Use of circTMEM165 for the preparation of a product for the diagnosis and/or treatment of cardiovascular diseases.

2. The use according to claim 1, wherein circTMEM165 comprises the nucleotide sequence shown in SEQ ID No. 1.

3. The use according to claim 1, wherein the cardiovascular disease is atherosclerosis.

4. The use of claim 1, wherein the product comprises a kit and a medicament.

5. A kit for diagnosing atherosclerosis, comprising a marker that recognizes circTMEM 165;

the circTMEM165 comprises a nucleotide sequence shown as SEQ ID NO. 1.

6. The kit according to claim 5, wherein the label recognizing circTMEM165 is selected from at least one of a primer binding to circTMEM165 and a biomacromolecule binding to circTMEM 165.

7. The kit according to claim 6, wherein said circTMEM165 binding biomacromolecule is selected from at least one of an antibody, a functional fragment of an RNA binding protein or a functional fragment of an RNA binding protein.

8. The kit according to claim 6, wherein the primer binding to circTMEM165 has the nucleotide sequence shown as SEQ ID No.2 and SEQ ID No. 3.

9. A medicament for treating atherosclerosis, comprising at least one of circTMEM165, a recombinant vector containing a gene encoding circTMEM165, a recombinant virus containing a gene encoding circTMEM165, and a recombinant viral vector containing a gene encoding circTMEM 165;

the circTMEM165 comprises a nucleotide sequence shown as SEQ ID NO. 1.

10. The medicament of claim 9, further comprising a pharmaceutically acceptable carrier;

preferably the carrier comprises at least one of chitosan, cholesterol, liposomes, and lipid nanoparticles;

preferably, the pharmaceutical dosage forms include tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions, oral emulsions and injections.

Background

Cardiovascular diseases (CVD) are one of the most serious diseases threatening humans in the world today, and the morbidity and mortality of the world population has jumped over the tumor by killers. The report of the Chinese cardiovascular disease report 2019 states that at present, cardiovascular disease deaths in China account for the first cause of total death of urban and rural residents, 45.5% in rural areas and 43.16% in cities. That is, 2 out of every 5 deaths die from cardiovascular disease. The coronary heart disease death rate of residents in cities and rural areas in China keeps on rising trend since 2012, and the coronary heart disease death rate in rural areas has obvious rising trend. At present, the prevention and treatment of atherosclerosis still remains a medical problem to be solved urgently, and particularly, the pathogenesis and molecular mechanism of atherosclerosis are still not completely clear.

Atherosclerosis is a chronic inflammatory disease of the arterial vessel wall characterized by intimal lipid deposition and fibrous cap formation. Atherosclerosis, a type of cardiovascular disease (CVD), is a major cause of morbidity and mortality in western countries. Statistically, nearly 1 million americans have one or more forms of cardiovascular disease each year. There is increasing evidence that dysfunction of Endothelial Cells (ECs) and Vascular Smooth Muscle Cells (VSMCs) is critical for atherogenesis. The abnormal proliferation and migration of these cells will contribute to the progression of atherosclerosis. Vascular smooth muscle cell migration and proliferation will form a stable fibrous cap, encapsulating the atherosclerotic plaque. During the development and progression of inflammation, ECs and VSMCs produce various types of cytokines including Tumor Necrosis Factor (TNF), Interleukins (IL), adhesion molecules, interferons, and adhesion factors associated with atherosclerosis.

Circular RNA (circrna) is a special subclass of endogenous non-coding RNA molecules with a covalently closed loop structure that renders them resistant to exonuclease degradation relative to their linear RNA decay. At present, there is no detailed study of circRNA in the detection and treatment of atherosclerosis.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide application of circTMEM165 in preparation of products for diagnosing and/or treating cardiovascular diseases, wherein the circTMEM165 can regulate and control inflammatory adhesion and mitochondrion division of human umbilical vein endothelial cells and promote apoptosis of the endothelial cells, and further can realize detection and treatment of atherosclerosis.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention provides application of circTMEM165 in preparation of products for diagnosing and/or treating cardiovascular diseases.

Research shows that circTMEM165 can inhibit inflammatory adhesion and mitochondrion division of human umbilical vein endothelial cells and promote apoptosis of human umbilical vein endothelial cells, and further can detect and treat atherosclerosis.

Preferably, the circTMEM165 contains a nucleotide sequence shown as SEQ ID NO. 1.

Preferably, the cardiovascular disease is atherosclerosis.

Preferably, the product comprises a kit and a medicament.

In a second aspect the invention provides a kit for diagnosing atherosclerosis, said kit comprising a marker which recognises circTMEM 165;

the circTMEM165 comprises a nucleotide sequence shown as SEQ ID NO. 1.

Preferably, the label recognizing circTMEM165 is selected from at least one of a primer binding to circTMEM165 and a biomacromolecule binding to circTMEM 165.

Preferably, the circTMEM165 binding biomacromolecule is selected from at least one of an antibody, a functional fragment of an RNA binding protein or a functional fragment of an RNA binding protein.

Preferably, the primer binding to circTMEM165 has the nucleotide sequence shown as SEQ ID NO.2 and SEQ ID NO. 3.

In a third aspect, the present invention provides a medicament for treating atherosclerosis, said medicament comprising at least one of circTMEM165, a recombinant vector comprising a gene encoding circTMEM165, a recombinant virus comprising a gene encoding circTMEM165, and a recombinant viral vector comprising a gene encoding circTMEM 165;

the circTMEM165 comprises a nucleotide sequence shown as SEQ ID NO. 1.

Preferably, the medicament further comprises a pharmaceutically acceptable carrier;

preferably the carrier comprises at least one of chitosan, cholesterol, liposomes, and lipid nanoparticles;

preferably, the pharmaceutical dosage forms include tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions, oral emulsions and injections.

Compared with the prior art, the invention has the beneficial effects that at least:

the circTMEM165 can play a role in combining with a downstream target gene miR-192-3p, and can well repair cell damage caused by miR-192-3p to a downstream target protein SCP2 of miR-192-3 p; so that circTMEM165 regulates the inflammatory adhesion, mitochondrion division and endothelial cell apoptosis through the circTMEM165/miR-192-3p/SCP2 axis; thereby providing a new target and thought for the prevention, treatment and diagnosis of atherosclerosis.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.

FIG. 1 is a graph showing the results of the study of the downregulation of circTMEM165 expression in clinical specimens and in animal models of atherosclerosis in example 1 of the present invention;

FIG. 2 shows the structure verification result of circTMEM165 in example 2 of the present invention;

FIG. 3 is a graph showing the results of the study of the inhibition of inflammation and adhesion of HUVEC by circTMEM165 in example 3 of the present invention;

FIG. 4 is the results of the study of the inhibition of apoptosis and mitochondrion division of HUVEC by circTMEM165 in example 4 of the present invention;

FIG. 5 shows the results of the study of the effect of circTMEM165 as a sponge of miR-192-3p in example 5 of the present invention;

FIG. 6 is a study result of the regulation of inflammatory adhesion, apoptosis and mitochondrial division of HUVEC by miR-192-3p of circTMEM165 in example 6 of the present invention;

FIG. 7 shows the results of the study of the effect of circTMEM165 on cells via the miR-192-3p-SCP2 axis in example 7 of the present invention;

FIG. 8 shows the results of the study of the regulation of inflammatory adhesion, apoptosis and mitochondrial division of HUVEC by miR-192-3p-SCP2 axis in circTMEM165 of example 8 of the present invention;

FIG. 9 is a graph showing the expression of the circTMEM165/miR-192-3p/SCP2 axis in clinical samples and animal models in example 9 of the present invention;

in the figure:

FIG. 1A is a graph showing the expression analysis of circular RNA TMEM165 in the serum of an atherosclerotic patient and a healthy patient according to an embodiment of the present invention.

FIG. 1B is a graph showing the expression analysis of circular RNA TMEM165 in the ascending aorta tissue of an atherosclerotic patient and a healthy patient according to an embodiment of the present invention.

FIG. 1C provides a graph of the expression analysis of circular RNA TMEM165 in the aorta of carotid balloon injured SD rats and healthy SD rats according to an embodiment of the present invention.

FIG. 1D is a graph showing the expression analysis and quantitative analysis of circTMEM165 in healthy SD rats and SD rats with carotid balloon loss according to the practice of the present invention.

FIG. 1E is a graph showing the expression analysis of circTMEM165 in VSMC/THP-1/HUVEC according to the present invention.

FIG. 1F is a graph showing the expression results of circTMEM165 induced by LPS in the present invention.

FIG. 2A is a graph showing the result of sanger sequencing of circTMEM165 according to the present invention.

FIG. 2B provides a structural analysis diagram of linear TMEM165 and circTMEM165, in accordance with embodiments of the present invention.

FIG. 2C is a graph showing the results of validation of the circular structure of circTMEM165 in gDNA and cDNA, respectively, in a human umbilical vein endothelial cell line according to an embodiment of the present invention.

FIG. 2D is a graph showing the results of detection of circular TMEM165 and linear TMEM165, respectively, by RNA endonuclease in accordance with an embodiment of the present invention.

FIG. 2E is a graph showing the results of detecting circular TMEM165 and linear TMEM165, respectively, under the action of actinomycin according to an embodiment of the present invention.

FIG. 2F is a graph showing the results of the localization of circTMEM165 in HUVEC according to the present invention.

FIG. 3A is a diagram showing the construction of circTMEM165 overexpression vector according to the embodiment of the present invention.

FIG. 3B is a graph that provides results of circTMEM165 overexpression and knockdown efficiency in accordance with an embodiment of the present invention.

FIG. 3C is a graph showing the results of observation of the adhesion effect of HUVEC by CFSE staining the interaction of THP-1 with HUVEC transfected with NC/si-circTMEM165/vector/circTMEM165, and a quantitative analysis thereof, according to an embodiment of the present invention.

FIG. 3D is a graph showing the results of the fluorescent quantitative PCR method for detecting VCAM expression in HUVEC transfected with NC/si-circTMEM165/vector/circTMEM165 in the example of the present invention.

FIG. 3E is a graph showing the results of the fluorescent quantitative PCR method for detecting MCP-1 expression in HUVEC transfected with NC/si-circTMEM165/vector/circTMEM165 according to the embodiment of the present invention.

FIG. 3F is a graph showing the results of fluorescent quantitative PCR assay for TNF expression in HUVEC transfected with NC/si-circTMEM165/vector/circTMEM165 according to the present invention.

FIG. 3G is a graph showing the results of fluorescent quantitative PCR method for detecting IL-1 expression in HUVEC transfected with NC/si-circTMEM165/vector/circTMEM165 according to the present invention.

FIG. 3H is the result and quantitative graph of p-AKT/AKT/p-IKB/IKB/p-65 in HUVEC transfected with NC/si-circTMEM165/vector/circTMEM165 detected by Western blotting, and-actin is used as an internal reference protein.

FIG. 3I is the expression result and quantitative graph of p-ERK/ERK/p-p 38/p-JNK/JNK/p-c-Jun in HUVEC transfected with NC/si-circTMEM165/vector/circTMEM165 detected by Western blotting method according to the present invention.

FIG. 4A is a graph showing the results of cell flow assay for detecting apoptosis in HUVECs transfected with NC/si-circTMEM165/vector/circTMEM165 according to the present invention.

FIG. 4B is a graph showing the results and quantitative analysis of the apoptosis of HUVECs transfected with NC/si-circTMEM165/vector/circTMEM165 by TUNEL staining.

FIG. 4C is the result and the quantitative analysis chart of p53/caspase 3/closed-caspase 3 in HUVEC transfected with NC/si-circTMEM165/vector/circTMEM165 by Western blotting method and using-actin as the reference protein.

FIG. 4D is a chart showing the results and quantitative analysis of mitochondrial MitoTracker staining for HUVECs transfected with NC/si-circTMEM165/vector/circTMEM165 in the examples of the present invention.

FIG. 4E is the result and the quantitative analysis chart of MFN1/DRP1/OPA1/FIS1 in HUVEC transfected with NC/si-circTMEM165/vector/circTMEM165 by Western blotting and the expression of-actin as an internal reference protein according to the present invention.

Fig. 5A provides a wien diagram for predicting a downstream target miRNA of circTMEM165 in an embodiment of the invention.

Fig. 5B is a diagram of the results of expression analysis of 7 mirnas detected by the probe pull-down miRNA experiment of biotin-labeled circTMEM165 according to the embodiment of the present invention.

FIG. 5C provides a binding site map of circTMEM165 and miR-192-3p in accordance with an embodiment of the present invention.

FIG. 5D is a diagram providing the results of knocking down over-expressed circTMEM165 and detecting miR-192-3p in the embodiments of the present invention.

FIG. 5E is a diagram of results of cell fluorescence in situ hybridization experiments for detecting co-localization of circTMEM165 and miR-192-3p in embodiments of the present invention.

FIG. 5F is a diagram showing the construction of a luciferase reporter gene vector according to an embodiment of the present invention.

FIG. 5G is a graph showing the results of the luciferase reporter gene method for verifying the binding effect of circTMEM165 and miR-192-3 p.

FIG. 6A provides a graph of results of designing and validating mimics and inhibitors of miR-192-3p, in accordance with an embodiment of the present invention.

FIG. 6B is a diagram of the results and quantitative analysis of the HUVEC adhesion effect observed by the interaction of CFSE-stained THP-1 with the HUVEC transfected with NC/si-circTMEM165/si-circTMEM165+ miR-192-3p inhibitor according to the embodiment of the present invention.

FIG. 6C is a diagram of the results and quantitative analysis of the HUVEC adhesion effect observed by the interaction between CFSE-stained THP-1 and transfected vector/circTMEM165/circTMEM165+ miR-192-3p micic HUVEC provided in the present invention.

FIG. 6D is a diagram showing the result of expression and quantitative analysis of protein by Western blotting method for detecting p-AKT/p-p65 in HUVEC transfected with NC/si-circTMEM165/si-circTMEM165+ miR-192-3p inhibitor and vector/circTMEM165/circTMEM165+ miR-192-3p mice treated by LPS, with-actin as the reference protein.

FIG. 6E is a result chart of cell flow assay for detecting apoptosis of HUVECs transfected with NC/si-circTMEM165/si-circTMEM165+ miR-192-3p inhibitor and vector/circTMEM165/circTMEM165+ miR-192-3p imic.

FIG. 6F is a graph showing the results of mitochondrial MitoTracker staining of HUVECs transfected with NC/si-circTMEM165/si-circTMEM165+ miR-192-3p inhibitor and vector/circTMEM165/circTMEM165+ miR-192-3p mici by LPS treatment in the examples of the present invention.

FIG. 6G is the result and the quantitative analysis chart of the expression of-actin as an internal reference protein in HUVEC obtained by Western immunoblotting for detecting the presence of DRP1 in LPS-treated transfected NC/si-circTMEM165/si-circTMEM165+ miR-192-3p inhibitor.

FIG. 6H is the result of expression and quantitative analysis of-actin as an internal reference protein for detecting DRP1 in HUVEC treated with LPS and transfected vector/circTMEM165/circTMEM165+ miR-192-3p micic by Western blotting method in the embodiment of the present invention.

FIG. 7A provides a Wien diagram predicting a downstream target protein of miR-192-3p in an embodiment of the invention.

FIG. 7B is a graph of the expression analysis result of the miR-192-3p knock-down and over-expression and downstream protein detection by fluorescent quantitative PCR in the embodiment of the invention.

FIG. 7C provides a binding site map of miR-192-3p and its downstream SCP2 for an embodiment of the invention.

FIG. 7D is a graph of expression analysis results of separately detecting 2 downstream proteins in a probe pull-down downstream protein experiment of miR-192-3p labeled with biotin according to an embodiment of the present invention.

FIG. 7E is the result and quantitative analysis chart of the present invention, which provides the expression result of knocking down the over-expressed miR-192-3p, detecting SCP2 by Western blotting, and using-actin as the reference protein.

FIG. 7F is the expression result and quantitative analysis chart of LPS induced HUVEC detecting SCP2, actin as internal reference protein.

FIG. 8A is a graph showing the results of an over-expression knockdown of SCP2 according to an embodiment of the present invention.

FIG. 8B is a set of results and a quantitative analysis chart for observing the adhesion effect of HUVECs by interaction of CFSE-stained THP-1, LPS treatment and HUVECs transfected with NC/si-circTMEM165/si-circTMEM165+ miR-192-3p inhibitor/si-circTMEM 165+ miR-192-3p inhibitor + si-SCP2 according to the embodiment of the present invention.

FIG. 8C is a diagram showing the result of expression and quantitative analysis of protein by Western blotting method for detecting p-AKT/p-p65 in HUVEC transfected with NC/si-circTMEM165/si-circTMEM165+ miR-192-3p inhibitor/si-circTMEM 165+ miR-192-3p inhibitor + si-SCP2 by LPS treatment, and using-actin as reference protein.

FIG. 8D is a diagram showing the results of cell flow assay for detecting the apoptosis of HUVECs transfected with NC/si-circTMEM165/si-circTMEM165+ miR-192-3p inhibitor/si-circTMEM 165+ miR-192-3p inhibitor + si-SCP 2.

FIG. 8E is the result and the quantitative analysis chart of the expression of-actin as an internal reference protein in HUVEC obtained by Western blotting method for detecting the clear-caspase 3 in LPS treated transfected NC/si-circTMEM165/si-circTMEM165+ miR-192-3p inhibitor/si-circTMEM 165+ miR-192-3p inhibitor + si-SCP 2.

FIG. 8F is a graph showing the results and quantitative analysis of mitochondrial MitoTracker staining by HUVECs transfected with NC/si-circTMEM165/si-circTMEM165+ miR-192-3p inhibitor/si-circTMEM 165+ miR-192-3p inhibitor + si-SCP2 after LPS treatment.

FIG. 8G is the result of expression and quantitative analysis of-actin as an internal reference protein for the detection of DRP1 in HUVEC transfected with NC/si-circTMEM165/si-circTMEM165+ miR-192-3p inhibitor/si-circTMEM 165+ miR-192-3p inhibitor + si-SCP2 by Western blotting.

FIG. 9A is a graph of HE staining analysis of healthy SD rats, SD rats with loss of carotid balloon and SD rats treated with circTMEM165 nucleic acid via tail vein injection in accordance with an embodiment of the present invention.

FIG. 9B is a graph showing the expression analysis of circTMEM165 in SD rats, SD rats with loss of carotid balloon and SD rats treated with circTMEM165 nucleic acid by tail vein injection according to an embodiment of the present invention.

FIG. 9C is a graph showing the expression analysis of miR-192-3p in SD rats, SD rats with carotid balloon loss, and SD rats treated by tail vein injection of circTMEM165 nucleic acid according to an embodiment of the present invention.

FIG. 9D is a graph showing the expression analysis of SCP2 in SD rats, SD rats with carotid balloon loss and SD rats treated with circTMEM165 nucleic acid by tail vein injection according to an embodiment of the present invention.

FIG. 9E is an analysis chart of the fluorescence in situ hybridization assay for detecting miR-192-3p and circTMEM165 expression in healthy SD rats, SD rats with carotid balloon loss and SD rats treated by tail vein injection of circTMEM165 nucleic acid according to the embodiment of the invention.

FIG. 9F is an analysis chart of the expression result of DRP1 in rats as an immunohistochemical analysis model according to the present invention.

FIG. 9G is a graph showing the analysis of the expression of SCP2 in a rat, which is an immunohistochemical analysis model, according to an embodiment of the present invention.

FIG. 9H is a graph showing the results of analysis of SCP2 expression in the ascending aorta of immunohistochemical healthy and atherosclerotic patients according to an embodiment of the present invention.

FIG. 9I is the result and quantitative analysis chart of the expression of SCP2 in ascending aorta of healthy and atherosclerotic patients detected by Western blotting and the expression of actin as an internal reference protein according to the embodiment of the present invention.

FIG. 9J is a graph of fluorescence in situ hybridization assay for detecting the expression of miR-192-3p and circTMEM165 in the ascending aorta of healthy people and atherosclerotic patients and its quantitative analysis.

FIG. 9K is a graph showing the results of detecting miR-192-3p expression in tissues of healthy people and atherosclerotic patients by a fluorescence quantitative PCR method according to an embodiment of the present invention.

Fig. 9L provides a diagram of the pathways involved in this study, in accordance with an embodiment of the present invention.

Detailed Description

The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, unless otherwise indicated, the use of the term "including" and other forms is not limiting.

Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

The embodiment of the invention provides application of circTMEM165 in preparation of a product for diagnosing and/or treating cardiovascular diseases.

In some embodiments, the circTMEM165 comprises the nucleotide sequence set forth in SEQ ID No. 1.

In the present invention, the inventors examined the expression of circTMEM165 in tissues and sera of healthy humans and atherosclerotic patients, tissues of healthy SD rats and rats injured by carotid balloon, and healthy C57 mice and APOE-/-mice, and found that the expression of circTMEM165 was significantly down-regulated in all disease groups compared to the control group; further research shows that circTMEM165 can inhibit inflammatory adhesion and mitochondrion division of human umbilical vein endothelial cells and promote apoptosis; subsequently, the inventor discovers that circTMEM165 can play a binding role with a downstream target gene miR-192-3p through combined bioinformatics prediction analysis, and the circTMEM165 can well repair cell damage caused by miR-192-3p to a downstream target protein SCP2 of miR-192-3 p; the inflammatory adhesion and mitochondrion apoptosis of endothelial cells are regulated and controlled through a circTMEM165/miR-192-3p/SCP2 axis; therefore, the research provides a new theoretical basis for the regulation and control mechanism of circTMEM165 on the development and development of atherosclerosis, and provides a new target point and thought for the prevention, treatment and diagnosis of atherosclerosis.

In addition, in the present invention, it should be noted that, in the present invention, circTMEM165 contains the nucleotide sequence shown in SEQ ID No.1, which means that circTMEM165 may contain other functional sequences, such as a tag sequence or a linker sequence, in addition to the nucleotide sequence shown in SEQ ID No. 1.

In some embodiments, the cardiovascular disease is atherosclerosis.

In the present invention, diagnosing and/or treating atherosclerosis means that circTMEM165 provided by the present invention can be used for diagnosing atherosclerosis, or for treating atherosclerosis, or for both diagnosing atherosclerosis and treating atherosclerosis;

further, atherosclerosis refers to a disease caused by damage to vascular endothelial cells. Atherosclerosis occurs as a result of an excessive chronic inflammatory proliferative response locally produced in the blood vessel due to damage of vascular endothelial cells and smooth muscle cells by various risk factors. The inflammatory response of vascular endothelial cells and their mitotic fusion therefore play an important role in the progression of atherosclerosis.

In some embodiments, the above products include kits and medicaments.

The embodiment of the invention also provides a kit for diagnosing atherosclerosis, which comprises a marker for identifying circTMEM 165;

the circTMEM165 contains a nucleotide sequence shown as SEQ ID NO. 1.

The circTMEM165 is detected in clinical atherosclerotic plaque samples and found to be remarkably reduced in expression level. The kit provided by the invention takes circTMEM165 as a detection target, and can realize the diagnosis of atherosclerosis to a certain extent by detecting the expression level of the circTMEM165 in cells.

In some embodiments, the label that recognizes circTMEM165 is selected from at least one of a primer that binds to circTMEM165 and a biomacromolecule that binds to circTMEM 165.

Further, the biological macromolecule bound to circTMEM165 is selected from at least one of an antibody, a functional fragment of an RNA binding protein, and a functional fragment of an RNA binding protein.

Further, the primer binding to circTMEM165 has the nucleotide sequence shown as SEQ ID NO.2 and SEQ ID NO. 3.

Using the above markers, the expression level of circTMEM165 in the cells can be quantitatively detected.

The embodiment of the invention also provides a medicament for treating atherosclerosis, which comprises at least one of circTMEM165, a recombinant vector containing a coding gene of circTMEM165, a recombinant virus containing a coding gene of circTMEM165 and a recombinant viral vector containing a coding gene of circTMEM 165;

circTMEM165 contains a nucleotide sequence shown as SEQ ID NO. 1.

It should be noted that the drug for treating atherosclerosis provided by the present invention may include one or two or more of circTMEM165, a recombinant virus containing a gene encoding circTMEM165, or a recombinant viral vector containing a gene encoding circTMEM165, such as a recombinant vector including only circTMEM165, or only a recombinant vector including a gene encoding circTMEM165, or a recombinant viral vector including both circTMEM165 and a gene encoding circTMEM165, or a recombinant vector including a gene encoding circTMEM165, a recombinant virus including a gene encoding circTMEM165, and a recombinant viral vector including a gene encoding circTMEM165, or a recombinant virus including both circTMEM165, a recombinant virus including a gene encoding circTMEM165, and a recombinant viral vector including a gene encoding circTMEM 165.

In some embodiments, the above medicament further comprises a pharmaceutically acceptable carrier;

preferably, the carrier comprises at least one of chitosan, cholesterol, liposomes, and lipid nanoparticles.

The carriers can effectively wrap one or more of circTMEM165, recombinant viruses containing the coding gene of circTMEM165 or recombinant viral vectors containing the coding gene of circTMEM165, and carry the active ingredients into the body and release the active ingredients so as to achieve the purpose of administration and treatment; furthermore, the carrier can further play a role in targeted drug delivery by further modifying the carrier, such as connecting binding sites and the like.

The dosage form of the drug in the present invention is not strictly limited, and for example, the above drug dosage forms include tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions, oral emulsions, and injections;

among them, carriers for tablets generally include lactose and corn starch, and additionally, lubricating agents such as magnesium stearate; diluents for use in capsules generally include lactose and dried corn starch; oral suspensions are generally prepared by mixing the active ingredient with suitable emulsifying and suspending agents;

optionally, some sweetener, aromatic or coloring agent may be added into the above oral preparation (except for injection).

When the medicine is administered in the form of injection, the medicine can be prepared into any preparation form acceptable for injection, such as, but not limited to, injection solution or powder injection;

among the carriers and solvents that may be used are water, ringer's solution and isotonic sodium chloride solution; in addition, the sterilized fixed oil may also be employed as a solvent or suspending medium, such as a monoglyceride or diglyceride.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Example 1

This example is a study of the down-regulation of circTMEM165 expression in clinical specimens and animal models of atherosclerosis, the results of which are shown in figure 1;

the expression of circTMEM165 was examined by qRT-PCR in 8 pairs of healthy and atherosclerotic patients in ascending aorta tissue and 20 pairs of human serum (P <0.05), respectively, and a clear down-regulation trend was found (FIGS. 1A-B). Then, the expression level of circTMEM165 was significantly reduced (P <0.05) by qRT-PCR in healthy SD rats and rats with carotid balloon injury (FIG. 1C). It was preliminarily concluded that circTMEM165 may have a protective effect on atherosclerosis.

The inventors labeled healthy SD rats and rats injured via the carotid balloon with a red fluorescent probe labeled with circTMEM165 of cy5 and found that circTMEM165 was significantly down-regulated in the injured rats (FIG. 1D). Taking RNA of each cell line, and detecting the expression quantity of circTMEM165 in various human cell lines by qRT-PCR (P < 0.05). As the expression level of circTMEM165 in each cell line was measured by qRT-PCR, it was found that Human Umbilical Vein Endothelial (HUVEC) expression was most abundant, so the HUVEC cell line was selected as the study subject (FIG. 1E, wherein VSMC: human vascular smooth muscle cell line; HUVEC: human endothelial cell line; THP-1: human monocyte cell line). Meanwhile, HUVEC is induced by LPS, total RNA is extracted, and the expression condition of circTMEM165 is detected by qRT-PCR (P <0.05), and the expression of circTMEM165 is found to be reduced after LPS induction (FIG. 1F).

Example 2

In this embodiment, the structure of the annular TMEM165 is verified, and the verification result is shown in fig. 2;

to further determine the head-to-tail loop structure of circTMEM165, sanger sequencing was performed first, and it was found that the head-to-tail loop structure of circTMEM165 was confirmed from the sequence (FIGS. 2A-B). Subsequently, gDNA and cDNA of HUVEC were extracted separately, and qRT-PCR examined the expression of circular TMEM165 and linear TMEM165, and no significant linear TMEM165 was found in gDNA (fig. 2C). The total RNA of HUVEC was treated with RNA exonuclease and actinomycin B, respectively, and the expression of circular TMEM165 and linear TMEM165 was re-extracted and examined, and it was found that the circular RNA was not degraded due to its stability, while the linear RNA was significantly degraded (FIGS. 2D-E).

To better visualize the localization of circTMEM165 in cells, probes for circTMEM165 were designed and FISH stained to find that circTMEM165 was mainly localized in the nuclei of HUVEC (fig. 3F).

Example 3

This example is a study of circTMEM165 inhibition of HUVEC inflammation and adhesion, the results of which are shown in figure 3;

the inventors designed an overexpression vector for circTMEM165 (fig. 3A) and examined the knockdown and overexpression efficiency of circTMEM165 using qRT-PCR found that the expression of circTMEM165 was significantly reduced in HUVECs transfected with si-circTMEM165, while the expression of circTMEM165 was significantly increased in HUVECs transfected with the overexpression vector for circTMEM165, (P <0.05) (fig. 3B). Subsequently, CFSE was used to stain THP-1 cells for interaction with HUVEC, and fluorescence microscopy was used to observe the adhesion effect of HUVEC, and it was found that knocked-down circTMEM165 promoted the adhesion of endothelial cells, while circTMEM165 inhibited the adhesion effect of endothelial cells, and was quantitatively analyzed using Image J (FIG. 3C). Transfection of NC/si-circTMEM165/vector/circTMEM165 HUVEC, RNA extraction, qRT-PCR detection of VCAM/MCP-1/TNF α/IL-1 β expression respectively, finding that knocking down circTMEM165 promotes VCAM/MCP-1/TNF α/IL-1 β expression (FIG. 4D-G, P < 0.05). In order to further verify that circTMEM165 is involved in the inflammation regulation process of HUVEC, the expression of inflammation pathway related protein p-AKT/p-p 38/p-IKAB/p-ERK/p-p 65/p-JNK/p-c-JUN is detected by using a protein immunoblotting method, and the good protection effect of circTMEM165 in the three proteins p-AKT/p-p65/p-c-JUN is found (FIG. 4H-I).

Example 4

This example is a study of the inhibition of apoptosis and mitochondrion division of HUVEC by circTMEM165, the results of which are shown in FIG. 4;

taking HUVEC transfected with NC/si-circTMEM165/vector/circTMEM165, washing and using a cell flow apoptosis kit to detect apoptosis of cells, the knocked down circTMEM165 promotes apoptosis, and in contrast, circTMEM165 inhibits HUVEC apoptosis (FIG. 4A). Subsequently, the apoptotic effect of circTMEM165 on HUVEC was further verified using TUNEL staining kit, and it was clearly observed that knocked-down circTMEM165 promoted apoptosis, whereas circTMEM165 inhibited HUVEC apoptosis (FIG. 4B, red: TUNEL dye labeled apoptotic cells, blue: DAPI labeled nuclei). Meanwhile, the expression conditions of apoptosis-related proteins p53, caspase3 and cleved-caspase3 are respectively detected by using a protein immunoblotting method, and circTMEM165 is found to be capable of inhibiting HUVEC apoptosis (FIG. 4C). Subsequently, mitochondrial staining of HUVEC using mitoTracker found that circTMEM165 could inhibit HUVEC from mitochondrial fission (fig. 4D), while western immunoblotting detected the expression of mitochondrial fission fusion-related proteins MFN1, DRP1, OPA1 and FIS1, respectively, effectively demonstrating this, with the effect being exerted mainly by the DRP1 pathway (fig. 4E).

Example 5

This example is a study of the role of circTMEM165 as a miR-192-3p sponge, the results of which are shown in fig. 5;

prediction of downstream target genes of circTMEM165 by bioinformatics methods were: miR-1305, miR-4659a, miR-4659b, miR-885, miR-192-3p, miR-1225-5p and miR-1251 (FIG. 5A). In order to further determine the downstream target gene of circTMEM165, a biotin-labeled circTMEM165 was designed to perform RNA pull-down experiment on miRNA, and miR-192-3p is found to be well pulled down by the biotin-labeled circTMEM165 (FIG. 5B). The binding sites for circTMEM165 and miR-192-3p were then predicted (FIG. 5C), and the expression of miR-192-3p, which was found to be negatively correlated with circTMEM165, was detected by transfecting NC/si-circTMEM165/vector/circTMEM165 with HUVEC, qRT-PCR (FIG. 5D). The above results show that miR-192-3p is selected as the downstream target gene of circTMEM 165. Fluorescent probes for miR-192-3p were then designed and found co-localized with circTMEM165, both acting within the nuclei of HUVEC (FIG. 5E). To demonstrate the direct binding effect of the two, the inventors designed luciferase reporter experiments and showed that circTMEM165 binds directly to miR-192-3p (FIGS. 5F-G).

Example 6

The embodiment is a research that circTMEM165 regulates and controls inflammatory adhesion, apoptosis and mitochondrion division of HUVEC through miR-192-3p, and the research result is shown in figure 6; a mimic and an inhibitor of miR-192-3p are designed, and transfection of HUVEC verifies the knocking-down and over-expression efficiency of miR-192-3p, and the knocking-down is about 1/2, but the over-expression is about 10 times (FIG. 6A). HUVECs were transfected with NC/si-circTMEM165/si-circTMEM165+ miR-192-3p inhibitor/vector/circTMEM165/circTMEM165+ miR-192-3p imic, respectively, and treated with LPS, and transfected HUVECs were stained with CFSE, and miR-192-3p was found to restore HUVEC adhesion by si-circTMEM165 (FIGS. 6B-C). Using Western immunoblotting to detect the expression of p-AKT/p-p65, respectively, miR-192-3p was found to be able to restore the inflammatory response and mitochondrial fission caused by si-circTMEM165 (FIG. 6D). Cell flow experiments found that miR-192-3p was able to restore HUVEC apoptosis induced by si-circTMEM165 (FIG. 6E). HUVEC mitochondria were stained with MitoTracker and observed for mitochondrial disruption using confocal microscopy, and it was found that miR-192-3p could restore mitochondrial disruption caused by si-circTMEM165 (FIG. 6F). The above results show that circTMEM165 can play a role in tissues and cells through miR-192-3 p.

Example 7

The present example is a study of the effect of circTMEM165 in cells via miR-192-3p-SCP2 axis, and the results are shown in FIG. 7;

bioinformatics predicted the downstream target protein of miR-192-3p, and the Weinn graph showed 22 candidate target genes predicted by three websites of mirTartar base, Targetscan and mirDB (FIG. 7A). Subsequently, HUVEC transfected by over-expressed miR-192-3p is knocked down, RNA qRT-PCR is extracted to detect the expression condition of downstream target genes, and the expression of GALNT1/SCP2 is found to be in negative correlation with miR-192-3p (FIG. 7B). Next, the inventors predicted the binding sites of miR-192-3p and SCP2 (FIG. 7C), and verified the binding effect of miR-192-3p to SCP2 using a biotin-labeled miR-192-3p probe pull-down experiment (FIG. 7D). And the miR-192-3p analogue and the inhibitor are transfected respectively, and the expression of SCP2 is detected, so that the expression of SCP2 is down-regulated when the miR-192-3p analogue is transfected, and is up-regulated when the miR-192-3p analogue is transfected (figure 7E). Subsequently, HUVEC was induced with LPS, total protein was extracted, and SCP2 was assayed to find that the expression level of SCP2 was significantly reduced with the duration of LPS treatment (FIG. 7F).

Example 8

The embodiment is a research that circTMEM165 regulates and controls inflammatory adhesion, apoptosis and mitochondrion division of HUVEC through miR-192-3p-SCP2 axis, and the research result is shown in figure 8;

to further determine the function of SCP2, the inventors designed and tested two knockdowns of SCP2 to detect miR-192-3p downstream regulatory mechanisms, named si-SCP2-1 and si-SCP2-2, respectively. The QRT-PCR was used to test the knockout efficiency of SCP2, and both were found to be effectively knocked down, with the knockout efficiency of SCP2-2 being more pronounced (FIG. 8A). To reveal the effect of SCP2, experiments were designed to observe the effect of SCP2 on cell adhesion and it was found that the knock-out of SCP2 disrupts the protective effect of miR-192-3p inhibitors on HUVEC adhesion (fig. 8B). At the protein level, the regulation effect of circTMEM165 in the inflammatory NF-kB pathway is realized by axis circTMEM165/miR-192-3p/SCP2, the expression levels of p-p65 and p-AKT are detected, a miR-192-3p inhibitor is found to inhibit the inflammatory response caused by si-circTMEM165, and si-SCP2 can reversely promote the expression of the two proteins (FIG. 8C). Next, apoptosis of HUVEC was detected using flow cytometry, and it was found that knockout of SCP2 can induce HUVEC apoptosis again (fig. 8D). The same effect was observed with clear-caspase 3 at the protein level (FIG. 8E). On the basis of the above results, the role of TMEM165/miR-192-3p/SCP2 axis in HUVEC mitochondria was further determined. Knock down of SCP2 was found to result in some mitochondrion based on mitoTracker staining (fig. 8F). Meanwhile, the expression of DRP1 was examined, and it was found that SCP2 also promoted the expression of DRP1 (fig. 8G).

Example 9

This example shows the expression of circTMEM165/miR-192-3p/SCP2 axis in clinical samples and animal models, and the expression results are shown in FIG. 9;

to further determine the function of circTMEM165, in vivo transfection experiments were performed based on the construction of a rat carotid balloon injury model using plasmid/polyethylenimine (pei)/Polyethylenol (PEG) cocktail injection of circTMEM165 into rats. pcDNA3.1-circTMEM165/PEI/PEG or pcDNA3.1/PEI/PEG cocktail is injected once every seven days after balloon injury, and the injection is carried out three times in total, so as to achieve the treatment effect. To verify the success of the model, the carotid arteries of healthy rats, carotid artery injured rats and rats treated with circTMEM165 nucleic acid were taken. HE staining results showed that the damaged group had significantly thicker carotid than the other two groups (fig. 9A). Data by QRT-PCR showed that the expression of circTMEM165 was upregulated in pcDNA3.1-circTMEM165/PEI/PEG treated rats compared to the pcDNA3.1/PEI/PEG cocktail group (FIG. 9B). In contrast, in the treatment group, the target gene miR-192-3p downstream of circTMEM165 was significantly down-regulated (fig. 9C) and SCP2 was effectively up-regulated (fig. 9D). Furthermore, down-regulation of circTMEM165 expression following PCDNA3.1-circTMEM165/PEI/PEG treatment was observed in fluorescence in situ hybridization of rat carotid arteries (FIG. 9E). The expression of the mitochondrial fission-related protein DRP1 and the apoptosis-related protein clear-caspase 3 was determined by immunohistochemical method, and the expression of DRP1 and clear-caspase 3 in the lesion group was significantly increased and the expression in the treatment group was decreased (FIG. 9F). To determine the function of the downstream target of miR-192-3p, the expression of SCP2 in the carotid arteries of healthy rats, injured rats and treated rats was examined using immunohistochemical methods, and it was found that the expression of SCP2 in injured rats was significantly lower than in the other two groups (FIG. 9G). And was detected in healthy and atherosclerotic tissues, SCP2 was significantly upregulated in healthy tissues (fig. 9H). To further determine the relationship of axis circTMEM165/miR-192-3p/SCP2 to atherosclerosis, axis circTMEM165/miR-192-3p/SCP2 was applied to clinical specimens for testing and found to significantly reduce the expression of SCP2 in tissue samples from atherosclerotic patients compared to healthy humans (FIG. 9I). Similarly, a miR-192-3p green probe and a circTMEM165 red probe are respectively designed. By detecting the expression of the two nucleic acids through FISH, the expression of miR-192-3p in an atherosclerotic tissue sample is obviously increased. The expression of circTMEM165 is remarkably reduced, and the protective function in atherosclerosis is further verified (FIG. 9J: red: probe-labeled circTMEM 165; green: probe-labeled miR-192-3 p; blue: DAPI-labeled nucleus). Finally, the expression of miR-192-3P was examined in healthy and atherosclerotic tissue samples and was found to be upregulated in atherosclerosis (fig. 9K), with a significant difference (P < 0.05). According to the results, the circTMEM165/miR-192-3p/SCP2 axis regulates the occurrence and development of atherosclerosis through the pathways of inflammation and adhesion, apoptosis, mitochondrion and the like, and provides a potential target for preventing and treating atherosclerosis (figure 9L).

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

SEQUENCE LISTING

<110> affiliated Hospital of Qingdao university

Application of <120> circTMEM165 in preparation of products for diagnosing and/or treating cardiovascular diseases

<130> 2021

<160> 3

<170> PatentIn version 3.3

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