CN116694630B

CN116694630B - Sequence combination for promoting overexpression of circular RNA and application thereof

Info

Publication number: CN116694630B
Application number: CN202310967440.3A
Authority: CN
Inventors: 李宏宇; 王宇琛; 苏冰; 谭帅帅; 吴理达; 顾雨春
Original assignee: Chengnuo Regenerative Medical Technology Beijing Co ltd
Current assignee: Chengnuo Regenerative Medical Technology Beijing Co ltd
Priority date: 2023-08-03
Filing date: 2023-08-03
Publication date: 2023-10-31
Anticipated expiration: 2043-08-03
Also published as: CN116694630A

Abstract

The invention belongs to the field of biotechnology and biological medicine, and particularly relates to a sequence combination structure for promoting overexpression of circular RNA and application thereof. The invention provides a sequence combination for promoting the expression of circular RNA, and the sequence is shown as SEQ ID NO.1-2. The sequence combination can improve the expression efficiency and cyclization efficiency of circular RNA (circRNA), and the expression quantity of the circRNA can be improved by at least thousand times through the sequence combination provided by the invention.

Description

Sequence combination for promoting overexpression of circular RNA and application thereof

Technical Field

The invention belongs to the field of biotechnology and biological medicine, and particularly relates to a sequence combination for promoting overexpression of circular RNA and application thereof.

Background

Circular RNAs (circRNAs) are a class of non-coding RNA molecules that do not have a5 'terminal cap and a 3' terminal poly (a) tail and form a Circular structure by covalent bonds. Unlike traditional RNAs, circRNAs are produced in a circular transcriptional fashion, are not readily degraded by exonuclease RNaseR, are more stable than linear RNAs, are highly sequence-conserved and have tissue expression differences, and are thus considered to be new tumor detection markers. In 2012, salzman reported about 80 circular RNAs for the first time by the RNA-Seq method, after which a large number of circRNAs molecules were found successively. Up to now reports have found that the main functions of 4 circular RNAs, namely the cavernous body of miRNA, regulation of in vitro transcription and variable cleavage, regulation of RNA binding proteins and direct translation into proteins, respectively, are vital regulatory functions in the life process of cells due to their wide range of biological functions.

Because the expression vector has important biological functions, the over-expression technology in cells becomes a key problem which needs to be solved by researchers at first, the over-expression quantity of the annular RNA of the existing annular RNA over-expression technology is generally low, and CN113278635B discloses an annular RNA over-expression technology which has high and stable expression efficiency and can be applied to various expression systems; but its looping efficiency is low. Therefore, how to provide a method for expressing a circular RNA molecule with stable expression efficiency and high cyclization efficiency has been a problem to be solved.

Disclosure of Invention

In order to improve the expression efficiency and cyclization efficiency of the circular RNA, the invention provides a sequence combination for promoting the overexpression of the circular RNA and application thereof, wherein the sequence combination enables the circular RNA to be efficiently expressed in vitro, and conditions are created for the application and theoretical research of the circular RNA.

In one aspect, the invention provides a combination of sequences that promote expression of a circular RNA, the sequences comprising any one of the following:

1) DNA shown in SEQ ID NO.1-2 or reverse complement thereof;

2) The sequences of group 1) combine the transcribed RNAs.

Wherein the DNA shown in SEQ ID No.1 or RNA transcribed therefrom is used as a5 'loop forming region (5' circle), the DNA shown in SEQ ID No.2 or RNA transcribed therefrom is used as a3 'loop forming region (3' circle), or the reverse complement of the DNA shown in SEQ ID No.2 or RNA transcribed therefrom is used as a5 'loop forming region, and the reverse complement of the DNA shown in SEQ ID No.1 or RNA transcribed therefrom is used as a 3' loop forming region.

In another aspect, the invention provides a nucleic acid sequence for expressing a circular RNA, the nucleic acid sequence comprising:

1) The two ends of the circularized circRNA sequence are respectively connected with DNA shown in SEQ ID NO.1-2 or the reverse complementary sequence thereof;

2) The two ends of the circRNA sequence are respectively connected with the DNA shown in SEQ ID NO.1-2 or the reverse complementary sequence thereof or the transcribed RNA sequence.

The term "circRNA", namely, loop RNA (circular RNA), is a novel non-coding RNA with regulatory functions and has a closed loop structure.

The "circRNA sequence to be cyclized" according to the present invention may also be referred to as "circRNA gene sequence", i.e.the DNA sequence from which the circRNA loop forming (speed) sequence or the complete sequence (full length sequence) is transcribed, which may be naturally occurring or artificially synthesized, and within this range are all DNA molecules from which the complete circRNA loop sequence of the circRNA can be transcribed. For example, SEQ ID No.3 is the gene sequence of circ-MTO1, the sequence to be cyclized of circ-MTO1 in the present invention.

The "circRNA sequence" in the present invention is an RNA molecule, including a circRNA loop forming (speed) sequence or a full sequence (full length sequence), and all RNA sequences which can be connected end to form the corresponding circRNA can be called the circRNA sequence.

The term "nucleic acid sequence" as used interchangeably herein includes polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides or analogs or modified forms thereof. They include single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybridization, and include purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

The nucleic acid sequence for expressing a circular RNA according to the present invention may be referred to as a nucleic acid sequence for producing a circular RNA or a nucleic acid sequence capable of expressing a circular RNA.

In the present invention, after determining the target circRNA, the "to-be-circularized circRNA sequence" or "circRNA sequence" of the target circRNA is referred to based on the circRNA data, and it is within the ability of those skilled in the art to refer to the specific sequence. The application of the invention is applicable to any circularisable RNA and is not limited to a certain circRNA.

In another aspect, the present invention provides a vector for expressing circular RNA, wherein the vector comprises DNA shown as SEQ ID NO.1-2 or the reverse complement thereof.

More specifically, the DNA shown in SEQ ID No.1 and SEQ ID No.2 are ligated in sequence on the vector, more specifically, SEQ ID No.1 is upstream of SEQ ID No. 2.

More preferably, the SEQ ID NO.1 and SEQ ID NO.2 are separated by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and all fragments which can be excised by technical means are within this range.

Preferably, the vector comprises a cloning vector, an expression vector.

Preferably, the vector comprises a DNA vector, an RNA vector.

Preferably, the vector is a viral-derived vector.

Preferably, the viral-derived vector comprises a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a herpesviral vector.

In the specific embodiment of the invention, pcDNA3.1-circ is a vector for expressing circular RNA constructed by taking pcDNA3.1 expression vector as a framework, and pLenti-EF1 alpha-circ-MTO 1-GFP-puro is a vector for expressing circular RNA constructed by taking pLenti-EF1 alpha-GFP-puro lentiviral vector as a framework.

Preferably, the vector further comprises a circRNA sequence to be circularized.

More preferably, the circularized circRNA sequence is linked between SEQ ID NO.1 and SEQ ID NO. 2. More specifically, there is no tight connection of the spaces.

In another aspect, the invention provides a host cell comprising a combination of the foregoing sequences, a nucleic acid sequence or a vector.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.

Preferably, the host cell comprises a stem cell, a somatic cell or an immune cell of human origin.

Preferably, the somatic cardiomyocytes, chondrocytes, endothelial cells, epithelial cells, fibroblasts, hair follicle dermal papilla cells, hepatocytes, renal cells, keratinocytes, melanocytes, osteoblasts, preadipocytes, skeletal muscle cells, smooth muscle cells.

The term "somatic cell" can be classified into embryonic stem cells and adult stem cells according to the developmental stage in which they are located. Pluripotent stem cells and multipotent stem cells can be classified according to their developmental potential. The adult stem cells can be classified into neural stem cells, hematopoietic stem cells, bone marrow mesenchymal stem cells, skin stem cells, adipose stem cells, etc., according to their functions.

The term "immune cells" includes T cells, B cells, NK cells, macrophages, dendritic cells, and the like.

In a specific embodiment, the cell is an ex vivo cell line.

In a specific embodiment, the cells do not include embryonic cells.

In another aspect, the invention provides a composition for promoting expression of a circular RNA, the composition comprising the vector described above.

Preferably, the composition comprises a vector composition or an expression system composition,

preferably, the vector composition further comprises a packaging plasmid capable of providing the desired protein of the viral particle in trans.

Preferably, the packaging plasmid capable of providing the desired protein of the viral particle in trans may be a plasmid, or may be divided into a packaging plasmid, an envelope plasmid and a vector plasmid.

Preferably, the expression system composition further comprises an agent for gene transfer.

In particular, said causing gene transfer comprises causing the aforementioned product of the invention to enter a cell.

Preferably, the agents include lipids and liposomes (DOTMA, DOGS, DOPE, etc.), calcium phosphate, polyethylene glycol (polyethylene glycol, PEG), cationic polymers (such as Polyethylenimine (PEI)), DEAE-dextran, nanoparticles, or lipo series commercial transfection agents (such as lipo3000 used in the present invention).

In another aspect, the invention provides a pharmaceutical composition comprising a combination of the foregoing sequences, a nucleic acid sequence that expresses a circular RNA, a vector or host cell, and a pharmaceutically acceptable carrier.

As used herein, a "pharmaceutically acceptable carrier" shall be compatible with the aforementioned sequence combinations, nucleic acid sequences, vectors, host cells of the invention, i.e., capable of being blended therewith without substantially reducing the efficacy of the pharmaceutical composition in general. Pharmaceutically acceptable carriers may include, but are not limited to, buffers, excipients, stabilizers, or preservatives. Examples of pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, such as salts, buffers, sugars, antioxidants, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifiers, or combinations thereof. The amount of pharmaceutically acceptable carrier in the pharmaceutical composition can be determined experimentally based on the activity of the carrier and the desired characteristics of the formulation, such as stability and/or minimal oxidation.

In another aspect, the invention provides a method of increasing the expression level of circRNA in a cell, the method comprising the step of expressing in the cell a nucleic acid sequence or vector as described above that expresses a circular RNA.

This method improves the circularization efficiency of the circRNA, and thus may also be referred to as a method for improving the circularization efficiency of the circRNA. In cancers caused by a decrease in the expression level of the circRNA, the increase in the expression level of the circRNA can inhibit the proliferation and migration of cancer cells, and thus, the increase in the expression level of the circRNA can be also referred to as a method for treating cancers caused by the low expression level of the circRNA.

Preferably, the cell is an ex vivo cell.

Preferably, the method is performed in vitro.

Preferably, the method is non-therapeutic.

Preferably, the increasing comprises increasing the expression level by at least 1.5-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 1500-fold, 2000-fold, 2500-fold, 3000-fold, 3500-fold, 4000-fold, 5000-fold or more than the original expression level.

Preferably, when the cell is a cell in a human body and the decrease in the expression amount of the target circRNA results in a disease, the method of the present invention may also be referred to as a method for treating a disease, in which the decrease in the expression amount of the target circRNA results in a disease, by elevating the circRNA in the cell.

In another aspect, the invention also provides a method of producing circRNA, comprising expressing the aforementioned nucleic acid sequence, vector, composition, or culturing the aforementioned host cell.

In another aspect, the invention provides a circRNA produced by the aforementioned method of producing a circRNA.

On the other hand, the invention also provides a use method of the sequence combination, which comprises the steps of respectively connecting the sequence combination at two ends of a sequence to be cyclized to form a fusion sequence, and enabling the fusion sequence to be expressed, so that the sequence to be cyclized forms a loop.

The sequence combination can achieve the purposes of improving the expression quantity of the circular RNA, improving the looping efficiency, looping the RNA and enabling the target gene to be expressed efficiently and stably.

In the present invention, the expression includes transient expression and stable expression.

Preferably, said transient expression is effected by transfection of the vector.

Preferably, the method of transfection comprises a physical method, a chemical method.

Preferably, the physical method comprises calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation.

Preferably, the chemical method comprises a colloidal dispersion system, a lipid-based system.

Most preferably, the colloidal dispersion system comprises macromolecular complexes, nanocapsules, microspheres, beads.

Most preferably, the lipid-based system comprises an oil-in-water emulsion, a micelle, a mixed micelle, a liposome.

The term "stable transfection" refers to the permanent introduction of recombinant DNA into a host cell, where the gene of interest is integrated into the chromosomal DNA of the host cell and directs the expression of the corresponding large protein in the cell, and the stably transfected cell is reusable.

Preferably, the stable expression is achieved by integrating the sequence of interest into the host genome using transposons, viral vectors, recombinant enzymes or gene editing techniques. In the present invention, the target sequence is the aforementioned nucleic acid sequence for expressing the circular RNA.

Preferably, the viral vector comprises a lentiviral vector, a poxviral vector, a herpes simplex viral vector, an adenoviral vector, an adeno-associated viral vector.

In particular, the gene editing techniques include large fragment knock-in techniques implemented based on Cas proteins.

In particular, the present invention uses lentiviral vectors in particular embodiments to integrate into cells the aforementioned nucleic acid sequences that cause expression of circular RNAs.

In another aspect, the invention also provides the use of one or more of the above sequence combinations, nucleic acid sequences, vectors, host cells, compositions, pharmaceutical compositions for the preparation of a product for the treatment of cancer caused by a reduced expression of cirRNA.

The pharmaceutical compositions of the present disclosure may be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will depend on factors such as the condition of the subject, the type and severity of the disease in the subject, although appropriate dosages may be determined by clinical trials.

Preferably, the cancer comprises cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, cutaneous squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, esophageal cancer, thyroid cancer, transitional bladder epithelial cancer, leukemia, brain tumor, stomach cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, female genital tract cancer, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma.

Preferably, the cancer is liver cancer, and the invention is tested against the liver cancer cell line HepG 2.

In another aspect, the invention provides the use of one or more of the above sequence combinations, nucleic acid sequences, vectors, host cells, compositions, pharmaceutical compositions for the preparation of circularized RNA (circRNA).

Preferably, the introducing of the vector into the expression system may take into account any method known in the art for delivering genetic material into the expression system. Non-limiting examples include viral transduction, electroporation transfection, liposome delivery, polymeric carriers, chemical carriers, lipid complexes, polymeric complexes, dendrimers, nanoparticles, emulsions, natural endocytic or phagocytic pathways, cell penetrating peptides, microinjection, microneedle delivery, particle bombardment, and the like.

Drawings

FIG. 1 is a schematic diagram of the structure of a transient expression vector (pcDNA3.1-circ) for promoting overexpression of circular RNA.

FIG. 2 is a fluorescent image of 293 cells transfected with pcDNA3.1-circ-MTO1 overexpression vector for 48 h.

FIG. 3 is a sequencing map of the circularized splice sites of circ-MTO 1.

FIG. 4 is an agarose gel electrophoresis of the products of PCR amplification of the linear MTO1 primer and the circ-MTO1 specific primer 48h after transfection of the pcDNA3.1-circ-MTO1 over-expression vector into 293 cells.

FIG. 5 is a graph showing the results of detecting the change in the expression level of circ-MTO1 by RT-PCR after transfection of HepG2 cell with pcDNA3.1-circ-MTO1 over-expression vector for 48 hours.

FIG. 6 shows the change in the expression level of circ-SMARCA5 by RT-PCR after transfection of HepG2 cell for 48h with the pcDNA3.1-circ-SMARCA5 overexpression vector.

FIG. 7 is a sequencing map of the circularized splice site of pcDNA3.1-circ-SMARCA 5.

FIG. 8 is a schematic representation of the structure of lentiviral vectors promoting overexpression of circular RNAs.

FIG. 9 is a fluorescent image of 48h after transfection of 293FT cells with the pLenti-EF 1. Alpha. -circ-MTO1-GFP-puro vector.

FIG. 10 is a fluorescent image of 48h after transfection of HepG2 cell with the pLenti-EF 1. Alpha. -circ-MTO1-GFP-puro vector.

FIG. 11 is a fluorescent diagram of a cell line (HepG 2-MTO 1) for stable overexpression of circ-MTO1 by HepG2 constructed after packaging lentivirus with circ-MTO1 lentivirus overexpression vector.

FIG. 12 shows the measurement of the expression level of circ-MTO1 in HepG2-MTO1 and normal HepG2 cell lines.

Detailed Description

The present invention is further described in terms of the following examples, which are given by way of illustration only, and not by way of limitation, of the present invention, and any person skilled in the art may make any modifications to the equivalent examples using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.

Vectors used in the experiments were all supplied by general biological company, 293FT cells and HepG2 cells were purchased from North Nanopsis, and lipo3000 transfection reagent was purchased from thermofisher company.

Example 1 construction of pcDNA3.1-circ overexpression vector

Step 1, constructing pcDNA3.1-circ

Constructing a vector pcDNA3.1-circ for promoting the ring formation of the circular RNA through a framework vector pcDNA3.1, specifically: the nucleotide sequence shown in SEQ ID No.1-2 for promoting the loop formation of the circular RNA is connected into the MCS region of the framework vector through an oligo assembly, wherein the nucleotide sequence shown in SEQ ID No.1 is used as a5 'loop formation region (5' circle), the nucleotide sequence shown in SEQ ID No.2 is used as a3 'loop formation region (3' circle), and NheI and BmtI sites are reserved between the 5 'circle and the 3' circle so as to facilitate the subsequent insertion and assembly of the circular RNA.

And 2, constructing a pcDNA3.1-circ-MTO1 over-expression vector.

The sequence SEQ ID No.3 to be cyclized of circ-MTO1 (CircBaseID: hsa_circ_ 0007874) was inserted between the NheI and BmtI multiple cloning sites reserved on the pcDNA3.1-circ vector to give a pcDNA3.1-circ-MTO1 overexpression vector.

Verification 1, transfection of 293FT and HepG2 cells and immunofluorescence detection

The specific method comprises the following steps:

(1) 293FT and HepG2 cells were cultured according to 1.5X10 ⁶ cell/10cm ² Carrying out passage;

(2) 293FT and HepG2 cells were cultured according to 3X 10 ⁵ cell density of cells/well was seeded into 6-well plates;

(3) the cells are uniformly inoculated, transfection is carried out when the fusion degree reaches 20% -30%, the original culture solution is removed, and 2mL of fresh culture solution is added;

(4) a1.5 mL EP tube was prepared, 125. Mu.L of Opti-MEM broth and 2. Mu.g of plasmid were added, and mixed well upside down; preparing another 1.5mL EP tube, adding 125. Mu.L of Opti-MEM culture solution and 5. Mu.L of Lipo3000 reagent, gently mixing upside down, and incubating at room temperature for 5min;

(5) adding the Opti-MEM culture solution containing plasmid DNA into the Opti-MEM culture solution containing Lipo2000, gently reversing and mixing uniformly, and incubating for 10min at room temperature;

(6) adding the DNA-Lipo3000 complex to 293FT cells, gently shaking the plate to mix the complex, and standing at 37deg.C with 5% CO ₂ Culturing in a saturated humidity incubator;

(7) after 4 hours, the fresh medium was changed and the culture was continued. After 48 hours, intracellular fluorescence was observed, and the results are shown in FIG. 2. It shows that the circ-MTO1 over-expression vector can be transcribed and translated normally in 293FT cells, and the vector is normal.

Verification 2, verification of splice sequence of cyclization site and verification of cyclization efficiency

293 cells transfected and untransfected with pcDNA3.1-circ-MTO1 overexpression vectors are taken as an overexpression group and a control group, RNA extraction is carried out, PCR is carried out, and PCR products are sequenced to detect the splicing condition. Meanwhile, the control group and the over-expression group are subjected to PCR by using the linear MTO1 specific primer and the circ-MTO1 specific primer, and then cyclization efficiency is verified by agarose gel electrophoresis, and the specific operation method is as follows:

(1) the transient cells are collected, RNA is extracted, and the extraction steps are as follows: inoculating the cells to a six-hole plate, and extracting RNA when the cells grow to 90% fusion; sucking the culture solution, adding 1mL Trizol, standing at room temperature for 5min, and repeatedly blowing the cells; the cell lysate was transferred into a 1.5mL EP tube, 0.2 times the volume of chloroform was added thereto, the mixture was shaken for 15 seconds, left at room temperature for 3 minutes, and then centrifuged at 12000rpm for 10 minutes; sucking the upper water phase into a new EP pipe, adding isopropyl alcohol with equal volume, and mixing reversely; centrifuging at 12000rpm for 5min, and discarding filtrate in the collecting pipe; adding 1ml of seventy percent ice-bath ethanol into the centrifuge tube, centrifuging at 12000rpm for 5min, and discarding the filtrate in the collecting tube; residual ethanol and airing at room temperature, adding 50 mu L DEPC water, covering a cover, standing at room temperature for 3min to dissolve RNA and precipitate; the concentration and purity of the extracted RNA were determined by an ultra-micro spectrophotometer and stored at-80 ℃.

(2) The extracted RNA was used in subsequent reverse transcription experiments. Reverse transcription was performed using the extracted RNA, and the reaction system is shown in Table 1:

TABLE 1 reverse transcription reaction System

(3) The inverted cDNA was used for subsequent PCR amplification. Wherein, the primer sequences for detecting the circ-MTO1 product are shown in SEQ ID No. 4-5, and the PCR amplification system for detecting the linear MTO1 product is shown in SEQ ID No. 6-7 as shown in Table 2:

SEQ ID No.4：ACTGTTCAGGAGGGAGCTGTA

SEQ ID No.5：TCCGATGCCACCAAAGGAAG

SEQ ID No.6：TTAAACCGGCGTAAGGGACC

SEQ ID No.7：CAGTGTGTTCAGGCTCTGGT

TABLE 2 PCR amplification System

The sequencing results are shown in FIG. 3, in which the circularized splice site of circ-MTO1 was correct. And it was confirmed by agarose gel electrophoresis that the PCR products of the specific circularized primers were found to have distinct bands in the overexpressed group (FIG. 4), while there was no band in the control group, while the ratio of band gray values of the PCR products of the linear primers to those of the overexpressed group and the control group was found to be 87.1% (calculation formula (circular primer-overexpressed group-circular primer-control group)/[ (circular primer-overexpressed group-circular primer-control group) + (linear primer-overexpressed group-linear primer-control group) ]).

TABLE 3 contrast of band gray values

Verification 3 overexpression efficiency verification on HepG2

Since the presence of circ-MTO1 in HepG2 and low expression levels relative to normal hepatocytes have been reported in the relevant literature (circMTO 1 suppresses hepatocellular carcinoma progression via the miR-541-5p/ZIC1 axis by regulating Wnt/. Beta. -catenin signaling pathway and epithelial-to-mesenchymal transition, PMID: 34930906), the pcDNA3.1-circ-MTO1 overexpression vector was introduced into HepG2 cells to examine its overexpression efficiency in HepG2 cells, as in the previous detailed transfection method.

Using HepG2 cells not transfected with the circ-MTO1 overexpression vector as a control group, it was found that the expression level of circ-MTO1 in the HepG2 cells transfected with the circ-MTO1 overexpression vector was increased 3125-fold by RT-PCR detection (FIG. 5).

Example 2 construction and validation of pcDNA3.1-circ-SMARCA5 overexpression vector

To further verify whether the pcDNA3.1-circ vector of example 1 can overexpress other circular RNAs, the vector pcDNA3.1-circ described in example 1 was used to construct a pcDNA3.1-circ-SMARCA5 over-expression vector, and it has been reported that circ-SMARCA5 is a circRNA that is under-expressed in HepG2 cells (Circular RNA cSMARCA5 inhibits growth and metastasis in hepatocellular carcinoma, PMID: 29378234).

Verification of expression efficiency of the 1, pcDNA3.1-circ-SMARCA5 overexpression vector on HepG2 cells

The sequence SEQ ID No.8 to be cyclized of the circ-SMARCA5 (CircBase ID: hsa_circ_ 0001445) was inserted between the NheI and BmtI multiple cloning sites reserved on the pcDNA3.1-circ vector to give a pcDNA3.1-circ-SMARCA5 overexpression vector. And the expression efficiency of pcDNA3.1-circ-SMARCA5 over-expression vector is verified by introducing the vector into HepG2 cells.

As a result, as shown in FIG. 6, the expression level of the transfected pcDNA3.1-circ-SMARCA5 over-expression vector group was 2315-fold higher than that of the control group not transfected with the over-expression vector (FIG. 6).

Verification 2, circularized sequence splice site verification of pcDNA3.1-circ-SMARCA5 overexpression vector

RNA from the HerG 2 cells overexpressing circ-SMARCA5 was extracted, reverse transcribed, and the products obtained by PCR using circularized specific primers were subjected to a first generation of sequencing, and the splice sites were found to be completely correct (FIG. 7), and the specific procedure was the same as in example 1. The sequence of the used circ-SMARCA5 specific primer is shown in SEQ ID No. 9-10:

SEQ ID No.9：TCTCCAAGATGGGCGAAAGTT

SEQ ID No.10：TGTGTTGCTCCATGTCTAATCA

EXAMPLE 3 construction and validation of pLenti-EF 1. Alpha. -circ-MTO1-GFP-puro over-expression lentiviral vector

Step 1, construction of pLenti-EF 1. Alpha. -circ-GFP-puro

The structure of pLenti-EF1 alpha-circ-GFP-puro is shown in FIG. 8, and the skeleton carrier is pLenti-EF1 alpha-GFP-puro, and the skeleton carrier also contains a nucleotide sequence combination for promoting the ring formation of the circular RNA shown in SEQ ID No.1-2. Wherein the nucleotide sequence shown in SEQ ID No.1 serves as a5 'loop forming region (5' circle), the nucleotide sequence shown in SEQ ID No.2 serves as a3 'loop forming region (3' circle), and BsiWI sites are reserved between the 5 'circle and the 3' circle for subsequent insertion and assembly of the circRNA.

Step 2 construction of pLenti-EF 1. Alpha. -circ-MTO1-GFP-puro

The pLenti-EF1 alpha-circ-GFP-puro vector constructed in the step 1 is used for constructing a pLenti-EF1 alpha-circ-MTO 1-GFP-puro over-expression lentiviral vector.

Specifically, the sequence SEQ ID No.3 to be cyclized of circ-MTO1 (circBase ID: hsa_circ_ 0007874) is inserted into the reserved BsiWI site on the pLenti-EF1 alpha-circ-GFP-puro vector to construct the circ-MTO1 over-expression lentiviral vector pLenti-EF1 alpha-circ-MTO 1-GFP-puro.

Verification 1, expression verification in 293FT cells

The pLenti-EF1 alpha-circ-MTO 1-GFP-puro vector constructed in the step 2 was transfected into 293 cells according to the method of example 1, and a fluorescence map in 48h of cells after transfection is shown in FIG. 9, which shows that the pLenti-EF1 alpha-circ-MTO 1-GFP-puro vector can be normally expressed in 293FT cells.

Verification 2, verification of expression in HepG2 cells

The pLenti-EF1 alpha-circ-MTO 1-GFP-puro vector constructed in the step 2 is transiently transfected into HepG2 cells through a transfection reagent lipo3000, and the expression level change of circ-MTO1 in an over-expression group transferred into the pLenti-EF1 alpha-circ-MTO 1-GFP-puro vector and a control group not transfected with the vector is detected by means of RNA extraction, reverse transcription and PCR.

It was found that the expression level of circ-MTO1 was increased 2000-fold compared to the control group, demonstrating that pLenti-EF 1. Alpha. -circ-MTO1-GFP-puro overexpressing lentiviral vectors were able to efficiently overexpress circ-MTO1 (FIG. 10).

EXAMPLE 4 construction and validation of a HepG2 cell line stably overexpressing circ-MTO1

The circular RNA over-expression lentiviral vector pLenti-EF 1. Alpha. -circ-MTO1-GFP-puro constructed in example 3 was used to construct stably expressed cells, and the evaluation was performed by stably over-expressing the circ-MTO1 expression level change in the HepG2 cell line, as follows:

1. lentivirus preparation

(1) The pLenti-EF 1. Alpha. -circ-MTO1-GFP-puro and PLP1, PLP2 and pLP-VSVG plasmids were co-transfected into 293T cells, and the transfection procedure was the same as in example 1;

(2) after 48h of incubation, the cells were observed, the cell culture supernatant was collected, centrifuged at 3000rpm for 15min at 4℃and re-filtered using a 0.45 μm filter;

(3) after 48h of incubation, the cells were observed, the cell culture supernatant was collected, centrifuged at 3000rpm for 15min at 4℃and re-filtered using a 0.45 μm filter;

(4) adding culture medium, shaking uniformly with PEG concentrate, concentrating overnight, centrifuging at 4deg.C at 1500rpm for 30min, discarding supernatant, centrifuging at 4deg.C at 1500rpm for 4min, removing excessive supernatant, adding 1mL HBSS solution to resuspend virus precipitate, blowing, mixing, and packaging;

2. construction of stable over-expression cell lines

(1) HepG2 cells grown in log phase were grown at 3X 10 ⁵ cells/well were seeded into 6-well plates;

(2) adding the lentivirus prepared in the step (1) into a HepG2 cell culture medium;

(3) after 48h of culture, puro 1mg/ml was added to the uninfected virus HepG2 cells and the infected lentivirus HepG2 cells, respectively, for drug killing, and continuous observation was performed

(4) After seven days of drug killing, all HepG2 cells infected by viruses are found to express fluorescence, and the cell line construction is completed.

Expression level verification

The constructed stable and over-expressed circ-MTO1 HepG2 cell strains all have fluorescent expression (figure 11), which shows that the HepG2-MTO1 cell strains are successfully constructed, and meanwhile, compared with the wild cell strain HepG2, the expression quantity of the circ-MTO1 is improved by 52 times by using RT-PCR detection.

In summary, the present invention provides a sequence combination for promoting loop formation of circular RNA, wherein the sequence has the function of promoting loop formation of circular RNA; the vector for promoting the ring formation of the annular RNA is connected into a skeleton vector, the constructed vector for promoting the ring formation of the annular RNA can realize the over-expression of the annular RNA while promoting the ring formation of the annular RNA, the over-expression quantity is not less than 2000 times, the ring formation efficiency is not less than 87.1 percent, and the effect is excellent; the method for promoting the cyclic RNA to form the ring and over-express is simple to operate, mature in technology and wide in application prospect in related researches of the functions of the cyclic RNA.

Claims

1. A nucleic acid combination for promoting the expression of circular RNA, wherein the sequence of the nucleic acid combination is DNA shown as SEQ ID NO.1-2 or RNA transcribed from SEQ ID NO.1-2.

2. A nucleic acid for expressing a circular RNA, the nucleic acid comprising the sequence of:

1) The two ends of the circularized circRNA sequence are respectively connected with DNA shown in SEQ ID NO. 1-2;

2) The RNA sequences transcribed from the DNA shown in SEQ ID No.1-2 are linked to both ends of the circRNA sequence.

3. A vector for expressing circular RNA, wherein the vector contains DNA shown in SEQ ID NO.1-2.

4. The vector of claim 3, comprising a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a herpesviral vector.

5. The vector of claim 3, further comprising a circRNA sequence to be circularized thereon.

6. The vector of claim 5, wherein the circRNA sequence to be circularized is linked between SEQ ID NO.1 and SEQ ID NO. 2.

7. A host cell comprising one or more of the nucleic acid combination of claim 1, the nucleic acid of claim 2, or the vector of claim 3.

8. The host cell of claim 7, comprising a human stem cell, a somatic cell, or an immune cell.

9. A composition for promoting expression of a circular RNA, comprising the nucleic acid combination of claim 1, the nucleic acid of claim 2, the vector of claim 3, or the host cell of claim 7, further comprising one or more of the following:

1) Packaging plasmid capable of providing in trans the proteins required for the viral particles,

2) Packaging plasmid, envelope plasmid and vector plasmid,

3) A reagent for gene transfer,

4) Pharmaceutically acceptable carriers.

10. A method of increasing the expression of circRNA in a cell, said method comprising the step of expressing in a cell the nucleic acid of claim 2, the vector of claim 3, said method being of non-therapeutic or diagnostic interest.

11. A method of producing circRNA comprising expressing the nucleic acid of claim 2, the vector of claim 3, the composition of claim 9, or culturing the host cell of claim 7.

12. A method of using the nucleic acid combination of claim 1, comprising ligating the nucleic acid combination of claim 1 to both ends of the sequence to be circularized to form a fusion sequence, and expressing the fusion sequence, whereby the sequence to be circularized is circularized, said method being of non-therapeutic or diagnostic interest.

13. The method of any one of claims 10-12, wherein said expressing comprises transiently expressing or stably expressing.

14. The method of claim 13, wherein said transient expression is achieved by a transfection vector and said stable expression comprises integration of the sequence of interest into the host genome by using transposons, viral vectors, recombinant enzymes or gene editing techniques.

15. Use of one or more of the nucleic acid combination of claim 1, the nucleic acid of claim 2, the vector of claim 3, the host cell of claim 7 or the composition of claim 9 for the preparation of circularized RNA.

16. Use of one or more of the nucleic acid combination of claim 1, the nucleic acid of claim 2, the vector of claim 3, the host cell of claim 7 or the composition of claim 9 for the preparation of a product for increasing the expression of circRNA in a cell.