Nucleotide for inhibiting DDX17 related RNA expression product and application thereof

文档序号：758702 发布日期：2021-04-06 浏览：14次中文

阅读说明：本技术 一种抑制ddx17相关rna表达产物的核苷酸及其应用 (Nucleotide for inhibiting DDX17 related RNA expression product and application thereof ) 是由何越峰蒋成兰毛一竹谭婧文周倩尹锦瑶孙明军于 2021-01-17 设计创作，主要内容包括：本发明公开了一种抑制DDX17相关RNA表达产物的核苷酸及其应用,其特征是：所述的核苷酸特异性的抑制DDX17基因的mRNA和相关的四个环状RNA,所述核苷酸的序列是：CGCCGAAAGAAGGAGAUUA或GGGAGAUGUUUGUCCUAAA或者GGUUGAUGAGCUACGCCGA及其互补序列。本发明核苷酸能够有效抑制A549和MDA-MB-231细胞中DDX17基因的mRNA和相关的四个环状RNA表达,并减少A549和MDA-MB-231细胞生存,与抗肿瘤药物联合可以增加抗肿瘤的效果。(The invention discloses a nucleotide for inhibiting DDX17 related RNA expression products and application thereof, which is characterized in that: the nucleotide specifically inhibits mRNA of DDX17 gene and related four circular RNAs, and the sequence of the nucleotide is as follows: CGCCGAAAGAAGGAGAUUA or GGGAGAUGUUUGUCCUAAA or GGUUGAUGAGCUACGCCGA and their complements. The nucleotide can effectively inhibit mRNA of DDX17 gene and expression of four related circular RNAs in A549 and MDA-MB-231 cells, reduce survival of the A549 and MDA-MB-231 cells, and can increase anti-tumor effect by combining with anti-tumor drugs.)

1. A nucleotide for inhibiting DDX17 related RNA expression products, which is characterized by a sequence CGCCGAAAGAAGGAGAUUA (SEQ ID NO.1) or GGGAGAUGUUUGUCCUAAA (SEQ ID NO.2) or GGUUGAUGAGCUACGCCGA (SEQ ID NO.3) and a complementary sequence thereof.

2. The nucleotide according to claim 1, characterized in that the nucleotide can be modified by one or more of fluoro modification, thio modification, methoxy modification and cholesterol modification.

3. Use of a nucleotide according to claims 1 to 2, characterized by the use in the preparation of an anti-tumor medicament.

4. Use according to claim 3, characterized in that said tumor is lung cancer.

5. Use according to claim 3, characterized in that said tumor is breast cancer.

6. A pharmaceutical composition characterized by comprising the nucleotide of any one of claims 1-2 and other anti-tumor therapeutic agents.

7. The pharmaceutical composition of claim 6, wherein the other anti-neoplastic therapeutic agent is arsenic trioxide.

Technical Field

The invention belongs to the field of molecular biology and medicines, and particularly relates to a nucleotide for inhibiting DDX17 related RNA expression products and application thereof. The nucleotide can inhibit the expression of DDX17mRNA, hsa _ circ _0001230, hsa _ circ _0063328, hsa _ circ _0002211 and hsa _ circ _00633315 RNAs and can also be combined with other antitumor drugs for use.

Background

Single-chain covalently closed rings were first reported in 1976 as viroids, being pathogens of certain plants, and were first detected in 1979 by electron microscopy in human HeLa cells. With the development of high-throughput RNA sequencing and bioinformatics tools, scientists found that circRNA is a ubiquitous feature of the human transcriptome and is ubiquitous in many other metazoan. Recently, more and more studies have found various functions of circRNAs, including acting as protein scaffolds or miR sponges and being translated into polypeptides.

The unique structure of circRNAs makes their half-life longer and resistance to RNase R longer than linear RNAs, making them potential candidates for diagnostic biomarkers and therapeutic targets. Numerous studies have revealed their unique expression profiles and important biological roles in a variety of diseases, such as cancer, cardiovascular disease, neurological disease and autoimmune disease.

The DEAD box protein, which DDX17 is characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), is a putative RNA helicase. They are involved in many cellular processes involving changes in RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. This gene encodes the DEAD box protein, an ATPase that is activated by multiple RNAs rather than dsDNA. This protein, as well as the protein encoded by the DDX5 gene, are more closely related to each other than to other members of the DEAD box family. Due to the alternative splicing and the use of alternative translation initiation codons, the gene can encode multiple isoforms. Plays an important role in the growth of cancer.

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. Recently, RNA interfering drugs have gradually returned to the historical stage.

Disclosure of Invention

The invention aims to provide a nucleotide for inhibiting DDX17 related RNA expression products and application thereof.

The object of the present invention is achieved by a sequence comprising synthetic nucleotides consisting of the complement of CGCCGAAAGAAGGAGAUUA (SEQ ID NO.1) or GGGAGAUGUUUGUCCUAAA (SEQ ID NO.2) or GGUUGAUGAGCUACGCCGA (SEQ ID NO. 3). The nucleotide can be modified by one or more of fluoro modification, sulfo modification, methoxy modification and cholesterol modification. The nucleotide can be applied to preparing anti-tumor drugs, such as lung cancer and breast cancer. It can also be used in combination with other antitumor drugs, such as arsenic trioxide. The research is funded by an innovative team cultivation project (202005AE160002) in Yunnan province.

The invention (advantage): the nucleotide has good DDX17 related RNA inhibition effect, acts on a specific target site, acts on 5 related RNAs simultaneously, has strong action, strong specificity, low toxicity and small side effect, is modified to have long half-life period, and can be used together with various antitumor drugs.

Drawings

In the figure, DDX17mRNA is abbreviated as mRNA, hsa _ circ _0001230 is abbreviated as RNA30, hsa _ circ _0063328 is abbreviated as RNA28, hsa _ circ _0002211 is abbreviated as RNA11, and hsa _ circ _0063331 is abbreviated as RNA31, and the added arsenic is arsenic trioxide. Fragment 1: CGCCGAAAGAAGGAGAUUA (SEQ ID NO.1) or fragment 2: GGAGAUGUUUGUCCUAAA (SEQ ID NO.2) or fragment 3: GUUGAUGAGCUACGCCGA (SEQ ID NO. 3).

Figure 1 silencing efficiency of three fragments in a549 cells, each group consisting of 4 wells.

FIG. 2 silencing of three fragments in MDA-MB-231 cells, efficiency each group consisted of 4 wells.

FIG. 3 comparison of cell viability in control, silent (three fragments), plus arsenic (arsenic added to each fragment), plus arsenic silencing (arsenic added to each fragment). The specific values of A549 cell viability are respectively 100.00, 63.55, 55.11, 68.41, 95.12, 44.22, 32.01 and 46.32, and the specific values of MDA-MB-231 cell viability are respectively 100.00, 70.12, 63.02, 71.52, 96.45, 55.34, 54.31 and 54.12. Each group consisted of 6 wells.

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.

Examples

The RNA sequence in this example is: fragment 1 CGCCGAAAGAAGGAGAUUA (SEQ ID NO.1) or fragment 2GGAGAUGUUUGUCCUAAA (SEQ ID NO.2) or fragment 3GUUGAUGAGCUACGCCGA (SEQ ID NO.3) and its complementary sequence, the reference sequence is GUAUGUCGACGCGCUCAUA (all sequences of SEQ ID NO.4 are from 5 to 3, all sirna sequences synthesized by Shanghai Jima pharmaceutical technology Limited are added with dTdT. DDX17mRNA, hsa _ circ _0001230, hsa _ circ _0063328, hsa _ circ _0002211, and hsa _ circ _0063331 RNA sequence information in UCSC database during the synthesis.

A549 and MDA-MB-231 cells were cultured in 1640 medium containing 1% double antibody and 10% fetal bovine serum under the condition of 37 ℃ and 5% CO2 in a carbon dioxide box. Plated in 96-well plates at 3000 cells and 4000 cells per well.

A549 cells are transfected by using an Rfect transfection reagent of a hundred generation company, MDA-MB-231 cells are transfected by using a transfection reagent of a Biyunsday company, the cells are transfected by using a method according to a specification, the activity of the cells is detected by using a method of CCK8 of the Biyunsday company after about 72 hours of transfection, and the RNA interference efficiency is detected by RNA extraction and fluorescence quantification.

Perfluorogenic quantitative PCR for frequent RNA interference efficiency data 2^ s^-ΔΔCtAnd (4) showing. The primers are respectively as follows: primers for detecting DDX17 were ACCCAGATCAACGTAGGCA (SEQ ID NO.5) TCTCTGCGCATCCTTCGAG (SEQ ID NO.6, and detection RNAs 30 GGAGCTCACTCAAATCCCAC (SEQ ID NO.7) and ATGCTAACTTCCCACATTTGG (SEQ ID NO.7)ID NO.8), sense RNAs 28 CGCTCCCCAGGATTACCA (SEQ ID NO.9) and GAGACTTGGAAAGAGATTTGG (SEQ ID NO.10), sense RNAs 11 CGCTCCCCAGGATTACCA (SEQ ID NO.9) and GTGAAAAAGACCACAAATTTGGA (SEQ ID NO.11), sense RNAs 31 GGAGCTCACTCAAATCCCAC (SEQ ID NO.7) and GTGAAAAAGACCACAAATTTGGA (SEQ ID NO.12), and an annealing temperature of 60 degrees.

The combined effect detection adopts a control group, an arsenic adding group, a silent group and an arsenic adding silent comparison, and the cell activity of the control group is 100%.

SEQUENCE LISTING

<110> university of Kunming medical science

<120> nucleotide for inhibiting DDX17 related RNA expression product and application thereof

<130> 1

<140> 1

<141> 2020-12-20

<160> 12

<170> PatentIn version 3.3

<210> 1

<211> 19

<212> RNA