CN114350785A

CN114350785A - Application of circular RNA hsa _ circ _0000734 gene

Info

Publication number: CN114350785A
Application number: CN202210018948.4A
Authority: CN
Inventors: 刘健; 孙艳秋; 文建庭; 忻凌; 姜辉; 万磊
Original assignee: First Affiliated Hospital of AHUTCM
Current assignee: First Affiliated Hospital of AHUTCM
Priority date: 2022-01-05
Filing date: 2022-01-05
Publication date: 2022-04-15
Anticipated expiration: 2042-01-05
Also published as: CN114350785B

Abstract

The invention relates to an application of a circular RNA hsa _ circ _0000734 gene, wherein a specific PCR primer is designed and synthesized according to an extracted total RNA sequence, and an RT-qPCR technology is utilized to find that the expression of hsa _ circ _0000734 in peripheral blood mononuclear cells of a Rheumatoid Arthritis (RA) patient is obviously increased and has a positive correlation with the course of disease and disease activity indexes of the RA patient, so that the hsa _ circ _0000734 gene can be applied to the preparation of a reagent for auxiliary diagnosis, treatment or prognosis evaluation of the rheumatoid arthritis; the invention designs and synthesizes a specific overexpression and interference sequence simultaneously, can inhibit inflammation, proliferation and migration of RA patient synovial Fibroblast (FLS) induced by TNF-alpha by regulating NF-kB signal path, promotes apoptosis of cells, and can be used as a tool for molecular targeted therapy of RA.

Description

Application of circular RNA hsa _ circ _0000734 gene

Technical Field

The invention belongs to the field of rheumatism molecular biology, and particularly relates to application of a circular RNA hsa _ circ _0000734 gene. Specifically, the invention relates to application of the circular RNA hsa _ circ _0000734 gene in preparation of RA auxiliary diagnostic reagents, molecular targeted therapeutic reagents or prognostic evaluation reagents.

Background

Rheumatoid Arthritis (RA) is characterized by symmetrical, peripheral and polyarthritis as the main component, and has chronic, progressive and invasive characteristics, which may lead to systemic multiple system damage, an autoimmune and inflammatory disease, and finally joint deformity and loss of function (Fraenkel Liana, Bathon Joan M, England Bryant R, et al.2021 American College of Rheumatologic guidelines for the Treatment of Rheumatoid Arthritis [ J ]. Arthritis Rheumatoid Arthritis, 2021,73:1108 3; Zhajianan, Guo Shining, Schrodi vessel J, et al.112molecular and immune surgery in Rheumatoid Arthritis [ J.: listing J.20212). RA is complicated in pathogenesis, and its occurrence and development may be closely related to genetics, the surrounding environment, immune function disorder, platelet activation, etc. The prevalence rate of RA is 1:3, the peak prevalence is 30-50 years old, the prevalence rate of RA in mainland areas of China is 0.2-0.4%, the global prevalence rate is 0.5-1%, and the annual incidence rate is 20/100-50/100 thousands of people (Zeng Q Y, Ren C, Darmawan J, et al, Rheomatic Diseases in China [ J ]. Therits Research & Therapy,2008,10 (1); Pan L, Wang T.Features of cardiac remodelling in Patients with acid core Syndrome combined with Rheomatoid disease [ J ]. Sci Rep,2017,7 (1)). The exact etiology and pathogenesis of RA is currently unclear. Environmental factors, genetic background, environmental-genetic interactions and epigenetic modifications determine the onset of RA (McInnes IB, Schett G. the pathogenesis of rhematoid arthritis. N Engl J Med.2011; 365(23): 2205-). 2219). The development of non-steroidal anti-inflammatory drugs, glucocorticoids, biologicals, and the like has had a great effect on the treatment of RA, but RA has not been cured so far. Therefore, the search for biomarkers for early diagnosis, targeted therapy and prognosis evaluation of RA is of great significance for the treatment and prognosis of RA.

In recent years, with the development and wide application of sequencing technologies and bioinformatics, more and more non-coding RNAs (non-coding RNAs), such as circular RNAs (circular RNAs), long non-coding RNAs (long non-coding RNAs), micro RNAs (micro RNAs, mirnas), and the like, play an important role in RA pathogenesis. The circular RNA is a novel non-coding RNA without a 5 'end and a 3' end and a covalent closed loop structure, and has the characteristics of sequence conservation and structural stability among species, specific expression of cells or tissues and the like (Wen Jianting, Liu Jian, Wang Xin, et al]Int Immunopharmacol,2021,92: 107366). It plays an important role in miRNA molecular sponge, regulation of gene expression, interaction with protein and other aspects (Zhang Juan, Zhang Yue, Ma Yeye, et al]Int J Nanomedicine,2021,16: 7977-. In addition, circular RNA is involved in the development of various diseases and can be used as a biological marker for early diagnosis and prognosis judgment of diseases (Chen Yufeng, Xu Xianghe, Li Xuegang, et al.identification of circular RNAs hsa _ circ _0140271in peripheral blood across cell as an animal diagnostic biomarker for a large rheum arthritis, [ J ] J]J Orthop Surg Res,2021,16: 647). The cirRNAs are differentially expressed in various autoimmune diseases and are shown to play important regulatory roles, such as Rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, ankylosing spondylitis, etc. (Ouyang Q, Liu C, Lu X, et al. identification of Circular RNAs Circuit 0005008and Circuit 0005198in plasmid as Novel Biomarkers for New-Onset Rheumatoid Arthroitis. front Pharmacol.2021; 12: 722017; Luo Q, Li X, Fu B, et al. expression profile and diagnostic value of Circular RNAs in peripheralblood from patients with systemic lupus erythematosus.Mol Med Rep.2021；23(1):1；Li F,Liu Z,Zhang B,et al.Circular RNA sequencing indicates circ-IQGAP2 and circ-ZC3H6 as noninvasive biomarkers of primary

syndrome. rheologic (Oxford) 2020; 59(9) 2603-2615; tang YP, Zhang QB, Dai F, et al.circular RNAs in periheral blood monnouceller cells from bottom alkyl voiding. Chin Med J (Engl). 2021; 134(21):2573-2582). Some studies at present indicate that the cirRNAs are involved in processes such as RA immune inflammation, synovial cell proliferation, migration, apoptosis, etc., but the mechanism thereof is less studied. The human hsa _ circ _0000734 gene is located on chromosome 17, and the mature sequence after splicing is 328 bp. At present, no report is found on the application of hsa _ circ _0000734 gene and RA in detection and treatment.

Disclosure of Invention

Aiming at the technical defects of lack of diagnosis markers, lack of targeting and pertinence of treatment medicines and the like of the existing rheumatoid arthritis, the invention provides application of a circular RNA hsa _ circ _0000734 gene in the aspects of preparing RA detection reagents, RA prognosis evaluation reagents and the like.

First, the present invention provides a pair of primer pairs for detecting the hsa _ circ _0000734 gene of circular RNA, the nucleotide sequences of the primer pairs are shown as SEQ ID NO.1 and SEQ ID NO. 2.

Secondly, the invention provides a reagent for detecting the expression quantity of hsa _ circ _0000734 gene of circular RNA, which comprises a primer pair with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2; further, the detection reagent comprises: 5uL2 XSYBR Green mix, 1uL Forward primer (10uM), 1uL Reverse primer (10uM), 1ug/100uL of cDNA 1uL, 1uL each of SEQ ID NO.1 and SEQ ID NO.2 at a concentration of 10umol/L, RNase Free water2 uL.

Thirdly, the invention also provides the application of the circular RNA hsa _ circ _0000734 gene in preparing the RA targeted therapeutic agent, in particular to preparing a specific over-expression sequence SEQ ID NO.3 and a small interference RAN sequence according to the circular RNA hsa _ circ _0000734 gene, and the sequence is used for over-expressing and interfering the hsa _ circ _0000734 gene in synovial fibroblasts.

Fourthly, the present invention provides a rheumatoid arthritis RA diagnostic reagent, which comprises a specific over-expression sequence pcDNA3.1-hsa _ circ _0000734 and a small interference sequence si-hsa _ circ _0000734, wherein the nucleotide sequence of the specific over-expression sequence pcDNA3.1-hsa _ circ _0000734 is shown in SEQ ID NO.3, and the specific over-expression sequence pcDNA3.1-hsa _ circ _0000734 and the small interference sequence si-hsa _ circ _0000734 can be used for transfecting synovial fibroblasts, so that the hsa _ circ _0000734 gene is over-expressed and small-interfered in the cells.

The result shows that the transfection of pcDNA3.1-hsa _ circ _0000734 can obviously improve the expression level of hsa _ circ _0000734 in FLS induced by TNF-alpha, promote the generation of inflammatory cytokines, improve the proliferation and migration capacity of cells, activate NF-kB signal channels and reduce the apoptosis of the cells. And the transfected siRNA-hsa _ circ _0000734 can obviously reduce the expression of hsa _ circ _0000734 in RA-FLS induced by TNF-alpha, inhibit the generation of inflammatory cytokines, inhibit the activation of NF-kB signal channels, reduce the proliferation and migration capacity of cells and promote the apoptosis of the cells.

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

the present application, which systematically explores the expression of the hsa _ circ _0000734 gene of circular RNA in RA, confirms that: compared with a healthy control group, the expression of hsa _ circ _0000734 in PBMCs of the RA patients is obviously increased, and the expression is positively correlated with disease activity indexes of ESR, CRP, RF, anti-CCP and DAS 28. The overexpression of hsa _ circ _0000734 obviously increases the inflammatory reaction of TNF-alpha induced RA-FLS, improves the proliferation and migration capacity of cells, activates NF-kB signal channels, and reduces the apoptosis of the cells; the small interference hsa _ circ _0000734 can obviously inhibit RA-FLS inflammatory reaction induced by TNF-alpha, reduce the proliferation and migration capacity of cells, inhibit the activation of NF-kB signal channel and promote the apoptosis of the cells. The expression and function of hsa _ circ _0000734 gene in RA are discovered and verified. The discovery is expected to further enrich and perfect the research of the pathogenesis of RA, and also bring hope for developing novel biomarkers for early diagnosis, targeted treatment and prognosis evaluation of RA.

Drawings

FIG. 1 shows that circular RNA hsa-circ _0000734 is expressed in PBMCs of RA patients.

FIG. 2 shows the correlation analysis of hsa-circ _0000734 levels in PBMCs of RA patients with clinical markers.

FIG. 3 shows RT-qPCR detection of expression and transfection efficiency of hsa-circ _0000734 in FLS.

FIG. 4 shows the ELISA assay for the effects of hsa-circ _0000734 on the inflammatory factors IL-6, IL-17, and IL-23.

FIG. 5 shows the effect of the CCK8 method on the proliferative capacity of cells tested by hsa-circ-0000734.

FIG. 6 shows the effect of Transwell on the migratory capacity of cells tested by hsa-circ _ 0000734.

FIG. 7 shows the Western Blot method to examine the effect of hsa-circ _0000734 on NF-. kappa.B signaling pathway.

FIG. 8 shows the immunofluorescence assay to detect the effect of hsa-circ _0000734 on the NF-. kappa.B signaling pathway.

FIG. 9 shows flow cytometry to examine the effect of hsa-circ _0000734 on apoptosis.

Detailed Description

The present invention will be further described with reference to specific embodiments, which are provided for the purpose of illustrating the principles and procedures of the present invention and are not to be construed as limiting the invention. Those skilled in the art will appreciate that various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention.

The following examples are examples of experimental methods in which specific conditions are not specified, and the tests are usually carried out under conventional conditions or under conditions recommended by the manufacturers.

Example 1

The expression of circular RNA hsa _ circ _0000734 in PBMCs of RA patients was explored.

After the project is examined by the ethical committee, the applicant systematically studies new diagnosis and treatment targets of human RA, extracts PBMCs by extracting peripheral blood of RA patients, and detects the expression of hsa _ circ _0000734 gene by RT-qPCR method.

In this example, 30 blood samples of RA patients, 30 blood samples of healthy persons in a control group, and 60 samples in total were collected (for 30 RA patients, see Table 1 for specific information and Table 2 for sample characteristics)

TABLE 1 sample information

TABLE 2 sample characterization

30 RA cases and 30 healthy control group PBMCs are detected by adopting RT-qPCR technology, and the specific detection steps are as follows:

1. extraction of PBMCs from RA patients

At room temperature, 4ml of venous blood is collected aseptically by using an EDTA-K2 anticoagulation tube, and the venous blood is immediately and gently shaken up to be anticoagulated. Sucking whole blood into a sterile 15ml centrifuge tube, adding phosphate buffer saline solution PBS with the same volume with the whole blood into the sterile centrifuge tube, and fully mixing the whole blood and the phosphate buffer saline solution to ensure that the blood is diluted by equal times, reduce the agglutination of red blood cells and improve the separation effect. The Ficoll fraction (whole blood: PBS: Ficoll fraction 1: 1: 1.5 by volume) was placed in a 50ml sterile centrifuge tube, and the diluted blood was slowly added to the Ficoll fraction by attaching to the centrifuge tube wall, taking care not to mix the blood into the Ficoll fraction. After equilibration, the cells were centrifuged at 400g/min for 35 minutes at room temperature in a centrifuge. After centrifugation, the tube is divided into three layers, wherein the upper layer is plasma and Hank's liquid, and the lower layer is mainly red blood cells and granulocytes. The middle layer is lymphocyte separation liquid, and a white cloud layer narrow band mainly comprising mononuclear cells including lymphocytes and monocytes is arranged at the interface between the upper layer and the middle layer. And inserting the gun head into the cloud layer to suck the mononuclear cells. Put into another sterile 50ml centrifuge tube, add PBS with more than 5 times volume, centrifuge for 20 minutes at 400g/min, and wash the cells twice. After washing and twice centrifugation, the red precipitate at the bottom of the centrifuge tube is more, about 5ml of erythrocyte lysate is added to resuspend the cells, and after standing for 5 minutes, 400g/min is centrifuged for 20 minutes. The supernatant was discarded, the cells resuspended in 1ml PBS, and added to a fresh sterile 1.5ml centrifuge tube and centrifuged at 3500r/min for 10 minutes. Removing supernatant, precipitating to obtain PBMC, and storing in a refrigerator at-80 deg.C.

2. RNA extraction

The cell pellet was collected and lysed by adding 1ml TRIzol. After completion of the lysis, 0.2mL of chloroform was added, followed by vigorous shaking for 15 seconds, and the mixture was left at room temperature for 5 minutes. Centrifuge at 12000rpm for 10 minutes at 4 ℃ and take the supernatant (approximately 500ul) and add to another EP tube. 0.5mL of pre-cooled isopropanol was added, gently mixed and incubated at-20 ℃ for 30 minutes. Centrifugation was carried out at 12000rpm at 4 ℃ for 15 minutes, and the supernatant was discarded. 1mL of pre-chilled 75% ethanol (absolute ethanol diluted with DEPC water) was added. Centrifuge at 12000rpm for 5 minutes at 4 ℃ and discard the supernatant. Repeating the above steps, drying RNA precipitate at room temperature, adding 20-50 μ L DEPC water, and storing at-80 deg.C for use.

3. Determination of RNA concentration and purity

mu.L of the RNA sample was pipetted into the buffer and the absorbance at 260nm and 280nm was determined. Based on the ratio of OD260/OD280, RNA quality was estimated. The ratio of OD260/OD280 is 1.8-2.0, and can meet the experimental requirements. When OD260/OD280 is less than 1.8, the pollution of protein in the solution is obvious; when OD260/OD280>2.2, it indicates that RNA has been degraded.

4. RT reaction

Reverse transcription Kit PrimeScriptRT reagent Kit. The method comprises two steps, wherein the first step is to remove genome DNA, and the reaction system comprises the following steps: add total RNA (1. mu.g), 5 Xg DNA Eraser Buffer 2.0. mu.L, gDNA Eraser 1.0. mu.L, RNase Free water to 10. mu.L, then on PCR instrument at 42 ℃ for 2min, immediately ice-wash for 1 min. A second step of reverse transcription reaction system: PrimeScript RT Enzyme Mix I1.0. mu.L, RT Primer Mix 1.0. mu.L, RNase Free dH2O 4.0.0. mu.L, RevertaID TM M-MuLV Reverse Transcnriptase 4.0. mu.L, RNase Free dH2O 4.0.0. mu.L, 37 ℃, 15 min; taking out the reaction solution at 85 ℃ for 5s to obtain cDNA, and storing at-80 ℃ for later use. The reaction system and reaction conditions are shown in the following table.

1) Removing genome DNA reaction: to a 0.2mL EP tube was added:

the reaction conditions were as follows:

2) the reverse transcription reaction system is as follows:

the reaction conditions were as follows:

5. fluorescent quantitative PCR reaction

Taking out cDNA as a template for fluorescence quantification, wherein the reaction system comprises: 2 × SYBR Green mix 5uL, forward and reverse primers 1uL, template cDNA 1uL, RNase Free water2uL, PCR amplification reaction is carried out on a fluorescent quantitative PCR instrument under the reaction condition of 95 ℃ for 1min, 40 cycles are carried out at 95 ℃ for 20s and 60 ℃ for 1min to obtain the Ct value of each sample, beta-actin is used as an internal reference gene, and 2 samples are adopted as experimental results^-△△CTThe method of (3) for analysis.

1) Taking out cDNA as a template for fluorescence quantification, wherein the reaction system is as follows:

2) the reaction conditions were as follows:

3) primer information for the detection index is as follows:

the experimental results are shown in figure 1, and the results show that the expression of the circular RNA hsa-circ _0000734 of the RA patients is obviously up-regulated (P <0.001) compared with the healthy control group, and the difference has statistical significance.

As shown in FIG. 2, Spearman correlation analysis showed that the expression level of circular RNAhsa-circ _0000734 in RA patients was positively correlated with the disease course, disease activity index ESR, CRP, RF, anti-CCP, DAS28 score of RA patients, with no significant correlation with age (P > 0.05).

Example 2

Circular RNA hsa-circ _0000734 overexpression sequence and small interference sequence and application thereof

This example is designed for the mature sequence of circular RNA hsa-circ _0000734, and synthesizes its specific over-expression sequence pcDNA3.1-hsa-circ _0000734 and small interference sequence si-hsa-circ _0000734, transfects FLS, so that hsa-circ _0000734 gene is over-expressed and small interference in the cell, and further applies the over-expression and small interference gene vector to regulate the inflammation, proliferation, migration and apoptosis of FLS.

Wherein, the nucleotide sequence of the specific overexpression sequence pcDNA3.1-hsa _ circ _0000734 is shown in SEQ ID NO. 3. The nucleotide sequence of the small interference sequence si-hsa _ circ _0000734 is:

Sense：GCCCUAUAAUGAAGGCCAUTT

Antisense：AUGGCCUUCAUUAUAGGGCTT

1. the transfection conditions and procedure were as follows:

(1) FLS cells were cultured in 6cm dish to 80-90% confluency, the culture broth was decanted, and the cells were washed twice with 3ml PBS; (2) adding 1mL of Trypsin-EDTA solution, mixing uniformly, carefully absorbing pancreatin solution, and standing at 37 ℃ for 3 minutes; (3) adding 2mL of DMEM culture solution containing 10% FBS, and blowing to make the cells form single cell suspension; (4) counting with a blood counting plate, diluting the cells to3×10⁵cell/mL; (5) by 5X 10³Inoculating 96-well plates with the cell/well concentration, uniformly mixing, and culturing at 37 ℃ for 24h by using 5% CO 2; (6) the transfection reagent Lipofectamin2000 was used to transfect the overexpression plasmids at the following doses, each dose setting three more wells;

Lipo(uL)	0.2	0.3	0.4
				plasmid (ug)	0.2	0.3	0.5

(7) Adding 75 mu L (25 mu L/well x 3well) of serum-free DMEM into a 1.5mL EP tube, adding plasmids with different dosages calculated according to the table, uniformly mixing, taking another 1.5mL EP tube, adding 75 mu L (25 mu L/well x 3well) of serum-free DMEM, adding Lipofectamin2000 with corresponding dosages calculated according to the table, uniformly mixing, standing at room temperature for 5 minutes, mixing the two groups of tubes, standing at room temperature for 20 minutes, sucking out the culture solution in a 96-well plate, and adding 50 mu L of serum-free DMEM culture solution into each well; (8) adding the transfection mixture dropwise into a 96-well plate, uniformly mixing, and incubating for 5 hours in an incubator; (9) and (4) transfecting the plasmid group, sucking and discarding the transfection solution, replacing the transfection solution with a complete culture medium, and observing the transfection solution after incubating in an incubator for 24 hours. (10) RT-qPCR was used to examine the transfection efficiency of the cells. Add 100. mu. mol (5. mu.l) siRNA to 250. mu.l serum-free medium and mix gently; lipofectamine 2000 was gently shaken before use, and then 5ul of Lipofectamine 2000 was diluted in 250. mu.l of serum-free medium and incubated for 5 minutes at room temperature. The diluted siRNA and Lipofectamine 2000 were mixed (to make the total volume 500. mu.l), gently mixed, and left at room temperature for 20 minutes. Add 500. mu.l of transfection solution to each well of cells and shake gently. After culturing at 37 ℃ for 24 hours, gene expression is detected, and after transfection for 4-6 hours, culture medium is replaced for culture. After 24 hours of transfection, cells were trypsinized, washed 2 times with PBS, cell pellets were collected and stored in a-80 ℃ freezer.

2. Grouping of cells

Digesting normal FLS, RA-FLS cells, terminating digestion, centrifuging, discarding culture medium, washing with PBS 2 times, resuspending with culture medium, and performing 5 × 10⁵Each cell/well was seeded in a 6-well plate, and after the cells were attached to the wall, they were stimulated with TNF-. alpha. (20ng/ml) and placed in a 5% incubator at 37 ℃ overnight for culture. The grouping is as follows:

grouping 1:

grouping 2:

as shown in FIG. 3, the RT-qPCR method detects the expression of hsa-circ _ 0000734. Compared with normal FLS (NC-FLS), the expression level of hsa-circ _0000734 in RA-FLS is remarkably increased; after TNF-alpha induced RA-FLS, expression of hsa-circ _0000734 was further increased (FIG. 3A). The expression of hsa-circ _0000734 was significantly increased after transfection of the hsa-circ _0000734 overexpression plasmid, and the expression of hsa-circ _0000734 was significantly decreased after transfection of the si-circ _0000734 small interfering RNA (FIG. 3B), indicating that the overexpression plasmid and siRNA of hsa-circ _0000734 were successfully transfected.

FIG. 4 shows the results of ELISA kit to detect the effect of hsa-circ _0000734 on the inflammatory factors IL-6, IL-17, IL-23. Compared with the RA-FLS group, the expression of IL-6, IL-17 and IL-23 in the TNF-alpha + RA-FLS group is increased, and compared with the pcDNA3.1-NC group, the expression of IL-6, IL-17 and IL-23 in the pcDNA3.1-circ _0000734 group is obviously increased; compared with the si-NC group, the expression of IL-6, IL-17 and IL-23 in the si-circ-0000734 group is obviously reduced.

FIG. 5 shows the results of CCK8 kit to determine the effect of hsa-circ _0000734 on the proliferative capacity of cells. The proliferation of cells in the TNF-alpha + RA-FLS group was significantly increased compared to the RA-FLS group; compared with the pcDNA3.1-NC group, the proliferation capacity of the cells of the pcDNA3.1-circ _0000734 group is obviously improved; the proliferation potency of the cells in the si-circ _0000734 group was significantly reduced compared to the si-NC group.

FIG. 6 shows the results of Transwell testing the effect of hsa-circ _0000734 on the migratory capacity of cells. The migration of cells of the TNF-alpha + RA-FLS group was significantly increased compared to the RA-FLS group; compared with the pcDNA3.1-NC group, the migration capacity of the cells of the pcDNA3.1-circ _0000734 group is obviously improved; the migration capacity of the cells in the si-circ _0000734 group was significantly reduced compared to the si-NC group.

FIG. 7 shows the results of Western Blot to examine the effect of hsa-circ _0000734 on NF-. kappa.B signaling pathway. Compared with the RA-FLS group, the expression of IKK alpha and P-P65 protein in the TNF-alpha + RA-FLS group is obviously increased; compared with the pcDNA3.1-NC group, the expression of IKK alpha and P-P65 proteins of the pcDNA3.1-circ _0000734 group is obviously increased; IKK alpha, P-P65 protein expression was significantly reduced in the si-circ _0000734 group compared to the si-NC group.

FIG. 8 shows the results of immunofluorescence assays to detect the effect of hsa-circ _0000734 on the NF-. kappa.B signaling pathway. Compared with the RA-FLS group, the optical density of the IKK alpha and P-P65 proteins in the TNF-alpha + RA-FLS group is obviously enhanced; compared with the pcDNA3.1-NC group, the pcDNA3.1-circ _0000734 group has enhanced IKK alpha and P-P65 protein optical density; the optical density of IKK α, P-P65 proteins was significantly reduced in the si-circ _0000734 group compared to the si-NC group.

The results in FIG. 9 show that flow cytometry examined the effect of hsa-circ _0000734 on apoptosis. Compared with the RA-FLS group, the apoptosis of the TNF-alpha + RA-FLS group is obviously reduced; compared with pcDNA3.1-NC, the pcDNA3.1-circ _0000734 group has obviously reduced apoptosis; apoptosis was significantly increased in the si-circ _0000734 group compared to the siRNA-NC group.

The experiments show that the annular RNA hsa-circ _0000734 inhibits inflammation, proliferation and migration of RA-FLS cells induced by TNF-alpha by regulating NF-kB signal channels, promotes apoptosis of the cells, and reduces the over-proliferation state of the RA-FLS. The discovery is expected to further enrich and perfect the research of the pathogenesis of RA, and also bring hope for developing novel biomarkers for early diagnosis, targeted treatment and prognosis evaluation of RA.

The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.

Sequence listing

<110> Anhui TCM university first subsidiary hospital (Anhui province TCM college)

<120> application of circular RNA hsa _ circ _0000734 gene

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

attggcaatc cagtgcccta 20

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

acgagagggt tcaactgtg 19

<210> 3

<211> 328

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gccatcatgc agaaggggga tacaaacata aagcccatcc tccaagtcat caacatccgt 60

cccattacta cggggaatag tccgccgcgt tatcgactgc tcatgagtga tggattgaac 120

actctatcct ctttcatgtt ggcgacacag ttgaaccctc tcgtggagga agaacaattg 180

tccagcaact gtgtatgcca gattcacaga tttattgtga acactctgaa agacggaagg 240

agagtagtta tcttgatgga attagaagtt ttgaagtcag ctgaagcagt tggagtgaag 300

attggcaatc cagtgcccta taatgaag 328

Claims

1. The application of the reagent for detecting the expression quantity of the hsa _ circ _0000734 gene in the preparation of the rheumatoid arthritis detection reagent is characterized in that the reagent for detecting the expression quantity of the hsa _ circ _0000734 gene comprises a primer pair with nucleotide sequences shown in SEQ ID No.1 and SEQ ID No. 2.

2. The use of claim 1, wherein the reagent for detecting the expression level of the hsa _ circ _0000734 gene of the circular RNA further comprises: 5uL of 2 XSSYBR Green mix, 1uL of Forward primer (10uM), 1uL of Reverse primer (10uM), 1ug/100uL of cDNA 1uL, 1uL each of SEQ ID NO.1 and SEQ ID NO.2 at a concentration of 10umol/L, and 2uL of RNase Free water.

3. The application of the circular RNA hsa _ circ _0000734 gene in preparing the RA (rheumatoid arthritis) targeted therapeutic agent is characterized in that a specific overexpression sequence SEQ ID NO.3 and a small interference RAN sequence are prepared according to the circular RNA hsa _ circ _0000734 gene and used for overexpression and interference of the hsa _ circ _0000734 gene in synovial fibroblasts.

4. A rheumatoid arthritis RA diagnostic reagent, which comprises a specific over-expression sequence pcDNA3.1-hsa _ circ _0000734 and a small interference sequence si-hsa _ circ _0000734, wherein the nucleotide sequence of the specific over-expression sequence pcDNA3.1-hsa _ circ _0000734 is shown in SEQ ID NO.3, and the specific over-expression sequence pcDNA3.1-hsa _ circ _0000734 and the small interference sequence si-hsa _ circ _0000734 can be used for synovial membrane transfection of fibroblast, so that the circular RNA hsa _ circ _0000734 gene is over-expressed and small-interfered in the cell.