WO2018170619A1 - Dual-mirna inhibition expression vector and application thereof - Google Patents

Abstract

Provided is a dual-miRNA inhibition expression vector and an application thereof. The expression vector is a dual-miRNA inhibition expression vector pLKO-Tud-148a-152 obtained after connecting a Tud RNA targeting miR148a and miR-152 to a pLKO.1-puro cloning vector, and has the function of inhibiting the activity of has-miR-148a and has-miR-152.

Description

 Title of Invention: Vector for Inhibiting Expression of Dual miRNAs and Application Thereof

 [0001] The present invention relates to the field of molecular biology, and more particularly to a vector for inhibiting expression of a dual miRNA and an application thereof.

 Background technique

 [0002] RNA is an important substance in living organisms and plays various functions in life activities. RNA can be classified into messenger RNA (mRNA) and non-coding RNA depending on whether the protein is encoded or not.

(non-coding RNA, ncRNA). Small RNA (small

 RNA, smRNA) is an important class of ncRNAs. miRNAs are endogenous small RNAs in organisms that are typically 20-24 nt in length. The miRNA is part of the pri-miRNA (primary RNA) and is initially expressed in the nucleus by RNA polymerase Π. The mature miRNA acts as a guiding molecule, binds to the target gene mRNA according to the principle of base pairing, and directs the silencing complex (RISC) to degrade mRNA or hinder its translation, thereby exerting a negative regulation effect on the expression of the target gene.

 [0003] miR-148a is a microRNA that has been studied more in recent years. It is reported that miR-148a is closely related to the metabolism of exogenous substances, apoptosis, the occurrence, development and epigenetics of various cancers, so it is important to study the function of miR-148a; miR-152 is a kind of With multifunctional miRNAs, it was found that miR-152 is associated with methylation, such as methyltransferase DNMT1 content and enzyme activity, miR-152 can be methylated by endometrial cancer DNA into a silent gene, and its Associated with the development of a variety of cancers, it is a tumor suppressor microRNA that is associated with many diseases such as pre-eclampsia, trophoblastic tumors, bladder cancer, gastrointestinal cancer, and ovarian cancer. By controlling the expression of miR-148a and miR-152, peers and other drugs work together to provide new epigenetic ideas for the treatment of cancer.

 technical problem

 [0004] MiRNA functional studies often require miRNA silencing techniques, including anti-miR, antago miR, miRNA

Sponge et al., these technologies are all transient transfection techniques, unable to maintain long-term stable silencing of the target miRNA, and the silencing effect is far from optimal. [0005] Tough Decoy RNA (Tud RNA) is a novel miRNA-inhibiting miRNA that inhibits miRNA by introducing double-stranded RNA to adsorb target miRNAs. Since the inserted RNA is double-stranded and has a secondary structure of a stem loop, it is resistant to intracellular nuclease degradation and can inhibit miRNA for a long-term, stable, and efficient manner.

 Problem solution

 Technical solution

 The technical problem to be solved by the present invention is to provide a dual miRNA suppression expression vector which is simple in structure, low in cost and simple in operation.

 [0007] A Tud RNA targeting a dual miRNA, the nucleotide sequence of which is shown in SEQ ID NO: 1 in the Sequence Listing.

A dual miRNA-inhibiting expression vector comprising the sequence of the invention SEQ ID NO: 1.

[0009] The use of the dual miRNA-inhibiting expression vector of the present invention in tumor therapeutic research, the double miRNA-inhibiting expression vector can inhibit the expression of miR-148a and miR-152 by transforming Hela cells.

[0010] A method for constructing a dual miRNA suppression expression vector according to the present invention comprises the following steps:

(1) Designing a dual miRNA-targeted Tud RNA Seql according to the miR-148a and miR-152 sequences and the principle of Tud RNA technology, the nucleosides of the miR-148a and miR-152 sequences The acid sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the Sequence Listing, respectively, and the nucleotide sequence of the Seq1 sequence is shown in SEQ ID NO: 1 of the Sequence Listing, and was commissioned by Shanghai Biotech for synthesis.

(2) The synthesized sequence is two complementary single-stranded DNAs. The two single-stranded DNAs were dissolved in ddH ₂ 0, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and then allowed to cool to room temperature at room temperature.

[0013] (3) extraction vector pLK0.1-puro, double digestion with Age I and Eco RI enzyme 1

 After h, the digested vector was recovered using the MinElute Reaction Cleanup Kit, and the TuD obtained in the previous step was further treated with T4 DNA ligase.

The RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-TuD-148a-152, and finally the ligation product was transformed into competent E. coli Stbl3 and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Single colonies were picked and sequenced. The bacteria with the correct sequencing were expanded and extracted with a small amount of extraction kit without endotoxin plasmid. The extracted plasmid was the plasmid for the homologous interference pLKO-TuD-148a-152 required by the present invention. Advantageous effects of the invention

 Beneficial effect

 [0014] The homologous interference miR-148a and miR-152 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the currently used single-stranded miRNA sponge. And the same target for two targets, can better achieve the interference of two miRNAs, improve the efficiency of miRNA function research.

 Brief description of the drawing

 DRAWINGS

 Figure 1 shows the miRNA expression levels of 16HBE cells and TuD-148a-152 cells, wherein, a.

 Expression of miR-148a, b. expression of miR-152.

 BEST MODE FOR CARRYING OUT THE INVENTION

 BEST MODE FOR CARRYING OUT THE INVENTION

[0016] The present invention can be better understood in light of the following examples. However, those skilled in the art will readily understand

The specific material ratios, process conditions, and results of the examples are merely illustrative of the invention and should not be construed as limiting the invention as described in the claims.

The lentiviral plasmid pLKO.l-puro vector used in the present invention was purchased from Addgene; the human bronchial epithelial cells (16HBE cell strain) used in the present invention were purchased from ATCC, USA.

[0018] Example 1 Design and Synthesis of TuD RNA Targeting miR-148a and miR-152

[0019] Based on the sequence information of miR-148a and miR-152 provided in the TuD RNA design sequence and miRBase, a TuD RNA oligonucleotide sequence targeting miR-148a and miR-152 was designed, and its sequence is SEQ ID.

ΝΟ:1, commissioned by Shanghai Biotech to synthesize by means of gene synthesis.

[0020] Example 2 annealing of the sequence

[0021] The synthesized sequence is two complementary single stranded DNAs. The two single-stranded DNAs were dissolved in ddH ₂ 0, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and allowed to stand at room temperature to allow them to naturally cool to room temperature.

Example 3 Recombinant Construction of pLKO-Tud-148a-152 Lentiviral Recombinant Vector

[0023] The vector pLK0.1-puro was extracted, and after digestion with Ag I and Eco RI for 16 h, the digested vector was recovered with MinElute Reaction Cleanup Kit, and T4 DNA was used. Ligase will get the TuD from the previous step

 The RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-Tud-148a-152, and finally the ligation product was transformed into competent E. coli Stbl3 and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Single colonies were picked and sequenced. The correct sequencing bacteria were expanded and extracted with a non-endotoxin plasmid miniprep kit. The extracted plasmid was the plasmid of the same interference interference pLKO-TuD-148a-152 required by the present invention.

Example 4 Plasmid of pLKO-TuD-148a-152 16HBE cell

 [0025] 16HBE cells were seeded in 6-well plates, 1000000 cells per well, and the cell density was about 60% after 18 hours. The plasmid pLKO-TuD-148a-152 was transduced into 16HBE cells with Lipfectamine 2000, and culture was continued for 48 h. Thereafter, the cells were cultured for 3 days in DMEM medium containing 1.0 g/ml puromycin, and the cell line obtained by the screening was named TuD-148a-152 cell line.

 Example 5 Fluorescence Quantitative PCR Detection of Changes in Expression Levels of miRNAs

 [0027] Normal 16HBE cells and TuD-148a-152 cells were inoculated into 6-well plates, respectively, and the cells were cultured for about 24 hours to a degree of fusion of 80%. The miRNAs of these cells were extracted using the miRcute miRNA extraction and isolation kit, and the corresponding cDNA was obtained by reverse transcription. Take 2 kinds of cells of cDNA 2

 As a template, real-time PCR was used to detect the changes in the expression levels of miR-148a and miR-152, respectively. The experiment was repeated three times, and three parallel samples were set per well to snord.

44 as an internal reference. The results are shown in Figure 1. It can be seen that the expression level of miR-148a in TuD-148a-152 cells is 52% lower than that in 16HBE cells, and the expression level of 1^1 -152 is 61 ^< 3⁄4 lower than that in 1613⁄4 cells. Statistical significance (/?<0.01), indicating that TuD-148a-152 cell line was successfully constructed.

 Industrial applicability

 [0028] The homologous interference miR-148a and miR-152 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the currently used single-stranded miRNA sponge. And the same target for two targets, can better achieve the interference of two miRNAs, improve the efficiency of miRNA function research.

Claims

Claim  [Claim 1] A Tud RNA targeting a dual miRNA, characterized in that the nucleotide sequence thereof is shown in SEQ ID NO: 1 of the Sequence Listing.  [Claim 2] A vector for inhibiting expression of a dual miRNA, which comprises the sequence of claim 1.  [Claim 3] The use of the dual miRNA-inhibiting expression vector of claim 2 for tumor therapeutic research, wherein the dual miRNA-inhibiting expression vector inhibits the expression of human miR-148a and miR-152.  [Claim 4] A method for constructing a dual miRNA-inhibiting expression vector according to claim 2, comprising the steps of:  (1) Designing a dual miRNA-targeted Tud RNA Seq1 according to the miR-148a and miR-152 sequences and the principle of Tud RNA technology, wherein the nucleotide sequences of the miR-148a and miR-152 sequences are respectively As shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the Sequence Listing, the nucleotide sequence of the Seq1 sequence is shown in SEQ ID NO: 1 of the Sequence Listing, and was commissioned by Shanghai Biotech for synthesis. (2) The synthesized sequence is two complementary single-stranded DNAs. The two single-stranded DNAs were dissolved in ddH ₂ 0, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and allowed to stand at room temperature to allow them to naturally cool to room temperature.  (3) The extraction vector pLK0.1-puro, treated with Age I and Eco RI enzymes for 1 h The recombinant vector was ligated with the T4 DNA ligase, and the TuD RNA sequence obtained in the previous step was ligated into the vector pLKO.l-puro to form a recombinant vector pLKO-TuD-148a-152, and finally the ligated product was transformed into a sensation. The cells were plated in E. coli Stbl3 and plated on ampicillin-containing LB medium and cultured at 37 °C for 14 h. Pick a single colony and sequence it. The correct bacteria were amplified and cultured and extracted with a small amount of extraction kit without endotoxin plasmid. The extracted plasmid was the plasmid of the same interference interference pLKO-TuD-148a-152 required by the present invention.