阅读说明：本技术 miRNA表达载体及其应用 (MiRNA expression vector and application thereof ) 是由 刘晓萍 罗霭玲 徐令 江华 刘晓丹 杨旭 蔡蔓斯 于 2020-05-07 设计创作，主要内容包括：本发明涉及一种miRNA表达载体及其应用,所述miRNA表达载体从5’端到3’端依次具有以下元件：第一启动子、目标序列、第二启动子和线粒体定位序列,所述目标序列由待表达miRNA的DNA序列重复多次得到。通过实验测试证明,相比于已知载体,利用本申请的miRNA表达载体能够使待表达miRNA在宿主细胞的线粒体中定向显著表达,从而有助于调节线粒体中miRNA的表达,实现线粒体功能调控,为靶向线粒体的药物开发提供基础。(The invention relates to a miRNA expression vector and application thereof, wherein the miRNA expression vector sequentially comprises the following elements from a 5 'end to a 3' end: the miRNA expression vector comprises a first promoter, a target sequence, a second promoter and a mitochondrial positioning sequence, wherein the target sequence is obtained by repeating a DNA sequence of miRNA to be expressed for multiple times. Experimental tests prove that compared with known vectors, the miRNA expression vector can be used for realizing the directional and significant expression of the miRNA to be expressed in the mitochondria of host cells, so that the regulation of the expression of the miRNA in the mitochondria is facilitated, the regulation of mitochondrial function is realized, and a foundation is provided for the development of drugs targeting mitochondria.)

1. A miRNA expression vector having, in order from 5 'to 3': the miRNA expression vector comprises a first promoter, a target sequence, a second promoter and a mitochondrial positioning sequence, wherein the target sequence is obtained by repeating a DNA sequence of miRNA to be expressed for multiple times.

2. The miRNA expression vector of claim 1, wherein the target sequence is obtained by repeating a DNA sequence of the miRNA to be expressed three or more times.

3. The miRNA expression vector of claim 1, wherein the miRNA to be expressed is miR-92b-5p, miR-25-5p or miR-34a-5p, and the DNA sequences of the miRNA are shown in SEQ ID NO: 1 to SEQ ID NO: 3, respectively.

4. The miRNA expression vector of claim 1, wherein the first promoter is a U6 promoter and the second promoter is a CMV promoter.

5. The miRNA expression vector of claim 1, wherein the mitochondrial localization sequence is COX8 sequence, and the sequence is as shown in SEQ ID NO: 4, respectively.

6. The miRNA expression vector of claim 1, wherein the mitochondrial localization sequence further comprises a tag protein sequence at the 3' end.

7. The miRNA expression vector of claim 1, wherein the miRNA expression vector has a sequence as set forth in SEQ ID NO: 5, respectively.

8. Use of the miRNA expression vector of any one of claims 1-7 for mitochondrially targeted expression of miRNA.

9. A recombinant cell comprising the miRNA expression vector of any one of claims 1-7, or having the miRNA expression vector integrated into the genome of the recombinant cell.

10. An antitumor drug comprising the miRNA expression vector of any one of claims 1 to 7 and a pharmaceutically acceptable carrier or excipient.

Technical Field

The invention relates to the technical field of biochemical molecules, in particular to a miRNA expression vector and application thereof.

Background

Mitochondria are organelles having a double-layered membrane structure in the cytoplasm, and are sites where ATP is synthesized by oxidative phosphorylation of cells. Mitochondria participate in the regulation of eukaryotic cell differentiation, energy metabolism, signal transduction, calcium ion homeostasis, active oxygen generation, apoptosis, aging, and other functions.

Gliomas are the most common tumors of the nervous system and are classified according to WHO classification as grade I-IV, with the most malignant being glioblastoma (grade IV). Chemotherapy, radiotherapy and surgical treatment have no obvious effect on improving the survival rate and reducing the death rate of gliomas, particularly glioblastoma. Mitochondrial dysfunction in gliomas includes: structural abnormalities, mitochondrial membrane potential changes, disturbed apoptotic signal pathways, mtDNA mutations, tricarboxylic acid cycle-related enzyme mutations, and the like. Increased mitochondrial fusion, decreased division, and the appearance of mitochondria in the glioma cells pseudopoda and dendritic pseudopoda. Mitochondrial fat metabolism in glioma cells promotes abnormal increases in reactive oxygen species, leading to apoptosis. It was found that patients with glioblastoma have mutations in the enzyme IDH1 which catalyzes the reduction of NADP + to NADPH. Studies have demonstrated that various chemotherapeutic drugs (e.g., paclitaxel) can inhibit gliomas by targeting mitochondrial function. In a variety of tumors, including gliomas, the cellular metabolism changes from oxidative phosphorylation to aerobic glycolysis, and this metabolic remodeling is often accompanied by mitochondrial hyperpolarization. Mitochondrial hyperpolarization in glioma cells can be reversed by the small molecule oral drug dichloroacetic acid, which in turn leads to restricted tumor cell growth. Tricyclic antidepressants can promote the reduction of oxygen consumption of glioma cells, the release of cytochrome C and induce apoptosis, but are limited in application due to side effects. Therefore, the development of mitochondria-targeted drugs will be a potentially effective approach to the treatment of gliomas.

Micro RNA (microRNA, miRNA) is a type of non-coding RNAs with endogenous size of 18 nt-24 nt, and thousands of miRNAs are discovered at present. It has been confirmed that miRNA plays an important role in cell differentiation, proliferation, apoptosis, etc., and regulates translation and degradation of mRNA by binding to 3 'untranslated region (3' UTR) of target gene mRNA. mirnas play important roles in multiple stages in the development of tumors due to deregulated expression. The miRNA expression and activity can be regulated and controlled by the miRNA mimetibody or the inhibitor, so that a new method can be provided for anti-tumor treatment. There are a variety of non-coding RNAs in mitochondria, including miRNAs, snornas, srpsrnas, pirnas, and repeat-related small RNAs, which generally function like oncogenes or oncogenes in human tumors and are involved in regulation of mitochondrial function.

In order to further research the function of miRNA in glioblastoma in mitochondria, miRNA needs to be overexpressed in glioblastoma mitochondria, but neither the existing commercial vectors nor the vectors reported in the literature can be effectively realized.

Disclosure of Invention

Based on this, there is a need for a miRNA expression vector that can efficiently express miRNA in mitochondria.

A miRNA expression vector having, in order from 5 'to 3': the miRNA expression vector comprises a first promoter, a target sequence, a second promoter and a mitochondrial positioning sequence, wherein the target sequence is obtained by repeating a DNA sequence of miRNA to be expressed for multiple times.

In one embodiment, the target sequence is obtained by repeating the DNA sequence of the miRNA to be expressed more than three times.

In one embodiment, the miRNA to be expressed is miR-92b-5p, miR-25-5p or miR-34a-5p, and the DNA sequences of the miRNA are respectively shown in SEQ ID NO: 1 to SEQ ID NO: 3, respectively.

In one embodiment, the first promoter is the U6 promoter and the second promoter is the CMV promoter.

In one embodiment, the mitochondrial localization sequence is a COX8 sequence, which is set forth in SEQ ID NO: 4, respectively.

In one embodiment, the mitochondrial localization sequence further comprises a tag protein sequence at the 3' end.

In one embodiment, the miRNA expression vector has a sequence as set forth in SEQ ID NO: 5, respectively.

The invention also provides an application of the miRNA expression vector in the mitochondria directional expression of miRNA.

The invention also provides a recombinant cell, wherein the recombinant cell contains the miRNA expression vector, or the genome of the recombinant cell is integrated with the miRNA expression vector.

The invention also provides an anti-tumor medicament which contains the miRNA expression vector and a pharmaceutically acceptable carrier or excipient.

The miRNA expression vector is obtained by long-term screening optimization construction, and is sequentially provided with a first promoter, a target sequence, a second promoter and a mitochondrial positioning sequence from a 5 'end to a 3' end, wherein the target sequence is obtained by repeating a DNA sequence of miRNA to be expressed, namely mature miRNA for multiple times. Experimental tests prove that compared with known vectors, the miRNA expression vector can be used for realizing the directional and significant expression of the miRNA to be expressed in the mitochondria of host cells, so that the regulation of the expression of the miRNA in the mitochondria is facilitated, the regulation of mitochondrial function is realized, and a foundation is provided for the development of drugs targeting mitochondria.

Drawings

FIG. 1 is an electrophoretogram for the purification and identification of PCR amplification products of COX8 sequence and hsa-miR-92b-5p mature triplet sequence; wherein, Lane 1 is marker, Lane 2 is hsa-miR-92b-5p mature triplet sequence, and Lane 3 is COX8 sequence;

FIG. 2 is a diagram showing the result of electrophoresis detection of pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO by AfeI enzyme digestion; wherein, Lane 1 is marker, Lane 2 is pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO before enzyme digestion, Lane 3 is pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO after enzyme digestion;

FIG. 3 shows the sequencing results of Sanger sequencing to verify whether COX8 was successfully cloned into pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector;

FIG. 4 is a sequencing result of Sanger sequencing to verify whether the has-miR-92b-5p mature triplet is successfully cloned into pLB vector;

FIG. 5 is a diagram showing the results of electrophoretic detection of BamHI and EcoRI double digestion of the ha-miR-92 b-5p mature triplet PCR product and pHBLV-U6-MCS-CMV-COX8-ZsGreen-PGK-PURO vector; wherein, A is a detection result of a has-miR-92b-5p mature body triplet, a Lane 1 is marker, a Lane 2 is before enzyme digestion, and a Lane 3 is after enzyme digestion; b is the detection result of pHBLV-U6-MCS-CMV-COX8-ZsGreen-PGK-PURO vector, lane 1 is marker, lane 2 is before enzyme digestion, and lane 3 is after enzyme digestion;

FIG. 6 is the sequencing result of Sanger sequencing to verify whether the sequence of the has-miR-92b-5p mature triplet was successfully cloned into pHBLV-U6-MCS-CMV-COX8-ZsGreen-PGK-PURO vector;

FIG. 7 is a graph showing the result of detecting mitochondrial outer membrane protein VDAC by Western blot to determine the purity of isolated mitochondria;

FIG. 8 is a graph of the results of qRT-PCR detection of expression of has-miR-92b-5p in mitochondria of U251 cells; wherein 1 is pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO, 2 is pHBLV-U6-MCS-CMV-COX8-ZsGreen-PGK-PURO, and 3 is pHBLV-U6-has-miR-92b-5p-CMV-COX 8-ZsGreen-PGK-PURO;

FIG. 9 is a graph of results of qRT-PCR detection of expression of has-miR-92b-5p in mitochondria of U251 cells of a pri-miR-92b-5p group (pri-miRNA), a miR-92b-5p maturate group (miR-92b-5p), a miR-92b-5p triplet group (miR-92b-5p-3) and a miR-92b-5p quintet group (miR-92b-5 p-5);

FIG. 10 is a photomicrograph of fluorescence in situ hybridization detection of miR-92b-5p localization and expression in cells;

FIG. 11 is a graph showing the results of miR-92b-5p expression in mitochondria of U251 cells transfected with a pmcherry-COX8-miR-92b-5p-N1 expression vector;

FIG. 12 is the sequencing result of Sanger sequencing to verify whether the sequence of miR-25-5p mature body triplet is successfully cloned to pHBLV-U6-MCS-CMV-COX8-ZsGreen-PGK-PURO vector;

FIG. 13 is the sequencing result of Sanger sequencing to verify whether the sequence of miR-34a-5p mature triplet was successfully cloned into pHBLV-U6-MCS-CMV-COX8-ZsGreen-PGK-PURO vector;

FIG. 14 is a graph of the results of qRT-PCR detection of expression of has-miR-25-5p in mitochondria of U251 cells; wherein 1 is pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO, 2 is pHBLV-U6-MCS-CMV-COX8-ZsGreen-PGK-PURO, and 3 is pHBLV-U6-has-miR-25-5p-CMV-COX 8-ZsGreen-PGK-PURO;

FIG. 15 is a graph showing the results of qRT-PCR detection of expression of has-miR-34a-5p in mitochondria of U251 cells; wherein 1 is pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO, 2 is pHBLV-U6-MCS-CMV-COX8-ZsGreen-PGK-PURO, and 3 is pHBLV-U6-has-has-miR-34a-5p-CMV-COX 8-ZsGreen-PGK-PURO;

FIG. 16 is a photomicrograph of fluorescence in situ hybridization detection of miR-25-5p localization and expression in cells;

FIG. 17 is a micrograph of the location and expression of miR-34a-5p in cells by fluorescence in situ hybridization detection.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein.

A promoter refers to a DNA sequence located upstream of the 5' end of a structural gene and capable of activating RNA polymerase, allowing it to bind to a template DNA precisely and having specificity for transcription initiation. An expression vector refers to a vehicle into which a DNA or RNA sequence can be introduced into a host cell in order to transform the host and facilitate expression of the introduced sequence. MicroRNA (miRNA) is a non-coding single-stranded RNA molecule with the length of about 22 nucleotides, which is coded by endogenous genes and is involved in the regulation and control of the expression of genes after transcription in animals and plants.

The miRNA expression vector of one embodiment of the invention has the following elements from the 5 'end to the 3' end in sequence: the miRNA expression vector comprises a first promoter, a target sequence, a second promoter and a mitochondrial positioning sequence, wherein the target sequence is obtained by repeating a DNA sequence of miRNA to be expressed for multiple times.

Generally, when constructing an miRNA over-expression vector, a pri-miRNA sequence is cloned into the expression vector, but the inventor does not successfully express a target miRNA in mitochondria through a large amount of previous attempts. At present, the known mitochondria-specific expression vector clones the gene COX8 coded by mitochondria DNA into a gene expression vector to achieve the purpose of mitochondria-specific expression, but the vector can only over-express mRNA with protein coding function, but not miRNA, and the known miRNA over-expression vector can not be specifically positioned in mitochondria. The miRNA expression vector is obtained by long-term screening optimization construction, and is sequentially provided with a first promoter, a target sequence, a second promoter and a mitochondrial positioning sequence from a 5 'end to a 3' end, wherein the target sequence is obtained by repeating a DNA sequence of miRNA to be expressed, namely mature miRNA for multiple times. Experimental tests prove that compared with known vectors, the miRNA expression vector can be used for realizing the directional and significant expression of the miRNA to be expressed in the mitochondria of host cells, so that the regulation of the expression of the miRNA in the mitochondria is facilitated, the regulation of mitochondrial function is realized, and a foundation is provided for the development of drugs targeting mitochondria.

In a specific example, the target sequence is obtained by repeating the DNA sequence of the miRNA to be expressed more than three times, and the expression effect is more remarkable.

In a specific example, the miRNA to be expressed is miR-92b-5p, miR-25-5p or miR-34a-5p, and the DNA sequences of the miRNA are respectively shown in SEQ ID NO: 1 to SEQ ID NO: 3, respectively. It will be appreciated that the particular miRNA is not so limited and may be adjusted as desired.

In one specific example, the first promoter is the U6 promoter and the second promoter is the CMV promoter. It is to be understood that the promoter is not limited thereto, and other promoters that can be used for eukaryotic expression may be selected as desired.

In a specific example, the mitochondrial localization sequence is a COX8 sequence, which is shown in SEQ ID NO: 4, respectively. It is understood that the mitochondrial localization sequence is not limited thereto, and may be COX10 sequence, OPA1 sequence, or the like. It will be appreciated that other elements such as resistance selection genes and the like may also be included in the miRNA expression vector.

In a specific example, the sequence of the miRNA expression vector is set forth in SEQ ID NO: 5, the target sequence is obtained by repeating three times the DNA sequence with the target sequence of miR-92b-5p based on the modification of the pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO expression vector.

In the recombinant cell according to an embodiment of the present invention, the miRNA expression vector is contained in the recombinant cell, or the miRNA expression vector is integrated into a genome of the recombinant cell. It is understood that the recombinant cell may be a prokaryotic cell such as DH5 a or the like, or a eukaryotic cell such as U251, U138 cells or the like.

The anti-tumor drug of one embodiment of the invention contains the miRNA expression vector and a pharmaceutically acceptable carrier or excipient. Preferably, the anti-tumor drug is used for treating mitochondria-related tumors, such as gliomas and the like.

In particular, a pharmaceutically acceptable carrier or excipient refers to one or more compatible solid or liquid fillers or gel substances, suitable for human use, and must be of sufficient purity and sufficiently low toxicity. Optionally, the pharmaceutically acceptable carrier is selected from one or more of sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, gelatin, magnesium stearate, glycerol, mannitol, sodium lauryl sulfate, coloring agents, flavoring agents, stabilizing agents, antioxidants and preservatives.

The preparation method of the miRNA expression vector of an embodiment of the invention comprises the following steps: providing a basic expression vector which is provided with a first promoter and a second promoter along the 5 'end to the 3' end in sequence, then connecting a mitochondrion positioning sequence to the 3 'end of the second promoter, and connecting a target sequence to the 3' end of the first promoter to obtain the miRNA expression vector. It is understood that the preparation method is not limited thereto, and those skilled in the art can adjust the preparation steps as needed.

The following are specific examples.