CN116287194A

CN116287194A - Application of miR-199a-3p as molecular marker for diagnosing lipid metabolic disorder and treatment of hyperlipidemia and complications thereof

Info

Publication number: CN116287194A
Application number: CN202310079567.1A
Authority: CN
Inventors: 陈卫东; 周云; 王艳东; 李惟国; 丁学智
Original assignee: Peoples Hospital of Hebi
Current assignee: Peoples Hospital of Hebi
Priority date: 2023-02-08
Filing date: 2023-02-08
Publication date: 2023-06-23

Abstract

The invention belongs to the field of biological medicines, and relates to application of miR-199a-3p as a molecular marker for diagnosing lipid metabolic disorders in treatment of hyperlipidemia and complications thereof. Experimental results show that hsa-miR-199a-3p is obviously increased in LDLR-/-and APOE-/-mice, is also obviously increased in patients with hyperlipidemia, and the molecular level of the hsa-miR-199a-3p is positively correlated with the total cholesterol level of a tested person. The miRNA-199a-3p can be rapidly, conveniently and sensitively used for diagnosing or evaluating lipid metabolism disorder and diseases characterized by dyslipidemia. The detection method provided by the invention is simple and convenient to operate, has small trauma to the detected person, high sensitivity and strong specificity, and can be used for diagnosing early hyperlipidemia.

Description

Application of miR-199a-3p as molecular marker for diagnosing lipid metabolic disorder and treatment of hyperlipidemia and complications thereof

Technical Field

The invention belongs to the field of biological medicine, and relates to miRNA-199a-3p as a molecular marker for diagnosing lipid metabolism disorder and application thereof.

Background

With the development of technology and the change of life style, the damage of hyperlipidemia to society is increasingly aggravated. Hyperlipidemia is a global chronic disease with increasing incidence year by year, plays an important role in the development of type II diabetes, hypertension, nonalcoholic fatty liver, cardiovascular diseases, atherosclerosis and other diseases, and is characterized clinically by elevated levels of triglycerides and cholesterol in plasma. Bad lifestyles such as unreasonable diet, lack of exercise, smoking, etc. can severely affect the lipid content in blood. In addition, other factors affecting blood lipid levels include gender, age, and diabetes and obesity.

There is growing evidence that non-coding RNAs, such as lncRNA, microRNA, act on the occurrence of hyperlipidemia. mirnas have been the focus of research in recent years as important targets for regulating fat, cholesterol metabolism and homeostasis. MicroRNAs (miRNAs) is a single stranded non-coding RNA consisting of 20-22 nucleotides (nt) that modulates gene expression by targeting the 3' translated region (UTR) of a target gene, resulting in translational inhibition and/or mRNA destabilization. Studies have shown that many mirnas can act as mediators of different stages of lipid homeostasis, including cholesterol/fatty acid biosynthesis, degradation, transport, storage, and Low Density (LDL) and High Density Lipoprotein (HDL) formation, playing a vital role in cardiovascular physiology and pathophysiology.

miR-199 is a highly conserved miRNA family consisting of miR-199a and miR-199 b. miRNA-199a was cloned in 2003 from human osteosarcoma cells and mouse skin. Two sites in the human genome were found to encode miR-199a, one on chromosome 1 and the other on chromosome 19. At present, research on miR-199a-3p mainly relates to tumors, infection, transplantation, regeneration, fibrosis and the like, and diagnostic and therapeutic effects in lipid metabolism disorders have not been found.

We found that miRNA-199a-3p was significantly elevated in plasma of patients with lipid metabolism disorder and correlated positively with cholesterol levels. Mouse model apoE for lipid metabolism disorder ^-/- And LDLR ^-/- We found that miRNA-199a-3p levels were significantly elevated, however FXR in a mouse model of bile acid metabolism disorders ^-/- And TGR5 ^-/- There was no significant change in miRNA-199a-3 p. Thus, miRNA-199a-3p may be used as a biomarker for lipid metabolism disorders.

Disclosure of Invention

Aiming at the problems and defects existing in the prior art, the invention provides miRNA-199a-3p, the nucleic acid sequence of which is SEQ ID NO.1, and the specific information is as follows: ACAGUAGUCU GCACAUUGGU UA.

The invention provides an application of miRNA-199a-3p molecules as diagnostic markers in the preparation of products for diagnosing lipid metabolic disorders.

As a preferred technical scheme of the invention, the product for diagnosing lipid metabolism disorder at least comprises PCR amplification primers used when SYBR Green, taqMan probes, double hybridization probes or composite probes are used for detecting the miRNA-199a-3p expression level.

In a specific embodiment of the invention, the specific information of the PCR amplification primer sequences is as follows:

Forward primer AGCAGATTCC TACGGTTCGT CG

Reverse primer CGCATCTATC GATGTCAGTC GCT

further, the product for diagnosing lipid metabolism disorder comprises a chip, a kit, test paper or a high-throughput sequencing platform and the like; the preparation methods of the product are all technical methods known to those skilled in the art.

The kit comprises a reagent for detecting the expression level of miRNA-199a-3p, wherein the reagent comprises nucleic acid combined with miRNA-199a-3p or a DNA sequence thereof, and the nucleic acid comprises PCR amplification primers used when SYBR Green, taqMan probes, molecular beacons, double hybridization probes or composite probes detect the expression level of miRNA-199a-3 p.

The chip or the test paper comprises a reagent for detecting the expression level of miRNA-199a-3p, wherein the reagent comprises nucleic acid combined with miRNA-199a-3p or a DNA sequence thereof, and the nucleic acid comprises a probe capable of detecting the expression level of miRNA-199a-3 p.

Such disorders and complications of lipid metabolism include, but are not limited to, hyperlipidemia, hypercholesterolemia, atherosclerosis, coronary heart disease, or diabetes, etc.

The diagnosis steps of the novel diagnostic product for lipid metabolism disorder are as follows: (1) Extracting miRNA and preparing cDNA in blood of patients with lipid metabolic disorder or hyperlipidemia; (2) fluorescent quantitative PCR detection of miR-199a-3p levels; (3) The miRNA molecular marker is used for evaluating and analyzing lipid metabolism disorder and drug effect.

Compared with the prior art, miR-199a-3p is used as a molecular diagnosis marker, and has the advantages of high detection sensitivity, good specificity, low cost and the like. miR-199a-3p not only serves as a biomarker for early screening and early diagnosis, but also can be used for disease diagnosis, prognosis and drug effect evaluation indexes.

Drawings

FIG. 1 is a schematic diagram of the has-miR-199a-3p levels of a normal population and a hyperlipidemia patient;

FIG. 2 is a schematic diagram of an analysis of the correlation between the has-miR-199a-3p level and the blood Total Cholesterol (TC) level in a detection sample;

FIG. 3WT and LDLR ^-/- And APOE ^-/- Schematic representation of mouse mma-miR-199 a-3p levels;

FIG. 4miR-199a-3p Agomir vs. Western diet 6 weeks APOE ^-/- Effects of mouse plasma TG, TC, LDL-C, HDL-C; wherein: (a) total cholesterol concentration, (b) triglyceride concentration, (c) low density lipoprotein cholesterol concentration, (d) high density lipoprotein cholesterol concentration.

FIG. 5miR-199a-3p Agomir vs. Western diet 6 weeks APOE ^-/- Mouse atherosclerotic plaque effect; wherein: (a) control group, (b) Agomir group, (c) plaque area.

Detailed Description

The invention is further described by the following examples, which include the materials used, as well as the particular sources. It should be understood that these are merely exemplary and are not limiting of the present invention. Materials similar or identical to the type and model of reagents, instruments, or properties or functions described below may be used in the practice of the invention. The reagents used in the present invention may be any suitable commercially available reagents unless otherwise specified.

Example 1

The main materials used in the invention are as follows:

tir reagent Jiangsu Enmo Alsai Biotechnology Co., ltd

miRNA cDNA first strand synthesis kit Beijing Baitaike biotechnology Co

2X TSINGKE Master qPCR Mix Beijing Optimu Corp

miRNA primer Beijing Optimu Biotech Co

1. Clinical sample collection

The research process of the invention collects the peripheral blood samples of the hyperlipidemia and normal people belonging to the hospitals. The process strictly complies with medical ethics requirements and strictly complies with patient information confidentiality principles. The storage conditions of the samples were consistent. And selecting samples for fluorescence quantitative PCR detection by sorting medical record data. ( Total cholesterol is more than or equal to 6.2mmol/L; the triglyceride is more than or equal to 2.3mmol/L; low density lipoprotein > 3.37mmol/L can be diagnosed as dyslipidemia )

2. Extraction of plasma miRNA

500 μl Tri-reagent vortex was added to each 200 μl blood sample. Standing at room temperature for 5 min, adding 50 μl of BCP into the sample, vortex shaking for 15 s, changing the color of the solution from pink to milky, standing at room temperature for 8 min, and centrifuging at 4deg.C and 13500rpm for 15 min. After centrifugation, the liquid in the centrifuge tube was found to separate, the supernatant containing RNA was transferred, and the supernatant was added with an equal amount of isopropanol solution and mixed well by reversing upside down ten times over-80 degrees overnight. The following day 4℃and 13500rpm, for 60 minutes. The supernatant was discarded and a white precipitate was retained. The centrifuge tube was gently shaken with a pre-chilled 75% ethanol solution without nuclease water to blow up the pellet, and centrifuged at 13500rpm at 4℃for 5 minutes. Repeating the steps, and cleaning the precipitate to improve the purity of RNA. And discarding the supernatant, placing the centrifuge tube in an ultra-clean bench, and airing at room temperature until the white precipitate becomes transparent, thus ending. Proper amount of DEPC water was added to dissolve RNA. RNA denaturation: the dissolved RNA was heated on a constant temperature dry heater at 55℃for 10 minutes.

3. Fluorescent quantitative PCR (polymerase chain reaction) detection of has-miR-199a-3p level

RNA concentration was determined using a NanoDrop200 spectrophotometer, with 500ng RNA as template. After being gently mixed, the mixture is placed in a PCR instrument to carry out the first-step reaction, the temperature is 37 ℃ for 60 minutes, and the components of a reaction system are shown in Table 1.

Table 1: one component table of reverse transcription reaction system

After the reaction of the first step is completed, the second part of the required reagents are respectively added, and the specific details are shown in Table 2. Gently mixing, placing in a PCR instrument at 37 ℃ for 60 minutes, and reversely transcribing into cDNA.

Table 2: two component table of reverse transcription reaction system

The cDNA was diluted to 6ng per microliter and used as a fluorescent quantitative PCR template, and the reaction system is shown in Table 3.

Table 3: composition table of PCR reaction system

PCR was adjusted to 50℃for 2 minutes; 95 ℃ for 2 minutes; 95 ℃ for 15 seconds; 60 ℃ for 30 seconds.

4. Data processing and analysis

Analysis data calculated has-miR-199a-3p relative expression data. Formula rq=2 ^-△△CT Wherein% Δct= [ CT _Patient(s) (miRNA) -CT (internal reference)]-[CT _{Normal person} (miRNA) -CT (internal reference)]. Analyzing the real-time fluorescence quantitative PCR detection result by using SPSS20.0 software, wherein the measurement data are expressed by mean ± standard deviation, the comparison between groups adopts t test, and P<The difference of 0.05 is statistically significant, expressed as a.

The has-miR-199a-3p level of the normal population and the hyperlipidemia patients is shown in figure 1. The has-miR-199a-3p levels were positively correlated with cholesterol levels, as shown in FIG. 2.LDLR (Low Density polyethylene) ^-/- And APOE ^-/- The mouse mmu-miR-199a-3p level was higher than that of the wild type mice, as shown in FIG. 3.

Effect of miR-199a-3p on mouse lipid metabolism

APOE at 6-8 weeks ^-/- Mice were divided into two groups, one group with miR-199a-3p Agominr injected into the tail vein, the other group with NC-Agominr injected into the tail vein, and the rest conditions were completely consistent, and each group was given Western diet and free water. Mice were sacrificed 6 weeks after feeding and each index was tested. After intraperitoneal injection of tribromoethanol for anesthesia, the mice were anesthetized with a sterile capillary and were collected from the intraorbital side-entry needles. Incubation was carried out at 37℃for 1h, and then centrifugation was carried out at 8000rpm for 10min, and the separated serum was assayed for TC, TG, HDL-C and LDL-C levels (Nanjing Seiko technologies Co.). The results of TG, TC, LDL-C, HDL-C changes are shown in FIG. 4, in which plasma TG, TC, LDL-C levels were significantly reduced and HDL-C levels were increased in mice injected with miR-199a-3p Antagomir. Indicating that miR-199a-3p can inhibit lipid metabolism disorder.

(1) Working fluid is respectively added into blank holes, calibration holes and sample holes of the 96-well plate, and distilled water, calibration liquid and serum samples are sequentially added. After mixing, the mixture was incubated in an incubator at 37℃for 10 minutes, and the values were measured by a spectrophotometer. And calculating the level of each biochemical index according to a formula.

(2) Oil red O staining: preparing a dye liquor, namely 5g, 100ml of 50% ethanol, filtering to dissolve oil red O in the ethanol, and continuously stirring until the oil red O is completely dissolved. The dye solution is evenly dripped on a slide, the dye solution is sucked and removed after 10 minutes, the slide is washed by distilled water, and the observation is carried out under a microscope.

The heart and main artery of the mice were fixed in paraformaldehyde with a mass fraction of 4%, paraffin-embedded sections were stained with oil red, and the lesion areas were analyzed by ImageJ software as shown in fig. 5. Mice injected with 199a-3pAgomir group had decreased aortic atherosclerotic plaques, indicating that miR-199a-3 had a cardiovascular protective effect.

Claims

1. An application of miRNA-199a-3p as a molecular diagnosis marker in diagnosis of lipid metabolic disorders and complications.

2. The use of miRNA-199a-3p as a molecular diagnostic marker for diagnosis of lipid metabolic disorders and complications according to claim 1, wherein the nucleic acid sequence of miRNA-199a-3p is SEQ id No.1.

3. The use of the miRNA-199a-3p as a molecular diagnostic marker in diagnosing lipid metabolism disorder diseases and complications, wherein the diagnostic lipid metabolism disorder products comprise PCR amplification primers used when detecting the expression quantity of the miRNA-199a-3p by using SYBR Green, taqMan probes, double hybridization probes or composite probes, and the design of the primers is shown as SEQ ID NO. 2.

4. The use of miRNA-199a-3p as a molecular diagnostic marker for the diagnosis of lipid metabolic disorders and complications according to claim 1, wherein the product for diagnosing lipid metabolic disorders and hyperlipidemia further comprises a chip, a kit, a test paper or a high throughput sequencing platform.

5. Use of miRNA-199a-3p as a marker for molecular diagnostics in the diagnosis of lipid metabolic disorders and complications according to claim 1, including but not limited to hyperlipidemia, hypercholesterolemia, atherosclerosis, coronary heart disease or diabetes.