CN111394351A - 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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- CN111394351A CN111394351A CN202010189773.4A CN202010189773A CN111394351A CN 111394351 A CN111394351 A CN 111394351A CN 202010189773 A CN202010189773 A CN 202010189773A CN 111394351 A CN111394351 A CN 111394351A
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- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 9
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- 239000003814 drug Substances 0.000 claims description 7
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- 230000000259 anti-tumor effect Effects 0.000 claims description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 6
- 239000005547 deoxyribonucleotide Substances 0.000 claims description 6
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- 239000008194 pharmaceutical composition Substances 0.000 claims description 6
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- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 3
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 3
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 3
- 235000012000 cholesterol Nutrition 0.000 claims description 3
- 125000001153 fluoro group Chemical group F* 0.000 claims description 3
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 3
- 229940124597 therapeutic agent Drugs 0.000 claims description 3
- 125000000446 sulfanediyl group Chemical group *S* 0.000 claims 1
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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
-
- 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 the expression of human DICER1-AS1 and application thereof, which is characterized in that the nucleotide is specifically combined with human DICER1-AS1, the sequence of the nucleotide consists of AAACACAG AAGAUCUUGCUUCUCUCCC sequences or 18-22 continuous nucleotide sequences in complementary sequences, particularly comprises the sequences of AAACACAGAAGAUCUUGCU and AAGAUCUUGCUCUCUCCC, the invention can effectively inhibit the expression of DICER1-AS1 in cells of A549 and XW L C-05 by inhibiting human DICER1-AS1, and can increase the inhibition of the growth and the proliferation of the cells by combining with an antitumor drug, thereby effectively treating various tumors.
Description
Technical Field
The invention belongs to the field of medical material technology and medicine, in particular to cyclic RNAs nucleotide, and especially relates to human cyclic RNAs nucleotide. The nucleotide can be complemented with DICER1-AS1, thereby inhibiting the expression of human DICER1-AS1, and playing a role in resisting tumors with other antitumor drugs.
Background
However, there is increasing evidence that lncRNA is involved in gene regulation, cell cycle regulation, cell differentiation, immune response, tumor metabolism and other processes, L ncRNA generally performs different functions in the cytoplasm and nucleus, and environmental changes or infections can also induce changes in the localization of lncRNA in the nucleus and cytoplasm.
The RNA interference (RNAi) technology utilizes small double-stranded RNA to efficiently and specifically degrade intracellular homologous RNA to silence a target gene, thereby achieving the function of interfering the target gene.
Disclosure of Invention
The invention aims to provide siRNA capable of specifically and efficiently inhibiting DICER1-AS1 expression and application thereof.
The object of the invention is achieved in that the sequence comprising synthetic nucleotides consists of a sequence of 18 to 22 consecutive nucleotides of the sequence AAACACAGAAGAUCUUGCUUCUCUCC (SEQ ID NO.1) or a sequence of 18 to 22 consecutive nucleotides of the complementary sequence. In particular it comprises the sequence: AAACACAGAAGAUCUUGCU (SEQ ID NO.2) and AAGAUCUUGCUUCUCUCCC (SEQ ID NO.3) or the complementary sequences. The nucleotide is ribonucleotide, deoxyribonucleotide or chimera of ribonucleotide and deoxyribonucleotide. The nucleotide is further modified by one or more of ribose modification, base modification, phosphate backbone modification, fluoro modification, sulfo modification, methoxy modification and cholesterol modification. Nucleotides and other antineoplastic therapeutic agents, particularly trivalent inorganic arsenic. Application in preparing medicine for treating cancer. Cancers include: liver cancer, cardiac cancer, colon cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, chronic or acute leukemia, brain tumor, esophageal cancer, oral cancer, urethral 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, colorectal cancer, testicular cancer, cancer of the endocrine system, and lymphocytic lymphoma. The study was supported by the national 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 together with various antitumor drugs.
Detailed Description
The present invention is further illustrated but not limited in any way by the following description, and any alterations or substitutions based on the teachings of the present invention are intended to fall within the scope of the present invention.
The nucleotide (siRNA) for inhibiting the expression of DICER1-AS1 has a sequence consisting of 18-22 continuous nucleotide sequences in a AAACACAG AAGAUCUUGCUUCUCUCC sequence or a complementary sequence.
The sequence of the nucleotide is 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 modification, base modification, phosphate backbone modification, fluoro modification, sulfo modification, methoxy modification and cholesterol modification.
The application of the nucleotide is the application of the nucleotide in preparing anti-tumor drugs.
The tumor is lung cancer.
The pharmaceutical composition comprises the nucleotide and other anti-tumor therapeutic drugs.
The other anti-tumor therapeutic drug is sodium arsenite.
The application of the pharmaceutical composition 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 complementary sequences, all from 5 to 3, with dTdT added to the ends. The RNA sequence information of DICER1-AS1 is present in the UCSC database. The subject is supported by the national science foundation under this study (81860572).
Control Synthesis
First, nucleotides were synthesized by jima pharmaceutical technology ltd, shanghai, and a double-stranded RNA sequence including a complementary sequence thereof was synthesized, and negative control group using a sequence having a cargo number a06001 of negative control siRNA of jima pharmaceutical technology ltd as a negative control of the control group was used.
The cell culture comprises that A549 cells or XW L C-05 cells are cultured in 1640 medium containing 10% fetal calf serum at 37 ℃ under the condition of 5% CO2, 2500 cells are paved in a 96-well plate, RNA transfection is carried out by adopting Rfect transfection reagent of the hundred generation company, the efficiency of RNA interference is often achieved by fluorescence quantitative PCR, the silencing efficiencies of a control group, a silencing group 1 and a silencing group are respectively achieved, the A549 cells are respectively 1.014 +/-0.091, 0.241 +/-0.028 and 0.151 +/-0.041, the XW L C-05 cells are respectively 1.019 +/-0.08, 0.250 +/-0.017 and 0.112 +/-0.015, and the data use 2^ 0-ΔΔCtAnd (4) showing.
Primers used for fluorescence quantification: DICER1-AS1 upstream: TGGACCATAAAGACAAGGACG (SEQ ID NO. 4): AGAGAGACAGTCCTGCTTGG (SEQ ID NO.5), the internal reference adopts ACTB gene in the article "Chengjiang, Zhangluo, Zhang Twai, Juan, Zhoume, He Yufeng. arsenic and its metabolite influence on P21 gene expression [ J ]. occupation and health, 2018, 34(23): 3213-3216 ].
Arsenic is added into two cells 48 hours after transfection by using a 96-well plate, sodium arsenite is dissolved in a culture medium, the same amount of the culture medium is not added, the final concentration of the sodium arsenite is 60 micromole per liter, the final concentration of the arsenic in XW L C-05 cells is 40 micromole per liter, and the cell activity is detected by MTS after 48 hours of culture.
Control group without arsenic addition: in order to use the negative control fragment without adding sodium arsenite, the activity of this group was determined to be 100.0%. Non-arsenic test group 1: results of nucleotide fragment 1 silencing. Non-arsenic test group 2: silencing nucleotide fragment 2 silencing results. Arsenic addition control group: the result of adding sodium arsenite after using the negative control fragment was obtained. Arsenic addition experimental group 1: silencing nucleotide fragment 1 was followed by addition of sodium arsenite. Arsenic addition experimental group 2: silencing nucleotide fragment 2 was followed by addition of sodium arsenite. The cell viability of each group was in percent (%):
as a control group without arsenic, the A549 is 99.8 plus or minus 5.1, the XW L C-05 is 100.89 plus or minus 3.9,
in the experimental group 1 without arsenic, A549 is 91.3 +/-2.9, XW L C-05 is 93.4 +/-3.4,
in the experiment group 2 without arsenic, A549 is 81.3 +/-3.4, XW L C-05 is 82.6 +/-2.8,
as a control group, A549 is 91.2 + -4.1, XW L C-05 is 84.4 + -2.9,
as addition experiment group 1, A549 is 55.32 + -4.6, XW L C-05 is 53.1 + -4.8,
as in the arsenic adding experimental group 2, A549 is 49.4 +/-3.1, and XW L C-05 is 48.1 +/-3.9.
SEQUENCE LISTING
<110> university of Kunming medical science
<120> siRNA for inhibiting DICER1-AS1 expression and application 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 (10)
1. A nucleotide for inhibiting the expression of DICER1-AS1, wherein the sequence of the nucleotide consists of 18 to 22 contiguous nucleotides of the AAACACAGAAGAUCUUGCUUCUCUCC sequence or the complementary sequence.
2. The nucleotide according to claim 1, characterized in that its sequence is AAACACAGAAGAUCUUGCU or AAGAUCUUGCUUCUCUCCC and its complement.
3. The nucleotide according to claim 1, characterized in that the nucleotide is a ribonucleotide, a deoxyribonucleotide or a chimera of a ribonucleotide and a deoxyribonucleotide.
4. The nucleotide according to claim 1, wherein the nucleotide is further modified by one or more of ribose modification, base modification, phosphate backbone modification, fluoro modification, thio modification, methoxy modification and cholesterol modification.
5. The use of the nucleotide as claimed in any one of claims 1 to 4, wherein the nucleotide is used in the preparation of an anti-tumor medicament.
6. Use according to claim 5, characterized in that said tumor is lung cancer.
7. A pharmaceutical composition comprising the nucleotide of any one of claims 1-4 and an additional anti-tumor therapeutic agent.
8. The pharmaceutical composition of claim 7, wherein said other anti-tumor therapeutic agent is sodium arsenite.
9. Use of a pharmaceutical composition according to claim 7 or 8 for the preparation of an anti-tumor medicament.
10. Use according to claim 9, characterized in that said tumor is lung cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010189773.4A CN111394351B (en) | 2020-03-18 | 2020-03-18 | siRNA for inhibiting DICER1-AS1 expression and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010189773.4A CN111394351B (en) | 2020-03-18 | 2020-03-18 | siRNA for inhibiting DICER1-AS1 expression and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111394351A true CN111394351A (en) | 2020-07-10 |
| CN111394351B CN111394351B (en) | 2023-11-07 |
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| CN202010189773.4A Active CN111394351B (en) | 2020-03-18 | 2020-03-18 | siRNA for inhibiting DICER1-AS1 expression and application thereof |
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2020
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