CN111394351B - siRNA for inhibiting DICER1-AS1 expression and application thereof - Google Patents
siRNA for inhibiting DICER1-AS1 expression and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
Abstract
The invention discloses a nucleotide for inhibiting expression of human DICER1-AS1 and application thereof, which is characterized in that: the nucleotide specifically binds to human DICER1-AS1, and the sequence of the nucleotide consists of a AAACACAG AAGAUCUUGCUUCUCUCCC sequence or 18-22 consecutive nucleotide sequences in the complementary sequence, in particular the nucleotide sequence comprises: AAACACAGAAGAUCUUGCU and AAGAUCUUGCUUCUCUCCC. The invention can inhibit human DICER1-AS1, can effectively inhibit DICER1-AS1 expression in A549 and XWLC-05 cells, and can increase the growth and proliferation inhibition by combining with anti-tumor drugs, thereby effectively treating various tumors.
Description
Technical Field
The invention belongs to the technical field of medical materials and medicines, and particularly relates to cyclic RNAs (ribonucleic acid), in particular to human cyclic RNAs. The nucleotide can be complementary with DICER1-AS1, so AS to inhibit expression of human DICER1-AS1, and has anti-tumor effect with other anti-tumor drugs.
Background
Long non-coding RNAs (lncrnas) are a class of RNA molecules that are less than 200 a bp a long. LncRNA is typically transcribed by RNA polymerase II and undergoes 5' end capping, RNA splicing and polyadenylation procedures. LncRNA does not encode or has very low protein-encoding capacity (3), and has been considered to be "transcriptional noise" in the past without any biological impact. However, there is growing evidence that lncRNA is involved in gene regulation, cell cycle regulation, cell differentiation, immune response, tumor metabolism and other processes. LncRNA generally performs different functions in the cytoplasm and nucleus. Interestingly, environmental changes or infections can also induce changes in the localization of lncRNA in the nucleus and cytoplasm. In tumor microenvironments, lncRNA may exhibit oncogenic or tumor-inhibiting functions due to changes in its expression. Hypoxia, low pH and energy stress in the tumor microenvironment are all factors that lead to alterations in lncRNA.
RNA interference (RNAi) technology utilizes double-stranded small RNA to efficiently and specifically degrade intracellular homologous RNA so as to silence a target gene, thereby realizing the function of interfering with the target gene.
Disclosure of Invention
The invention aims to provide an siRNA capable of specifically and efficiently inhibiting expression of DICER1-AS1 and application thereof.
The object of the invention is achieved in that the sequence comprising the synthetic nucleotides consists of 18-22 consecutive nucleotide sequences in the sequence of AAACACAG AAGAUCUUGCUUCUCUCC (SEQ ID NO. 1) or 18-22 consecutive nucleotide sequences in the complementary sequence. In particular it comprises the sequence: AAACACAGAAGAUCUUGCU (SEQ ID NO. 2) and AAGAUCUUGCUUCUCUCCC (SEQ ID NO. 3) or a complementary sequence. The nucleotide is ribonucleotide, deoxyribonucleotide or chimera of ribonucleotide and deoxyribonucleotide. The nucleotide is further modified by one or more of ribose, base, phosphate skeleton, fluoro, thio, methoxy and cholesterol. Nucleotides and other antitumor therapeutic agents, in particular trivalent inorganic arsenic. The application of the composition in preparing medicines for treating cancers. The cancers include: liver cancer, cardiac cancer, gastric cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, chronic or acute leukemia, brain tumor, esophageal cancer, oral cancer, urinary tract cancer, skin cancer, rectal cancer, middle ear cancer, bone cancer, intestinal cancer, gallbladder cancer, laryngeal cancer, gingival cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, carcinoma of large intestine, testicular cancer, cancer of the endocrine system, lymphocytic lymphoma. The study was supported by national natural science foundation (81860572).
The invention (advantage): the nucleotide has good DICER1-AS1 inhibition effect, acts on specific target sites, has strong specificity, low toxicity, small side effect and long modification half-life, and can be used with various antitumor drugs.
Detailed Description
The invention is further described below without limiting the invention in any way, and any alterations or substitutions based on the teachings of the invention are within the scope of the invention.
The nucleotide (siRNA) for inhibiting expression of DICER1-AS1 provided by the invention comprises a AAACACAG AAGAUCUUGCUUCUCUCC sequence or 18-22 continuous nucleotide sequences in a complementary sequence.
The nucleotide has a sequence of AAACACAGAAGAUCUUGCU or AAGAUCUUGCUUCUCUCCC and the complementary sequence thereof.
The nucleotide is ribonucleotide, deoxyribonucleotide or chimera of ribonucleotide and deoxyribonucleotide.
The nucleotide is further modified by one or more of ribose, base, phosphate skeleton, fluoro, thio, methoxy and cholesterol.
The application of the nucleotide is the application of the nucleotide in preparing antitumor drugs.
The tumor is lung cancer.
The pharmaceutical composition of the invention comprises the nucleotide and other anti-tumor therapeutic drugs.
The other antitumor therapeutic drug is sodium arsenite.
The application of the pharmaceutical composition provided by the invention is the application of the pharmaceutical composition in preparing antitumor drugs.
The tumor is lung cancer.
Example 1
The silencing RNA sequences in this example are: AAACACAGAAGAUCUUGCU (SEQ ID NO. 2) and AAGAUCUUGCUUCUCUCCC (SEQ ID NO. 3) and the complement, all from 5-to 3-terminal, all with dTdT added at the end. RNA sequence information for DICER1-AS1 is present in the UCSC database. The subject was supported by the study by national natural science foundation (81860572).
Control synthesis
First, a nucleotide was synthesized by Shanghai Ji Ma pharmaceutical technology Co., ltd, and a double-stranded RNA sequence comprising the complementary sequence thereof was synthesized, and a negative control having a negative control siRNA accession number A06001 sequence of Ji Ma pharmaceutical technology Co., ltd was used as a control group.
Cell culture: a549 or XWLC-05 cells were cultured in 1640 containing 10% fetal bovine serum at 37℃under 5% CO 2. 2500 wells per well were plated in 96-well plates. RNA transfection is performed by using the reagent for transfection by the hundred-code company Rfect, and the efficiency of RNA interference is often improved by fluorescent quantitative PCR. The following are respectively a control group, a silencing group 1 and a silencing groupThe efficiency of the cells was found to be a 549: 1.014+ -0.091,0.241 + -0.028,0.151 + -0.041; XWLC-05 cells were 1.019.+ -. 0.08,0.250.+ -. 0.017, 0.112.+ -. 0.015, respectively, data using 2 -ΔΔCt And (3) representing.
Primer for fluorescent quantification: DICER1-AS1 upstream: TGGACCATAAAGACAAGGACG (SEQ ID NO. 4) downstream: AGAGAGACAGTCCTGCTTGG (SEQ ID NO. 5) reference uses the ACTB gene from the article "Chen Jiangrong, zhang Rebing, zhang Yuan, hu Juan, zhou Mei, he Yuefeng. Influence of arsenic and its metabolites on P21 gene expression [ J ]. Profession and health, 2018, 34 (23)".
Arsenic is added to two cells 48 hours after transfection using a 96-well plate, sodium arsenite is dissolved in the medium, the same amount of medium is added without arsenic, the final concentration of sodium arsenite is 60 micromoles per liter, the final concentration of arsenic in XWLC-05 cells is 40 micromoles per liter, and MTS is used for detecting the cell viability after further culturing for 48 hours. Cell viability was calculated using the Promega MTS assay using absorbance at 490 nm.
Control group without arsenic: to avoid the addition of sodium arsenite after the negative control fragment was used, the viability of this group was set at 100.0%. Arsenic-free experimental group 1: results of silencing of nucleotide fragment 1. Arsenic-free experimental group 2: silencing nucleotide fragment 2 silencing results. Arsenic addition control group: results of adding sodium arsenite after using negative control fragment. Arsenic addition experimental group 1: silencing nucleotide fragment 1 results from the addition of sodium arsenite after silencing. Arsenic addition experimental group 2: silencing nucleotide fragment 2 results from the addition of sodium arsenite after silencing. The following are the cell viability for each group in percent (%):
control group without arsenic: a549 is 99.8+ -5.1, XWLC-05 is 100.89 + -3.9,
arsenic-free experimental group 1: a549 is 91.3+ -2.9, XWLC-05 is 93.4+ -3.4,
in the experiment group 2 without arsenic, A549 is 81.3 plus or minus 3.4, XWLC-05 is 82.6 plus or minus 2.8,
arsenic addition control group: a549 is 91.2+ -4.1, xwlc-05 is 84.4+ -2.9,
arsenic addition experimental group 1: a549 is 55.32 + -4.6, xWLC-05 is 53.1+ -4.8,
arsenic addition experimental group 2: a549 was 49.4±3.1 and xwlc-05 was 48.1±3.9.
SEQUENCE LISTING
<110> university of Kunming medical science
<120> an siRNA for inhibiting expression of DICER1-AS1 and use thereof
<130>
<141> 2019-12-24
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 27
<212> RNA
<213> Artificial sequence (Artificial sequence)
<400> 1
aaacacagaa gaucuugcuu cucuccc 27
<210> 2
<211> 19
<212> RNA
<213> Artificial sequence (Artificial sequence)
<400> 2
aaacacagaa gaucuugcu 19
<210> 3
<211> 19
<212> RNA
<213> Artificial sequence (Artificial sequence)
<400> 3
aagaucuugc uucucuccc 19
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
tggaccataa agacaaggac g 21
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
agagagacag tcctgcttgg 20
Claims (3)
1. An application of the nucleotide for inhibiting the expression of human DICER1-AS1 in preparing the medicine for treating lung cancer features that its sequence is AAACACAGAAGAUCUUGCU or AAGAUCUUGCUUCUCUCCC and its complementary sequence.
2. A pharmaceutical composition comprising the nucleotide of claim 1 and sodium arsenite.
3. The pharmaceutical composition according to claim 2, characterized in that the concentration of sodium arsenite is 40-60uM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202010189773.4A CN111394351B (en) | 2020-03-18 | 2020-03-18 | siRNA for inhibiting DICER1-AS1 expression and application thereof |
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| CN202010189773.4A CN111394351B (en) | 2020-03-18 | 2020-03-18 | siRNA for inhibiting DICER1-AS1 expression and application thereof |
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| CN111394351A CN111394351A (en) | 2020-07-10 |
| CN111394351B true CN111394351B (en) | 2023-11-07 |
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