WO2017219170A1

WO2017219170A1 - Construction and application of lentiviral vector for specifically inhibiting human mirna-29a and mir-424 expression

Info

Publication number: WO2017219170A1
Application number: PCT/CN2016/086336
Authority: WO
Inventors: 毛侃琅
Original assignee: 毛侃琅
Priority date: 2016-06-19
Filing date: 2016-06-19
Publication date: 2017-12-28

Abstract

A construction and application of a lentiviral vector for specifically inhibiting human miRNA-29a and miR-424 expression. The lentiviral vector comprises a fundamental sequence, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence, and an oligonucleotide sequence targeting miR-29a and miR-424 of a pLKO.1-puro expression vector. A multiple cloning site comprises an Age I enzyme cutting site and an EcoR I enzyme cutting site; the oligonucleotide sequence targeting miR-29a and miR-424 is inserted into the multiple cloning site in sense orientation. A pLKO-Tud-29a-424 lentiviral expression vector has the advantages of high transfection efficiency, a small amount required, and continuous, efficient, stable, and specific inhibition in human miRNA-29a-424 expression, and may serve as a powerful tool for preparing drugs for treating diseases related to abnormal miRNA-29a-424 expression.

Description

Construction and application of lentiviral vector specifically inhibiting the expression of human miRNA-29a and miR-424

Technical field

[0001] The present invention relates to the field of gene editing and epigenetics, and in particular to the construction and application of a lentiviral vector which specifically inhibits the expression of human mi RNA-29a and miR-424.

Background technique

[0002] MicroRNAs (miRNAs) are a class of endogenous, non-coding RNAs found in eukaryotes, typically between 22 and 25 nt in size. miRNAs are widely distributed in plants, animals, and multicellular organisms, and can Play an important regulatory role, and in the study of human miRNAs, it is found that the expression of miRNA in normal tissues and tumor tissues is significantly different, some miRNAs are lowly expressed in tumor tissues, and some are highly expressed in tumor tissues. This suggests that miRNAs play a crucial role in tumorigenesis.

[0003] miR-29a is a small RNA closely related to cell proliferation. It is involved in many diseases and can act as a tumor suppressor gene in a variety of tumors. It has the ability to grow and invade cells such as human gastric cancer and bladder cancer. Correlation, its expression level is an important reference for evaluating the benign and malignant gliomas. It is also associated with diseases such as atherosclerosis and liver fibrosis. It has important potential application value for the treatment of various tumors; miR-424 is found in recent years. A miRNA that affects the biological effects and development of tumor cells by acting on target genes by acting on target genes in a variety of tumors, exerting effects similar to oncogenes, tumor suppressor genes, or promoting, Inhibit the invasion and metastasis of tumors. Studies have shown that miR-424 is a multifunctional miR NA, which is associated with cell invasion and metastasis of cervical cancer, pancreatic cancer, etc.; it is associated with the expression of inflammatory factors such as IL-6 and TNF-oc; since the miR-424 promoter region has CpG Island, which is also associated with methylation-induced gene silencing. By controlling the expression of miR-29a and miR-424, peers can work synergistically with other drugs to provide new epigenetic ideas for the treatment of cancer.

technical problem

[0004] Currently, miRNA function studies are generally achieved by miRNA overexpression and silencing. miRNA silencing is the presentation of synthetic oligonucleotides into cells, with endogenous miRNAs.

The formation of a heteroduplex, the miRNA reduces the inhibition of the target gene, in order to achieve the regulation of gene function . Silencing of miRNAs, especially long-term stable silencing, is currently difficult to achieve. Commonly used methods for silencing miRNA are anti-miR, antagomiR, miRNA sponge, etc. Among them, anti-miR and antagomiR are transient transfection techniques, and the interference effect cannot be stably maintained, while the miRNA sponge effect is far from optimal. There are no reports of homologous optimization of miR-29a and miR-424 interference to enhance its effect.

[0005] Tough Decoy RNA (Tud RNA) is a novel miRNA-inhibiting miRNA that inhibits miRNA by introducing double-stranded RNA to target miRNAs. Because the inserted RNA is double-stranded and has a secondary structure of stem-loops, it is resistant to intracellular nuclease degradation and inhibits miRNAs in a long-term, stable, and efficient manner.

Problem solution

Technical solution

[0006] The object of the present invention is to provide a lentiviral vector for constructing homologous interference miR-29a and miR-424, and constructing a lentiviral vector capable of stably maintaining interference effects, in view of the deficiencies in the prior art, Applied to the field of gene editing.

[0007] In order to solve the above technical problem, the technical solution adopted by the present invention is as follows:

The gene interference sequence of the corresponding TuD RNA against miR-29a and miR-424 was designed and synthesized, and the nucleotide sequence thereof is shown in SEQ ID NO.: 1. This sequence was ligated to the lentiviral vector pLKO.l-puro to obtain a lentiviral vector pLKO-T _U d-29a-424 lentiviral vector which stably maintains the interference effect, and its nucleotide sequence 歹IJ is as SEQ ID NO.: 2 is shown.

The present invention constructs and synthesizes the gene interference sequence of the corresponding TuD RNA of miR-29a and miR-424, and is ligated to the lentiviral vector pLKO.l-puro, and the resulting vector has the same interference with miR-29a. And the role of m iR-424, the specific integration steps are as follows:

[1010] S10, Peer Design and Synthesis of TuD for miR-29a and miR-424d: According to TuD

The sequence information of miR-29a and miR-424 provided in miRBase was designed to design the same TuD RNA oligonucleotide sequence for mi R-29a and miR-424, and its sequence is as SEQ ID NO.: 1 Show, commissioned Shanghai Biotech to synthesize the sequence by means of primers.

[0011] S20. The synthesized sequence is two complementary single-stranded DNAs. The two single-stranded DNAs were dissolved in ddH20, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and then allowed to cool to room temperature at room temperature. [0012] S30, the extraction vector pLK0.1-puro, after digestion with Ag I and Eco RI enzyme for 16 h, the enzymatically digested vector was recovered by MinElute Reaction Cleanup Kit, and the previous step was obtained by using T4 DNA ligase. TuD

The RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-TuD-29a-424, and finally the ligation product was transformed into competent E. coli ToplO and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Five single colonies were picked from the plates and added to 5 tubes containing ampicillin in liquid LB medium for 8 hours at 37 °C. The bacteria were sent to Shanghai Biotech for sequencing. Sequencing was performed on the correct strain and extracted with a small amount of endotoxin-free extraction kit. The extracted plasmid was the plasmid for miR-29a and miR-424 which were required for the present invention.

Advantageous effects of the invention

Beneficial effect

[0013] The homologous interference miR-29a and miR-424 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

1 is a schematic view showing the structure of a pLKO-TuD-29a-424 lentiviral expression vector according to an embodiment;

2 is a flow diagram showing the steps required to transform the lentiviral expression vector shown in FIG. 1 into the lentiviral vector of the present invention;

3 is a miRNA expression level of 16HBE cells and TuD-29a-424 cells in Example 6, wherein

, a. expression of miR-29a, b. expression of miR-424.

BEST MODE FOR CARRYING OUT THE INVENTION

[0017] The present invention can be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions, and results described in the examples are merely illustrative of the invention and are not intended to limit the invention as described in the claims. . The square used in the following examples The method is a conventional method unless otherwise specified; the reagents are all commercially available products unless otherwise specified. Specific steps can be found in: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor)

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 the United States ATCC; S-Poly(T) hsa-miR- 29a qPCR-assay primer set and S-Poly(T) hsa-miR-424 qPCR-assay primer set The miRNA reverse transcription and fluorescence quantification kit was purchased from Shenzhen Anran Biotechnology Co., Ltd.

[0019] Example 1 Design and Synthesis of TuD RNA for miR-29a and miR-424a

[0020] Based on the TuD RNA design sequence and the sequence information of miR-29a and miR-424 provided in miRBase, the TuD RNA oligonucleotide sequence targeting miR-29a and miR-424 was designed, and its sequence is SEQ ID. ΝΟ.: 1, commissioned by Shanghai Biotech to synthesize by means of gene synthesis.

[0021] Example 2 annealing of the sequence

[0022] The synthesized sequence is two complementary single-stranded DNA. The two single-stranded DNAs were dissolved in ddH20, mixed at an equimolar ratio, treated at 95 ° C for 5 min, and allowed to cool to room temperature by allowing them to stand at room temperature.

Example 3 Construction of Recombinant Vector of pLKO-Tud-29a Lentivirus

[0024] The vector pLK0.1-puro was extracted and digested with Age I and Eco RI for 16 h, and the digested vector was recovered with MinElute Reaction Cleanup Kit, and then T4 DNA was used.

Ligase will get TuD from the previous step

The RNA sequence was ligated into the vector pLKO. l-puro to form the recombinant vector pLKO-Tud-29a-424, and finally the ligation product was transformed into competent E. coli ToplO and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Five single colonies were picked from the plate and added to 5 test tubes of liquid LB medium containing ampicillin for 8 h at 37 °C. The bacteria were sent to Shanghai Biotech for sequencing. The sequencing results were completely correct. This is the pLKO-Tud-29a-424 lentiviral recombinant vector.

Example 4 Lentivirus titer determination

On the first day, 293FT cells were seeded into a multi-well plate, and 200,000 cells were seeded per well, 500 cells were added to each well, and cultured at 37 ° C, 5 < 3⁄4 CO 2 overnight;

[0027] The next day, the dilution ratio of the solution of the recombinant lentivirus is 1, 10, 100, 1000, 10000, 1 00000, 1000000, 10000000 and 100000000, the solution of the recombinant lentivirus was serially diluted with a medium, and then 100 gradient-diluted solutions of the recombinant lentivirus and 10 (L perforated plate in a cell culture medium in a multiwell plate) Mixed transfection in different wells, 24 h after transfection, aspirate the medium and replace with 50 (L containing 5 U DNasel in fresh medium, incubate at 37 °C for 30 min to remove residual plasmid DNA that may attach to the cell surface Then, the medium was changed to 1 mL of normal medium, and the cultivation was continued for 48 hours;

[0028] On the fourth day, the medium in each well of the multiwell plate was aspirated, 50 (L-trypsin-EDTA solution was added to digest the cells, reacted at 37 ° C for 1 minute, and then the medium was added to terminate the digestion reaction. After the cells are purged, the cells of each well are collected by centrifugation, the total RNA of each well is extracted, and then the total cDNA of each well is reverse-transcribed; and the total cDNA of each of the obtained cells is separately fluorescent. Quantitative PCR was performed to obtain the ct value of each well of the cells, and the experimental group with the smallest difference from the α value of the control group but exceeding 2 was selected to obtain the dilution factor, and the lentivirus titer was calculated according to the following formula:

T = 20000 x R, where T is the lentivirus titer, T is in units of TU/mL, and R is the dilution factor.

[0030] After calculation, the lentivirus titer of the package is greater than 10000000 TU/mL, indicating that the packaging of the lentivirus is successful.

Example 5 Lentiviral Transduction 16HBE Cells

[0032] 16HBE cells were seeded in 6-well plates, 1000000 cells per well, and the cell density was about 50% after 12 hours. The virus solution was taken separately, and the virus was diluted 10 times with DMEM complete medium, and then polyglycolamine was added.

(polybrene) to a final concentration of 8 g/mL. The medium in the 6-well plate was removed, and the virus-containing DMEM complete medium (containing 10% fetal bovine serum) was added. After 24 hours, the virus-containing DMEM complete medium was discarded, and the fresh DMEM complete medium was replaced. After 24 hours, 0.5 was used. The cells were screened at a g/ml concentration of puromycin. After 10 days of screening, the medium was changed once every 3 days, and the concentration of puromycin was continuously increased to 1.0 g/ml. The cell line obtained by screening was named TuD-29a-424 cell line.

[0033] Example 6 Fluorescence quantitative PCR detection of miR-29a expression level changes

[0034] Normal 16HBE cells and TuD-29a-424 cells were inoculated into 6-well plates (about 300,000 per well), 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, followed by S-Poly(T) hsa-miR-29a qPCR-assay primer set and S-Poly(T) hsa-miR-424 qPCR-assay primer set kit. The miRNA is reverse transcribed and tailed to obtain the corresponding cDNA. Take 2 kinds of cells of cDNA 2 As a template, real-time PCR was used to detect the expression levels of miR-29a and miR-424. The experiment was repeated 3 times, and 3 parallel samples were set per well to snord.

44 as an internal reference. The results are shown in Figure 3. It can be seen that the expression level of miR-29a in TuD-29a-424 cells is 51% lower than that in 16HBE cells, and the expression level of miR-424 is 58% lower than that in 16HBE cells. The difference is statistically significant ( p<0.01), indicating that the TuD-29a-424 cell line was successfully constructed.

Industrial applicability

The homologous interference miR-29a and miR-424 TuD RNA sequences designed by the invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the commonly used single-stranded miRNA sponge, and the same吋 Targeting two targets can better achieve the interference of two miRNAs and improve the efficiency of miRNA function research.

Claims

Claim

A lentiviral vector that specifically inhibits the expression of human miRNA-29a and miR-424, and is characterized by comprising a basic sequence of a pLKO.l-puro expression vector, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence, and a targeting Oligonucleotide sequences of miR-29a and miR-424. The multiple cloning site includes an Age I cleavage site and an EcoR I cleavage site; the oligonucleotide sequence targeting miR-29a and miR-424 is mediated by the Age I cleavage site + the neck I sequence + Target nucleotide sequence + neck II sequence + loop + neck II sequence complementary sequence + target nucleotide sequence complementary sequence + neck I sequence complementary sequence + stop site sequence + EcoR I restriction site composition.

A lentiviral vector specific for inhibiting expression of human miRNA-29a and miR-424 according to claim 1, wherein said oligonucleotide sequences targeting miR-29a and miRNA-424 are positively inserted into said In the multiple cloning site, the nucleotide sequence is 5'-A

CCTTTTTGAATTC -3' (SEQ ID NO.: l).

A lentiviral vector specific for inhibiting expression of human miRNA-29a and miR-424 according to claims 1-2, characterized in that the oligonucleotide sequences targeting miR-29a and miR-424 are inserted into The steps in the pLKO.l-puro expression vector are as follows:

S1. Design and synthesis of TuD for miR-29a and miR-424: Based on the sequence information of miR-29a and miR-424 provided by the TuD design sequence and miRBase, homology was designed for miR-29a and miR- a TuD RNA oligonucleotide sequence of 424, the sequence of which is SEQ ID

N0.: 1 is entrusted to Shanghai Biotech to synthesize the sequence by means of primers; the synthesized sequence is two complementary single-stranded DNA. 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.

S3. The vector pLK0.1-puro was extracted and treated with Ag I and Eco RI for 16 h. The digested vector was recovered using the MinElute Reaction Cleanup Kit, and the TuD RNA sequence obtained in the previous step was ligated into the vector pLKO.1-puro with T4 DNA ligase to form a recombinant vector _P LKO-TuD-29a-424, and finally The ligation product was transformed into competent E. coli ToplO and plated onto a plate containing ampicillin LB medium, 37

Incubate at °C for 14 h. Five single colonies were picked from the plates and added to 5 tubes of ampicillin-containing liquid LB medium for 37 h at 37 °, and then the bacteria were sent to Shanghai for sequencing. The correct sequencing strain was taken and extracted with a small amount of endotoxin-free extraction kit, and the extracted plasmid was the plasmid of miR-29a and miR-424 which is required for the present invention.

[Claim 4] A method of using a lentiviral vector which specifically inhibits expression of human miRNA-29a and miR-424 according to claim 1, comprising the steps of:

51. Place ToplO E. coli containing pLKO-TuD-29a-424 vector in 10 mL LB medium, incubate at 37 ° C, 300 rpm for 12-16 h to OD ₆₀₀ =0.6-0.8 on a constant temperature air shaker.

The obtained bacterial liquid was placed in a centrifuge, centrifuged at 10,000 rpm for 1 min, the supernatant was discarded, the desired bacterial cells were obtained, and the plasmid was extracted with E.Z.N.A. Endo-free Plasmid DNA Mini Kit I.

52. Culture 293FT cells. The 293FT cells with good growth state were inoculated into a 10 cm culture dish, and 5 million cells were inoculated into each dish. The cells were cultured in DMEM-free medium for about 18 h, and the fusion degree reached 80-90%. The required lentiviral vector 10 g, pMDLg/pRRE, pRSV-Rev and pMD-G vectors are each 5

, transfected into 293FT cells with Lipofectamine 2000, 4-6 after transfection

h Replace with DMEM complete medium, continue to culture for 48 hours, collect the virus-containing supernatant medium, centrifuge at 6000 g for 10 min, then take the supernatant and filter with 0.45 μηι filter to obtain lentiviral solution.

53. 293FT cells were cultured, and 293FT cells in good growth state were inoculated into 24-well plates, 200,000 cells were inoculated into each well, and cultured in 500 medium, 3TC, 5<3⁄4CO ₂ overnight. The next day, according to the virus stock: the dilution ratio of the medium is 10-10000000. Slow disease drug serial dilutions were prepared of each 100 [mu] _L! Each well of the original culture medium was then drawn each 100 μL, then add 100 of each dilution lentivirus starts its transfection. 24 hours after the start of transfection, aspirate The lentiviral medium was replaced with 500 μL of fresh medium containing 5 U DNaseI and incubated at 37 °C for 30 min to remove residual plasmid DNA that may attach to the cell surface. The medium was then changed to 1 mL of normal medium and culture was continued for 48 h.

S4. Carefully aspirate all the medium in each well, digest the cells by adding 500 trypsin-EDTA solution, and react at 37 ° C for 1 minute. The medium was then added to terminate the digestion reaction and the cells were washed, and the cells of each well were collected by centrifugation. Total RNA from each well was extracted and then reversed for cDNA. The Ct values of the cells per well were obtained by fluorescence quantification of the total cDNA of the obtained cells per well, and the experimental group with the smallest difference from the Ct value of the control group but exceeding 2 was selected, and the dilution factor was obtained according to the following formula. Calculate the lentivirus titer:

T = 20000xR , where T is the lentivirus titer, T is in units of TU/mL, and R is the dilution factor.

As long as the lentivirus titer reaches 10,000,000 TU/mL or more, it is considered that the required lentiviral fluid is successfully obtained.