WO2018170652A1 - Tud rna for multiple knockdown of three mirnas and application thereof - Google Patents

Abstract

Provided is a Tud RNA for inhibiting human miRNA-29a, miR-140 and miR-185 expressions. The DNA sequence of the Tud RNA is represented by SEQ ID NO 1. Also provided is an application of the Tud RNA for inhibiting human miRNA-29a, miR-140 and miR-185 expressions. The application comprises: first designing the DNA sequence of the Tud RNA that inhibits human miRNA-29a, miR-140 and miR-185; then cloning the DNA sequence onto a pENTR/U6 vector so as to obtain a recombinant vector pENTR/U6-Tud-29a-140-185; and transferring the DNA sequence on the recombinant vector pENTR/U6-Tud-29a-140-185 to a lentiviral vector CS-RfA-EG by means of Gateway technology so as to obtain a recombinant plasmid CS-RfA-EG-Tud-29a-140-185. The recombinant plasmid can inhibit human miRNA-29a, miR-140 and miR-185 expressions.

Description

 Title of Invention: Multiple knockdown of three miRNA Tud RNAs and their applications

 [0001] The present invention relates to the field of gene editing and epigenetics, and specifically relates to a Tud RNA that knocks down three mi RNAs and its application.

 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. Related, its expression level is an important reference for evaluating the benign and malignant glioma, and it is also associated with diseases such as atherosclerotic liver fibrosis, and has important potential application value for the treatment of various tumors; miR-140 and various diseases The occurrence and development are closely related, such as bone and joint diseases, liver diseases, pituitary adenomas, testicular development, head and neck tumors, ovarian and breast diseases. miR-140 can inhibit the proliferation and invasion and metastasis of hepatocellular carcinoma by targeting TGFBR1 and other gene expression. miR-140 is highly expressed in articular cartilage and plays a vital role in the pathogenesis of osteoarthritis. Under various mechanisms, miR-140 plays an oncogene role in some tumors, and plays a tumor suppressor role in other tumors, and is involved in a variety of tumor chemotherapy resistance; miR-185 is a 22 nt miRNA , located in human chromosome 22ql l.21, plays an important role as a tumor suppressor gene in the occurrence and invasion of tumors such as colon cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer, etc. In addition, it is a methylation-related tumor suppressor. Sexual miRNAs can affect the methylation level of the whole genome by directly targeting the expression of DNMT1, thereby regulating the methylation status of certain genes and affecting gene expression. By controlling the expression of miR-29a, miR-140 and miR-185, the synergy with other drugs can 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 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.

[0006] Lentiviral vector is currently used to construct a stable cell line. Compared with expression vectors such as retrovirus and adenovirus, it can simultaneously infect dividing cells and non-dividing cells, and has high transfection efficiency. And high stability, can make the experimental target gene long-term stable expression

 Problem solution

 Technical solution

 The primary object of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a Tud RNA that multiple knockdown of three mi RNAs.

 Another object of the present invention is to provide the use of the Tud RNA of the multiple knockdown of three miRNAs.

 A further object of the present invention is to provide a recombinant vector containing the TudRNA.

 [0010] The object of the present invention is achieved by the following technical scheme: a Tud RNA with multiple knockdown of three miRNAs, the DNA sequence ij ij is shown as SEQ ID NO 1;

[0011] the Tud of multiple knockdown of three miRNAs

 The RNA is applied to inhibit the expression of miRNA-29a, miR-140 and miR-185, and the Tud RNA is preferably constructed on a lentiviral vector, and the recombinant plasmid consisting of the Tud RNA and the lentiviral vector is applied to the T24 cell. The purpose of inhibiting the expression of miRNA-29a, miR-140 and miR-185 is achieved;

[0012] The lentiviral vector is preferably a CS-RfA-EG lentiviral vector;

[0013] The Tud RNA is constructed on a CS-RfA-EG lentiviral vector, and comprises the following steps:

(1) Designing a DNA sequence ij ij of Tud RNA containing multiple knockdown three miRNAs, as shown in SEQ ID NO: 1; for cloning into a pENTR/U6 vector, the following DNA sequences were synthesized:

[0015] F: 5,-CACC GCCAATCAAGATGATCCTAGCGCCACCTTTTT -3'

 R: 5'-AAAA

 ATTTCGGTCGGGGAGTTGATGATCCTAGCGCC -3';

[0017] (2) Mixing the DNA sequence FP and RP in equimolar amounts, treating at 95 ° C for 5 min, then pressing 0.1

The temperature of ° C / s was cooled to room temperature, and precipitated to obtain a double-stranded DNA fragment;

[0018] (3) The pENTR/U6 vector is ligated with the double-stranded DNA fragment obtained in the step (2) to obtain a recombinant vector pENTR/U6-Tud-29a-140-185;

[0019] (4) The Gateway technology will be located in the recombination carrier pENTR/

 Transfer of the double-stranded DNA fragment on U6-Tud-29a-140-185 to the lentiviral vector CS-RfA-EG to obtain CS

-RfA-EG-Tud-29a-140-185 recombinant plasmid.

 Advantageous effects of the invention

 Beneficial effect

 The TuD RNA sequence designed to inhibit human miR-29a, miR-140 and miR-185 has a stem-loop structure and is not easily degraded, and the double-stranded Tud RNA is relatively common with the currently used single-stranded miRNA sponge. The binding efficiency is higher, and the same target can better achieve the interference of the three miRNAs for the three targets, and improve the efficiency of the miRNA function research.

 Brief description of the drawing

 DRAWINGS

1 is a schematic diagram showing the structure of a CS-RfA-EG lentiviral vector; FIG. 2 is an example of T24 cells transfected with T24 cells transfected with C S-RfA-EG-Tud-29a-140-185 lentivirus. miRNA expression level, wherein, a. Expression of miR-29a, b. expression of miR-140, c. expression of miR-185.

 BEST MODE FOR CARRYING OUT THE INVENTION

 BEST MODE FOR CARRYING OUT THE INVENTION

[0022] The present invention will be further described in detail below with reference to the embodiments and drawings, but the embodiments of the invention are not limited thereto.

[0023] Embodiment 1

 [0024] (1) Designing a DNA sequence ij ij of Tud RNA that multiplexes three miRNAs, as shown in SEQ ID NO: 1.

To clone it into the pENTR/U6 vector, the following DNA sequences were synthesized:

[0025] F: 5, -CACC

 GCCAATCAAGATGATCCTAGCGCCACCTTTTT -3'

 R: 5'-AAAA

 ATTTCGGTCGGGGAGTTGATGATCCTAGCGCC -3';

[0027] (2) Mixing the DNA sequence FP and RP in equimolar amounts, treating at 95 ° C for 5 min, then pressing 0.1

The temperature was lowered to room temperature at ° C/s, and 2 times of frozen absolute ethanol (added 0.1 times pH 5.6 of 3 mol/L NaAc) was precipitated to obtain a double-stranded DNA fragment;

[0028] (3) Will pENTR/U6 carrier (purchased from Thermo

 Fisher) is ligated with the double-stranded DNA fragment obtained in step (2) to obtain the recombinant vector pENTR/

U6-Tud-29a-140-185. The reaction system is as follows:

Double-stranded DNA fragment 100 ng

[0030] pENTR/U6 vector 20 ng T4 DNA ligase (purchased from NEB Corporation) Ιμΐ

[0032] Τ4 DNA ligase Buffer 2 μL

[0033] ddH20 is supplemented to 20μ1;

 [0034] After ligation at 16 ° C for 5 h, 5 μl of the ligation product was transformed into competent E. coli Stbl3 (purchased from Quantum Gold Co., Ltd.) and cultured in LB solid medium containing 5 (Vg/ml of Kanamycin). That is, a positive clone, and a positive clone was picked and cultured in an LB liquid medium containing 5 (Vg/ml of Kanamycin) prepared according to the molecular cloning formula, and sequenced to confirm that the human miR-29a, miR-140 and miR- were inhibited. Recombinant vector pENTR/U6-Tud-29a-140-185 of 1 85 TuD RNA.

 (4) Transfer of the double-stranded DNA fragment located on the recombinant vector pENTR/U6-Tud-29a-140-185 to the lentiviral vector CS-RfA-EG by Gateway technology, CS-RfA-EG-Tud- 29a-140-185 recombinant plasmid, the specific operation process is as follows:

 [0036] pENTR/U6-Tud-29a- 140- 185 100 ng

 CS-RfA-EG vector 150ng

 [0038] LR clonase II enzyme mix (purchased from Invitrogen) Ιμΐ

[0039] TE total 9μ1

 [0040] The reaction was carried out at 25 ° C for 6 hours, and the reaction was carried out at 37 ° C for 10 minutes to remove excess LR.

 Clonase II enzyme. Then, the Ιμΐ competent Escherichia coli Stbl3 (purchased from Quanjin Company) was cultured at 30 ° C for 22 to 24 hours in LB solid medium containing 10 (Vg/ml Ampicillin), and the clone that grew was a positive clone. Positive clones were picked and cultured in LB liquid medium containing 10 (Vg/ml Ampicillin, cultured at 30 ° C for 20 hours, and the plasmid was purified and sent for sequencing. The correct sequencing result was obtained by correct sequencing. This recombinant plasmid was defined as: CS-RfA-EG-Tud-29a-140-185, a Tud RNA lentiviral plasmid that inhibits the expression of human mi RNA-29a, miR-140 and miR-185. Extraction with endotoxin-free plasmid The recombinant plasmid was extracted from the box (purchased from Tiangen Biochemical).

 (5) Packaging of lentivirus

[0042] CS-RfA-EG-Tud-29a-140-185, pCMV-VSV-G-RSV-Rev and pCAG-HIVgp plasmid were introduced into human embryonic kidney 293T cells in a ratio of 5:2:2 with Lipofectamine 2000. The supernatant of the cell culture was collected, and filtered with a 0.45 μηι filter to obtain a virus containing the CS-RfA-EG-Tud-29a-140-185 plasmid. (6) virus transfection of T24 cells

 T24 cells were infected with a virus containing the CS-RfA-EG-Tud-29a-140-185 plasmid. The infection steps are as follows: 50,000 T24 cells and 100,000 TU virus solution were suspended in DMEM medium, and prepared according to the amount of ΙΟΟμΙ mixture/well. Each ΙΟΟμΙ mixture also contained 10% FBS and 8 g/ml. The amine was mixed and placed in a 96-well plate. The reaction was carried out at 37 ° C for 24 h. The culture medium was replaced with fresh medium (DMEM + 10% FBS), and the culture was continued for 24 h and subcultured.

 [0045] (7) Quantitative PCR detection of changes in miRNA expression levels

 [0046] The normal T24 cells were uncontrolled, and the miRNAs of normal T24 cells and T24 cells transfected with CS-Rf A-EG-Tud-29a-140-185 lentivirus were extracted with miRcute miRNA extraction and isolation kit, respectively, and reverse transcription and After tailing, the corresponding cDNA was obtained. Take 2 kinds of cells of cDNA 2

 As a template, the expression levels of miR-29a, miR-140 and miR-185 were detected by real-time PCR, and the experiment was repeated 3 times with 3 parallel samples per well, with snord 44 as an internal reference. As a result, as shown in Fig. 3, it can be seen that the expression level of miR-29a with TuD-29a-140-185 cells is 58<3⁄4 lower than that of T24 cells, and the expression level of miR-140 is 54% lower than that of T24 cells, miR-185 The expression level is 67% lower than that of T24 cells. The difference was statistically significant (/?<0.01), indicating that the TuD-29a-140-185 cell line was successfully constructed.

 Industrial applicability

 The TuD RNA sequence designed to inhibit human miR-29a, miR-140 and miR-185 of the present invention has a stem-loop structure and is not easily degraded, and the double-stranded Tud RNA is relatively common with the currently used single-stranded miRNA sponge. The binding efficiency is higher, and the same target can better achieve the interference of the three miRNAs for the three targets, and improve the efficiency of the miRNA function research.

Claims

Claim  [Claim 1] A Tud RNA that multiple knocks down three miRNAs, characterized by: encoding the Tud  The DNA sequence RNA ij of RNA is shown as SEQ ID NO 1. [Claim 2] The use of Tud RNA for multiple knockdown of three miRNAs according to claim 1, characterized in that : The Tud RNA is applied to inhibit the expression of human miRNA-29a, miR-140 and miR-152.  [Claim 3] The use according to claim 2, wherein: the Tud RNA is first constructed on a lentiviral vector, and the recombinant plasmid consisting of the Tud RNA and the lentiviral vector is applied to inhibit miRNA- Expression of 29a, miR-140 and miR-152.  [Application 4] The application according to claim 3, wherein: the lentiviral vector is CS-Rf  A-EG lentiviral vector.  [Claim 5] The use according to claim 4, wherein: the shRNA is constructed in CS- On the RfA-EG lentiviral vector, the following steps are included:  (1) Designing a DNA sequence of Tud RNA containing multiple knockdown three miRNAs, as shown in SEQ ID NO: 1; to clone it into the pENTR/U6 vector, the following DNA sequences were synthesized:  F: 5'-CACC

 CCTAGCGCCACCTTTTT -3' R: 5'-AAAA TGATGATCCTAGCGCC -3';  (2) The DNA sequence FP and RP were mixed in an equimolar amount, treated at 95 ° C for 5 min, then cooled to room temperature at a rate of O.rC / s, and precipitated to obtain a double-stranded DNA fragment;  (3) ligating the pENTR/U6 vector with the double-stranded DNA fragment obtained in the step (2) to obtain a recombinant vector pENTR/U6-Tud-29a-140-152;  (4) The double-stranded DNA fragment located on the recombinant vector pENTR/ U6-Tud-29a-140-152 was transferred to the lentiviral vector CS-Rf A-EG by Gateway technology to obtain CS-Rf A-EG-Tud- 29a-140-152 recombinant plasmid.