heart diseases is saR-ATP2A2:saR-CAND1.

[0026] saR-ATP2A2:saR-CAND1 sequence: 5’-UUGAAGCUGCAGAAGUGAU (SEQ ID NO:1)-dTdT-3’---CAND1.

[0027] 3’-dTdT-CACUUCGACCUCUUCACUU (SEQ ID NO:2)-5’---ATP2A2。

[0028] Specifically, the heart diseases include heart failure, myocardial infarction, atherosclerotic heart disease, myocarditis, and angina pectoris.

[0029] Further, the heart disease is heart failure.

[0030] On the other hand, the present invention provides a small nucleic acid drug for treating cancer, the small nucleic acid drug includes saRNA targeting TP53 and saRNA targeting CEBPA, the sequence of the saRNA targeting TP53 is as shown in SEQ ID NO:5, and the sequence of the saRNA targeting CEBPA is as shown in SEQ ID NO:6.

[0031] Specifically, the cancers include liver cancer, lung cancer, pancreatic cancer, leukemia, oral cancer, skin cancer, head and neck cancer, laryngeal cancer, esophageal cancer, lymphoma, gastric cancer, bone cancer, kidney cancer, neuroblastoma, glioblastoma, cholangiocarcinoma, 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, uterine cancer.

[0032] Further, the target diseases include liver cancer, lung cancer, and pancreatic cancer.

[0033] Specifically, the small nucleic acid drug for treating cancer is saR-p53:saR-CEBPA.

[0034] saR-p53:saR-CEBPA sequence: 5’-UUAGGAAGGCUCUCUGGAA (SEQ ID NO:5)-dTdT-3’---p53.

[0035] 3’-dTdT-CAUCCUUAACAGUGACCAG (SEQ ID NO:6)-5’---CEBPA.

[0036] Specifically, the above-mentioned small nucleic acid drugs are all obtained by using the above-mentioned preparation method.

[0037] On the other hand, the present invention provides the application of the above-mentioned small nucleic acid drug in the preparation of a drug for treating heart diseases and / or cancers.

[0038] Specifically, the drug may further contain other active ingredients and pharmaceutically acceptable excipients.

[0039] Furthermore, the pharmaceutically acceptable excipients are selected from excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH regulators, cryoprotectants, and flavoring agents.

[0040] Specifically, the excipients are selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, mannitol, magnesium stearate, starch, calcium phosphate, ethyl cellulose, methyl cellulose, alginic acid, gelatin, gum arabic, glyceryl monostearate, sodium starch glycolate, guar gum, glycerol, and propylene glycol.

[0041] Specifically, the buffers are selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0042] Specifically, the emulsifiers are selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, glyceryl oleate, and polyglyceryl-3 polyricinoleate.

[0043] Specifically, the stabilizers are selected from at least one of acacia gum, agar, alginic acid, cellulose ether, and carboxymethyl chitin ester.

[0044] Specifically, the diluents are selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0045] Specifically, the binders are selected from at least one of ethanol, starch paste, syrup, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, sodium alginate, and polyvinylpyrrolidone.

[0046] Specifically, the preservatives are selected from at least one of methyl paraben, propyl paraben, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, chlorobutanol, thimerosal, mercuric oxycyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, parachlorometacresol, benzalkonium bromide, benzalkonium chloride, and ethyl paraben.

[0047] Specifically, the lubricants are selected from at least one of magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, and poloxamer.

[0048] Specifically, the pH regulators are selected from at least one of citric acid, fumaric acid, succinic acid, tartaric acid, malic acid, and ascorbic acid.

[0049] Specifically, the cryoprotectants are selected from at least one of sucrose, glucose, mannitol, fructose, trehalose, dextran, lactose, glycerol, methanol, ethanol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), acetamide, or formamide.

[0050] Specifically, the flavoring agent is selected from at least one of sweet orange flavor essence, vanilla flavor essence, strawberry flavor essence, milk flavor essence, banana flavor essence, and cherry flavor essence.

[0051] Specifically, the dosage form of the drug is drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, powder, emulsion, solution, dripping pill, pill, buccal tablet, lyophilized powder injection, gel, suppository or aerosol.

[0052] On the other hand, the present invention provides the use of the above small nucleic acid drug in the preparation of a preparation for activating or upregulating the expression of a target gene in cells.

[0053] Specifically, the small nucleic acid drug is directly introduced into the cells.

[0054] Specifically, the target gene includes but is not limited to the ATP2A2 gene, CAND1 gene, TP53 gene, and CEBPA gene.

[0055] The beneficial effects of the present invention are as follows: The present invention provides a preparation method of a multi-target small nucleic acid drug based on the small activating RNA technology, and according to this method, small nucleic acid drugs for heart diseases and cancers are obtained, namely saR-ATP2A2:saR-CAND1 and saR-p53:saR-CEBPA. saR-ATP2A2:saR-CAND1 has extremely strong myocardial protection, reduces cell death, can reverse the cardiac function and the process of injurious electrophysiological and structural remodeling in heart failure mice, and even has the ability to stimulate myocardial regeneration. saR-p53:saR-CEBPA has extremely strong cancer cell killing ability and the ability to inhibit cancer cell migration, can reverse and prevent the growth and organ metastasis of tumors, and the inhibitory effect of saR-p53:saR-CEBPA on three types of cancer cells, namely liver cancer, lung cancer, and pancreatic cancer, and the ability to induce tumor growth and metastasis. Description of the Drawings

[0056] Figure 1 Effects of saRNA on ATP2A2 and CAND1 mRNA in AC16 cells (n = 4); in the figure, Sa50 is the drug concentration of 50 nM, Sa100 is the drug concentration of 100 nM, and Sa200 is the 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.

[0057] Figure 2Effect 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.

[0058] Figure 3 Effect of saRNA on TP53 and CEBPA mRNAs 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.

[0059] Figure 4 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.

[0060] Figure 5 Effect of saRNA on TP53 and CEBPA mRNAs 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.

[0061] Figure 6 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.

[0062] Figure 7 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.

[0063] Figure 8 The effect of saRNA on HepG2 cell migration (n=4); in the figure, Sa50 is a drug concentration of 50nM, Sa100 is a drug concentration of 100nM, and Sa200 is a drug concentration of 200nM; compared with the Scramble control group, *P<0.05.

[0064] Figure 9 Tunel detected the effect of saRNA on HepG2 cell apoptosis (n=4); in the figure, Sa50 is a drug concentration of 50nM, Sa100 is a drug concentration of 100nM, and Sa200 is a drug concentration of 200nM; compared with the Scramble control group, *P<0.05, **P<0.01; compared with the Sa50 concentration group, #P<0.05.

[0065] Figure 10 Flow cytometry was used to detect the effect of saRNA on apoptosis of 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. DETAILED DESCRIPTION

[0066] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0067] Design steps of the present invention: (1) According to the target disease, two key disease resistance genes that play a decisive role in the occurrence and development of the target disease can be selected. For example, for heart failure, the ATP2A2 gene encoding the SERCA2a protein and the CAND1 gene encoding the CAND1 protein can be selected; for cancer, the TP53 gene encoding the p53 protein and the CEBPA gene encoding the C / EBP-α protein can be selected.

[0068] (2) Search the GenBank website for the selected gene sequence and extract the 1000 base pair sequence of the promoter region upstream of the gene coding region.

[0069] (3) Based on the promoter sequence obtained above, avoid CpG islands, locate the species-conserved segment (especially the highly conserved sequence between humans and mice), and extract a 19-base sequence as the sense strand (SS), the GC content of which should not exceed 60%.

[0070] (4) Based on the sense strand obtained above, design an antisense strand (antisense or AS). The antisense strand must be completely complementary to the 19 bases of the sense strand.

[0071] (5) Add two deoxythymine (dT) or uracil (U) to the 3' end of each chain.

[0072] Example 1 saR-ATP2A2: saR-CAND1 According to the above multi-target saRNA design steps, the present invention designs the following double-stranded oligonucleotide fragment (dsRNA), namely saR-ATP2A2: saR-CAND1. One of the chains can recognize and bind to the promoter region of the ATP2A2 gene, inducing the transcription process of this gene; the other chain can recognize and bind to the promoter region of the CAND1 gene, and induce the transcription process of the CAND1 gene.

[0073] saR-ATP2A2:saR-CAND1 sequence: 5'-UUGAAGCUGCAGAAGUGAU (SEQ ID NO: 1)-dTdT-3'---CAND1.

[0074] 3'-dTdT-CACUUCGACCUCUUCACUU (SEQ ID NO: 2)-5'---ATP2A2.

[0075] Control group saRNA sequence: 5'-CUUACGCUGAGUACUUCGA (SEQ ID NO: 3)-dTdT-3'.

[0076] 3'-dTdT-GAAUGCGACUCAUGAAGCU (SEQ ID NO: 4)-5'.

[0077] Experimental Example 1 Effect of saR-ATP2A2:saR-CAND1 on chronic heart failure 1 Experimental methods: The saR-ATP2A2:saR-CAND1 was transfected into the AC16 human cardiomyocyte cell line using lipofectamine. The specific operation was as follows: Dilute 10 μL of lipofectamine in 100 μL of serum-free DMEM medium, then mix it with 1 μg of saR-ATP2A2:saR-CAND1 and incubate at room temperature for 15 min, and then add it to the AC16 human cardiomyocyte cell line. After 48 hours, qRT-PCR detection was performed.

[0078] Quantitative real-time PCR (qRT-PCR): Lyse the transfected AC16 human cardiomyocytes, extract the total RNA using RNAzol reagent, quantify the RNA yield with Qubit, and reverse transcribe the RNA using reverse transcriptase with Oligodt primers. Analyze the cDNA samples with pre-designed real-time PCR primers to detect the ATP2A2 and CAND1 mRNA levels.

[0079] Western blot: Prepare the polyacrylamide gel according to the instructions of the 12.5% ExpressCast PAGE Color Gel Quick Kit. The concentration of the separating gel is 12.5%, the sample loading volume is 20 μL, the electrophoresis conditions for the stacking gel are 80 V for 30 min; the electrophoresis conditions for the separating gel are 120 V for 60 min.

[0080] Electrotransfer: Use a polyvinylidene fluoride membrane and transfer the membrane at 25 V for 10 min using the semi-dry transfer method.

[0081] Blocking: Block with 5% skim milk for 1 h.

[0082] Primary antibody incubation: Dilute the primary antibody with 5% skim milk at a ratio of 1:2000 and incubate overnight at 4°C. Wash with TBST 3 times, 10 min each time.

[0083] Secondary antibody incubation: Dilute the secondary antibody with 5% skim milk at a ratio of 1:1000 and incubate at room temperature for 2 h. Wash with TBST 3 times, 10 min each time.

[0084] Developing: Prepare the developing solution by mixing solution A and solution B of the developer in a ratio of 1:1. Use the ChemiDocMP Imaging System to develop the PVDF membrane, and then perform gray-scale analysis on the developed image using ImageJ.

[0085] The intracellular calcium content was detected using a kit purchased from Nanjing Jiancheng Bioengineering Institute.

[0086] Detection method for cell apoptosis: After 48 hours of transfection, the culture medium was aspirated, and the cells were digested. The cells were centrifuged at 1000 rpm for 1 minute, and then 400 μl of binding solution was added. 2.5 μl of V-FITC dye was added to the cells, and the cells were gently pipetted to mix evenly. The cells were incubated at 4°C in the dark for 15 minutes. Then, 5 μl of PI dye was added, and the cells were gently pipetted to mix evenly. The cells were incubated at 4°C for 5 minutes. Flow cytometry was used to detect cell apoptosis.

[0087] 2 Experimental results: 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 were also significantly upregulated ( Figure 2 ); Under hypoxic conditions, calcium overload occurred in AC16 cells, and cell apoptosis occurred. 48 hours after transfection with saR-ATP2A2:saR-CAND1, calcium overload in the cells was reversed to normal, and cell apoptosis was inhibited.

[0088] The above experimental results were further confirmed in primary cultured mouse cells.

[0089] 3 Experimental methods: Lipid nanoparticles encapsulated with saR-ATP2A2:saR-CAND1 (LNP-PCM / saR-ATP2A2:saR-CAND1) treated with the cardiomyocyte-specific targeting peptide PCM (WLSEAGPVVTVRALRGTGSW, SEQ ID NO:9) were injected into mice via the tail vein.

[0090] The experimental procedures for qRT-PCR and Western blot were the same as above.

[0091] Detection method: Echocardiography was performed using a high-resolution Vevo 770 imaging system platform. A 30 MHz probe was used for M-Mode echocardiography to measure the ejection fraction, left ventricular fractional shortening, end-diastolic diameter of the ventricle, end-systolic diameter of the ventricle, and the thickness of the anterior and posterior walls at end-diastole. Each mouse was examined 2 times at each section, and the values of 5 consecutive cardiac cycles were read each time, and the average value was taken.

[0092] 4 Experimental results: 48 hours later, the levels of ATP2A2 and CAND1 transcripts (mRNA) were measured by qRT-PCR, and both were found to be significantly upregulated (more than +3 times); Western blot experiments confirmed that the proteins SERCA2a and CAND1 encoded by ATP2A2 and CAND1 were also significantly upregulated.

[0093] The mouse aortic arch constriction (TAC) was used as a classic surgical hypertrophic cardiomyopathy model and a decompensated transition to a chronic heart failure model (similar to human heart failure caused by hypertension or aortic valve stenosis) to detect cardiac systolic and diastolic functions. It was found that heart failure cardiac function indexes such as ejection fraction and left ventricular shortening fraction decreased significantly.

[0094] Indexes reflecting ventricular anatomical size, including left ventricular end-diastolic diameter and left ventricular end-systolic diameter, increased significantly, while values such as left ventricular end-diastolic anterior and posterior wall thicknesses and left ventricular end-systolic anterior and posterior wall thicknesses decreased significantly; cardiomyocyte apoptosis and pyroptosis increased significantly, and autophagy decreased; the incidence of arrhythmia induction increased substantially.

[0095] After 48 h of treatment with LNP-PCM / saR-ATP2A2:saR-CAND1, the above-mentioned heart failure cardiac function decline, adverse structural remodeling, cell death, and autophagy impairment were significantly reversed and improved. Notably, the regenerative capacity of some cardiomyocytes was activated, which was one of the mechanisms for the partial recovery of the reduced left ventricular anterior and posterior wall thicknesses.

[0096] In all the above experiments, compared with the control saRNA and the negative control (Mock-control) group, no significant effect was produced. All statistical data were obtained by comparing the saR-ATP2A2:saR-CAND1 group with the control saRNA and the negative control (Mock-control) group respectively.

[0097] As can be seen from the above, saR-ATP2A2:saR-CAND1 has a strong cardioprotective effect, reduces cell death, can reverse the cardiac function and the process of injurious electrophysiological and structural remodeling 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.

[0098] Example 2 saR-p53:saR-CEBPA To further verify the feasibility of multi-target saRNA as a small nucleic acid drug, according to the method in the above multi-target saRNA design steps, the following double-stranded oligonucleotide was designed, namely saR-p53:saR-CEBPA. One strand can recognize and bind to the promoter region of the p53 gene and induce the transcription process of this gene; the other strand can recognize and bind to the promoter region of the CEBPA gene and induce the transcription process of the CEBPA gene.

[0099] saR-p53:saR-CEBPA sequence: 5’-UUAGGAAGGCUCUCUGGAA (SEQ ID NO:5)-dTdT-3’---p53。

[0100] 3’-dTdT-CAUCCUUAACAGUGACCAG (SEQ ID NO:6)-5’---CEBPA。

[0101] Control group saRNA sequence: 5’-CUUACGCUGAGUACUUCGA (SEQ ID NO:7)-dTdT-3’。

[0102] 3’-dTdT-GAAUGCGACUCAUGAAGCU (SEQ ID NO:8)-5’。

[0103] 1 Experimental method: Use liposomes to transfect saR-p53:saR-CEBPA into human hepatocellular carcinoma cell line HEPG2 and human non-small cell lung cancer cell line A549 respectively. The specific operation is the same as that in Example 1.

[0104] The detection methods of qRT-PCR, Western blot, and cell apoptosis are the same as those in Example 1.

[0105] Detection method of cell viability: At 48 h after transfection, add CCK-8 reagent, incubate for 2 h at 37 °C in the dark, use a microplate reader to detect the absorbance value at a wavelength of 450 nm, and perform statistics to analyze the effect of saR-p53:saR-CEBPA on cell viability.

[0106] Cell apoptosis was detected using a TUNEL detection kit (Vazyme).

[0107] The detection method of cell migration ability includes the following steps: For cell migration ability analysis, seed and transfect A549 cells in a 24-well culture plate. After 48 hours, scratch the cells and expose them to normal glucose (5 mM) in a normoxic (21% O2) environment. The cells are in serum starvation state, and mitomycin-C (10 μg / mL, Roche) is added to inhibit cell proliferation. Calculate the migration using the area of the cell-free region at 0 hour and 24 hours.

[0108] 2 Experimental results: Lipofectamine was used to transfect saR-p53:saR-CEBPA into the human hepatocellular carcinoma cell line HEPG2 and the human non-small cell lung cancer cell line A549 respectively. After 48 hours, the 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 were also significantly upregulated ( Figure 4 、 Figure 6 ).

[0109] Cell-level experiments found that compared with the scramble control group, saR-p53:saR-CEBPA induced a visibly and statistically significant decrease in cell viability (cck-8 assay) in three different cancer cell lines ( Figure 7 ), induced a large number of cell apoptoses (TUNEL staining) ( Figure 9 、 Figure 10 ), inhibited cancer cell proliferation (Edu assay), inhibited the cancer cell migration ability (Transwell assay) ( Figure 8 ), inhibited the cancer cell invasion ability (healing assay), and inhibited the cancer cell colony formation ability (colony formation assay).

[0110] 3 Experimental methods: In the nude mouse tumor-bearing model, we implanted EPG2, PANC-1, and A549 cells into the backs of nude mice respectively to induce tumor formation.

[0111] 4 Experimental results: Compared with the control group, directly injecting saR-p53:saR-CEBPA into the tumor tissue significantly reduced the size, weight, and volume of the tumor and inhibited tumor growth. The EPG2, PANC-1, and A549 cells 60 hours after transfection with saR-p53:saR-CEBPA almost lost the ability to form tumors in nude mice.

[0112] When EPG2, PANC-1, and A549 cells were injected into nude mice via the tail vein respectively, obvious cross-tissue and organ metastasis phenomena were found in each. After injecting saR-p53:saR-CEBPA via the tail vein, the cancer cell metastasis was significantly reduced or even disappeared.

[0113] EPG2, PANC-1, and A549 cells were pre-transfected with saR-p53:saR-CEBPA for 60 h and then injected into mice via the tail vein. It was found that the phenomenon of tissue and organ metastasis of these cells was significantly reduced or even disappeared.

[0114] In all the above experiments, the control group of saRNA showed no 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 of saRNA and the Mock-control group respectively.

[0115] Conclusion: saR-p53:saR-CEBPA has extremely strong cancer cell killing ability and the ability to inhibit cancer cell migration, reverse and prevent tumor growth and organ metastasis. In particular, we verified the inhibitory effect of saR-p53:saR-CEBPA on three types of cancer cells, namely 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 anti-cancer small nucleotide drug.

[0116] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a multi-target small nucleic acid drug based on small activating RNA technology, characterized in that, The following steps are involved: (1) Select at least two disease resistance genes based on the target disease; (2) extracting a 1000 base pair sequence of the promoter region upstream of the coding region of the disease resistance gene in step (1); (3) Based on the promoter sequence, avoid CpG islands and locate species-conserved segments, and extract a 19-base sequence as the sense strand; (4) Based on the sense strand obtained in step (3), design an antisense strand that is completely complementary to the 19 bases of the sense strand; (5) Add two deoxythymine or uracil residues to the 3' end of each chain.

2. A small nucleic acid drug for treating heart diseases prepared by the preparation method according to claim 1, characterized in that, The small nucleic acid drug includes saRNA targeting CAND1 and 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.

3. The small nucleic acid drug according to claim 2, characterized in that, The heart disease includes heart failure, myocardial infarction, filled atherosclerotic heart disease, myocarditis or angina pectoris.

4. A small nucleic acid drug for treating cancer prepared by the preparation method according to claim 1, characterized in that, The small nucleic acid drug includes saRNA targeting TP53 and 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.

5. The small nucleic acid drug according to claim 4, wherein The cancers include liver cancer, lung cancer, pancreatic cancer, leukemia, oral cancer, skin cancer, head and neck cancer, laryngeal cancer, esophageal cancer, lymphoma, gastric cancer, bone cancer, kidney cancer, neuroblastoma, glioblastoma, bladder cancer, endometrial cancer, breast cancer, peritoneal cancer, cervical cancer, bile duct cancer, choriocarcinoma, colorectal cancer, melanoma, myeloma, ovarian cancer, prostate cancer, basal cell carcinoma, rectal cancer, squamous cell carcinoma, thyroid cancer or uterine cancer.

6. Use of the small nucleic acid drug according to any one of claims 2 to 5 in the preparation of drugs for treating heart diseases and / or cancer.

7. The application according to claim 6, wherein The medicine also contains other active ingredients and pharmaceutically acceptable excipients.

8. The application according to claim 6, characterized in that, The dosage form of the medicine is drops, tincture, powder, tablet, capsule, granule, ointment, powder, emulsion, pill, freeze-dried powder injection, gel, suppository or aerosol.

9. Use of the small nucleic acid drug according to any one of claims 2 to 5 in preparing a preparation for activating or upregulating the expression of a target gene in a cell.

10. The application according to claim 9, wherein The target genes include ATP2A2 gene, CAND1 gene, TP53 gene and CEBPA gene.

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