CN111635900A

CN111635900A - shRNA (short hairpin ribonucleic acid) of targeted circular RNA0089762 and application thereof

Info

Publication number: CN111635900A
Application number: CN202010573066.5A
Authority: CN
Inventors: 高志良; 赵绮毅; 梁蕙欣
Original assignee: Third Affiliated Hospital Sun Yat Sen University
Current assignee: Third Affiliated Hospital Sun Yat Sen University
Priority date: 2020-06-22
Filing date: 2020-06-22
Publication date: 2020-09-08
Anticipated expiration: 2040-06-22
Also published as: CN111635900B

Abstract

The invention relates to shRNA (short hairpin ribonucleic acid) targeting circular RNA0089762, wherein the nucleotide sequence of the shRNA is as follows, sh-circRNA-1: 5'-CCATGTCGCCTGGTTCTAGTT-3' or sh-circRNA-2: 5'-CGCTGCATGTGCCATGTCGTT-3', meanwhile, the invention also provides shRNA of the target circular RNA0089762, which is applied to cell experiments to promote metabolic inflammation; the metabolic diseases caused by the metabolic inflammation comprise obesity, type 2 diabetes, atherosclerosis or non-alcoholic fatty liver disease.

Description

shRNA (short hairpin ribonucleic acid) of targeted circular RNA0089762 and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to shRNA of a target circular RNA0089762 and application thereof in regulating metabolic inflammation.

Background

Due to the changes of modern living habits and environments, metabolic disorders and metabolites including free fatty acids and endotoxins are generated, macrophages are polarized and chronic low-grade inflammation, called metabolic inflammation, is induced, tissues and organs are damaged and metabolic diseases are caused; the metabolic inflammation is characterized by persistent low-level inflammation, and relates to molecules and signal transduction pathways similar to those of the classical inflammation, so that the expression and activity of various inflammatory molecules can be enhanced, the morphology and the function of related organs are further damaged, and the metabolic inflammation is closely related to various chronic metabolic diseases, such as non-alcoholic fatty liver disease (NAFLD), atherosclerosis, type 2 diabetes, obesity and the like.

Non-alcoholic fatty liver disease (NAFLD) refers to the clinical pathological syndrome characterized mainly by excessive fat deposition in liver cells due to other definite liver damage factors except alcohol, including non-alcoholic simple fatty liver (simplex), non-alcoholic steatohepatitis (NASH) and its associated cirrhosis (cirrhosis). In recent years, with the change of living habits and dietary structures of people, the prevalence rate of non-alcoholic fatty liver diseases is increasingly higher, and China already reaches 15.35% and is in a continuously rising trend; even more, up to 20-25% in western countries, has become the first major liver disease, and therefore, it is urgent to enhance the research on the pathogenesis of NAFLD and discover new prevention and treatment measures.

Circular RNA (circRNA) is a special endogenous non-coding RNA, and is a hot spot for research on RNA family following microRNA and long non-coding RNA. In recent years, with the rapid development of RNA sequencing technology and bioinformatics, it has been found through analysis of large-scale transcriptome data that circRNA exists in large quantities, accounting for about 10% of eukaryotic cell transcripts, and compared to miRNA and long-chain non-coding RNA, circRNA has some significant characteristics, such as: the circular closed structure is provided, no polyA tail exists, the circular closed structure is not easily degraded by exonuclease, the circular closed structure can stably exist in eukaryotic cells, most sequences are highly conserved, and certain tissue structure specificity is provided, so that the circRNA occupies an important position in gene expression and other biological processes. The circRNA is closely related to various diseases and plays a crucial role in regulation during the occurrence and development of the diseases, so the circRNA is likely to become a high-efficiency clinical diagnosis marker in the future.

Recent studies have demonstrated that mitochondrially localized circular RNA0089762 in liver fibroblasts is down-regulated in NAFLD patients and is closely linked to metabolic inflammation, and therefore circular RNA0089762 inhibits metabolic inflammation.

Disclosure of Invention

The invention provides shRNA targeting circular RNA0089762, and the shRNA can promote metabolic inflammation when being applied to a cell experiment.

The technical scheme adopted by the invention is as follows:

in a first aspect, the invention provides an shRNA targeting circular RNA0089762, the nucleotide sequence of the shRNA being as defined in sh-circRNA-1: 5'-CCATGTCGCCTGGTTCTAGTT-3' or sh-circRNA-2: 5'-CGCTGCATGTGCCATGTCGTT-3' are provided.

In a second aspect, the invention provides a shRNA targeting circular RNA0089762 for use in a cellular assay to promote metabolic inflammation.

Further, the metabolic diseases caused by the metabolic inflammation include obesity, type 2 diabetes, atherosclerosis or non-alcoholic fatty liver disease.

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

the invention designs shRNA of a target circular RNA0089762, which can effectively target circular RNA0089762 positioned by mitochondria in liver fibroblasts and reduce the expression of circular RNA0089762, so that the levels of mitochondria active oxygen and IL-1 beta, TNF-alpha, IL-8 and CCL-24 inflammatory cytokines in the liver fibroblasts exposed by lipid are increased, and further the effect of promoting metabolic inflammation is achieved, and a cell model related to the metabolic inflammation is constructed.

Drawings

FIG. 1 is a schematic diagram showing the comparison of the expression levels of shRNA (sh-circRNA-1, sh-circRNA-2) transfected to circRNA0089762 by palmitate treatment and the expression level of circRNA0089762 in fibroblasts as a control sh-Ctrl, and shRNA (sh-circRNA-1, sh-circRNA-2) transfected to circRNA0089762 and the expression level of circRNA0089762 in fibroblasts as a control sh-Ctrl without palmitate treatment in the example of the present invention;

FIG. 2 is a graph showing the comparison of the levels of mROS in fibroblasts treated with palmitate and in fibroblasts not treated with palmitate in the absence of shRNA treatment and the comparison of the levels of mROS in fibroblasts transfected with shRNA (sh-circRNA-1, sh-circRNA-2) against circRNA0089762 and used as a control sh-Ctrl in the presence of palmitate treatment in accordance with an embodiment of the present invention;

FIG. 3 is a graph showing the comparison of the levels of IL-1 β, TNF- α, IL-8 and CCL-24 cytokines in palmitate-treated and non-palmitate-treated fibroblasts and the levels of IL-1 β, TNF- α, IL-8 and CCL-2 cytokines in fibroblasts transfected with shRNA 0089762 (sh-circRNA-1, sh-circRNA-2) and sh-Ctrl as a control in the example of the present invention.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The invention provides shRNA (short hairpin ribonucleic acid) targeting circular RNA0089762, wherein the nucleotide sequence of the shRNA is as follows, sh-circRNA-1: 5'-CCATGTCGCCTGGTTCTAGTT-3' or sh-circRNA-2: 5'-CGCTGCATGTGCCATGTCGTT-3' are provided.

Wherein, the nucleotide sequence of the shRNA used as the comparison is as follows:

sh-Ctrl：5’-CTTTCTCCGAACGTGTCAC-3’

the invention also provides a method for applying shRNA of the target circular RNA0089762 to cell experiments to promote metabolic inflammation; the metabolic diseases caused by the metabolic inflammation comprise obesity, type 2 diabetes, atherosclerosis or non-alcoholic fatty liver disease, and a specific cell experiment method is as follows:

first, construct shRNA-stably transfected clones using the lentiviral vector plasmid pLKO.1-puro and transfect 5 × 10 in a 10cm dish with 10. mu.g of pLKO1-puro shRNA expression plasmid⁶Preparing lentivirus particles from HEK293FT cells; lentiviral particle-enriched supernatants were collected at 48h and 72h post-transfection, respectively, and filtered through a 0.22 μm needle filter (Millex-GPFilter Unit)；

Next, normal liver fibroblasts were treated with 250. mu.M palmitate (palmitate) for 24h, and then lentivirus particles (multiplicity of infection 30) and 10. mu.g/mL polybrene (polybrene) were transfected at 37 ℃ into 6-well plates at 5 × 10⁵Normal fibroblasts treated with palmitate overnight; transfecting normal fibroblasts without palmitate treatment in the same manner;

finally, the medium was aspirated and fresh growth medium was added.

And (3) data testing:

(1) expression detection of circRNA0089762 in fibroblasts

Extracting total RNA of fibroblast cells obtained after cell experiment treatment by using TRIzol reagent, and reversely transcribing the RNA into a cDNA template by using a random primer and a reverse transcription kit capable of removing genome pollution and amplifying the cDNA template. qRT-PCR is carried out through a dye method fluorescent quantitative kit and a Roche real-time fluorescent quantitative PCR instrument, and the relative expression quantity of the circular RNA0089762 is detected; the results are shown in fig. 1, in the absence of shRNA treatment, the circRNA0089762 expression level in the palmitate-treated group was much lower than in the non-palmitate-treated group, i.e. palmitate treatment could down-regulate the expression of circRNA0089762 in liver fibroblasts; the expression of circRNA0089762 in fibroblasts transfected with shRNA (sh-circRNA-1, sh-circRNA-2) against circRNA0089762 was significantly down-regulated regardless of palmitate treatment, whereas the expression of circRNA0089762 in fibroblasts transfected with sh-Ctrl as control was not significantly changed.

(2) Measurement of mROS levels in fibroblasts

Incubating fibroblasts obtained by cell experimental treatment with 5 μ M MitoSOX (mitochondrial superoxide red fluorescent probe) working solution at 37 ℃ in the dark for 20 min; the cells were washed 3 times with PBS and covered with 1. mu.g/ml Hoechst 33342 fluorescent dye; analyzing the fluorescence signal by using a CytoFlex flow cytometer; analyzing the Mean Fluorescence Intensity (MFI) of mitochondrial reactive oxygen species (mROS) by using FlowJo software; as shown in FIG. 2, the increased mROS level is one of the manifestations of metabolic inflammation, the level of mROS in palmitate treated group was much higher than that in non-palmitate treated group without shRNA treatment, the level of mROS in fibroblasts transfected with shRNA (sh-circRNA-1, sh-circRNA-2) against circRNA0089762 was significantly increased in palmitate treatment, while the level of mROS in fibroblasts transfected with sh-Ctrl as a control was not significantly changed.

(3) Detection of levels of inflammatory cytokines IL-1 beta, TNF-alpha, IL-8 and CCL-2

Collecting the culture solution of fibroblast obtained by cell experiment treatment, and centrifuging at 1000g for 20min to remove cell debris; collecting 200 ul of supernatant, and detecting the concentrations of inflammatory cytokines IL-1 beta, TNF-alpha, IL-8 and CCL-2 by using an ELISA kit according to instructions; the results are shown in FIG. 3, where elevated levels of the inflammatory cytokines IL-1 β, TNF- α, IL-8 and CCL-2 are also one of the manifestations of metabolic inflammation, and in the absence of shRNA treatment, the levels of the 4 cytokines in the palmitate-treated group were much higher than those in the non-palmitate-treated group; the levels of 4 cytokines in fibroblasts transfected with shrnas against circRNA0089762 (sh-circRNA-1, sh-circRNA-2) were significantly elevated when treated with palmitate, whereas the levels of 4 cytokines in fibroblasts transfected with sh-Ctrl as a control were not significantly changed.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Sequence listing

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Claims

1. An shRNA targeting circular RNA0089762, characterized in that: the nucleotide sequence of the shRNA is as shown in sh-circRNA-1: 5'-CCATGTCGCCTGGTTCTAGTT-3' or

sh-circRNA-2：5’- CGCTGCATGTGCCATGTCGTT-3’。

2. The shRNA targeting the circular RNA0089762, according to claim 1, applied in a cell experiment to promote metabolic inflammation.

3. The use of the shRNA targeting circular RNA0089762 in a cellular assay to promote metabolic inflammation according to claim 2, wherein: the metabolic diseases caused by the metabolic inflammation comprise obesity, type 2 diabetes, atherosclerosis or non-alcoholic fatty liver disease.