Method for activating p21 gene expression

Abstract

The invention provides a small activating RNA (Ribonucleic Acid) which consists of a sense oligonucleotide chain and an antisense oligonucleotide chain and is used for activating or up-regulating human p21 gene expression, and the sense oligonucleotide chain or the antisense oligonucleotide chain has more than 75% of homology with any continuous fragment with the length of 16-35 nucleotides in a target gene sequence of a human p21 gene promoter. Wherein the target sequence of the human p21 gene is selected from a group consisting of sequences as shown in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11 and 12.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 2021108412870, application date April 10, 2019, and invention name “A method for activating p21 gene expression”. Technical Field

[0002] The present invention relates to the field of molecular biology, and in particular to positively regulating gene expression by using double-stranded small RNA targeting gene promoter sequence. Background Art

[0003] Double-stranded small nucleic acid molecules, including chemically synthesized oligoribonucleotides such as small activating RNA (saRNA) and naturally occurring oligoribonucleotides such as microribonucleotides (miRNA), have been shown to be able to target regulatory sequences of protein-coding genes such as promoter sequences in a sequence-specific manner and positively regulate gene expression levels at the transcriptional and epigenetic levels. This phenomenon is called RNA activation (RNAa) (Li, Okino et al. (2006) Proc Natl Acad Sci USA 103:17337-17342; Janowski, Younger et al. (2007) Nat Chem Biol 3:166-173; Place, Li et al. (2008) Proc Natl Acad Sci USA 105:1608-1613; Huang, Place et al. (2012) Nucleic Acids Res 40:1695-1707; Li (2017) Adv Exp Med Biol 983:1-20). Studies have shown that RNA activation is an endogenous molecular mechanism that is evolutionarily conserved from Caenorhabditis elegans to humans (Huang, Qin et al. (2010) PLoS One 5:e8848; Seth, Shirayama et al. (2013) Dev Cell 27:656-663; Turner, Jiao et al. (2014) Cell Cycle 13:772-781).

[0004] How to safely and selectively enhance the expression of endogenous genes or proteins in human cells remains a huge challenge in the field of gene therapy. Traditional viral-based gene therapy systems have inherent defects, including the possibility of altering the host genome and causing side effects such as immune responses. RNA activation has the advantage of being able to activate endogenous gene expression without changing the genome, representing a new strategy for activating endogenous gene expression, and has great application value in disease treatment.

[0005] p21 WAF1 / CIP1 The (also known as CDKN1A, hereinafter referred to as p21) gene is a cyclin-dependent kinase (CDK) inhibitor and an important tumor suppressor gene (Harper, Adami et al. (1993) Cell 75:805-816; Fang, Igarashi et al. (1999) Oncogene 18:2789-2797). Studies have shown that overexpression of p21 by ectopic vectors or activation of endogenous p21 transcription can effectively inhibit the growth of tumor cells and in vivo tumors (Harper, Adami et al. (1993) Cell 75:805-816; Eastham, Hall et al. (1995) Cancer Res55:5151-5155; Wu, Bellas et al. (1998) J Exp Med 187:1671-1679; Harrington, Spitzweg et al. (2001) J Urol 166:1220-1233). Therefore, the method of targeted activation of the p21 gene may have a wide range of application value in the treatment of many diseases such as cancer. Summary of the invention

[0006] The present invention provides a small activating RNA, characterized in that one chain of the small activating RNA has at least 75% homology or complementarity with any continuous fragment of 16-35 nucleotides in length in the sequences shown in SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, and 12, and the small activating RNA activates or upregulates the expression of the p21 gene by targeting the human p21 gene promoter sequence.

[0007] In an embodiment of the present invention, the target gene of the human p21 gene is selected from the group consisting of sequences shown in SEQ ID NOs: 5-12, wherein the human p21 gene promoter sequence is located at -893~-801 bp (SEQ ID NO: 5), -717~-632 bp (SEQ ID NO: 6), -585~-551 bp (SEQ ID NO: 7), -554~-504 bp (SEQ ID NO: 8), -514~-485 bp (SEQ ID NO: 9), -442~-405 bp (SEQ ID NO: 10), -352~-313 bp (SEQ ID NO: 11), and -325~-260 bp (SEQ ID NO: 12) upstream of the transcription start site (TSS).

[0008] In one embodiment, the small activating RNA comprises a sense nucleic acid chain and an antisense nucleic acid chain, wherein the sense nucleic acid chain and the antisense nucleic acid chain contain complementary regions, and the complementary regions can form a double-stranded nucleic acid structure, wherein the sense nucleic acid chain or the antisense nucleic acid chain has more than 75%, more than 80%, more than 90%, more than 95%, more than 99% or 100% homology with any continuous segment of 16-35 nucleotides in the human p21 gene promoter sequence.

[0009] In one embodiment, as described above, the sense nucleic acid chain and the antisense nucleic acid chain exist on two different nucleic acid chains; the sense nucleic acid chain and the antisense nucleic acid chain exist on the same nucleic acid chain, which is a hairpin-type single-stranded nucleic acid molecule, wherein the complementary regions of the sense nucleic acid chain and the antisense nucleic acid chain form a double-stranded nucleic acid structure.

[0010] In a specific embodiment, at least one strand of the small activating RNA has a 3' overhang with a length of 0 to 6 nucleotides; both strands of the small activating RNA have a 3' overhang with a length of 2-3 nucleotides.

[0011] In one embodiment, the sense nucleic acid strand or the antisense nucleic acid strand has a length of 16-35 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.

[0012] In a specific embodiment, the sense nucleic acid strand or the antisense nucleic acid strand is SEQ ID NO: 13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108, 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 201-202, 209-212, 215-218, 225-240, 243-246, 249-258, 261-262, 265-270, 275-280, 283-3 00, 303-308, 317-320, 323-324, 329-348, 351-352, 357-358, 361-366, 371-392, 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, 463-468 have at least 75%, such as 80%, 85%, 90%, 95%, 99%, 100% homology.

[0013] In a specific embodiment, the sense nucleic acid strand or antisense nucleic acid strand sequence is as shown in SEQ ID NO: 13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108, 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 201-202, 209-212, 215-218, 225-240, 243-246, 249-258, 261-262, 265-2 70, 275-280, 283-300, 303-308, 317-320, 323-324, 329-348, 351-352, 357-358, 361-366, 371-392, 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, 463-468 as shown in any one of the nucleotide sequences.

[0014] The present invention also provides a method for preparing the small activating RNA as described in any of the preceding items, characterized in that the method comprises the following steps: 1) using the target gene promoter sequence as a template, selecting a sequence containing 19 bases as a target site; 2) synthesizing an RNA sequence having more than 75% homology with the target site sequence obtained in step 1) to obtain a sense oligonucleotide chain; 3) the sequence is complementary to the sense oligonucleotide chain obtained in step 2); 4) mixing the sense oligonucleotide chain obtained in step 2) and the antisense oligonucleotide chain obtained in step 3) in an RNA annealing buffer at the same molar number, heating, and then naturally cooling to room temperature to obtain a double-stranded small activating RNA; wherein the human p21 gene promoter sequence is selected from the group consisting of the sequences shown in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, and 12.

[0015] In one embodiment, at least one nucleotide in the small activating RNA is a chemically modified nucleotide, and the chemical modification is at least one of the following modifications: (1) Modification of the phosphodiester bonds connecting nucleotides in the nucleotide sequence of the small activating RNA; (2) modification of the 2'-OH of the ribose in the nucleotide sequence of the small activating RNA; (3) modification of bases in the nucleotide sequence of the small activating RNA; (4) At least one nucleotide in the nucleotide sequence of the small activating nucleic acid molecule is a locked nucleic acid.

[0016] In one embodiment, the activation or upregulation of the expression of the p21 gene is upregulation by at least 10%, such as 15%, 20%, 30%, 40%, 50%, 80%, 100%, 200% or more.

[0017] The present invention also provides the use of small activating RNA in preparing a preparation for activating or up-regulating the expression of p21 gene in cells. The small activating RNA is directly introduced into the cells, or is produced in the cells after the nucleotide sequence encoding the small activating RNA is introduced into the cells. The cells are mammalian cells, preferably human cells, more preferably human tumor cells. The human cells can be isolated human cell lines, or can exist in the human body.

[0018] In one embodiment, the human body is a patient suffering from a tumor caused by insufficient expression of p21 protein, and the small activating nucleic acid molecule is administered in an effective amount to achieve treatment of the tumor, and the tumor is preferably bladder cancer, prostate cancer, liver cancer or colorectal cancer.

[0019] In yet another aspect, the present invention also provides an isolated p21 gene small activating RNA target site, wherein the target site is any continuous 16-35 nucleotide sequence selected from any one of SEQ ID No 5-12.

[0020] In another aspect, the present invention discloses a method for activating or up-regulating the expression of human p21 gene in cells, wherein the method comprises administering the small activating RNA as described in any of the preceding items to a subject or a cell. The small activating RNA can be directly introduced into the cell, or it can be produced in the cell after the nucleotide sequence encoding the small activating RNA is introduced into the cell. The cell is a mammalian cell, preferably a human cell, more preferably a human tumor cell, and even more preferably a human bladder cancer, prostate cancer, liver cancer or colorectal cancer cell.

[0021] The present invention further discloses a composition comprising the aforementioned small activating RNA and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is a liposome, a high molecular polymer or a polypeptide.

[0022] The present invention also discloses the use of the aforementioned small activating RNA or the aforementioned composition in the preparation of a drug for activating or up-regulating the expression of the p21 gene, preferably in the preparation of a drug for anti-tumor or benign proliferative lesions, more preferably, the tumor is bladder cancer, prostate cancer, liver cancer or colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The p21 gene promoter sequence is shown, from -1000 bp upstream of the transcription start site (TSS) to 3 bp downstream of the TSS. The TSS is indicated by a curved arrow.

[0024] Figure 2 Screening of small activating RNA hotspots on the p21 gene promoter is shown. Figure 1 439 double-stranded RNA molecules were designed and chemically synthesized based on the p21 promoter sequence shown, and then transfected into PC3 human prostate cancer cells. The p21 mRNA level was analyzed using the QuantiGene 2.0 method 72 hours later. (A) The fold change of p21 mRNA level caused by each of the 439 double-stranded RNA molecules (X-axis) relative to the control treatment (Y-axis). The double-stranded RNA molecules on the X-axis are ranked according to their position relative to the TSS of the p21 gene (from RAG-898 at the most upstream to RAG-177 at the most downstream). Eight hotspot regions (light gray rectangular boxes) are marked in the figure. (B) The data in (A) are ranked from low to high according to the fold change of p21 mRNA expression induced by each double-stranded RNA molecule. The dotted lines in (A) and (B) indicate a 2-fold induction.

[0025] FIG. 3 shows the activation effect of double-stranded RNA molecules in hotspot regions 1 to 8 on the p21 gene.

[0026] Figure 4 The RT-qPCR method was used to analyze the p21 gene mRNA level to verify the experimental results of QuantiGene 2.0. (A) 439 double-stranded RNA molecules were divided into four regions (groups) according to their activity in inducing p21 mRNA expression. Five double-stranded RNA molecules were randomly selected from each group and transfected into PC3 cells at a concentration of 10 nM. After 72 hours, total cell RNA was extracted and the p21 gene mRNA level was analyzed by RT-qPCR after reverse transcription. (B) The correlation between the relative mRNA levels of p21 gene induced by double-stranded RNA molecules detected by QuantiGene 2.0 (X axis) and RT-qPCR (Y axis).

[0027] Figure 5 The effects of small activating RNAs on inducing p21 mRNA expression and inhibiting KU-7 cell proliferation are shown. The three small activating RNAs shown were transfected into KU-7 cells at 10 nM for 72 hours. (A) RT-qPCR analysis of p21 gene mRNA expression levels. (B) CCK-8 method was used to assess cell viability. The viability of cells in the saRNA-treated group was expressed as a percentage of the viability of cells in the control-treated group (Mock). (C) Representative cell images at the end of transfection (100×).

[0028] Figure 6 The effect of small activating RNA on inducing p21 mRNA expression and inhibiting HCT116 cell proliferation is shown. The three small activating RNAs shown were transfected into HCT116 cells at 10 nM for 72 hours. (A) RT-qPCR analysis of p21 gene mRNA expression levels. (B) CCK-8 method was used to evaluate cell viability, and the viability of cells in the saRNA-treated group was expressed as a percentage of cell viability relative to that of the control-treated group (Mock). (C) Representative cell images at the end of transfection (100×).

[0029] Figure 7 The effect of small activating RNA on inducing p21 mRNA expression and inhibiting HepG2 cell proliferation is shown. The three small activating RNAs shown were transfected into HepG2 cells at 10 nM for 72 hours. (A) RT-qPCR analysis of p21 gene mRNA expression levels. (B) CCK-8 method was used to evaluate cell viability, and the viability of cells in the saRNA-treated group was expressed as a percentage of cell viability relative to that of the control-treated group (Mock). (C) Representative cell images at the end of transfection (100×). DETAILED DESCRIPTION

[0030] The present invention will be further illustrated by detailed description below.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0032] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0033] definition As used herein, the term "complementarity" refers to the ability of two oligonucleotide chains to form base pairs with each other. Base pairs are usually formed by hydrogen bonds between nucleotide units in antiparallel oligonucleotide chains. Complementary oligonucleotide chains can be base paired in Watson-Crick mode (e.g., AT, AU, CG), or in any other mode (e.g., Hoogsteen type or reverse Hoogsteen type base pairing) that allows the formation of duplexes. "100% pairing" or "complete complementarity" refers to having 100% complementarity, i.e., the nucleotide units of the two chains are all hydrogen bonded to each other.

[0034] Complete complementarity or 100% pairing refers to the situation that each nucleotide unit from the first oligonucleotide chain in the double-stranded region of the double-stranded oligonucleotide molecule can form hydrogen bonds with the second oligonucleotide chain without "mismatching". Incomplete complementarity refers to the situation that the nucleotide units of the two chains cannot all hydrogen bond with each other. For example, for two oligonucleotide chains with a double-stranded region of 20 nucleotides in length, if only two base pairs on each chain can hydrogen bond with each other, the oligonucleotide chains show 10% complementarity. In the same example, if 18 base pairs on each chain can hydrogen bond with each other, the oligonucleotide chains show 90% complementarity. Basic complementarity refers to about 79%, about 80%, about 85%, about 90%, about 95% or more complementarity.

[0035] The term "oligonucleotide" as used herein refers to a polymer of nucleotides, and includes, but is not limited to, single-stranded or double-stranded molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains comprising regularly and irregularly alternating deoxyribose and ribose moieties, as well as modifications of these types of oligonucleotides and with naturally occurring or non-naturally occurring backbones.

[0036] The term "oligoribonucleotide" as used herein refers to an oligonucleotide comprising two or more modified or unmodified ribonucleotides and / or analogs thereof.

[0037] As used herein, the terms "oligonucleotide chain" and "oligonucleotide sequence" are interchangeable and refer to a general term for a short chain of nucleotides of less than 50 bases (the nucleotides may be deoxyribonucleic acid DNA or ribonucleic acid RNA). In the present invention, the length of the oligonucleotide chain may be any length from 17 to 30 nucleotides.

[0038] As used herein, the term "gene" refers to the entire nucleotide sequence required to encode a polypeptide chain or transcribe a functional RNA. "Gene" can be a gene that is endogenous or completely or partially recombinant to a host cell (for example, due to the introduction of exogenous oligonucleotides encoding promoters and coding sequences or the introduction of heterologous promoters adjacent to endogenous coding sequences into host cells). For example, the term "gene" includes nucleic acid sequences that can be composed of exons and introns. The sequence encoding a protein is, for example, a sequence within an exon in an open reading frame between a start codon and a stop codon, and as used herein, "gene" can refer to all other sequences including, for example, gene regulatory sequences such as promoters, enhancers, and transcription, expression or activity of another gene known in the art, regardless of whether the other gene contains a coding sequence or a non-coding sequence. In one case, for example, "gene" can be used to describe a functional nucleic acid comprising a regulatory sequence such as a promoter or an enhancer. The expression of a recombinant gene can be controlled by one or more heterologous regulatory sequences.

[0039] As used herein, the term "target gene" can be a nucleic acid sequence naturally present in an organism, a transgenic, a viral or bacterial sequence, a chromosome or extrachromosomal and / or transiently or stably transfected or incorporated into a cell and / or its chromatin. The target gene can be a protein coding gene or a non-protein coding gene (e.g., a microRNA gene, a long non-coding RNA gene). The target gene usually contains a promoter sequence, and a small activating RNA designed to have identity (also known as homology) with the promoter sequence can achieve positive regulation of the target gene, which is manifested as upregulation of target gene expression. "Target gene promoter sequence" refers to the non-coding sequence of the target gene. In the present invention, the target gene promoter sequence in "complementary to the target gene promoter sequence" refers to the coding strand of the sequence, also known as the non-template strand, that is, a nucleic acid sequence with the same sequence as the gene coding sequence. "Target sequence" refers to the sequence fragment homologous or complementary to the sense oligonucleotide strand or antisense oligonucleotide of the small activating RNA in the target gene promoter sequence.

[0040] As used herein, the terms "sense strand" and "sense oligonucleotide strand" are interchangeable, and the sense oligonucleotide strand of the small activating RNA refers to the first RNA strand in the small activating RNA duplex that contains the coding strand that is identical to the promoter sequence of the target gene.

[0041] As used herein, the terms "antisense strand" and "antisense oligonucleotide strand" are interchangeable, and the antisense oligonucleotide strand of the small activating RNA refers to the second ribonucleic acid strand complementary to the sense oligonucleotide strand in the small activating RNA duplex.

[0042] As used herein, the term "coding strand" refers to the DNA strand in the target gene that cannot be transcribed, and the nucleotide sequence of this strand is consistent with the sequence of the RNA generated by transcription (in RNA, U replaces T in DNA). The coding strand of the double-stranded DNA sequence of the target gene promoter described in the present invention refers to the promoter sequence on the same DNA strand as the coding strand of the target gene DNA.

[0043] As used herein, the term "template strand" refers to the other strand of the double-stranded DNA of the target gene that is complementary to the coding strand, and can be used as a template to transcribe into RNA, which is complementary to the transcribed RNA base (AU, GC). During transcription, RNA polymerase binds to the template strand and moves along the 3'→5' direction of the template strand, catalyzing the synthesis of RNA in the 5'→3' direction. The template strand of the double-stranded DNA sequence of the target gene promoter described in the present invention refers to the promoter sequence on the same DNA strand as the target gene DNA template strand.

[0044] As used herein, the term "promoter" refers to a nucleic acid sequence that does not encode a protein and regulates the transcription of a protein-coding or RNA-coding nucleic acid sequence by being positionally associated with the nucleic acid sequence. Typically, a eukaryotic promoter contains 100-5,000 base pairs, although this length range is not meant to limit the term "promoter" as used herein. Although promoter sequences are generally located at the 5' end of a protein-coding or RNA-coding sequence, in some cases promoter sequences are also present in exon and intron sequences.

[0045] As used herein, the term "transcription start site" refers to the nucleotide that marks the start of transcription on the template strand of a gene. The transcription start site may appear on the template strand of the promoter region. A gene may have more than one transcription start site.

[0046] As used herein, the term "identity" or "homology" means that one of the oligonucleotide strands (sense strand or antisense strand) of the small activating RNA has at least 75% similarity with the coding strand or template strand of a certain region of the promoter sequence of the target gene.

[0047] As used herein, the terms "overhang", "overhang" and "dangling" are interchangeable and refer to the non-base paired nucleotides at the end (5' or 3') of the oligonucleotide chain, which are generated by extending beyond one of the strands in the double-stranded oligonucleotide. The single-stranded region extending beyond the 3' and / or 5' end of the duplex is called an overhang.

[0048] As used herein, the terms "gene activation" or "activated gene" are interchangeable and refer to measuring the transcription, translation or expression or active increase of a certain nucleic acid by measuring gene transcription level, mRNA level, protein level, enzyme activity, methylation state, chromatin state or configuration, translation level or its activity or state in a cell or biological system. These activities or states can be measured directly or indirectly. In addition, "gene activation", "activated gene" refers to the increase in activity associated with a nucleic acid sequence, regardless of the mechanism by which this activation occurs, such as it plays a regulatory role as a regulatory sequence, is transcribed into RNA, is translated into protein and increases protein expression.

[0049] As used herein, the terms "small activating RNA", "saRNA", and "small activating nucleic acid molecules" are interchangeable and refer to ribonucleic acid molecules that can promote gene expression, and can be composed of a first ribonucleic acid chain (antisense chain, also called antisense oligonucleotide chain) comprising a ribonucleotide sequence having sequence identity with a non-coding nucleic acid sequence of a target gene (e.g., a promoter, an enhancer, etc.) and a second ribonucleic acid chain (sense chain, also called sense chain or sense oligonucleotide chain) comprising a nucleotide sequence complementary to the first chain, wherein the first chain and the second chain form a duplex. Small activating RNA can also be composed of a single-stranded RNA molecule that can form a double-stranded region hairpin structure, which is synthesized or expressed by a vector, wherein the first region comprises a nucleic acid sequence having sequence identity with a promoter target sequence of a gene, and the second region comprises a nucleic acid sequence that is complementary to the first region. The duplex region length of the small activating RNA molecule is generally about 10 to about 50 base pairs, about 12 to about 48 base pairs, about 14 to about 46 base pairs, about 16 to about 44 base pairs, about 18 to about 42 base pairs, about 20 and about 40 base pairs, about 22 and about 38 base pairs, about 24 and about 36 base pairs, about 26 and about 34 base pairs, about 28 and about 32 base pairs, typically about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50 base pairs. In addition, the terms "saRNA" and "small activating RNA", "small activating nucleic acid molecule" also contain nucleic acids other than ribonucleotide portions, including but not limited to modified nucleotides or analogs.

[0050] As used herein, the term "hotspot" refers to a gene promoter region of at least 30 bp in length, in which functional small activating nucleic acid molecule targets are clustered, that is, at least 60% of the small activating nucleic acid molecules targeting these hotspot regions can induce target gene mRNA expression by 1.5 times or more.

[0051] As used herein, the term "p21" refers to p21 WAF1 / CIP1The gene, also known as CDKN1A, is a cyclin-dependent kinase (CDK) inhibitor and an important tumor suppressor gene, sometimes also called a "target gene." Overexpression of p21 or activation of endogenous p21 transcription can effectively inhibit the growth of tumor cells and in vivo tumors.

[0052] As used herein, the term "synthesis" refers to the synthesis method of oligonucleotides, including any method capable of synthesizing RNA, such as chemical synthesis, in vitro transcription, vector expression, etc. The preparation method of the small activating nucleic acid molecule provided by the present invention includes sequence design and sequence synthesis. The synthesis of the small activating nucleic acid molecule sequence can be carried out by chemical synthesis, or it can be commissioned to a biotechnology company specializing in nucleic acid synthesis. Generally speaking, the chemical synthesis method includes the following four processes: (1) synthesis of oligoribonucleotides; (2) deprotection; (3) purification and separation; (4) desalting and annealing. For example, the specific steps of the chemical synthesis of double-stranded RNA molecules such as saRNA described in the present invention are as follows: (1) Synthesis of oligoribonucleotides The automatic DNA / RNA synthesizer (e.g., Applied Biosystems EXPEDITE8909) is set to synthesize 1 micromole of RNA, and the coupling time of each cycle is set to 10-15 minutes. The starting material is a solid-phase-linked 5'-O-p-dimethoxytrityl-thymidine support. In the first cycle, one base is linked to the solid support. Then, in the nth (19≥n≥2) cycle, one base is linked to the base linked in the n-1th cycle. This cycle is repeated until the synthesis of the entire nucleic acid sequence is completed.

[0053] (2) Deprotection The solid support connected with saRNA is added to the test tube, and 1 ml of ethanol / ammonia solution (volume ratio of 1:3) is added to the test tube, then sealed and placed in a 25-70°C incubator for 2-30 hours, the solution of the solid support containing saRNA is filtered and the filtrate is collected, the solid support is rinsed with double distilled water twice (1 ml each time) and the filtrate is collected, the eluate is combined and collected, and dried under vacuum conditions for 1-12 hours. Then, 1 ml of tetrabutylammonium fluoride tetrahydrofuran solution (1M) is added, and the mixture is placed at room temperature for 4-12 hours, and then 2 ml of n-butanol is added, and the precipitate is collected by high-speed centrifugation to obtain the crude product of saRNA single strand.

[0054] (3) Purification and separation The obtained crude saRNA product was dissolved in 2 ml of 1 mol / ml ammonium acetate aqueous solution, and then separated by high pressure liquid chromatography reverse phase C18 column to obtain a purified saRNA single-stranded product.

[0055] (4) Desalting and annealing The salt was removed by size exclusion gel filtration, and the single-stranded oligoribonucleic acid of the sense chain and the antisense chain were mixed in 1-2 ml of buffer (10 mM Tris, pH = 7.5-8.0, 50 mM NaCl) at the same molar ratio. The solution was heated to 95°C and then slowly cooled to room temperature to obtain a solution containing saRNA.

[0056] Materials and methods Cell culture and transfection Cell lines RT4, KU-7, T24, J82, TCCSUP, and HT-1197 were cultured in modified McCoy's 5A medium (Gibco); cell lines 5637, PC3, and Bel-7402 were cultured in RPMI1640 medium (Gibco); and UM-UC-3 cell line was cultured in basal medium (Gibco). All media contained 10% fetal calf serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco). Cells were cultured at 37°C in 5% CO2. Designed double-stranded RNA molecules were transfected at a concentration of 10 nM (unless otherwise specified) using RNAiMax (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

[0057] RNA isolation and reverse transcription polymerase chain reaction The cells were 2-3×10 5 Cells / well were seeded in 6-well plates and reverse transfected with oligonucleotide duplexes. Total cellular RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) according to its instructions. RNA (1 μg) was reverse transcribed into cDNA using the PrimeScript RT Kit with gDNA Eraser (Takara, Shlga, Japan). qPCR was performed using the ABI7500 Fast Real-time PCR System (Applied Biosystems) and SYBR Premix Ex Taq II (Takara, Shlga, Japan) reagents, with the following reaction conditions: 95°C for 3 seconds, 60°C for 30 seconds, and amplification for 40 cycles. GAPDH was used as an internal reference. All primer sequences are listed in Table 1.

[0058] Table 1. Primer sequences for qRT-PCR analysis

[0059] Cell proliferation assay The cells were cultured at 2-4 × 10 3Cells / well were plated in 96-well plates, cultured overnight, and transfected with oligonucleotide duplexes. Three days after transfection, cell proliferation was detected using CCK8 (Dojindo) according to its instructions. The experimental steps are briefly described as follows: 10 μL CCK8 solution was added to each well, incubated at 37°C for 1 hour, and then the absorbance at 450 nm was measured using a microplate reader.

[0060] QuantiGene 2.0 analysis Cells were plated in 96-well plates and transfected with oligonucleotide duplexes. 72 hours after transfection, the target gene mRNA levels were quantified using the QuantiGene 2.0 kit (AffyMetrix). The QuantiGene 2.0 kit is a hybridization-based method that uses gene-specific probes to directly quantify mRNA levels. The experimental steps are briefly described as follows: Lysis buffer was added to lyse the transfected cells, and the cell lysate was loaded into the capture well plate coated with CDKN1A (p21) and HPRT1 (housekeeping gene) probes and hybridized overnight at 55°C. To enhance the hybridization signal, 2.0 PreAMP, 2.0 AMP and 2.0 Label Probe were hybridized in sequence in 100 μL of the corresponding buffer (provided in the Quantigene 2.0 kit). All hybridizations were shaken at 50-55°C for 1 hour. After the final wash step, 2.0 Substrate was added and incubated at room temperature for 5 minutes. The light signal was then detected using an Infinite 200PRO plate reader (Tecan, Switzerland).

[0061] Statistical analysis The results are expressed as mean ± standard deviation. One-way ANOVA followed by Tukey's t test was used for statistical analysis using GraphPad Prism software (GraphPad Software). The standard for statistical significance was set at p<0.05, p<0.01 and p<0.001.

[0062] Example The present invention is further illustrated by the following examples. The examples are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present invention in any way.

[0063] Example 1: Screening for functional small activating RNA (saRNA) targeting the promoter region of the p21 gene In order to screen for functional small activating RNA that can activate p21 gene expression, the 1 kb promoter sequence of p21 gene was retrieved from the UCSC Genome database ( Figure 1). A target of 19 bp was selected starting from -1 kb upstream of the transcription start site (TSS), and a total of 982 target sequences were obtained by moving toward the TSS site by moving 1 bp each time. The target sequences were filtered to exclude target sequences with a GC content higher than 65% or lower than 35%, and containing 5 or more consecutive identical nucleotides. After filtering, the remaining 439 target sequences entered the screening process as candidates. Based on these candidate sequences, the corresponding double-stranded double-stranded RNA molecules were chemically synthesized. Among them, the length of the sense and antisense strands of the double-stranded RNA molecules used in this experiment were both 21 nucleotides, and the 19 nucleotides in the 5' region of the first RNA strand (sense strand) of the double-stranded RNA molecule, such as the double-stranded saRNA, had 100% identity with the promoter target sequence, and its 3' end contained dTdT overhang; the 19 nucleotides in the 5' region of the second RNA strand were completely complementary to the 19 nucleotides in the 3' region sequence of the first RNA strand, and its 3' end contained dTdT overhang.

[0064] The aforementioned double-stranded RNA molecules were transfected into PC3 prostate cancer cells at a final concentration of 10 nM. After 72 hours, the p21 gene mRNA level was detected using the QuantiGene 2.0 kit. The fold change of p21 mRNA level of each double-stranded RNA molecule relative to the blank control was calculated and plotted on Figure 2 The fold changes of p21 gene mRNA caused by all double-stranded RNA molecules in this study ranged from 0.66 (inhibition) to 8.12 (induction) ( Figure 2 B). 361 (82.2%) double-stranded RNA molecules induced p21 expression by 1.01 to 8.12 folds; 74 (16.9%) double-stranded RNA molecules showed an inhibitory effect (0.99 to 0.66 folds); and 4 double-stranded RNA molecules (0.9%) had no effect on the mRNA level of the p21 gene (1.0 fold).

[0065] Among the 439 double-stranded RNA molecules screened, 132 double-stranded RNA molecules (30.1%) were able to induce p21 mRNA at least 2 times; 229 (52.4%) double-stranded RNA molecules induced p21 mRNA at least 1.5 times, and these double-stranded RNA molecules that induced p21 mRNA expression to increase by more than 10% were saRNAs, that is, functional saRNAs. These functional saRNAs are scattered throughout the p21 promoter region. However, there are clusters of functional small activating RNAs in 8 free regions, which are called "hotspots." Hotspot regions are defined as regions containing at least 10 corresponding small activating RNAs, of which at least 60% of the small activating RNAs can induce p21 mRNA expression by 1.5 times or more ( Figure 2A and Figure 3). The target sequences of hotspots 1 to 8 and the corresponding small activating RNA sequences are listed in Tables 2 and 3, respectively.

[0066] Table 2 Target sequences of p21 promoter hotspot regions

[0067] Table 3 Designed double-stranded RNA molecules located in the hotspot region of p21 promoter

[0068] These hot spots include hot spot region 1, the corresponding target sequence is −893 bp to −801 bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: 93. In this region, 44 functional saRNAs were found ( Table 3 , Figure 3A ), namely RAG-834, RAG-845, RAG-892, RAG-846, RAG-821, RAG-884, RAG-864, RAG-843, RAG-854, RAG-844, RAG-887, RAG-838, RAG-858, RAG-835, RAG-876, RAG-870, RAG-853, RAG-881, RAG-828, and RAG-864. 72. RAG-841, RAG-831, RAG-829, RAG-820, RAG-822, RAG-868, RAG-849, RAG-862, RAG-865, RAG-893, RAG-848, RAG-824 , RAG-866, RAG-840, RAG-875,, RAG-880, RAG-871, RAG-888, RAG-885, RAG-894, RAG-833, RAG-825, RAG-889, RAG-823; The corresponding target sequence of hotspot region 2 (Table 3, Figure 3B) is -717 to -632 bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: As shown in Figure 94, 31 functional saRNAs were found in this region, namely RAG-693, RAG-692, RAG-688, RAG-696, RAG-694, RAG-687, RAG-691, RAG-690, RAG-689, RAG-682, RAG-686, RAG-662, RAG-695, RAG-654, RAG-658, RAG-685, RAG-704, RAG-714, RAG-705, RAG-661, RAG-656, RAG-698, RAG-697, RAG-657, RAG-715, RAG-652, RAG-651, RAG-650, RAG-716, RAG-717, and RAG-711; Hotspot 3 ( Table 3 , Figure 3C ), the corresponding target sequence is −585 bp to −551 bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: 95. In this region, it was found that it contains 9 functional saRNAs, namely RAG-580, RAG-577, RAG-569, RAG-576, RAG-570, RAG-574, RAG-585, RAG-579, and RAG-584; Hotspot 4 ( Table 3 , Figure 3D ), the corresponding target sequence is −554 bp to −505 bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: 96. In this region, 17 functional saRNAs were found, namely RAG-524, RAG-553, RAG-537, RAG-526, RAG-554, RAG-523, RAG-534, RAG-543, RAG-525, RAG-535, RAG-546, RAG-545, RAG-542, RAG-531, RAG-522, RAG-529, and RAG-552; Hotspot 5 ( Table 3 , Figure 3E ), the corresponding target sequence is −514 bp to −485 bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: 97 . Nine functional saRNAs were found in this region, namely RAG-503, RAG-504, RAG-505, RAG-506, RAG-507, RAG-508, RAG-509, RAG-510, RAG-511, RAG-512, RAG-513, and RAG-514; Hotspot 6 ( Table 3 , Figure 3F ), ​​the corresponding target sequence is −442 bp to −405 bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: 98. Twelve functional saRNAs were found in this region, namely RAG-427, RAG-430, RAG-431, RAG-423, RAG-425, RAG-433, RAG-435, RAG-434, RAG-439, RAG-426, RAG-428, and RAG-442; Hotspot 7 ( Table 3 , Figure 3G ), the corresponding target sequence is −352 bp to −313 bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: 99. Thirteen functional saRNAs were found in this region, namely RAG-335, RAG-351, RAG-352, RAG-331, RAG-344, RAG-342, RAG-341, RAG-333, RAG-345, RAG-346, RAG-336, RAG-332, and RAG-343; Hotspot 8 ( Table 3 , Figure 3H ), the corresponding target sequence is −325 bp to −260 bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: 100. In this region, 18 functional saRNAs were found, namely RAG-294, RAG-285, RAG-286, RAG-292, RAG-291, RAG-284, RAG-279, RAG-280, RAG-325, RAG-293, RAG-322, RAG-321, RAG-281, RAG-289, RAG-278, RAG-283, RAG-282, and RAG-295.

[0069] To verify the results of QuantiGene 2.0, 439 double-stranded RNA molecules were divided into four groups (bins) according to the activity of activating p21 mRNA expression. Five double-stranded RNA molecules were randomly selected from each group and transfected into PC3 cells at a concentration of 10 nM. After 72 hours, total cell RNA was extracted and reverse transcribed, and the p21 gene mRNA level was analyzed by RT-qPCR. The p21 gene mRNA expression levels revealed by the two methods showed a significant correlation (R 2 = 0.82)( Figure 4 All functional saRNAs obtained by the QuantiGene 2.0 method could be verified as true functional small activating RNAs by RT-qPCR, and some of them showed stronger induction of p21 mRNA expression in RT-qPCR analysis (Table 4).

[0070] Table 4. Validation of the QuantiGene 2.0 method

[0071] In summary, the above data indicate that many specific sites in the p21 promoter region can be used as saRNA target sequences to induce p21 expression, some of which are more sensitive and their corresponding saRNAs are more likely to activate p21 expression.

[0072] Example 2: saRNA induces p21 gene mRNA expression and inhibits cancer cell proliferation In order to further evaluate the effect of p21 saRNA in inducing p21 gene mRNA expression and inhibiting cancer cell proliferation, saRNA (RAG1-431, RAG1-553, RAG1-688) screened by QuantiGene 2.0 were transfected into cancer cell lines KU-7 (bladder cancer), HCT116 (colon cancer) and HepG2 (hepatocellular carcinoma). The results showed that in all the above cell lines, saRNA could induce at least two times the expression level of p21 gene mRNA and inhibit cell proliferation, revealing the efficacy of saRNA-mediated p21 induction. Specifically, RAG1-431, RAG1-553, and RAG1-688 were transfected into KU7 cells, respectively, to induce p21 mRNA expression by 14.0, 36.9, and 31.9 times, respectively, and produced survival rates of 71.7%, 60.7%, 67.4% ( Figure 5 ). RAG1-431, RAG1-553, and RAG1-688 were transfected into HCT116 cells, respectively, and induced p21 mRNA expression by 2.3, 3.5, and 2.4 times, respectively, and produced survival rates of 45.3%, 22.5%, and 38.5% relative to the blank treatment ( Figure 6 ). RAG1-431, RAG1-553, and RAG1-688 were transfected into HepG2 cells, respectively, and induced p21 mRNA expression by 2.2, 3.3, and 2.0 times, respectively, and produced survival rates of 76.7%, 64.9%, and 79.9% relative to blank treatment ( Figure 7 ).

[0073] Incorporated by Reference The entire disclosure of each patent and scientific article cited herein is incorporated by reference for all purposes.

[0074] Equivalence The present invention may be implemented in other specific forms without departing from its essential features. Therefore, the foregoing embodiments are considered to be illustrative rather than limiting of the present invention described herein. The scope of the present invention is indicated by the appended claims rather than by the foregoing description, and it is intended that all changes within the meaning and scope of the equivalents of the claims be included therein.

Claims

1. A small activating RNA, characterized in that One strand of the small activating RNA has at least 75% homology or complementarity with any continuous fragment of 16-35 nucleotides in length of the sequences shown in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, and 12, and the small activating RNA activates or upregulates the expression of the p21 gene by targeting the human p21 gene promoter sequence.

2. The small activating RNA according to claim 1, wherein the small activating RNA comprises a sense nucleic acid chain and an antisense nucleic acid chain, wherein the sense nucleic acid chain and the antisense nucleic acid chain contain complementary regions, and the complementary regions can form a double-stranded nucleic acid structure, wherein the sense nucleic acid chain or the antisense nucleic acid chain has a homology of more than 75%, more than 80%, more than 90%, more than 95%, more than 99% or 100% with any continuous segment of 16-35 nucleotides in length in the human p21 gene promoter sequence.

3. The small activating RNA of claim 2, wherein the sense nucleic acid strand and the antisense nucleic acid strand are present on two different nucleic acid strands.

4. The small activating RNA according to claim 2, wherein the sense nucleic acid strand and the antisense nucleic acid strand are present on the same nucleic acid strand, which is a hairpin-type single-stranded nucleic acid molecule, wherein the complementary regions of the sense nucleic acid strand and the antisense nucleic acid strand form a double-stranded nucleic acid structure.

5. The small activating RNA of claim 3, wherein at least one strand of the small activating RNA has a 3' overhang of 0 to 6 nucleotides in length.

6. The small activating RNA of claim 5, wherein both strands in the small activating RNA have a 3' overhang of 2-3 nucleotides in length.

7. The small activating RNA according to any one of claims 2 to 6, wherein the sense nucleic acid strand or the antisense nucleic acid strand has a length of 16-35 nucleotides.

8. The small activating RNA of claim 1, wherein the sense nucleic acid strand or the antisense nucleic acid strand is identical to SEQ ID NO: 13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108, 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 201-202, 209-212, 215-218, 225-240, 243-246, 249-258, 261-262, 265-270, The sequences shown in 275-280, 283-300, 303-308, 317-320, 323-324, 329-348, 351-352, 357-358, 361-366, 371-392, 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, and 463-468 have at least 75% homology.

9. The small activating RNA of claim 8, wherein the sense nucleic acid strand or the antisense nucleic acid strand sequence is as shown in SEQ ID NO: 13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108, 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 201-202, 209-212, 215-218, 225-240, 243-246, 249-258, 261-262, 265-266 270, 275-280, 283-300, 303-308, 317-320, 323-324, 329-348, 351-352, 357-358, 361-366, 371-392, 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, 463-468 as shown in any one of the nucleotide sequences.

10. The small activating RNA according to any one of claims 1 to 9, wherein at least one nucleotide in the small activating RNA is a chemically modified nucleotide, and the chemical modification is at least one of the following modifications: (1) Modification of the phosphodiester bonds connecting nucleotides in the nucleotide sequence of the small activating RNA; (2) modification of the 2'-OH of the ribose in the nucleotide sequence of the small activating RNA; (3) modification of bases in the nucleotide sequence of the small activating RNA; (4) At least one nucleotide in the nucleotide sequence of the small activating nucleic acid molecule is a locked nucleic acid.

11. The small activating RNA according to any one of claims 1 to 10, wherein the activation or upregulation of the expression of the p21 gene is upregulation by at least 10%.

12. Use of the small activating RNA according to any one of claims 1 to 11 in the preparation of a preparation for activating or up-regulating the expression of p21 gene in cells.

13. The use according to claim 12, wherein the small activating RNA is directly introduced into the cell.

14. The use according to claim 13, wherein the cells are mammalian cells, preferably human cells, more preferably human tumor cells.

15. The use according to claim 14, wherein the cells exist in the human body.

16. The use as claimed in claim 15, wherein the human body is a patient suffering from a tumor caused by insufficient expression of p21 protein, and the small activating nucleic acid molecule is administered in an effective amount to achieve treatment of the tumor, and the tumor is preferably bladder cancer, prostate cancer, liver cancer or colorectal cancer.

17. An isolated p21 gene small activating RNA target site, wherein the target site is any continuous 16-35 nucleotide sequence selected from any one of SEQ ID No 5-12.

18. A method for activating or upregulating the expression of p21 gene in a cell, wherein the method comprises administering the small activating RNA according to any one of claims 1 to 11 to the cell.

19. The method of claim 18, wherein the small activating RNA is introduced directly into the cell.

20. The method according to claim 18 or 19, wherein the cell is a mammalian cell, preferably a human cell, more preferably a human tumor cell, and still more preferably a human bladder cancer, prostate cancer, liver cancer or colorectal cancer cell.

21. A composition comprising the small activating RNA according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

22. The composition of claim 21, wherein the pharmaceutically acceptable carrier is a liposome, a high molecular weight polymer or a polypeptide.

23. Use of the small activating RNA according to any one of claims 1 to 11 or the composition according to claim 21 or 22 in the preparation of a drug for activating or up-regulating p21 gene expression, preferably in the preparation of a drug for anti-tumor or benign proliferative lesions, more preferably, the tumor is bladder cancer, prostate cancer, liver cancer or colorectal cancer.

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