Application of miRNA marker in prevention or treatment of hypoxia-related diseases

Abstract

The invention provides an application of a miRNA marker in prevention or treatment of hypoxia-related diseases, and belongs to the field of biological medicines. According to the present invention, the whole transcriptome ceRNA is screened by using the TED deletion cell so as to obtain a group of miRNA markers, and the miRNA markers comprise one or more of miR-199a-3p, miR-199b-3p and miR488-3p; the gene can regulate and control EPAS1 expression, can be applied to detection of hypoxia tolerance and prevention or treatment of diseases or injuries caused by hypoxia or hypoxia environments, and provides a new thought for research of a hypoxia tolerance molecular mechanism.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of miRNA markers in preventing or treating hypoxia-related diseases. Background Art

[0002] When people from the plains enter low-oxygen environments such as plateaus, high altitudes, and deep seas, the oxygen content in plateaus is significantly lower than that in low-altitude areas. In order to adapt to the plateau environment, the body will experience physiological changes such as increased hemoglobin content and increased oxygen carrying capacity. At the same time, there will also be adverse reactions such as increased circulatory system resistance, damaged immune system, and decreased arterial oxygen partial pressure, which will cause headaches, dizziness, fatigue, poor sleep, acute cerebral edema, pulmonary edema, and other life-threatening diseases or injuries. Residents who have lived in plateaus for a long time and animals in high altitudes and deep seas usually tolerate hypoxia without hypoxia injury reactions. At present, there has been no detailed research on the specific molecular mechanism of this tolerance to hypoxia. Research on the molecular mechanism of hypoxia tolerance is extremely important for the selection, prevention, and treatment of hypoxia injury reactions in special occupational groups.

[0003] MicroRNA (miRNA) is a type of non-coding single-stranded RNA molecule with a length of about 22 nucleotides encoded by endogenous genes. They participate in post-transcriptional gene expression regulation in animals and plants. So far, 28,645 miRNA molecules have been found in animals, plants and viruses. Most miRNA genes exist in the genome in the form of single copies, multiple copies or gene clusters.

[0004] The present invention discovered a group of miRNA markers through studies at the transcriptome and cell levels, which can regulate the expression of EPAS1, a key gene for hypoxia tolerance in plateau populations. Summary of the invention

[0005] To achieve the above purpose, the present invention uses TED (Tibetan-enriched deletion) knockout cells with hypoxic environment resistance characteristics in previous studies to conduct experiments, and proposes the following technical solutions based on the results of the embodiments: The first aspect of the present invention provides a group of miRNA markers, which include one or more of miR-199a-3p, miR-199b-3p and miR-488-3p.

[0006] In the present invention, miRNA is a type of endogenous small RNA with a length of about 20-24 nucleotides, which has a variety of important regulatory effects in cells. It is presumed that the identified miRNAs are mostly generated by a single-stranded RNA precursor with a hairpin structure of about 70 bases in size, which is processed by Dicer enzyme. It has a 5' end phosphate group and a 3' hydroxyl group, and a small molecule RNA fragment of about 21-25nt in size, which is located at the 3' end or 5' end of the RNA precursor. MicroRNA exists in many forms, the most primitive of which is pri-miRNA, which is about 300-1000 bases in length. After one processing, pri-miRNA becomes pre-miRNA, i.e., microRNA precursor, with a length of about 70-90 bases; pre-miRNA is then digested by Dicer enzyme to become a mature miRNA of about 20-24nt in length. Each miRNA can have multiple target genes, and several miRNAs can also regulate the same gene. This complex regulatory network can regulate the expression of multiple genes through one miRNA, or finely regulate the expression of a gene through a combination of several miRNAs. The group of miRNA markers provided by the present invention can finely regulate the expression of EPAS1.

[0007] The second aspect of the present invention provides the use of a reagent for detecting the level of a miRNA marker in the preparation of a product for detecting hypoxia tolerance, wherein the miRNA marker is the miRNA marker described in the first aspect of the present invention.

[0008] Furthermore, the detection is performed on the sample to be tested.

[0009] In the present invention, the sample to be tested for the detection of the miRNA marker refers to a composition obtained from or derived from a subject (e.g., an individual of interest), comprising cell entities and / or other molecular entities characterized and / or identified based on physical, biochemical, chemical and / or physiological characteristics. The sample to be tested can be from the subject's blood and other fluid samples and tissue samples of biological origin, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of the tissue sample can be solid tissue, such as from fresh, frozen and / or preserved organ or tissue samples, biopsy tissues or aspirates; blood or any blood component; body fluids; cells from any time of individual pregnancy or development; or plasma. The term sample to be tested includes biological samples that have been processed in any way after they are obtained, such as reagent treatment, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedded in a semi-solid or solid matrix for sectioning purposes. The cell is an animal cell, such as a cell from a human, gorilla, monkey, horse, cow, sheep, pig, donkey, camel, dog, rabbit, cat, rat, mouse, fish, bird or insect as is well known in the art. The subject includes a mammal, preferably a primate, and particularly preferably a human.

[0010] Furthermore, the reagents for detecting the level of miRNA markers include reagents used in any of the following methods: RNA extraction, miRNA sequence analysis, detection methods based on PCR principles, Southern hybridization methods, Northern hybridization methods, dot hybridization methods, fluorescence in situ hybridization methods, DNA microarray methods, ASO methods, and high-throughput sequencing platform methods.

[0011] Furthermore, the reagents may also include auxiliary detection reagents for miRNA expression, which include but are not limited to: reaction reagents that visualize the amplicon corresponding to the primer, such as reagents that visualize the amplicon by agarose gel electrophoresis, enzyme-linked gel electrophoresis, chemiluminescence, in situ hybridization, fluorescence detection, etc.; RNA extraction reagents; reverse transcription reagents; cDNA amplification reagents; standard substances used to prepare standard curves; and positive controls.

[0012] Furthermore, the product also includes reagents for processing the sample to be tested.

[0013] Furthermore, the product is selected from the group consisting of probe sets, primer sets, kits, chips, test strips, high-throughput sequencing, systems, equipment, and devices.

[0014] The third aspect of the present invention provides the use of an agent for enhancing the function of a miRNA marker in the preparation of a product for preventing or treating a disease or injury caused by a low oxygen or hypoxic environment, wherein the miRNA marker is the miRNA marker described in the first aspect of the present invention.

[0015] In the present invention, enhancement refers to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more in the expression level or function of the marker based on its original expression level or function.

[0016] Furthermore, the reagents for enhancing the function of miRNA markers include miRNA mimics and miRNA agomir.

[0017] In the present invention, miRNA mimics are double-stranded small RNA molecules designed according to the mature miRNA sequence, which are used to simulate the endogenous mature miRNA sequence. Mimics include a sequence consistent with the mature sequence of the target miRNA, and a sequence complementary to the mature miRNA sequence, which is transfected into cells to achieve the purpose of upregulating mature miRNA. MiRNA agomir is a double-stranded small RNA molecule obtained after modification of the mature miRNA sequence, including chemical group modification in the antisense strand. MiRNA agomir achieves the purpose of upregulating mature miRNA by transfecting cells or administering drugs in animals. The characteristic of miRNA agomir is that it has strong stability and can meet the needs of animal experiments.

[0018] Furthermore, the sequences of the miRNA mimics are shown in SEQ ID NO: 1-4.

[0019] Furthermore, the sequence of the miRNA agomir may be the same as SEQ ID NO: 1-4, but modified by chemical groups.

[0020] The chemical group modification includes cholesterol modification at the 3' end, two sulfide skeleton modifications at the 5' end, four sulfide skeleton modifications at the 3' end, and full-chain methoxy modification.

[0021] Furthermore, the disease or injury caused by the hypoxic or anoxic environment includes altitude sickness, and the altitude sickness is selected from acute altitude sickness and chronic altitude sickness caused in a plateau environment. The plateau environment is an altitude of 2000m or more, 2500m or more, or 4500m or more, with low pressure and hypoxia.

[0022] Furthermore, the diseases or injuries caused by the low oxygen or hypoxic environment include high altitude heart disease, high altitude depression, high altitude polycythemia, high altitude blood pressure abnormalities, high altitude pulmonary edema, high altitude cerebral edema, high altitude gastrointestinal stress, high altitude coma, diseases with mixed symptoms of high altitude pulmonary type and cerebral type, or diseases with mixed symptoms of high altitude heart disease and polycythemia.

[0023] Furthermore, the low oxygen or anoxic environment includes an oxygen content of less than 5%.

[0024] Furthermore, the low oxygen or anoxic environment includes an oxygen content of less than 4%.

[0025] Furthermore, the low oxygen or anoxic environment includes an oxygen content of less than 3%.

[0026] Furthermore, the low oxygen or anoxic environment includes an oxygen content of less than 2%.

[0027] Furthermore, the low oxygen or anoxic environment includes an oxygen content of less than 1%.

[0028] Furthermore, the product is selected from drugs, probe sets, primer sets, kits, chips, test strips, high-throughput sequencing, systems, equipment, and devices.

[0029] Furthermore, the medicine also includes pharmaceutical excipients.

[0030] A fourth aspect of the present invention provides a method for preventing or treating diseases or injuries caused by low oxygen or anoxic environment, the method comprising enhancing the function of miRNA markers.

[0031] Furthermore, the miRNA marker is the miRNA marker described in the first aspect of the present invention.

[0032] Furthermore, the enhancement of the function of the miRNA marker includes directly administering the miRNA marker, promoting the expression of the miRNA marker, promoting the function of the miRNA enhancer, and enhancing the activity of substances in the upstream and / or downstream pathways.

[0033] Furthermore, the directly administered miRNA marker may be natural or artificially synthesized, or may be obtained by transfecting cells with a vector suitable for expressing the miRNA marker.

[0034] Furthermore, the vector includes a viral vector, a prokaryotic vector, and a eukaryotic vector.

[0035] The term "vector" refers to an element that allows the vector to be integrated into the host cell genome or to replicate autonomously in the cell independently of the genome. The vector may contain any element that ensures self-replication. It usually carries genes that are not part of the central metabolism of the cell, and is usually in the form of double-stranded DNA. The vector of the present invention may refer to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention is not limited and may be an expression vector, a viral vector, etc. In certain embodiments, the vector comprises a target gene, a promoter, a terminator encoding the miRNA of the present invention, or may further comprise a marker gene. The vector may use a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0036] Furthermore, the promotion of miRNA marker expression is by administering miRNA mimics or miRNA agomir.

[0037] Furthermore, the sequences of the miRNA mimics are shown in SEQ ID NO: 1-4.

[0038] The fifth aspect of the present invention provides the use of a miRNA marker in preparing a product for regulating EPAS1 expression, wherein the miRNA marker is the miRNA marker described in the first aspect of the present invention.

[0039] Furthermore, the product is selected from the group consisting of probe sets, primer sets, kits, chips, test strips, high-throughput sequencing, systems, equipment, and devices.

[0040] Furthermore, the regulating EPAS1 expression refers to regulating EPAS1 mRNA expression level or protein expression level.

[0041] EPAS1 (Endothelial PAS domain-containing protein 1) is also known as hypoxia-inducible factor-2alpha (HIF-2α). EPAS1 in the present invention includes wild type, mutant type or fragments thereof. The term covers full-length, unprocessed EPAS1, as well as any form of EPAS1 derived from processing in cells. The term covers naturally occurring variants of EPAS1 (such as splice variants or allelic variants).

[0042] The sixth aspect of the present invention provides the use of miRNA markers in the study of the molecular mechanism of hypoxia tolerance, wherein the miRNA markers are the miRNA markers described in the first aspect of the present invention.

[0043] The WT cells described in the present invention are 293T cells, which are human embryonic kidney cell lines and are commonly used cell lines in biomedical technology. Wild-type 293T cells are less tolerant to hypoxic environments, which limits the application of the cells in simulating low-oxygen environments such as plateaus, high altitudes, and deep seas. The Tibetan-enriched deletion (TED) sequence is a non-coding sequence located on human chromosome 2. TED-deficient cells can be used to simulate low-oxygen environments such as plateaus, high altitudes, and deep seas.

[0044] Advantages and beneficial effects of the present invention: The present invention uses TED-deficient cells to screen the whole transcriptome ceRNA and obtain a group of miRNA markers, which can regulate EPAS1 expression and can be used to detect hypoxia tolerance, prevent or treat diseases or injuries caused by low oxygen or hypoxic environment, and provide a new idea for studying the molecular mechanism of hypoxia tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a graph showing the expression of miR-199a-3p detected by the probe method in normoxic and hypoxic environments for different durations.

[0046] Figure 2 This is a graph showing the expression of miR-199b-3p detected by the probe method in normoxic and hypoxic environments for different durations.

[0047] Figure 3 This is a graph showing the expression of miR-488-3p detected by the probe method in normoxic and hypoxic environments for different durations.

[0048] Figure 4 This figure shows the results of regulating EPAS1 expression after inhibiting miRNA expression in TED cells and enhancing miRNA function in WT cells. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto, and any technician familiar with the technical field within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, shall be equivalently replaced or changed, and all shall be covered within the protection scope of the present invention. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0050] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0051] Example 1. Verification of the results of whole transcriptome ceRNA analysis 1. 293T-WT cells and 293T-TED - / - The cells were subjected to whole transcriptome sequencing and ceRNA analysis. According to the results of whole transcriptome ceRNA screening, miR-199a-3p and miR-199b-3p of TED cells were upregulated compared with WT cells at normoxia, 2h and 24h of hypoxia, miR-488-3p was upregulated only at 2h and 24h of hypoxia, miR-383-5p was downregulated at normoxia and 24h, and miR-152-3p and miR-182-5p were downregulated at 2h and 24h of hypoxia, respectively.

[0052] Normoxia: 37℃, 5% CO2; Hypoxia: 37℃, 1% O2, 5% CO2, 94% N2.

[0053] 2. The probe-based Hairpin-it miRNAs qRT-PCR quantitative kit was used to verify the expression of miR-199a-3p, miR-199b-3p and miR-488-3p in 293T-WT and TED cells (the preparation method is shown in CN114940975B) at 2h, 24h and 48h of normoxia and hypoxia. The results are shown in Figure 1-3 Shown: miR-199a-3p ( Figure 1 ) in wild-type cells decreased with the extension of hypoxia time, but decreased in TED cells after 2 hours, upregulated after 24 hours, and decreased after 48 hours, but the expression level was higher than that in wild-type cells. Figure 2 ) is highly expressed in TED cells, significantly upregulated at 24 hours of hypoxia, and decreased at 48 hours. It is also decreased in wild-type cells after hypoxia treatment. Figure 3 ) was expressed at a low level in TED cells and was significantly upregulated after hypoxia treatment, most significantly at 24 h, but was significantly downregulated in wild-type cells.

[0054] 2. Functional verification of miR-199a-3p, miR-199b-3p and miR-488-3p in regulating EPAS1 According to the verification results of miRNA, after hypoxia treatment, the expression of miR-199a-3p, miR-199b-3p and miR-488-3p was significantly upregulated in TED cells and significantly downregulated in wild cells.

[0055] 1. Inhibit miRNA in TED cells: Transfect TED cells with miRNA inhibitor, which specifically binds to mature miRNA and prevents the complementary pairing of miRNA with its target gene, thereby inhibiting the function of miRNA. Then qPCR is used to detect the expression of EPAS1 to complete the loss-of-function study. This proves whether the three miRNAs regulate the downregulation of EPAS1 in TED cells. The results are as follows Figure 4 As shown, it can be seen that the expression level of EPAS1 is upregulated after inhibiting the expression of miRNA in TED cells.

[0056] 2. Enhance miRNA in WT cells: Use miRNA mimics to transfect WT cells. Specific miRNA mimics can be introduced into cells expressing the corresponding miRNA to simulate the effects of microRNA, or combined with a dual-fluorescence reporter system with miRNA binding sites to verify the regulatory relationship between miRNA and target genes. qPCR can be used to detect the expression of EPAS1. At the same time, the expression of protein levels can be detected to complete gain-of-function studies. Results are as follows Figure 4 As shown, it can be seen that the expression level of EPAS1 was downregulated after enhancing the function of miRNA in WT cells.

[0057] The above results indicate that miR-199a-3p, miR-199b-3p and miR-488-3p can regulate the expression of EPAS1 through TED sequences. The regulation of EPAS1 expression by miRNA markers through TED sequences provides a target for the treatment and prevention of chronic hypoxic diseases at high altitude (such as high altitude pulmonary hypertension).

[0058] Table 1. Sequence Listing

[0059]

[0060] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative work shall fall within the scope of protection of the present invention.

Claims

1. A group of miRNA markers, characterized in that: The miRNA markers include one or more of miR-199a-3p, miR-199b-3p and miR-488-3p.

2. The use of a reagent for detecting miRNA marker levels in the preparation of a product for detecting hypoxia tolerance, characterized in that: The miRNA marker is the miRNA marker according to claim 1.

3. The use according to claim 2, characterized in that: The test is performed on the sample to be tested; Preferably, the reagents for detecting the level of miRNA markers include reagents used in any of the following methods: RNA extraction, miRNA sequence analysis, PCR-based detection methods, Southern hybridization methods, Northern hybridization methods, dot hybridization methods, fluorescence in situ hybridization methods, DNA microarray methods, ASO methods, and high-throughput sequencing platform methods; Preferably, the product also includes reagents for treating the sample to be tested; Preferably, the product is selected from the group consisting of a probe set, a primer set, a kit, a chip, a test paper, a high-throughput sequencing, a system, a device, and an apparatus.

4. Use of a reagent for enhancing the function of miRNA markers in the preparation of a product for preventing or treating diseases or injuries caused by hypoxia or anoxic environments, characterized in that: The miRNA marker is the miRNA marker according to claim 1.

5. The use according to claim 4, characterized in that The reagents for enhancing the function of miRNA markers include miRNA mimics and miRNA agomir; Preferably, the sequence of the miRNA mimics is as shown in SEQ ID NO: 1-4; Preferably, the sequence of the miRNA agomir may be the same as SEQ ID NO: 1-4, but modified by chemical groups; Preferably, the diseases or injuries caused by the hypoxic or anoxic environment include high-altitude heart disease, high-altitude depression, high-altitude polycythemia, high-altitude hypertension, high-altitude pulmonary edema, high-altitude cerebral edema, high-altitude gastrointestinal stress, high-altitude coma, high-altitude pulmonary and cerebral mixed symptoms, or high-altitude heart disease and polycythemia mixed symptoms. Preferably, the hypoxic or anoxic environment includes an oxygen content of less than 5%; Preferably, the hypoxic or anoxic environment includes an oxygen content of less than 4%; Preferably, the hypoxic or anoxic environment includes an oxygen content of less than 3%; Preferably, the hypoxic or anoxic environment includes an oxygen content of less than 2%; Preferably, the hypoxic or anoxic environment includes an oxygen content of less than 1%; Preferably, the product is selected from drugs, probe sets, primer sets, kits, chips, test strips, high-throughput sequencing, systems, equipment, and devices; Preferably, the drug further comprises pharmaceutical excipients.

6. A method for preventing or treating diseases or injuries caused by low oxygen or hypoxia, characterized in that: The steps of the method include enhancing miRNA marker function; Preferably, the miRNA marker is the miRNA marker according to claim 1.

7. The method according to claim 6, characterized in that The enhancement of miRNA marker function includes directly administering miRNA markers, promoting miRNA marker expression, promoting the function of miRNA enhancers, and enhancing the activity of substances in upstream and / or downstream pathways; Preferably, the directly administered miRNA marker can be natural or artificially synthesized, or obtained by transfecting cells with a vector suitable for expressing the miRNA marker; Preferably, the vector includes a viral vector, a prokaryotic vector, or a eukaryotic vector; Preferably, the promotion of miRNA marker expression is by administering miRNA mimics, miRNA agomir; Preferably, the sequence of the miRNA mimics is shown in SEQ ID NO: 1-4.

8. Use of miRNA markers in the preparation of products for regulating EPAS1 expression, characterized in that: The miRNA marker is the miRNA marker according to claim 1.

9. The use according to claim 8, characterized in that: The product is selected from the group consisting of probe sets, primer sets, kits, chips, test strips, high-throughput sequencing, systems, equipment, and devices; Preferably, the regulating EPAS1 expression refers to regulating EPAS1 mRNA expression level or protein expression level.

10. Application of miRNA markers in the study of molecular mechanisms of hypoxia tolerance, characterized in that: The miRNA marker is the miRNA marker according to claim 1.

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