CN114645089A

CN114645089A - Annular RNA marker related to rheumatoid arthritis and application thereof

Info

Publication number: CN114645089A
Application number: CN202210515288.0A
Authority: CN
Inventors: 方征宇; 王庆文; 尹为华; 韦伟; 余光银; 韩馨乐; 林素雯; 吕姝妍
Original assignee: SHENZHEN PKU-HKUST MEDICAL CENTER
Current assignee: SHENZHEN PKU-HKUST MEDICAL CENTER
Priority date: 2022-05-12
Filing date: 2022-05-12
Publication date: 2022-06-21
Anticipated expiration: 2042-05-12
Also published as: CN114645089B

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses an application of a circular RNA marker in preparation of a rheumatoid arthritis detection kit, wherein the circular RNA marker is hsa _ circ _0000137, and the nucleotide sequence is shown as SEQ ID NO. 1; hsa _ circ _0000137 in synovial tissue and fluid can be used as a detection marker for rheumatoid arthritis. The circular RNA marker hsa _ circ _0000137 provided by the invention is closely related to the activation degree of rheumatoid arthritis fibroblast-like synovial cells, the concentration of the circular RNA marker in synovial tissue and synovial fluid can be used as an activity detection index of the rheumatoid arthritis fibroblast-like synovial cells, and the circular RNA marker hsa _ circ _0000137 not only can be applied to evaluation of the pathological degree and prognosis of the rheumatoid arthritis synovial cells, but also can provide a new way for regulation and control of the rheumatoid arthritis fibroblast-like synovial cells and research of molecular diagnosis and treatment strategies.

Description

Annular RNA marker related to rheumatoid arthritis and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a circular RNA marker related to rheumatoid arthritis and application thereof.

Background

The rheumatism immune disease belongs to the autoimmune system disease and is a general term of a large class of diseases. RA is a chronic systemic autoimmune disease with unknown etiology characterized by synovial membrane hyperplasia and articular cartilage destruction, with the main pathological manifestations of synovial membrane lining fibroblast hyperplasia, synovial membrane lining thickening, followed by intravascular transformation, ultimately leading to erosive damage to cartilage and bone.

Fibroblastic synoviocytes (FLS) are one of the major component cells of rheumatoid arthritis pathological synovial tissue, causing inflammation and damage to joints by secreting inflammatory mediators, metalloproteinases and cathepsins, and invading the cartilage and bone surrounding the joint. The degree of such cell activation is a key factor in the development and prognosis of rheumatoid arthritis.

Non-coding RNAs (non-coding RNAs) account for 99% of total cellular RNA and are critical for establishing and maintaining homeostatic balance within biological systems, including the regulation of signaling pathways and biological processes that control joint development. Imbalance of non-coding RNAs can lead to joint diseases such as Osteoarthritis (OA) and RA.

Circular RNAs (circrnas) are a class of non-coding RNAs that can act as competitive endogenous RNAs (cerrnas) or microrna (miRNA/miR) sponges, resulting in degradation or translational inhibition of mirnas. Circular RNAs that are deregulated in inflammatory diseases bind to the 3' untranslated region (UTR) of messenger RNA via miRNA response elements, affecting the activity of inflammatory cells.

In recent years, circular RNAs surrounding RA pathogenesis have been studied in the following directions:

(1) screening studies of circRNA in PBMC were performed on RA, comparing RA to healthy control mononuclear cell numbers. Researchers such as OUYANG Q Q Q reported no significant difference in the number of mononuclear cells in RA versus healthy controls, and the differences between circRNA-092516, circRNA-003524, circRNA-103047, circRNA-104871, and circRNA-101873 in PBMCs of RA patients and healthy controls were found by circRNA microarray analysis and qRT-PCR validation.

(2) Hierarchical clustering analysis is carried out on RA synovial tissue and normal synovial tissue to determine whether circRNA is an important factor causing gene expression difference. For example, hsa _ circ _0001859 acts as a decoy that regulates the expression of ATF2 by acting as a sponge to directly inhibit miR-204/211 transcription. ATF2 is a CREB family member, commonly associated with AP1 (c-Jun and c-Fos) and heterodimers, and plays an important role in the skeletal system, central nervous system, and inflammation. In SW982 cells, siRNA silencing hsa _ circ _0001859 was shown to inhibit ATF2 expression and reduce its level of inflammation (LOPEZ-BERGAMI P, LAU E, RONAI Z. Emerging roles of ATF2 and the dynamic AP1 network in Cancer [ J ]. Nat Rev Cancer, 2010, 10 (1): 65-76. DOI: 10.1038/nrc 2681).

In recent years, the association between circRNA and bone diseases is more and more studied, however, the expression and function of circRNA in osteoarthritis is a completely new field, further attention needs to be paid, a large number of researchers are still needed to deeply study the detailed mechanism of circRNA in osteoarthritis, and the discovery of any new circRNA related to RA is a great contribution to the field.

Disclosure of Invention

The invention aims to provide a circular RNA marker related to rheumatoid arthritis and application thereof, so as to solve the technical problems.

In order to achieve the above objects, one of the objects of the present invention is to provide a circular RNA marker associated with rheumatoid arthritis, wherein the circular RNA marker is hsa _ circ _0000137, and the nucleotide sequence is shown in SEQ ID NO. 1.

The second purpose of the invention is to provide the application of the circular RNA marker in the preparation of a rheumatoid arthritis detection kit. The rheumatoid arthritis detection kit comprises a forward primer and a reverse primer, wherein the sequence of the forward primer is shown as SEQ ID NO.2, and the sequence of the reverse primer is shown as SEQ ID NO. 3.

The principle and the beneficial effects of the invention are as follows:

(1) the invention names the firstly discovered circular RNA marker as "hsa _ circ _ 0000137", the specific position is chr1:155408117-155408859, and the nucleotide sequence is shown as SEQ ID NO. 1.

(2) The hsa _ circ _0000137 is related to the disease activity of RA, is closely related to the activation degree of rheumatoid arthritis fibroblast-like synovial cells, has high potential as a diagnostic biomarker, and can be used as an important index for the activity detection of novel rheumatoid arthritis fibroblast-like synovial cells (RA-FLS). The concentration of the compound in synovial tissue and synovial fluid can be used as a novel activity detection index of rheumatoid arthritis fibroblast synovial cells, can be applied to rheumatoid arthritis synovial lesion degree, prognosis evaluation and the like, and can also provide a new way for regulation and control of rheumatoid arthritis fibroblast synovial cells and research of molecular diagnosis and treatment strategies.

(3) The hsa _ circ _0000137 provided by the invention can promote the invasion of rheumatoid arthritis fibroblast-like synoviocytes by regulating the expression of collagenase MMP9, can regulate the expression of inflammatory factor IL-6 and chemokine CCL2 to influence the development of arthritis, and can be used as a pathogenic factor in the development of human RA.

(4) The content of circRNA in peripheral blood is low, the number of affected factors is large, and the value is not high; the synovial fluid is also a detection object which can be obtained by RA patients, is closely related to the disease part, has stable and accurate concentration and is more significant than the circRNA in peripheral blood.

Since the clinical samples used in the invention include synovial tissue and fluid of RA patients and synovial tissue and fluid of OA patients, and the expression profiles of circRNA in RA-FLS (rheumatoid arthritis fibroblast-like synovial cell) cell lines and normal FLS (fibroblast-like synovial cell) cell lines are compared by taking OA synovial tissue and fluid as control groups, and hsa _ circ _0000137 is found, further research is carried out on hsa _ circ _0000137, and the two diseases can be identified at the gene level.

Drawings

FIG. 1 is a volcano plot of differentially expressed genes;

FIG. 2 is a graph of the correlation of hsa _ circ _0000137 expression with clinical presentation, wherein,

a: detecting the expression of hsa _ circ _0000137 in synovial tissue of OA patients and RA patients by adopting real-time fluorescent quantitative PCR;

b: real-time fluorescent quantitative PCR was used to detect the expression level of hsa _ circ _0000137 in synovial fluid of RA patients, and pearson correlation analysis was performed with the expression level of CRP blood concentration in patient serum (from clinical cases of patients);

c: performing pearson correlation analysis on the expression level of hsa _ circ _0000137 in synovial fluid of RA patients and a patient disease severity index DAS28 (data from clinical electronic medical records);

FIG. 3 shows the results of the cell function assay of hsa _ circ _ 0000137; wherein the content of the first and second substances,

a: MH7A cells were transfected with PBS (Ctrl), nonsense strand (Si NC), and SiRNA for hsa _ circ _0000137 (Si hsa _ circ _ 0000137), and expression of hsa _ circ _0000137 was detected by real-time fluorescent quantitative PCR;

b: the WST-1 method detects the proliferation level of cells, and the experimental result shows that the proliferation level is obviously reduced after MH7A cells transfect SiRNA of hsa _ circ _ 0000137;

c: the cell invasion capacity is detected by a Transwell method, and the experimental result shows that the invasion level is obviously reduced after MH7A cells transfect SiRNA of hsa _ circ _ 0000137;

FIG. 4 is a correlation analysis of hsa _ circ _0000137 with cytokine expression; and detecting the expression level of the inflammatory factor related gene by adopting a real-time fluorescent quantitative PCR method.

Detailed Description

The following is further detailed by way of specific embodiments:

example 1

Design of experiments

Rheumatoid Arthritis (RA) and Osteoarthritis (OA) have different pathogenesis, but as OA is a degenerative disease, usually has mild clinical symptoms and pathology and is often used as a control for the research of rheumatoid arthritis pathology, so the clinical samples used in the experiment include synovial tissue and synovial fluid of RA patients and synovial tissue and synovial fluid of OA patients, and circRNA differentially expressed from RA synovial tissue and synovial fluid is screened by taking the synovial tissue and synovial fluid of OA as a control group.

Experimental methods

S1 collecting synovial tissue and membrane fluid of RA and OA patients

RA synovial tissue and synovial fluid are obtained from 21 RA patients in Rheumatoid immunology departments of Shenzhen Hospital, Beijing university, and meet classification standards of European antirheumatic Association of the American college of rheumatology (ACR/EULARRA) for RA in 2010.

OA synovial tissue and synovial fluid are from 18 OA patients in Shenzhen Hospital rheumatological department of Beijing university, and meet the gout classification standard of the American college of rheumatology/European antirheumatic alliance in 2015.

All enrolled RA patients were informed of the purpose of specimen collection and signed a written consent. The experiment was approved by the ethical committee of Shenzhen hospital, Beijing university, and was conducted according to the ethical guidance of the committee. Specific clinical characteristics and laboratory test data are shown in the following table:

using high throughput sequencing technology, the expression profiles of circRNA in RA-FLS (rheumatoid arthritis fibroblast-like synoviocytes) and OA-FLS cell lines were compared, and the circular RNA with the highest differential expression (shown in FIG. 1) hsa _ circ _0000137 was found.

FIG. 1 is a volcano plot of differentially expressed genes, with dashed lines dividing FIG. 1 into left, middle and right regions, respectively a down-regulated circRNA set, an invariant circRNA set and an up-regulated circRNA set.

The position of hsa _ circ _0000137 is chr1:155408117 and 155408859, and the nucleotide sequence is shown as SEQ ID NO. 1.

S2 detection of Gene expression

(1) Extraction of synovial tissue circRNA

Grinding the synovium tissue of rheumatoid arthritis lesion in liquid nitrogen, and adding 1ml of TRIzol into every 50-100 mg of synovium tissue^™Reagents (Invitrogen, Catalog No.: 15596018) were homogenized using a homogenizer, and each 1ml of TRIzol was added^™0.2ml of chloroform was added to the reagent, followed by vigorous shaking for 15 seconds and standing at room temperature for 5 min. Centrifuging at 10000 Xg for 15min at the temperature of 2-8 ℃.

The homogenate was separated into a clear upper aqueous phase (containing RNA), interphase and lower organic layer (containing DNA and protein), the aqueous phase was transferred to a fresh EP tube, an equal volume of isopropanol was added, and the mixture was allowed to stand at room temperature for 10min to precipitate RNA. Centrifugation is carried out at 2-8 ℃ and 10000 Xg for 10min, supernatant is aspirated, and RNA precipitation is washed with 5% ethanol after ice-cold.

At least 1ml 75% ethanol per 1ml TRIzol reagent was used. Centrifuging at 2-8 ℃ and no more than 7500 Xg for 5min, and discarding the supernatant. Air-drying in a clean bench for about 5-10 min, and adding 25-200 μ l RNase-free water to dissolve RNA.

After the RA sample and the OA sample are mixed respectively, the mixture is sent to Beijing Nuo cereal induced biotechnology limited to carry out RNA-seq detection and circRNA expression profile analysis.

RNA-seq: namely transcriptome sequencing technology, and adopts high-throughput sequencing technology to perform sequencing analysis so as to reflect the expression level of the circRNA. Standard work flow starts with RNA extraction, to mRNA enrichment or removal of ribosomal RNA, reverse transcription of cDNA and preparation of sequencing libraries linked by linker sequences, followed by sequencing using a high throughput sequencing platform, each sample typically being sequenced to 10000000-30000000 read length. Finally, the data obtained by the experiment are compared or spliced to be sequenced to be read to a transcriptome, the read length covering the transcriptome is quantified, the filtration and the normalization among samples are carried out, and the difference of the expression level of each gene among each sample group is described by using a statistical model.

(2) Extraction of synovial fluid circRNA

The extraction of circRNA in synovial fluid was performed using free RNA extraction Kit (BIOG cfRNA Easy Kit, hundredth biotech, inc.) according to the instructions.

(3) Reverse transcription

The reagents in the reverse transcription Kit Transcriptor First Strand cDNA Synthesis Kit were thawed and placed on ice. The reverse transcription system was prepared in a PCR tube.

Setting a PCR program: thermal denaturation of circRNA, Random primers, at 65 ℃, 10min, 4 ℃, forever, taken out and placed on ice, and the following ingredients were added successively in the PCR tube:

mix well with a pipette, collect the sample at the bottom of the tube using a centrifuge, and place it on a PCR instrument.

Setting a PCR program: the reaction was terminated at 50 ℃, 60 min, 85 ℃, 5min (reverse transcriptase inactivated), 4 ℃, forever, and the cDNA samples obtained by reverse transcription were stored to-20 ℃.

(4) Real-time fluorescent quantitative PCR

The real-time fluorescent quantitative PCR (RT-qPCR) reagent of FastStart Universal SYBR Green Master (Rox) was used, each sample was repeated three times, and the samples were loaded according to the system shown in the following table, where the cDNA could be diluted by the same fold and loaded.

After sample application on ice, the solution was collected to the bottom of the tube after centrifugation and amplified according to the following RT-qPCR amplification procedure:

designing a forward primer sequence (SEQ ID NO. 2) and a reverse primer sequence (SEQ ID NO. 3), wherein the SEQ ID NO.2 comprises a trans-cyclization site primer.

SEQ ID NO.2：TAAAAGTAAACGGAGTTCCC；

SEQ ID NO.3：AAGAAGGTGGTGCAGAGGCA。

And (3) data analysis: after the reaction, the amplification curve and the dissolution curve of RT-qPCR were confirmed, and a standard curve was prepared for PCR quantification. C of the sample_tValues are averaged over triplicates. By Delta C_tThe method can be used for relatively quantifying the target gene. Relative expression level of target gene =2^-ΔΔCt，ΔΔC_t= Experimental group (Gene of interest C)_tReference gene C_t) Control group (Gene of interest C)_tReference gene C_t). In the experiment, beta-actin is used as an internal reference gene, Con is used as a control group, and the target genes are relatively quantified.

The experimental results are as follows:

synovial tissue was examined in 21 RA patients and 18 OA patients using Real-time PCR and it was found that hsa _ circ _0000137 was generally increased in expression of RA-FLS (rheumatoid arthritis fibroblast-like synovial cells) compared to OA-FLS (osteoarthritis fibroblast-like synovial cells) (FIG. 2A), suggesting that hsa _ circ _0000137 is associated with disease activity of RA.

In addition, CRP blood concentration (clinical case from patient) which is a closely related index to disease activity is linearly related to the expression of hsa _ circ _0000137 in synovial fluid of RA patients (FIG. 2B, R²= 0.6974); further studies showed that expression of hsa _ circ _0000137 in synovial fluid is linearly related to the RA patient disease Activity index DAS28 (assessed by the clinician, data from the patient's electronic medical record) (FIG. 2C, R)²= 0.6237). These indicate that there is a linear correlation between hsa _ circ _0000137 in synovial fluid and disease activity, and that it can be used as an index for determining the activity of RA-FLS. These results show that hsa _ circ _0000137 is associated with disease activity of RA, can be used as a novel rheumatoid arthritis fibroblast-like synovial cell activity detection index, can be applied to assessment of rheumatoid arthritis synovial lesion degree and prognosis and the like, and can also be used as rheumatoid arthritis fibroblast-like synovial membrane fine synovial cell activity detection indexThe cell regulation and molecular diagnosis and treatment strategy research provides a new approach.

S3, biological function detection

Using high-glucose DMEM medium containing 20% fetal calf serum and 1% double antibody in 5% CO₂RA Synovial Fibroblasts MH7A (RA synovium Fibroblasts) and HFLS (Normal synovium Fibroblasts) were cultured in a 37 ℃ incubator.

MH7A cells and HFLS cells were purchased from ATCC, USA under the respective serial numbers AC337864 and AC 38586.

In vitro experiments, the cell line used was the immortalized RA-FLS cell line MH7A as the subject of study, the normal FLS cell line HFLS as the control cell line, and siRNA of hsa _ circ _0000137 was designed by Shanghai Biotechnology Ltd.

The experimental results are as follows:

FIG. 3 shows the results of the cell function assay of hsa _ circ _ 0000137; FIG. 3A shows that MH7A cells transfected with PBS (Ctrl), nonsense strand (Si NC) and SiRNA of hsa _ circ _0000137 (Si hsa _ circ _ 0000137) were tested for expression of hsa _ circ _0000137 by real-time fluorescent quantitative PCR, and the results of the experiment showed that siRNA silenced the target hsa _ circ _ 000013770% or more.

S4, cell viability assay

(1) Cell proliferation assay

Cells in the logarithmic growth phase are planted in a 96-well plate according to 3000 cells per well and divided into three groups, after the cells are attached to the wall, one group is given PBS (Ctrl), and the other two groups are respectively transfected with siRNA of nonsense chain (Si NC) and hsa _ circ _0000137 (Si hsa _ circ _ 0000137).

On

days

0, 1, 2, 3, and 4, the WST-1 cell proliferation and cytotoxicity assay kit (bi yun day biotechnology, product No. C0036L) was used according to the instructions. The absorbance value (OD value) at 450nm was measured with a microplate reader (Tecan, Switzerland) and the fine proliferation rate was calculated according to the formula.

The experimental result shows that: following down-regulation of hsa _ circ _0000137 expression in MH7A, the cell proliferation capacity was significantly reduced (fig. 3B).

(2) Transwell cell invasion capacity detection

A. Preparation of matrix-free gel Transwell cell

Coating a basement membrane:

matrigel (Chemical Book, CAS number 119978-18-6) was diluted 1:8 and coated on the top of the bottom membrane of a Transwell cell (corning, usa, cat # 3415) and air dried for 3 h.

Hydration of basement membrane: the residual liquid in the plate was aspirated, and 50. mu.L of 10g/L Serum-free medium of Bovine Serum Albumin (Bovine Serum Albumin, BSA, Sigma, USA; cat # A7030) was added to each well at 37 ℃ for 30 min.

B. Preparation of cell suspensions

MH7A cells were divided into three groups, one group was given PBS, the other two groups were transfected with siRNA of nonsense strand and hsa _ circ _0000137 for 24h, cells were trypsinized, digestion was terminated, the culture was centrifuged and the medium was discarded, washed 1-2 times with PBS, and resuspended in serum-free medium containing BSA. Adjusting cell density, too many cells crossing the membrane too fast to count if finally counted by counting; if too few, it may not be possible to detect the time point at which all cells have passed, and therefore it is at a minimum ensured that a certain amount of cells will still be present in the upper chamber at the time of sampling.

C. Seeding cells

200 μ L of the cell suspension was added to the upper chamber of a Transwell chamber and 600 μ L of medium containing 10% FBS was added to the lower chamber of a 24-well plate.

The plates were incubated at 37 ℃ CO₂And (5) culturing for 24 hours in an incubator.

The chamber was removed, rinsed 2 times with PBS, the cells in the upper microporous membrane layer of the chamber were carefully wiped off with a cotton swab, fixed in a 24-well plate with 4% paraformaldehyde (or 95% ethanol) for 20 min, and stained with crystal violet solution for 15 min.

The photographs were taken under an inverted microscope, 10 fields were counted at random for each sample, averaged, and statistically analyzed.

D. Statistical analysis

The Student's t test was used to assess the significance of differences for the two sets of data-to-data analyses, and one-way analysis of variance (ANOVA) was used for the multiple sets of sample-to-sample analyses. A p value <0.05 considered significant for the differences (, p < 0.05;, p < 0.01).

The results are shown in FIG. 3C, which shows that the level of invasion is significantly reduced after the expression level of hsa _ circ _0000137 in MH7A cells is knocked down by SiRNA, and the results have statistical significance (P < 0.01)

S5, detection of expression levels of some inflammatory factors closely related to RA development in MH7A after downregulation of hsa _ circ _0000137 by Real-time PCR

In the course of the onset of RA, inflammatory cytokines such as TNF- α (tumor necrosis factor- α), IL-1 β, chemokine CCL2, interleukin 6 (IL-6), and matrix metalloproteinase 9 (MMP 9) are excessively secreted, which in turn causes tissue damage and bone erosion.

In this experiment, Real-time PCR was used to detect the expression levels of some of the inflammatory factors closely related to RA development in MH7A after downregulation of hsa _ circ _0000137, and the Real-time quantitative PCR protocol was as described above in S2.

The sequences of the other primers (synthesized by Shanghai Biotechnology, Ltd.) are shown in the following table:

the results are shown in fig. 4, after the expression level of hsa _ circ _0000137 is knocked down by SiRNA in MH7A cells, the expressions of chemokines CCL2, interleukin 6 (IL-6) and matrix metalloproteinase 9 (MMP 9) which are closely related to the development of RA disease course are also obviously down-regulated, and the important role of the hsa _ circ _0000137 in the development process of RA is confirmed again.

Example 2

The rheumatoid arthritis detection kit is used for detecting a circular RNA marker hsa _ circ _0000137 and comprises a forward primer and a reverse primer, wherein the sequence of the forward primer is shown as SEQ ID NO.2, and the sequence of the reverse primer is shown as SEQ ID NO. 3.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Sequence listing

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Claims

1. The application of the circular RNA marker in preparing the rheumatoid arthritis detection kit is characterized in that: the circular RNA marker is hsa _ circ _0000137, and the nucleotide sequence is shown in SEQ ID NO. 1; hsa _ circ _0000137 in synovial tissue and fluid can be used as a detection marker for rheumatoid arthritis.

2. Use according to claim 1, characterized in that: the rheumatoid arthritis detection kit comprises a forward primer and a reverse primer, wherein the sequence of the forward primer is shown as SEQ ID NO.2, and the sequence of the reverse primer is shown as SEQ ID NO. 3.