阅读说明：本技术 一种长链非编码rna及其干扰rna在治疗动脉粥样硬化中的应用 (Long-chain non-coding RNA and application of interference RNA thereof in treatment of atherosclerosis ) 是由 于波 田进伟 孙长斌 袭祥文 顾霞 刘新新 于 2019-10-21 设计创作，主要内容包括：本发明公开了一种长链非编码RNA及其干扰RNA在治疗动脉粥样硬化中的应用。本发明通过对动脉粥样硬化小鼠模型的主动脉组织进行高通量测序,发现了一种长链非编码RNA(lncRNA)分子在正常与晚期动脉粥样硬化小鼠主动脉组织中表达显著差异,同时该lncRNA分子在动脉粥样硬化组织中的高表达,将该lncRNA分子命名为lncRNA-AFIAR。实验证明lncRNA-AFIAR具有增强巨噬细胞增殖、抑制巨噬细胞凋亡等功能,参与了促进动脉粥样硬化的进展这一过程。通过对lncRNA-AFIAR进行抑制可以达到抑制巨噬细胞增殖、减少斑块形成进而达到抑制AS的进展的目的。因此,本发明的提出为诊疗动脉粥样硬化提供了新的分子标记和干预靶点,也为动脉粥样硬化的治疗提供了新的技术手段。(The invention discloses a long-chain non-coding RNA and application of an interference RNA thereof in treating atherosclerosis. According to the invention, through high-throughput sequencing on the aorta tissue of an atherosclerosis mouse model, a significant difference of the expression of a long-chain non-coding RNA (lncRNA) molecule in the aorta tissue of a normal atherosclerosis mouse and a late atherosclerosis mouse is found, and the lncRNA molecule is highly expressed in the atherosclerosis tissue and is named lncRNA-AFIAR. Experiments prove that the lncRNA-AFIAR has the functions of enhancing macrophage proliferation, inhibiting macrophage apoptosis and the like, and participates in the process of promoting the development of atherosclerosis. The purpose of inhibiting macrophage proliferation and reducing plaque formation and further inhibiting AS progression can be achieved by inhibiting lncRNA-AFIAR. Therefore, the invention provides a new molecular marker and an intervention target point for diagnosing and treating atherosclerosis, and also provides a new technical means for treating atherosclerosis.)

1. A long-chain non-coding RNA is named lncRNA-AFIAR, and the nucleotide sequence of the long-chain non-coding RNA is shown in SEQ ID NO. 1.

2. A reagent for detecting the long non-coding RNA of claim 1.

3. The reagent of claim 2, wherein the reagent is a primer, preferably wherein the primer sequence is as shown in SEQ ID No.2 and SEQ ID No. 3.

4. An agent for inhibiting the long non-coding RNA of claim 1.

5. The agent of claim 4, wherein the agent is an interfering RNA (siRNA), preferably wherein the sequence of the interfering RNA is as shown in SEQ ID No.4 and SEQ ID No. 5.

6. Use of the long non-coding RNA of claim 1 as a target for the preparation of a medicament for the diagnosis or treatment of atherosclerosis.

7. Use of an agent according to claim 2 or 3 for the manufacture of a medicament for the diagnosis of atherosclerosis.

8. Use of an agent according to claim 4 or 5 in the manufacture of a medicament for the treatment of atherosclerosis.

Technical Field

The invention relates to a long-chain non-coding RNA and application of an interference RNA thereof in treating atherosclerosis. The invention belongs to the technical field of biological medicines.

Background

In 2018, in 1 month, the national cardiovascular disease center issued "report 2017 on cardiovascular diseases in China". It indicates that in 2017, the number of patients with coronary atherosclerotic heart disease in China is 1100 million, the prevalence rate and death rate of cardiovascular disease in China are still in the rising stage in general, and the number of patients with cardiovascular disease will still increase rapidly in the next 10 years. Coronary heart disease is one of the diseases with the highest global mortality as a high morbidity of cardiovascular diseases, and according to the report of the world health organization in 2011, the number of deaths of coronary heart disease in China is listed in the second world.

Atherosclerosis is believed to be a chronic inflammatory response in which macrophages progressively accumulate on the walls of dilated arteries, partially within the lesion, engulf lipids, produce multiple mediators of inflammation, and exacerbate the disease. Macrophage accumulation is not actually dependent on monocyte recruitment, but rather on local macrophage proliferation. Therefore, the proliferation and apoptosis of macrophages are indistinguishable from the development and progression of atherosclerosis.

Long non-coding RNA (lncRNA) is a type of transcript with the length of more than 200nt (nucleotide) and cannot be translated into functional RNA molecules of protein, and most of lncRNA is transcribed by RNA polymerase II catalysis and shows stronger specificity in tissues and cells. Research on lncRNA has progressed rapidly in recent years, but the function of the vast majority of lncRNA remains unclear. According to the invention, an lncRNA molecule with high expression quantity and large difference is screened from the whole transcriptome high-throughput sequencing of atherosclerotic arterial tissue, named lncRNA-AFIAR, and the function detection of the lncRNA-AFIAR further discovers the relationship between the lncRNA-AFIAR and atherosclerotic diseases, so that the disease mechanism can be better understood, and a new diagnosis and treatment target point is provided for atherosclerosis from the RNA level.

Disclosure of Invention

The invention aims to provide lncRNA with remarkably increased expression level in atherosclerotic tissues relative to normal arterial tissues, interference RNA thereof and application of the lncRNA in treating atherosclerosis as a new target.

In order to achieve the purpose, the invention adopts the following technical means:

according to the invention, a long-chain non-coding RNA is screened by performing high-throughput sequencing on an artery tissue in a mouse atherosclerosis model, and is named as lncRNA-AFIAR, the nucleotide sequence of the long-chain non-coding RNA is shown in SEQ ID NO.1, and the expression quantity of the lncRNA-AFIAR in the atherosclerosis tissue is obviously increased compared with that in a non-atherosclerosis tissue. Designing primer sequences related to the incRNA-AFIAR fluorescent quantitative PCR amplification and silencing incRNA-AFIAR (siRNA) sequences. Fluorescent quantitative PCR experiments further prove that high-throughput sequencing results are obtained by extracting RNA from mouse atherosclerotic tissues, performing reverse transcription to synthesize cDNA, performing PCR amplification by using a designed lncRNA-AFIAR PCR primer, and obtaining an amplified product for the first time. The lncRNA-AFIAR is constructed to silence the macrophage RAW264.7 of a mouse, and the function of the lncRNA-AFIAR in the functions of proliferation, apoptosis and the like of RAW264.7 cells is researched, so that the action mechanism of the lncRNA-AFIAR in atherosclerosis is explored. Through the regulation and control function of lncRNA-AFIAR on macrophages, the lncRNA-AFIAR plays an important role in the whole occurrence and development process of atherosclerosis, and LncRNA-AFIAR siRNA inhibits the progress of AS by inhibiting the proliferation of macrophages and reducing the formation of plaques.

On the basis of the research, the invention provides a long-chain non-coding RNA, which is named lncRNA-AFIAR, and the nucleotide sequence of the long-chain non-coding RNA is shown in SEQ ID NO. 1.

Reagents for detecting the long non-coding RNA are also within the scope of the invention. Preferably, the reagent is a primer, and more preferably, the primer sequence is shown as SEQ ID NO.2 and SEQ ID NO. 3.

Agents for inhibiting the long non-coding RNA are also within the scope of the invention. Preferably, the agent is interfering RNA (siRNA), and more preferably, the sequence of the interfering RNA (siRNA) is shown as SEQ ID NO.4 and SEQ ID NO. 5.

Furthermore, the invention also provides application of the long-chain non-coding RNA as a target spot in preparation of medicines for diagnosing or treating atherosclerosis.

Furthermore, the invention also provides the application of the reagent for detecting the long-chain non-coding RNA in preparing the medicine for diagnosing atherosclerosis.

Furthermore, the invention also provides the application of the reagent for inhibiting the long-chain non-coding RNA in preparing the medicine for treating atherosclerosis.

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

experiments prove that the lncRNA-AFIAR has the regulation and control function on macrophages, and the lncRNA-AFIAR plays an important role in the whole occurrence and development process of atherosclerosis. Through screening the target spot in clinic, the early atherosclerosis timely drug intervention can be realized, and the disease can be delayed or even reversed. In the screening of the advanced atherosclerosis, the development condition of the pathological changes of the patient can be known, and the intervention is carried out in time to avoid the occurrence of clinical time. Therefore, the invention provides a new molecular marker and an intervention target point for diagnosing and treating atherosclerosis, and also provides a new technical means for treating atherosclerosis.

Drawings

FIG. 1 is a pathological staining established for an atherosclerotic mouse model;

A-D are ApoE mouse aortic atherosclerosis model aortic whole oil red O staining; A. b: an early AS model; C. d: a late AS model;

E-F is oil red O staining of a frozen section of the aortic root of a mouse atherosclerosis model; e: an early AS model; f: a late AS model;

FIG. 2 is a graph of the distribution of lncRNA differentially expressed from advanced mouse aortic tissue (ADV) and normal mouse aortic tissue (NOR) by high throughput sequencing;

FIG. 3 is a graph showing the expression of aortic tissue (ADV) in advanced atherosclerosis mice and aortic tissue (NOR) in normal mice by high throughput sequencing of IncRNA-AFIAR;

FIG. 4 is a diagram showing that Real-time PCR detects the expression of IncRNA-AFIAR in aortic tissue (ADV) of mice in advanced atherosclerosis and aortic tissue (NOR) of normal mice;

FIG. 5 shows the proliferation of RAW264.7 cells 48 hours after EDU staining detection of silencing sequences si-AFIAR of transfection-independent sequences si-NC and lncRNA-AFIAR; after lncRNA-AFIAR is silenced, RAW264.7 cell proliferation is obviously inhibited;

FIG. 6 shows the apoptosis of RAW264.7 cells 48 hours after Hochest33342 staining detection of silencing sequences si-AFIAR of transfection-independent sequences si-NC and lncRNA-AFIAR; after lncRNA-AFIAR is silenced, the apoptosis rate of RAW264.7 cells is obviously increased;

FIG. 7 shows that after Apoe-/-mice are subjected to high fat for 16 weeks, the AAV2/9-NC group and the AAV2/9-sh-AFIAR group are pathologically stained by the oil red O at the aortic root, and the aortic root plaques of the mice are stained by the oil red O staining, so that the AAV2/9-sh-AFIAR group is obviously reduced and reduced in atherosclerotic plaques (red) compared with the AAV2/9-NC group.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.