SaRNA for activating AMBRA1 transcription, modified saRNA and application thereof

Abstract

The invention discloses saRNA for activating AMBRA1 transcription, modified saRNA and application of the saRNA and the modified saRNA. AMBRA1 is used as a target spot, saRNA sequences targeting an AMBRA1 promoter region are designed, 31 saRNA sequences and saRNA modified by the saRNA sequences are obtained through screening, and the saRNA sequences and the saRNA modified by the saRNA sequences can up-regulate the expression level of AMBRA1 in tumor cells so as to regulate the cell cycle and inhibit the proliferation, migration and invasion ability of the tumor cells. The invention further studies the feasibility of the saRNA combined chemotherapeutic drug as a tumor combined treatment scheme, and the combined administration of the saRNA and the chemotherapeutic drug further significantly inhibits the proliferation ability of tumor cells, increases apoptosis, and enhances the sensitivity of the tumor cells to the chemotherapeutic drug, so that the prognosis of tumor patients is expected to be improved; the saRNA or the modified saRNA provided by the invention has an application prospect in the aspects of preparation of anti-tumor drugs and preparation of anti-tumor drug combined therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to small activating RNA (saRNA) and its application, in particular to saRNA activating AMBRA1 transcription and its application in preparing anti-tumor drugs, and belongs to the field of small activating RNA and its application. BACKGROUND

[0002] Small activating RNA (saRNA) is a kind of double-stranded small RNA with a length of 19-21 bp targeting the promoter region of the target gene. saRNA and small interfering RNA (siRNA) have exactly the same structure and chemical composition, but their biological functions are completely opposite. The key difference between saRNA and siRNA lies in the mechanism of action: siRNA exerts its effect by binding to AGO2-4, while saRNA only specifically binds to AGO2. After AGO2 binds to saRNA to form an AGO-saRNA complex, it enters the nucleus through importin-8, and the guide strand of saRNA targets the gene promoter, further recruiting key proteins for transcription initiation, such as RNA helicase A (RHA), RNA polymerase-associated protein CTR9 homolog (CTR9), and RNA polymerase II-associated protein 1 (PAF1) to form an RNA-mediated transcriptional activation (RITA) complex, which changes the chromatin structure to initiate transcriptional activation.

[0003] In many diseases, the decrease in expression of some key proteins is the main cause of pathogenesis. saRNA is very effective in inducing up-regulation of target gene expression, and has the advantages of specific activation, no change in genome, high and long-acting effect, and low toxicity, etc. In addition, saRNA can be easily synthesized by chemical modification such as (2'-OMe, 2'-F), which has been proved to reduce its off-target possibility, increase endonuclease resistance and serum stability. Therefore, this technology has a good development prospect, and is applied to restore the expression of pathogenic genes to achieve the purpose of treating diseases that cannot be targeted at present. At present, the up-regulation of genes in animals is mainly achieved by genetically engineered mice and virus vector-mediated transfection in animals. Its limitations and adverse side effects include a cumbersome construction process and various immune responses, etc. saRNA activates endogenous target gene expression in a site-specific manner, providing a safer and more concise method of gene therapy.

[0004] As a new drug development approach, saRNA is one of the few gene regulation technologies that can activate the expression of tumor suppressor genes. By targeting tumor suppressor genes, saRNA can enhance their transcriptional activation and restore normal cell phenotype. saRNA can induce cell cycle arrest, cell senescence, apoptosis, inhibit cell proliferation, metastasis and increase drug sensitivity to exert its anticancer effect. AMBRA1 (activating molecule in Beclin1-regulated autophagy) is involved in the regulation of many key physiological activities, such as post-translational modification of proteins, cell cycle, cell proliferation and cell death. As an important upstream regulator of the transition from G1 to S phase, AMBRA1 regulates its abundance by mediating the degradation of D-type cyclins, thereby slowing down the replication stress. In addition, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain the integrity of the genome during DNA replication, thereby inhibiting dysplasia and tumor growth. AMBRA1 is abnormally expressed in many tumor tissues, such as lung cancer patients with low expression of AMBRA1 have a poorer prognosis, and the level of AMBRA1 in the skin tissue of melanoma patients is negatively correlated with the risk of their spread. Experimental studies have shown that in a mouse model of melanoma with Braf / Pten mutations, AMBRA1 deletion can accelerate tumor growth and enhance the invasiveness and metastasis of melanoma by increasing cell motility / invasion, activating EMT-like processes and altering extracellular matrix remodeling, and reducing overall survival. AMBRA1 can also regulate the dephosphorylation of proto-oncogene c-MYC by interacting with phosphatase PP2A, thereby further preventing tumorigenesis and tumor hyperproliferation. In lung cancer, glioma, breast invasive carcinoma, renal clear cell carcinoma, renal chromophobe carcinoma, thymus carcinoma, esophageal carcinoma, mesothelioma, rectal adenocarcinoma, ovarian serous cystadenocarcinoma, thyroid carcinoma and other tumors, AMBRA1 acts as a tumor suppressor and its low expression is usually associated with poor prognosis. Therefore, AMBRA1 will be able to serve as a key target for tumor therapy to provide new ideas for drug development and clinical treatment programs.

[0005] AMBRA1 is also considered to be an autophagy-related protein, and is an important factor in the process of autophagosome formation. AMBRA1 is involved in immune regulation, and can regulate the activity and proliferation of various T cell subtypes such as precursor T cells and naive T cells in an autophagy-dependent manner and an autophagy-independent manner. AMBRA1 can activate the transcription of FOXP3 to promote the differentiation and maintenance of regulatory T cells. AMBRA1 is also one of the key genes for the development of the central nervous system, and plays a role in embryonic development and nervous system diseases. AMBRA1 mutant mice show impaired autophagy, ubiquitinated protein accumulation, and cell proliferation disorders, leading to spinal malformation and embryonic death. And AMBRA1 can produce a certain protective effect on lead-induced nerve damage. In both zebrafish and mouse animal models, reduced expression of AMBRA1 leads to muscle morphological abnormalities, structural and functional defects of muscle fibers, and other phenotypes.

[0006] In summary, AMBRA1 will be able to serve as a key target for the treatment of various diseases such as tumors, immunity and nervous system, and provide new ideas for the drug research and development and clinical treatment plan of related diseases. Screening of saRNA sequences targeting AMBRA1 and activating AMBRA1 transcription based on AMBRA1 as a research target has important application prospects for the treatment of various diseases such as anti-tumor. SUMMARY

[0007] One of the purposes of the present application is to provide saRNA activating AMBRA1 transcription;

[0008] The second purpose of the present application is to provide modified saRNA activating AMBRA1 transcription;

[0009] The third purpose of the present application is to apply the saRNA activating AMBRA1 transcription or the modified saRNA activating AMBRA1 to the preparation of anti-tumor drugs.

[0010] The fourth purpose of the present application is to combine the saRNA nucleic acid drug activating AMBRA1 transcription with other chemotherapy and immunotherapy drugs for tumor combination therapy.

[0011] The above purposes of the present application are achieved by the following technical solutions:

[0012] One aspect of the present application is to provide saRNA activating AMBRA1 transcription, which is selected from any one of the saRNAs consisting of a sense strand and an antisense strand shown in (1) to (31) below:

[0013] (1) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 1 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 2; (2) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 3 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 4; (3) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 5 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 6; (4) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 7 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 8; (5) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 9 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 10; (6) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 11 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 12; (7) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 13 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 14; (8) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 15 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 16; (9) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 17 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 18; (10) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 19 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 20; (11) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 21 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 22; (12) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 23 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 24; (13) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 25 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 26; (14) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 27 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 28; (15) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 29 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 30; (16) saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 31 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No.32 the antisense strand containing the nucleotide sequence shown in SEQ ID No. 34; (18) an saRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 35 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 36; (19) an saRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 37 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 38; (20) an saRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 39 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 40; (21) an saRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 41 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 42; (22) an saRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 43 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 44; (23) an saRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 45 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 46; (24) an saRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 47 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 48; (25) an saRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 49 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 50; (26) an saRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 51 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 52; (27) an saRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 53 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 54; (28) an saRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 55 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 56; (29) an saRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 57 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 58; (30) an saRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 59 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 60; (31) an saRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 61 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 62.

[0014] The present application also provides a saRNA sequence having 15 or more consecutive identical nucleotide sequences in succession with the nucleotide sequence of any one of saAMBRA1-1 to saAMBRA1-31 shown in (1) to (31) above; or, the TT base at the 3' end of the sense strand or the antisense strand of the saRNA is replaced with two bases that are complementary to the promoter region of the AMBRA1 gene.

[0015] In order to improve the stability of the saRNA in vivo, reduce the degradation of exonuclease, or promote the delivery of the saRNA in vivo to better exert the effect of activating the AMBRA1 gene, another aspect of the present application provides a modified saRNA obtained by modifying the saRNA provided by the present application. The saRNA provided by the present application can be modified in various ways to obtain a modified saRNA with improved stability or improved effect of activating the AMBRA1 gene. Those skilled in the art can modify the saRNA as described above according to the needs, including but not limited to one or more of the following modification types: (1) substitution of the 2' carbon position of the sugar ring structure of the nucleotide, including but not limited to substitution of the -OH group of the 2' hydroxyl group of the sugar ring with a methoxy group, a methylethoxy group, a methyl group, an amino group, a fluorine group, an -O-2-methylthioethyl group, an -O-3-aminopropyl group, or an -O-3-dimethylaminopropyl group; (2) modification of the phosphodiester bond between nucleotides, including but not limited to substitution of the bond with a phosphorothioate, a boranophosphonate, or a methylphosphonate; (3) modification of the nucleosides of the sequence, including but not limited to substitution of the nucleosides in the saRNA with peptide nucleosides, locked nucleosides, or unlocked nucleosides; (4) base modification, including but not limited to base modification or substitution of the 5-methylcytosine, 6-methyladenine, ribavirin, pseudouracil, and / or inosine of the nucleoside base; and (5) no modification or modification of the terminal end of the saRNA sequence, including but not limited to phosphorylation, phosphorothioation, vinylphosphonation, or other phosphorylation analogs and phosphate prodrugs.

[0016] In order to improve the strand selectivity of the saRNA to the target gene and the effect of activating the target gene, the 5' end of the antisense strand of the saRNA can be modified with phosphorylation or phosphorylation analogs or phosphate prodrugs, including but not limited to the following modification methods:

[0017]

[0018] In one preferred embodiment of the present application, the modified saRNA is obtained by modifying the saRNA with at least one modification selected from the following (1)-(6): (1) converting one non-ester oxygen bond between the phosphodiester into a sulfur; (2) substituting the -OH group at the 2' carbon position of the nucleobase with, without limitation, CH3 (methyl), -OCH3 (methoxy), -OCH2CH2OCH3 (methoxyethyl), -F (fluorine), NH2, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl, and / or deoxy-modifying the 2'-OH; preferably, the -OH group at the 2' carbon position of the nucleobase is modified by alternately substituting with methoxy and fluorine; (3) two or more phosphorothioate modifications at the end of the sense strand or the antisense strand of the saRNA; (4) replacing the TT base at the 3' end of the sense strand or the antisense strand of the saRNA with two bases that originally and complementarily pair with the promoter region of the AMBRA1 gene; (5) no modification or modification of the phosphate, phosphorothioate, or phosphate analog and phosphate prodrug modification at the 5' end of the antisense strand of the RNA sequence, and no modification or inverted abasis sugar cap modification at the 5' end of the sense strand.

[0019] The antisense strand described in the present application can be a polynucleotide complementary to the promoter region of the target gene, and the length thereof can be 19 nt or more; in one preferred embodiment, the length of the antisense strand can comprise the sequence and be between 19 nt and 26 nt; in addition, the antisense strand described in the present application can also have a nucleotide sequence partially complementary to the sense strand.

[0020] The antisense strand of saAMBRA1-4 is preferably chemically modified in the present application, and the nucleotide sequence of the modified antisense strand is as follows: (Phos)mU*fC*fUfAmCfUmGfUmCmAmCfUmCfAmGfUmAfGmU*dT*dT; wherein, * represents phosphorothioate modification, m represents 2'-methoxy modification, f represents 2'-fluorination modification, and Phos represents 5' phosphorylation modification.

[0021] Another aspect of the present application provides a delivery system for double-stranded RNA or hairpin double-stranded RNA or its transcription vector containing the saRNA sequence. The saRNA is delivered into cells in vivo by the delivery system; the vector system is selected from nucleic acid-lipid particles, liposomes, micelles, viral particles, nucleic acid complexes or mixtures consisting of more than one. In some cases, the modified saRNA molecule is complexed with lipids such as cationic lipids to form a lipid-nucleic acid complex; in one embodiment, the modified saRNA molecule is complexed with polymers such as cationic polymers to form a polymer-nucleic acid complex; in one embodiment, the modified saRNA molecule can also be complexed with cyclodextrin or its polymers; preferably, the modified saRNA molecule is encapsulated in nucleic acid-lipid particles.

[0022] Another aspect of the present application provides an anti-tumor pharmaceutical composition, wherein the pharmaceutical composition contains the saRNA or modified saRNA and a pharmaceutically acceptable carrier system and formulation adjuvant; wherein the carrier system contains any one selected from nucleic acid-lipid particles, liposomes, micelles, viral particles or nucleic acid complexes or mixtures consisting of more than one; the formulation adjuvant includes but is not limited to physiological saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol or ethanol; in addition, the pharmaceutical composition can be prepared into conventional formulations such as injection formulations, lyophilized formulations, tablets, capsules, powders, ointments or syrup dosage forms by further adding various formulation adjuvants or carriers, which all belong to the conventional formulation methods in the art.

[0023] The present application finds that the saRNA provided by the present application can effectively improve the inhibitory effect of chemotherapeutic drugs on tumors when used in combination with chemotherapeutic drugs; accordingly, in one preferred embodiment of the present application, the pharmaceutical composition can further contain chemotherapeutic drugs, which include antimetabolites, anti-tubulin drugs, alkylating agents, topoisomerase inhibitors, anti-tumor antibiotics or platinum drugs; preferably platinum drugs.

[0024] The tumor in the present application can be various tumors of digestive system, respiratory system, blood and lymphatic system or skin origin, preferably lung cancer.

[0025] The administration method of the pharmaceutical composition of the present application can be determined according to the symptoms or severity of the disease of the patient, and the administration method includes but is not limited to oral administration, intramuscular injection, intra-arterial injection, intravenous injection, subcutaneous injection, intracardiac injection or intrathecal injection, etc.; the administration dose of the pharmaceutical composition of the present application is determined according to the severity of the disease of the patient, administration time, administration method, age, body weight, gender and other factors.

[0026] The present application takes AMBRA1 as a research target, designs saRNA sequences targeting the promoter region of AMBRA1, and systematically screens 31 saRNA sequences of human AMBRA1 genes, and the results show that saRNA (saAMBRA1-4) can activate AMBRA1 expression in non-small cell lung cancer cells, thereby regulating cell cycle, inhibiting tumor cell proliferation, migration and invasion ability. In addition, the present application further studies the feasibility of chemically modified saRNA (saAMBRA1-4M) activating AMBRA1 expression combined with cisplatin as a treatment scheme for non-small cell lung cancer. Compared with the saAMBRA1-4M or cisplatin single administration group, the apoptosis of the combined administration group is further increased, the tumor cell proliferation ability is obviously inhibited, and the sensitivity of tumor cells to chemotherapeutic drugs is enhanced, thereby improving the prognosis of tumor patients. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 To reflect the activation effect of saRNA on AMBRA1 mRNA level in human non-small cell lung cancer A549 / H1299 cells.

[0028] Figure 2 To reflect the concentration effect of AMBRA1 mRNA level activation effect after transfection of saAMBRA1-4 in A549 / H1299 cells for 72h.

[0029] Figure 3 To evaluate the effect of AMBRA1 activation on A549 / H1299 cell cycle by flow cytometry after transfection of saAMBRA1-4 in A549 / H1299 cells for 72h.

[0030] Figure 4 To reflect the expression level of CCNA2, CCNE1, RAD51, E2F1 and other genes after transfection of saAMBRA1-4 to activate AMBRA1 expression in A549 / H1299 cells.

[0031] Figure 5 To evaluate the effect of AMBRA1 expression level on the proliferation ability, migration ability and invasion ability of non-small cell lung cancer cells; A: cell proliferation curve after transfection of saAMBRA1-4 to activate AMBRA1 expression in A549 / H1299 cells; B: evaluate the effect of AMBRA1 activation on the migration ability of A549 / H1299 cells by scratch test; C: detect the effect of saAMBRA1-4 activation of AMBRA1 transcription on the invasion ability of A549 / H1299 cells by Transwell experiment.

[0032] Figure 6To evaluate the effect of chemical modification of saAMBRA1-4 on the up-regulation of AMBRA1 transcription in A549 / H1299 cells.

[0033] Figure 7 To evaluate the effect of different concentrations of cisplatin, saAMBRA1-4M alone or in combination on the proliferation of A549 / H1299 cells.

[0034] Figure 8 To evaluate the effect of different concentrations of cisplatin and saAMBRA1-4M alone or in combination on apoptosis: Annexin V / PI double staining flow cytometry was used to detect the apoptosis of A549 / H1299 cells treated under different conditions.

[0035] Figure 9 To evaluate the effect of cisplatin and saAMBRA1-4M alone or in combination on the migration ability of H1299 cells. DETAILED DESCRIPTION

[0036] The present application will be further described below in conjunction with specific embodiments, and the advantages and features of the present application will become more apparent with the description. However, these embodiments are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the present application without departing from the spirit and scope of the present application, and such modifications and substitutions fall within the scope of protection of the present application.

[0037] Experimental methods

[0038] 1. Cell culture and cell transfection

[0039] A549 cells and H1299 cells were cultured in RPMI 1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin) and DMEM medium (10% fetal bovine serum, 1% penicillin-streptomycin) for 24 hours (24-well plate), respectively, and then the corresponding concentration of saRNA and 1 μL Lipo2000 were diluted into 50 uL OPTI-MEM medium according to the instructions of the transfection reagent. The two solutions were mixed and incubated for 15 minutes before being used for cell transfection. After incubation for 72 hours, the cells were collected for subsequent experiments.

[0040] 2. Total RNA extraction, reverse transcription and real-time fluorescent quantitative PCR detection

[0041] (1) Total RNA extraction: Add 500 μL of TRIzol reagent to the cells in the culture plate, let it stand at room temperature for 10 minutes, transfer the liquid to a 1.5 mL centrifuge tube, add 100 μL of chloroform, mix well and centrifuge, then take the upper aqueous phase solution to a new 1.5 mL centrifuge tube and add an equal volume of isopropanol, mix the solution well and centrifuge, then remove the liquid phase and wash the white precipitate with pre-cooled 75% ethanol twice; after removing the ethanol, let the precipitate air dry at room temperature; then add an appropriate amount of Nuclease-free water to dissolve the precipitate, and use a nucleic acid quantification instrument to determine the RNA concentration.

[0042] (2) Reverse transcription: RNA reverse transcription was performed using the HiScript II 1st Strand cDNA Synthesis Kit from Novozyme. Take 300 ng of RNA, add Nuclease-free water to make up to 8 μL, add 2 μL of 5×gDNA wiperMix, gently mix the reaction solution, and then place it at 42°C for 2 minutes. Then add 2 μL of 10×RTMix, 2 μL of HiScript III Enzyme Mix, 1 μL of Oligo(dT)20VN, 1 μL of Random hexamers, and 4 μL of Nuclease-free water to the above reaction solution. Gently mix the solution, and then perform amplification reaction in a PCR amplifier with the following program: 37°C for 15 minutes, 85°C for 5 seconds.

[0043] (3) Real-time fluorescent quantitative PCR reaction: Take 4 μL of the above reaction solution after dilution, add 2 μL of forward primer (2 μM), 2 μL of reverse primer (2 μM), 10 μL of 2×GoTaq qPCR Master Mix, and 4 μL of Nuclease-free water, respectively. The reaction was performed in an ABI QuantStudio6Flex real-time fluorescent quantitative PCR instrument. Each sample was run in triplicate, GAPDH was used as an internal reference, and the data was analyzed by relative quantification 2 －ΔΔCt method.

[0044] 3. Cell cycle detection

[0045] Add pre-cooled 75% ethanol to the centrifugally collected cells and fix overnight at 4°C. Then centrifuge to remove the ethanol solution and add pre-cooled PBS buffer for washing. Then add propidium iodide staining solution (ratio of 0.5 mL staining buffer, 25 μL propidium iodide staining, 10 μL RNase A) to the above cells to resuspend the cell precipitate, and incubate at 37°C in the dark for 30 minutes. Finally, collect the data using a flow cytometer and analyze the cell cycle results using ModFit software.

[0046] 4. Annexin V-FITC apoptosis detection

[0047] Add 195 μl Annexin V-FITC binding solution to centrifugally collected cells, then add 5 μl Annexin V-FITC and 10 μl propidium iodide staining solution in turn, and mix gently. Incubate at room temperature for 20 minutes in the dark, and detect cell apoptosis using a flow cytometer.

[0048] 5. Cell proliferation experiment

[0049] Centrifugally collect the transfected cells, resuspend and count them, and prepare a cell suspension of the same density to add to a 96-well plate (4-5 parallel wells are set for each sample). At a fixed time point, add 100 μl of a CCK-8 reagent and culture medium premix (CCK-8 reagent: culture medium = 1:9), continue to culture for 0.5-2 h (depending on the type of cell and culture medium), and then use an enzyme marker to measure the absorbance value at a wavelength of 450 nm.

[0050] 6. Scratch experiment

[0051] Prepare a cell suspension as above. Add 70 μl of the cell suspension to the wells of a Culture-Insert (2 wells) and culture. After observing cell adhesion and confluence under a microscope, remove the Culture-Insert. Then wash with PBS buffer to remove cell debris and non-adherent cells, and add fresh culture medium. Use an inverted microscope to take pictures at different time points (0, 12, 24 h) to detect cell migration.

[0052] 7. Invasion experiment

[0053] Dilute Matrigel with serum-free culture medium at a ratio of 1:50. Add 200 μL of the mixed solution to a Transwell chamber and incubate at 37°C for 1 h. Add 500 μL of complete culture medium to a 24-well plate, and then place the chamber in the 24-well plate. Then slowly drop the prepared cell suspension (prepared as above) into the chamber, and continue to culture for 24 h. Remove the chamber, wash it, fix and stain the lower surface of the chamber, and use an inverted microscope to take pictures and count to evaluate the cell invasion ability.

[0054] Experimental Example 1

[0055] 1. saRNA sequence design

[0056] According to the DNA genomic sequence located upstream of the transcription starting site (TSS) of the target gene and some promoter regions away from the TSS, 31 saRNA sequences that can activate the AMBRA1 gene were designed, and their gene activation activity was evaluated.

[0057] Table 1 saRNA sequences

[0058]

[0059]

[0060] Note: saCtr sequence is designed from published paper, Small dsRNAs induce transcriptional activation in human cells, which does not target any known human gene and is a common negative control without homology to the sequence of interest.

[0061] 2. Screening of saRNA targeting human AMBRA1

[0062] Firstly, lung cancer cells were selected as the research object to screen the saRNA sequences. After transfecting the saRNA at a working concentration of 50 nM into human non-small cell lung cancer A549 cells for 72 h, the activation effect of saRNA was reflected by detecting the AMBRA1 mRNA level by real-time fluorescent quantitative PCR. It can be seen from Table 1 that the sequences No. 4, 10, 25, 28, 30 and 31 have better activation effect (>1.5 times), and the rest of the saRNA does not show obvious activation effect. Figure 1 Figure 1 ) in another kind of human non-small cell lung cancer H1299 cells, the results showed that the sequences No. 4, 18, 22, 23, 28 and 30 have better activation effect (>1.5 times), and the rest of the saRNA does not show obvious activation effect.

[0063] Table 2 Activation effect of AMBRA1 on A549 and H1299 cells (50 nM)

[0064] Sequence No. A549 activation fold H1299 activation fold Sequence No. A549 activation fold saAMBRA1-1 saAMBRA1-17 1.39 1.41 saAMBRA1-2 1.21 1.10 saAMBRA1-18 1.18 1.04 saAMBRA1-3 1.38 1.52 saAMBRA1-19 1.15 1.37 saAMBRA1-4 1.41 1.37 saAMBRA1-20 1.87 1.63 saAMBRA1-5 1.17 1.49 saAMBRA1-21 1.20 1.19 saAMBRA1-6 1.07 1.05 saAMBRA1-22 1.25 1.17 saAMBRA1-7 1.27 1.58 saAMBRA1-23 1.12 1.05 saAMBRA1-8 1.29 1.56 saAMBRA1-24 1.16 1.05 saAMBRA1-9 1.30 0.70 saAMBRA1-25 1.32 1.28 saAMBRA1-10 1.57 1.29 saAMBRA1-26 1.68 1.34 saAMBRA1-11 1.33 0.76 saAMBRA1-27 1.15 1.19 saAMBRA1-12 1.15 0.97 saAMBRA1-28 0.94 1.09 saAMBRA1-13 1.55 1.93 saAMBRA1-29 1.29 1.14 saAMBRA1-14 1.31 1.06 saAMBRA1-30 1.36 1.40 saAMBRA1-15 1.56 1.51 saAMBRA1-31 1.42 1.07 saAMBRA1-16 1.56 1.10 Figure 2 1.10 1.16

[0065] It can be seen that in different tumor cells, the saRNA sequences activating the same gene may not be completely consistent. According to the above results, the sequences No. 4, 28 and 30 have better activation effect (>1.5 times) in two kinds of cells. Therefore, the sequence No. 4 (saAMBRA1-4) is preferred as an example for subsequent functional experiments. Subsequently, the concentration effect of sequence No. 4 on the activation effect of AMBRA1 mRNA level was further investigated. According to the results of real-time fluorescent quantitative PCR, after transfecting saAMBRA1-4 72 h in A549 cells, with the transfection concentration gradually increasing from 5 nM, 10 nM, 25 nM, 50 nM and 100 nM, the activation effect increased, showing a certain concentration effect, and reached a plateau at a working concentration of 25 nM. Figure 2 ​). In H1299 cells, saAMBRA1-4 also had a certain concentration effect 72h after transfection, with the transfection concentration increasing from 5nM, 10nM, 25nM, 50nM to 100nM, the activation effect increased, and the working concentration reached the platform when the working concentration was 25nM. Figure 3

[0066] Experimental Example 2: Evaluation of the effect of activation of AMBRA1 on cell cycle

[0067] AMBRA1 can negatively regulate the expression of many cell cycle proteins. The experiment further investigated the effect of activation of AMBRA1 on the cell cycle of A549 and H1299 cells by flow cytometry. The experimental results showed that the activation of AMBRA1 caused more cells to arrest in the G1 phase, the cell cycle was inhibited, leading to the inability of cells to divide normally, and the inhibition of tumor cell proliferation. Figure 4

[0068] Experimental Example 3: Evaluation of the regulation of downstream genes by activation of AMBRA1

[0069] AMBRA1 is involved in the regulation of cell cycle, DNA damage repair transcriptional regulation and other processes. The experiment detected the mRNA expression levels of multiple genes in A549 and H1299 cells after transfection of saAMBRA1-4. From the results of real-time fluorescence quantitative PCR, after transfection of saAMBRA1-4 to activate the expression of AMBRA1 in A549 and H1299 cells, the mRNA levels of CCNA2, CCNE1, RAD51 and E2F1 all decreased significantly Figure 5 ), among which, knocking down the level of CCNA2 can inhibit cell cycle progression and induce apoptosis, and significantly inhibit the growth of colon cancer cells. The decrease of CCNE1 expression can inhibit the malignant phenotype of lung adenocarcinoma cells, and weaken tumor growth and metastasis. The recombinase activity of the protein encoded by RAD51 gene is the core of catalyzing DNA double-strand break homologous recombination repair, and is involved in DNA damage response and other signaling pathways. In lung cancer cells, inhibition of RAD51 expression can significantly enhance drug-induced cell death and cell growth inhibition such as mitomycin. E2F1 as a classic transcription factor, it can activate MELTF, promote lung adenocarcinoma cell proliferation, migration and invasion.

[0070] Experimental Example 4: Evaluation of the effect of activation of AMBRA1 on cell proliferation

[0071] ​​The influence of AMBRA1 expression level on the proliferation ability of non-small cell lung cancer tumor cells was evaluated by CCK-8 experiment. In A549 and H1299 cells, after transfection of saAMBRA1-4 to activate AMBRA1 expression, the cell number was detected from day 1 to day 3, and the cell proliferation curve was drawn to detect the influence of the increase of AMBRA1 expression level on the proliferation ability of A549 and H1299 cells. The results showed that the proliferation ability of A549 and H1299 cells was significantly decreased after activation of AMBRA1 expression. Figure 5 A).

[0072] Example 5: Evaluation of the influence of activation of AMBRA1 on cell migration ability

[0073] The influence of AMBRA1 on the migration ability of tumor cells was further evaluated by scratch experiment. After transfection of saAMBRA1-4 to activate AMBRA1 expression in A549 and H1299 cells, the same number of cells were digested and reseeded into Culture-Insert, and after the cells adhered, the insert was removed, and the cell migration was detected by taking pictures at different time points (0, 12, 24 h) using an inverted microscope. The results showed that the migration rate of the experimental group was significantly lower than that of the control group after activation of AMBRA1 expression in A549 and H1299 cells, which could significantly inhibit the migration of non-small cell lung cancer cells.

[0074] Example 6: Evaluation of the influence of activation of AMBRA1 on cell invasion ability

[0075] The influence of AMBRA1 expression level on the invasion ability of cells was further detected by Transwell experiment in A549 and H1299 cells. After transfection of saAMBRA1-4 to activate AMBRA1 expression in A549 and H1299 cells, the same number of cells were digested and reseeded into the upper chamber of Transwell coated with Matrigel, and after 24 h, the cells that passed through the Matrigel were fixed and stained, and the invasion ability was evaluated by counting using an inverted microscope. The experimental results showed that Figure 6 C) After activation of AMBRA1 expression in A549 and H1299 cells, the number of cells that passed through the Matrigel to reach the lower chamber of Transwell in the experimental group was significantly less than that in the control group, which could significantly inhibit the invasion of non-small cell lung cancer cells.

[0076] Example 7: Evaluation of the influence of nucleic acid sequence modification on the activation effect of AMBRA1

[0077] To improve the stability of saRNA for subsequent evaluation at the animal level, the antisense strand of saAMBRA1-4 was chemically modified. The nucleotide sequence of the modified antisense strand is as follows: (Phos)mU*fC*fUfAmCfUmGfUmC mAmCfUmCfAmGfUmAfGmU*dT*dT; wherein * represents phosphorothioate modification, m represents 2'-methoxy modification, f represents 2'-fluoro modification, and Phos represents 5' phosphorylation modification.

[0078] The effect of chemically modified saRNA on the activation of AMBRA1 was evaluated in A549 and H1299 cells. After transfection of unmodified and modified saRNA (i.e. saAMBRA1-4 and saAMBRA1-4M) at a working concentration of 25 nM in A549 and H1299 cells for 72 h, the activation effect of saRNA was reflected by detecting the AMBRA1 mRNA level by real-time fluorescent quantitative PCR. A549 activation effect As can be seen from Table 3, in A549 cells, the chemically modified saAMBRA1-4M increased the expression of AMBRA1. In H1299 cells, the chemically modified saAMBRA1-4M had no effect on the activation effect of saRNA, which would be beneficial for subsequent evaluation of drug efficacy at the animal level.

[0079] Table 3 Effect of chemically modified saRNA at 25 nM on the activation of AMBRA1 in different cells

[0080] H1299 activation effect saAMBRA1-4 (25 nM) saAMBRA1-4M (25 nM) 1.94 1.68 Figure 7 2.79 1.76

[0081] Experimental Example 8 Evaluation of the effect of combination drug on cell proliferation

[0082] Traditional chemotherapy is still one of the main methods for treating cancer. Cisplatin and paclitaxel are widely used as first-line chemotherapy drugs for non-small cell lung cancer in clinical practice. Chemotherapy drugs are prone to cause a series of problems such as serious side effects and drug resistance. Therefore, the present experiment further explored the expression level of AMBRA1 and the sensitivity to chemotherapy drugs, as well as the feasibility of saAMBRA1-4M combined with cisplatin as a treatment regimen for non-small cell lung cancer.

[0083] A549 cells were treated with 5, 10, and 20 μM cisplatin alone or in combination with saAMBRA1-4M, and H1299 cells were treated with 10, 20, and 40 μM cisplatin alone or in combination with saAMBRA1-4M. CCK-8 experiment was used to detect the cell viability of A549 and H1299 cells at different concentrations of cisplatin and 25 nM saAMBRA1-4M alone or in combination. The experimental results showed that Figure 8), in A549, H1299 two kinds of cells, compared with the control group, cisplatin, saAMBRA1-4M alone after administration, cell viability decreased; At the same time, the combined use of drug group cell viability than single drug group also decreased significantly.

[0084] Experimental example 9 effect of combined administration on cell apoptosis evaluation

[0085] AMBRA1 is an important regulator of autophagy and apoptosis, can change the balance between autophagy and apoptosis to change the sensitivity of tumor cells to chemotherapy drugs. Therefore, the experiment was used 10 μM cisplatin alone or combined with 25 nM saAMBRA1-4M treatment of A549 cells, or 20 μM cisplatin alone or combined with 25 nM saAMBRA1-4M treatment of H1299 cells. By Annexin V / PI double staining flow cytometry to detect cell apoptosis; The results show that Figure 9 ), in A549, H1299 two kinds of cells, compared with the control group, cisplatin, saAMBRA1-4M alone after administration, cell apoptosis increased significantly; At the same time, the combined use of drug group cell apoptosis further increased.

[0086] Experimental example 10 effect of combined administration on cell migration ability

[0087] 20 μM cisplatin and 25 nM saAMBRA1-4M alone or combined use of H1299 cells, by scratch test to further evaluate the effect of combined administration on tumor cell migration ability; The experimental results show that ​ ), in H1299 cells, compared with the control group, cisplatin, saAMBRA1-4M alone after administration, cell migration ability decreased; At the same time, the combined use of drug group cell migration rate than single drug group also decreased significantly.

Claims

1. saRNA activating transcription of AMBRA1, characterized in that, The saRNA is selected from any one of the saRNAs consisting of a sense strand and an antisense strand represented by (1) to (31) below: (1) a saRNA consisting of a sense strand containing the nucleotide sequence represented by SEQ ID No. 1 and an antisense strand containing the nucleotide sequence represented by SEQ ID No. 2; (2) a saRNA consisting of a sense strand containing the nucleotide sequence represented by SEQ ID No. 3 and an antisense strand containing the nucleotide sequence represented by SEQ ID No. 4; (3) a saRNA consisting of a sense strand containing the nucleotide sequence represented by SEQ ID No. 5 and an antisense strand containing the nucleotide sequence represented by SEQ ID No. 6; (4) a saRNA consisting of a sense strand containing the nucleotide sequence represented by SEQ ID No. 7 and an antisense strand containing the nucleotide sequence represented by SEQ ID No. 8; (5) a saRNA consisting of a sense strand containing the nucleotide sequence represented by SEQ ID No. 9 and an antisense strand containing the nucleotide sequence represented by SEQ ID No. 10; (6) a saRNA consisting of a sense strand containing the nucleotide sequence represented by SEQ ID No. 11 and an antisense strand containing the nucleotide sequence represented by SEQ ID No. 12; (7) a saRNA consisting of a sense strand containing the nucleotide sequence represented by SEQ ID No. 13 and an antisense strand containing the nucleotide sequence represented by SEQ ID No. 14; (8) a saRNA consisting of a sense strand containing the nucleotide sequence represented by SEQ ID No. 15 and an antisense strand containing the nucleotide sequence represented by SEQ ID No. 16; (9) a saRNA consisting of a sense strand containing the nucleotide sequence represented by SEQ ID No. 17 and an antisense strand containing the nucleotide sequence represented by SEQ ID No. 18; (10) a saRNA consisting of a sense strand containing the nucleotide sequence represented by SEQ ID No. 19 and an antisense strand containing the nucleotide sequence represented by SEQ ID No. 20; (11) a saRNA consisting of a sense strand containing the nucleotide sequence represented by SEQ ID No. 21 and an antisense strand containing the nucleotide sequence represented by SEQ ID No. 22; (12) a saRNA consisting of a sense strand containing the nucleotide sequence represented by SEQ ID No. 23 and an antisense strand containing the nucleotide sequence represented by SEQ ID No. 24; (13) a saRNA consisting of a sense strand containing the nucleotide sequence represented by SEQ ID No. 25 and an antisense strand containing the nucleotide sequence represented by SEQ ID No. 26; (14) a saRNA consisting of a sense strand containing the nucleotide sequence represented by SEQ ID No. 27 and an antisense strand containing the nucleotide sequence represented by SEQ ID No. 28; (15) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 29 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 30; (16) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 31 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 32; (17) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 33 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 34; (18) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 35 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 36; (19) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 37 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 38; (20) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 39 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 40; (21) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 41 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 42; (22) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 43 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 44; (23) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 45 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 46; (24) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 47 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 48; (25) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 49 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 50; (26) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 51 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 52; (27) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 53 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 54; (28) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 55 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 56; (29) a saRNA consisting of a sense strand comprising the nucleotide sequence set forth in SEQ ID No. 57 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 58; (30) saRNA consisting of sense strand containing nucleotide sequence as shown in SEQ ID No. 59 and antisense strand containing nucleotide sequence as shown in SEQ ID No. 60; (31) saRNA consisting of sense strand containing nucleotide sequence as shown in SEQ ID No. 61 and antisense strand containing nucleotide sequence as shown in SEQ ID No.

62.

2. A saRNA, characterized in that, The saRNA has 15 or more than 15 continuous same nucleotide sequences with the saRNA sequence of claim 1; or, the TT base at the 3' end of the sense strand or the antisense strand of the saRNA is replaced by 2 bases which are complementary to the promoter region of AMBRA1 gene.

3. Modified saRNA modified by the saRNA of claim 1.

4. The modified saRNA according to claim 3, characterized in that, The modified saRNA is a modified saRNA obtained by modifying the saRNA sequence of claim 1 or 2 in the backbone, sugar ring, nucleoside or base; preferably, the modification is one or more than one of the following modifications: (1) substitution of the 2' carbon position of the sugar ring of the nucleotide, including but not limited to methoxy, methyl ethoxy, methyl, amino, fluorine, -O-2-methylthioethyl, -O-3-aminopropyl or -O-3-dimethylaminopropyl substitution; (2) modification of the phosphodiester bond between the nucleotides, including but not limited to phosphorothioate, boranophosphonate or methylphosphonate; (3) modification of the nucleosides of the sequence, including but not limited to peptide nucleic acid, locked nucleic acid or unlocked nucleic acid, morpholino nucleic acid; (4) base modification, including but not limited to base modification or substitution of 5-methylcytosine, 6-methyladenine, ribavirin, pseudouracil and / or inosine of the nucleoside base; (5) no modification or modification of the terminal of the saRNA sequence by phosphorylation, phosphorothioation, vinylphosphonation or other phosphonation analog modification or phosphate precursor; Preferably, the modified saRNA is a modified saRNA obtained by modification in at least one of the following (1)-(5): (1) conversion of one non-ester oxygen bond between the phosphodiester of the RNA to sulfur; (2) substitution of the -OH group at the 2' carbon position of the nucleic acid base by including but not limited to CH3, -OCH3, -OCH2CH2OCH3, -F, NH2 and / or deoxy modification of the 2'-OH; preferably, the -OH group at the 2' carbon position of the nucleic acid base is modified by alternately replacing methoxy and fluorine; (3) two or more than two phosphorothioate modifications at the terminal of the sense strand or the antisense strand of the saRNA; (4) replacement of the TT base at the 3' end of the sense strand or the antisense strand of the saRNA by 2 bases which are complementary to the promoter region of AMBRA gene; (5) no modification or modification of the 5' terminal of the antisense strand of the RNA sequence by phosphate, phosphorothioate or its phosphonic analog or phosphate prodrug modification, no modification or inverted non-base sugar cap modification at the 5' terminal of the sense strand.

5. The modified saRNA according to claim 4, characterized in that, The nucleotide sequences of the sense strand and the antisense strand of the modified saRNA are as follows: Sense strand: CCAUUGUUGUGGUGAUGAAdTdT; Antisense strand: PhosmU*fC*fUfAmCfUmGfUmCmAmCfUmCfAmGfUmAfGmU*dT*dT; wherein, * represents thiophosphoric acid modification, m represents 2'-methoxy modification, f represents 2'-fluorination modification, and Phos represents 5' phosphorylation modification.

6. Double-stranded RNA or hairpin double-stranded RNA containing the saRNA of claim 1 or 2 or containing the modified saRNA sequence of any one of claims 3-5, or a transcription vector system thereof.

7. The carrier system of claim 6, wherein, The vector system is selected from the group consisting of nucleic acid-lipid particles, liposomes, micelles, viral particles or nucleic acid complexes, or a mixture consisting of one or more.

8. Use of the saRNA of claim 1 or 2 or the modified saRNA of any one of claims 3-5 in the preparation of an antitumor drug.

9. An antitumor pharmaceutical composition, characterized by, The pharmaceutical composition contains the saRNA of claim 1 or 2 or the modified saRNA of any one of claims 3-5, and a pharmaceutically acceptable carrier system and formulation adjuvant.

10. The pharmaceutical composition of claim 9, wherein, The pharmaceutical composition further contains a chemotherapeutic drug or an immunosuppressant, etc., wherein the chemotherapeutic drug includes antimetabolites, anti-tubulin drugs, alkylating agents, topoisomerase inhibitors, antitumor antibiotics or platinum drugs, preferably platinum drugs; The tumor is various tumors derived from the digestive system, respiratory system, blood and lymphatic system or skin, preferably lung cancer.

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