WO2018170752A1

WO2018170752A1 - Tud rna for knocking down mirna-29a, mirna-148a, and mirna-424, and application thereof

Info

Publication number: WO2018170752A1
Application number: PCT/CN2017/077593
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2017-03-21
Filing date: 2017-03-21
Publication date: 2018-09-27

Abstract

Provided is a Tud RNA for knocking down miRNA-29a, miRNA-148a, and miRNA-424. A nucleotide sequence of the Tud RNA is represented by SEQ ID NO. 1. Also provided are a Tud RNA interference vector for suppressing expressions of miRNA-29a, miRNA-148a, and miRNA-424, and a preparation method therefor. The preparation method comprises: synthesizing, according to compared and analyzed human miR-29a, miR-148a, and miR-424 sequences, a fragment with BamHI and HindIII restriction enzyme cutting sites, connecting the fragment onto a plasmid vector subjected to BamHI and HindIII linearization, and filtering to obtain the interference vector.

Description

Title: A knockdown of miRNA-29a, miRNA-148a and miRNA-424

RNA and its application

Technical field

[0001] The present invention relates to a Tud RN^ application for knockdown of miRNA-29a, miRNA-148a and miRNA-424, and belongs to the field of genetic engineering technology.

Background technique

[0002] MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs found in eukaryotes, typically between 22 and 25 nt. miRNAs are widely distributed in plants, animals, and multicellular organisms, and are important. In the study of human miRNAs, it was found that the expression of miRNAs in normal tissues was significantly different. Some miRNAs were lowly expressed in tumor tissues and highly expressed in tumor tissues, indicating that miRNAs are involved in tumorigenesis. Has played a crucial role in the '

[0003] miR-29a is a small RNA closely related to cell proliferation. It is involved in many diseases, and it acts as a tumor suppressor gene in tumors. It is involved in the growth and invasion of cells such as human gastric cancer and bladder cancer. The level is an important reference for evaluating the benign and malignant gliomas. It is also associated with diseases such as atherosclerosis and has important potential applications for the treatment of various tumors; miR-148a has a more studied micr ₀ RNA. It has been reported that miR-148a is closely related to the metabolism, excretion, occurrence, development and epigenetic of various cancers; miR-424 is a miRNA in recent years, which acts in a variety of tumors. The target gene, which is involved in the regulation of target gene regulation ⁴ , affects the biological effects and development of tumor cells, plays a role similar to oncogenes, suppresses cancer, or promotes and inhibits tumor invasion and metastasis. Studies have shown that miR-424 is a multifunctional miRr associated with cervical cancer, pancreatic cancer and other cell invasion and metastasis; with inflammatory factors such as IL-6, TNF-oc; since the miR-424 promoter region has a CpG island, it Methylation-induced gene silencing also controls the expression of miR-29a, miR-148a, and miR-424, and synergizes with other drugs. miR, miRNA

Sponge et al., these technologies are all transient transfection techniques, unable to maintain the long silence of the target miRNA, and the silencing effect is far from optimal.

[0005] Tough Decoy RNA (Tud RNA) is a novel miRNA-inhibiting method for the adsorption of target miRNAs into double-stranded RNA to inhibit miRNAs. Since the bow I is double-stranded and has a secondary structure of a stem loop, it is resistant to intracellular nuclease degradation and stably and efficiently inhibits miRNA.

Problem solution

Technical solution

The present invention provides a Tud RN nucleotide sequence which knocks down miRNA-29a, miRNA-148a and miRNA-424 as shown in SEQ ID NO: 1, mainly in inhibiting human miR-29a, miR-148a and n Application in the expression product.

The present invention also provides a method for preparing a shRNA interference carrier, and the steps are as follows:

[0008] (1) adding a BamHI and Hir cleavage site to the nucleotide sequence shown in SEQ ID N0.1 to obtain a Tud RNA fragment;

(2) The interference fragment obtained in the step (1) was ligated to the BamHI and Hindlll linearized vector to obtain a Tud RNA vector.

[0010] The specific steps are as follows:

[0011] (1) adding a BamHI and Hir cleavage site to the nucleotide sequence shown in SEQ ID NO: 1 to obtain a Tud RNA fragment;

(2) The interference fragment obtained in the step (1) was ligated to the linearized nesil 1.0 plasmid vector via BamHI and Hindlll to obtain the Tud RNA vector p-Genesil-Tud-29a-148a-424.

[0013] Tud provided by the present invention

The preparation method of the RNA vector is used to inhibit the expression of human miR-29a, 11 11 -148 & and 1^1 -424 expression preparations. The single-stranded Tud RNA fragment at the cleavage site, after renatured into a double strand, was ligated into the p-Genesill.O vector via BamHI and Hindll. The effective interference fragment constructed and screened is Tud-29a-148a-424. Advantageous effects of the invention

Beneficial effect

The homology designed by the present invention interferes with the miR-29a, miR-148a and miR-424 TuD RNA sequence structures, which are not easily degraded, and the double-stranded Tud RNA is more efficient than the currently used single-stranded miRNA spons. And the same target for three targets, can better achieve the efficiency of the dry high-miRNA function of the three miRNAs.

Brief description of the drawing

DRAWINGS

[0017] Figure 1. miRNA expression levels of cells in each group, wherein a. miR-29a expression, b. miR-148a expression, c. miR-424 expression.

BEST MODE FOR CARRYING OUT THE INVENTION

[0018] The present invention will be further described in detail below with reference to the embodiments and drawings, but the invention is limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following 3⁄4 examples were purchased from the market unless otherwise stated.

[0019] The pGenesill.O vector was purchased from the Biovector Plasmid Vector Culture Cell Collection; Reverse Transcription -

SYBR fluorescence real-time quantitative PCR kits were purchased from Takara Reagent Co., Ltd.; endotoxin plasmid ί was purchased from Tiangen Biochemical.

SEQ ID NO 1: 5'-

Design and Synthesis of Tud RNA Targeting miR-29a, miR-148a and miR-424

[0023] TuD RNA oligonuclear 歹 ijTud targeting miR-29a, miR-148a and miR-424 was designed based on the tuD RNA design sequence and miR-29a, miR-148a and miR-4 column information provided in miRBase. -29a-148a-424, whose sequence 歹ij is shown in SEQ ID NO 1, was commissioned by Shanghai Biotech to be synthesized by gene.

[0024] Example 2:

[0025] Construction of p-Genesil-Tud-29a-148a-424 Vector

The Tud-29a-148a-424 sequence and p-Genesill.0 were separately treated with BamHI and Hindlll enzymes, and the fractions were recovered, ligated with T4 DNA ligase at 4 ° C overnight, and then transformed into competent E. coli ToplO colonies and cultured. Sended to Shanghai Shenggong for sequencing. The correctly sequenced p-Genesil-Τικ 8a-424 vector was constructed. Extraction of p-Genesil-Tud-29a-148a-424 using the endotoxin plasmid extraction kit ^:

[0027] Example 3:

[0028] p-Genesil-Tud-29a-148a-424 vector transduction Hela cells

[0029] HeLa cells were seeded in 6-well plates at 1000000 cells per well. After 18 h, the cell density was approximately transduced into Hela cells by jetPrime transfection reagent, p-Genesil-Tud-29a-148a-424 vector, β After that, the medium was changed to fresh DMEM complete medium, and then cultured for 24 h.

[0030] Example 4:

[0031] Detection of miR-29a, miR-148a and miR-424 expression levels

[0432] Hela cells and Hela cells transduced with p-Genesil-Tud-29a-148a-424 vector were used to extract miRNAs from these cells using the miRci miRNA extraction and isolation kit, followed by S-Poly(T) hsa-mil qPCR -assay primer set, S-Poly(T) hsa-miR-148a qPCR-assay primer set and SI hsa-miR-424 qPCR-assay primer set kit (both purchased from Shenzhen Anran Bio) Reverse transcription and tailing of miRI , the corresponding cDNA was obtained. Take 2 kinds of cells of cDNA 2

As a template, 3 experiments were performed to detect the expression levels of miR-29a, miR-148a and miR-424 by PCR, and 3 parallel samples were set per well, with snord 44 as an internal reference. The result is shown in Figure 1. Industrial applicability

The homology designed by the present invention interferes with the miR-29a, miR-148a and miR-424 TuD RNA sequences, and is not easily degraded. The double-stranded Tud RNA is more efficient than the commonly used single-stranded miRNA spons, and the same吋 For three targets, the efficiency of dry miRNA high miRNA function studies of three miRNAs can be well achieved.

Claims

Claims [Claim 1] A Tud RNA that knocks down miRNA-29a, miRNA-148a and miRNA-424, which is capable of knocking down the expression of human miR-29a, miR-148a and miR-424, sequences As shown in SEQ ID NO 1. [Claim 2] A method for producing a Tud RNA interference vector according to claim 1, wherein the steps are as follows:

(1) adding a BamHI and Hindll: point to the nucleotide sequence shown in SEQ ID NO: 1 to obtain a Tud RNA fragment;

(2) The interference fragment obtained in the step (1) was ligated to the BamHI and Hindlll † plasmid vector to obtain an interference vector.

[Claim 3] The method according to claim 2, wherein the specific steps are as follows:

(1) adding BamHI and Hindlll points to the nucleotide sequence shown in SEQ ID NO: 1 to obtain a Tud RNA fragment;

(2) The Tud RNA fragment obtained in the step (1) was ligated to the p-Genesill.O plasmid vector subjected to BamHI and Hindi, to obtain the interference vector p-Genesil-Tud-29a 24.