Application of miRNA markers in the prevention or treatment of hypoxia-related diseases

Abstract

This invention relates to the field of biomedicine and provides the use of miRNA markers for the prevention or treatment of hypoxia-related diseases. Using TED-deficient cells, the present invention screens transcriptome-wide ceRNAs (ceRNAs) to obtain a panel of miRNA markers, including one or more of miR-199a-3p, miR-199b-3p, and miR-488-3p. These markers can regulate EPAS1 expression and are applicable to detecting hypoxia tolerance, preventing or treating diseases or injuries caused by low or hypoxic environments, and providing new insights into the molecular mechanisms of hypoxia tolerance.

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 the deep sea, the oxygen content in plateaus is significantly lower than in low-altitude areas. To adapt to the plateau environment, the body undergoes physiological changes such as increased hemoglobin content and increased oxygen-carrying capacity. This can also lead to adverse reactions such as increased circulatory resistance, impaired immune system, and decreased arterial oxygen partial pressure. These reactions can cause headaches, dizziness, fatigue, poor sleep, acute cerebral edema, pulmonary edema, and other life-threatening illnesses or injuries. However, long-term residents of plateaus and animals living at high altitudes or in the deep sea typically tolerate hypoxia without developing hypoxic injury reactions. The specific molecular mechanisms underlying this hypoxia tolerance have not yet been fully studied. Studying the molecular mechanisms of hypoxia tolerance is extremely important for the selection, prevention, and treatment of hypoxic injury reactions in special occupational groups.

[0003] MicroRNA (miRNA) is a class of non-coding, single-stranded RNA molecules approximately 22 nucleotides long, encoded by endogenous genes. They participate in post-transcriptional gene expression regulation in plants and animals. To date, 28,645 miRNA molecules have been discovered in plants, animals, and viruses. Most miRNA genes exist in the genome as single copies, multiple copies, or gene clusters.

[0004] The present invention discovered a group of miRNA markers through transcriptome and cell level research, 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 objectives, 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 examples: In the first aspect, 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 class of endogenous small RNAs, approximately 20-24 nucleotides in length, that play a variety of important regulatory roles within the cell. Identified miRNAs are presumed to be primarily generated from a single-stranded RNA precursor with a hairpin structure, approximately 70 bases in length, which is processed by the Dicer enzyme. These small RNA fragments, approximately 21-25 nucleotides in length, have a 5' phosphate group and a 3' hydroxyl group, and are located at either the 3' or 5' end of the RNA precursor. MicroRNAs exist in various forms, the most primitive of which is pri-miRNA, approximately 300-1000 bases in length. Pri-miRNA undergoes a single processing step to become pre-miRNA, or microRNA precursor, approximately 70-90 bases in length. Pre-miRNAs are then cleaved by the Dicer enzyme to become mature miRNAs, approximately 20-24 nucleotides in length. Each miRNA can have multiple target genes, and several miRNAs can regulate the same gene. This complex regulatory network can regulate the expression of multiple genes through a single 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 miRNA marker levels 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 test sample for the detection of the miRNA marker refers to a composition obtained or derived from a subject (e.g., an individual of interest), comprising cellular entities and / or other molecular entities characterized and / or identified based on physical, biochemical, chemical, and / or physiological characteristics. The test sample can be derived 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 fresh, frozen, and / or preserved organ or tissue samples, biopsy tissues, or aspirates; blood or any blood component; body fluids; cells from any time during the individual's pregnancy or development; or plasma. The term test sample includes biological samples that have been processed in any way after their acquisition, such as by reagent treatment, stabilization, or enrichment for certain components (e.g., proteins or polynucleotides), or embedded in a semi-solid or solid matrix for sectioning purposes. The cells are animal cells, such as cells derived from humans, gorillas, monkeys, horses, cows, sheep, pigs, donkeys, camels, dogs, rabbits, cats, rats, mice, fish, birds, or insects, as are well known in the art. The subjects include mammals, preferably primates, and particularly preferably humans.

[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, 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.

[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 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 a reagent for enhancing the function of a miRNA marker in the preparation of a product for preventing or treating diseases or injuries caused by low oxygen or hypoxia, 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 based on the mature miRNA sequence and used to mimic the endogenous mature miRNA sequence. Mimics include a sequence consistent with the mature target miRNA sequence and a sequence complementary to the mature miRNA sequence, and are transfected into cells to achieve the purpose of upregulating mature miRNA. MiRNA agomir is a double-stranded small RNA molecule obtained by modifying the mature miRNA sequence, including chemical group modification of the antisense strand. MiRNA agomir achieves the purpose of upregulating mature miRNA by transfecting cells or administering it to animals. The characteristic of miRNA agomir is its high stability, which 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 backbone modifications at the 5' end, four sulfide backbone modifications at the 3' end, and full-chain methoxy modification.

[0021] Furthermore, the disease or injury caused by the low oxygen or hypoxic environment includes altitude sickness, which is selected from acute altitude sickness and chronic altitude sickness caused by high altitude environments. The high altitude environment is an altitude of 2000 meters or more, 2500 meters or more, or 4500 meters or more, and has low pressure and hypoxia conditions.

[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 and cerebral types, 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 lower 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 lower 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 environments, 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 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.

[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" is 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 can 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 optionally further comprises a marker gene. The vector can 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 achieved 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 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 probe sets, primer sets, kits, chips, test strips, high-throughput sequencing, systems, equipment, and devices.

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

[0041] EPAS1 (Endothelial PAS domain-containing protein 1), also known as hypoxia-inducible factor-2alpha (HIF-2α), is a protein that is expressed in the endothelial tissue. The term encompasses wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed EPAS1, as well as any form of EPAS1 derived from cellular processing. The term also encompasses naturally occurring variants of EPAS1 (e.g., splice variants or allelic variants).

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

[0043] The WT cells described in this paper are 293T cells, a human embryonic kidney cell line commonly used in biomedical technology. Wild-type 293T cells are less tolerant to hypoxia, limiting their use in simulated low-oxygen environments such as those found at high altitudes, 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 those found at high altitudes, high altitudes, and deep seas.

[0044] Advantages and beneficial effects of the present invention: The present invention uses TED-deficient cells to screen ceRNA in the entire transcriptome 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 environments, and provide new ideas for studying the molecular mechanism of hypoxia tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a probe method to detect the expression level of miR-199a-3p in normoxic and hypoxic environments for different lengths of time.

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

[0047] Figure 3 This is a probe method to detect the expression level of miR-488-3p in normoxic and hypoxic environments for different lengths of time.

[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 will be further described below with reference to specific examples, but the scope of protection of the present invention is not limited thereto. Any equivalent substitution or modification made by a person skilled in the art within the technical scope disclosed in the present invention and in accordance with the technical solution and inventive concept of the present invention shall be encompassed within the scope of protection of the present invention. Unless otherwise specified, the materials and reagents used in the following examples are all commercially available.

[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

[0052] 1. Verification of the results of whole-transcriptome ceRNA analysis

[0053] 1. 293T-WT cells and 293T-TED cells - / -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.

[0054] Normoxia: 37°C, 5% CO2; Hypoxia: 37°C, 1% O2, 5% CO2, 94% N2.

[0055] 2. The Hairpin-it miRNAs qRT-PCR quantification 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 described in CN114940975B) at 2h, 24h, and 48h of normoxia and hypoxia. The results are shown in Figure 2. Figure 1-3 Shown: miR-199a-3p ( Figure 1 ) in wild-type cells decreased with the extension of hypoxia time, while in TED cells it decreased after 2 hours, was upregulated after 24 hours, and decreased after 48 hours, but its 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, with the highest level at 24 h, but was significantly downregulated in wild-type cells.

[0056] 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.

[0057] 1. Inhibit miRNA in TED cells: Transfect TED cells with miRNA inhibitors, which specifically bind to mature miRNAs and prevent the complementary pairing of miRNAs with their target genes, thereby inhibiting the function of miRNAs. qPCR was then used to detect the expression of EPAS1 to complete the loss-of-function study. This demonstrated whether the three miRNAs regulated the downregulation of EPAS1 in TED cells. The results are as follows: Figure 4As shown, it can be seen that the expression level of EPAS1 is upregulated after inhibiting miRNA expression in TED cells.

[0058] 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 is 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. The results are as follows: Figure 4 As shown, the expression level of EPAS1 was downregulated after enhancing miRNA function in WT cells.

[0059] Taken together, these results suggest that miR-199a-3p, miR-199b-3p, and miR-488-3p regulate EPAS1 expression through the TED sequence. The regulation of EPAS1 expression by miRNA markers through the TED sequence provides a target for the treatment and prevention of high-altitude chronic hypoxic diseases, such as high-altitude pulmonary hypertension.

[0060] Table 1. Sequence Listing

[0061]

[0062] 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. Use of miRNA markers in the preparation of products for regulating EPAS1 gene expression, characterized in that: The miRNA markers are miR-199a-3p, miR-199b-3p and miR-488-3p, and the miRNA markers regulate EPAS1 gene expression.

2. The use according to claim 1, characterized in that The regulating EPAS1 gene expression refers to regulating EPAS1 mRNA expression level or protein expression level.

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