CN112522391B

CN112522391B - Application of hsa_circ_0008961 as gout diagnosis marker

Info

Publication number: CN112522391B
Application number: CN202011523907.8A
Authority: CN
Inventors: 青玉凤; 张全波; 戴菲; 黄玉琴; 郑建雄; 唐乙萍; 董曾荣; 周闻君
Original assignee: Affiliated Hospital of North Sichuan Medical College
Current assignee: Affiliated Hospital of North Sichuan Medical College
Priority date: 2020-12-10
Filing date: 2020-12-10
Publication date: 2023-06-16
Anticipated expiration: 2040-12-10
Also published as: CN112522391A

Abstract

The invention particularly relates to application of hsa_circ_0008961 as a gout diagnosis marker. The circRNA has important physiological effects in the occurrence and development processes of gout, and in order to further define the regulation mechanism of the circRNA in gout, the circRNAs with the diagnostic significance of gout are developed. According to the invention, the gene chip technology is used for screening and analyzing the circRNA differentially expressed in peripheral blood mononuclear cells of gout patients and healthy control persons, and the chip result is verified through real-time fluorescence quantitative PCR, so that hsa_circ_0008961 can be used as a biomarker for diagnosing gout, and the disclosed corresponding reagent or kit has high diagnostic sensitivity, good specificity and convenient detection for gout patients, and has good clinical application value.

Description

Application of hsa_circ_0008961 as gout diagnosis marker

Technical Field

The invention belongs to the technical field of molecular diagnosis, and particularly relates to a molecular marker for primary gout, which can be used for diagnosing primary gout.

Background

Gout is one of the common auto-inflammatory diseases of humans and is characterized by a disturbance of purine metabolism and/or uric acid excretion that increases uric acid levels in the body, forming monosodium urate crystals that deposit in tissues, leading to arthritis, soft tissue mass (i.e., tophus), uric acid nephropathy, and the like. Related to obvious geographical distribution, meta-analysis related to 17476 study subjects showed that the prevalence of gout in the chinese population is 1.1%; epidemiological investigation in the united states showed that the overall prevalence of gout was 3.9% during 2015-2016 and the overall affected population had an increasing trend year by year.

The pathogenesis of gout is based on hyperuricemia, but not all hyperuricemia progresses to gout. In fact, gout occurs in only 10% of patients with hyperuricemia. Serum uric acid may also fall to normal levels during gout flares. The diagnosis of gout at the present stage mostly depends on clinical manifestations, blood urine routine, blood uric acid measurement, joint ultrasound, joint cavity puncture examination and the like, and although the diagnosis has advantages, the diagnosis is either invasive examination, or has insufficient sensitivity or specificity, or has higher technical requirements, needs abundant clinical experience, and cannot meet clinical needs. Meanwhile, since gout not only can cause joint inflammation, long-term gout can also become an important risk factor of hypertension, hyperlipidemia, cardiovascular diseases and cerebrovascular diseases, so that searching a marker with high sensitivity and specificity for gout diagnosis and early treatment is necessary.

Circular RNAs (circrnas) are a class of closed circular non-coding RNAs whose main biological function is to exert a "sponging" effect by binding to micrornas (mirnas), and in addition, to regulate protein binding, participate in transcriptional regulation and encoding of protein polypeptides, participate in the occurrence of various diseases. In existing studies, circRNA was found to be differentially expressed between disease and normal populations and has the potential to be a biomarker. The inventor performs further excavation and integration analysis on gout sequencing data through a microarray chip technology, constructs a bioinformatic regulation network of the interaction of the circRNA and miRNA by a bioinformatic method, performs GO function analysis and Pathway analysis on a source gene of the circRNA respectively, searches for key molecules involved in regulation and control of gout, and discovers hsa_circ_0008961.

Disclosure of Invention

Application of circRNA markers for primary gout diagnosis in preparation of primary gout diagnosis kit.

The circRNA marker is SEQ ID NO: hsa_circ_0008961 shown in FIG. 1.

The expression level of hsa_circ_0008961 in the sample was detected using fluorescent quantitative PCR technique.

The hsa_circ_0008961 has an increased expression level in the peripheral blood of primary gout patients.

The primer pair of the circRNA marker is a specific primer aiming at hsa_circ_0008961.

The specific primer for hsa_circ_0008961 comprises a primer sequence shown in SEQ ID NO:2, and the upstream primer is shown as SEQ ID NO:3, and a downstream primer shown in 3.

The kit also includes enzymes and/or reagents required for reverse transcription and PCR reactions.

The kit also comprises reverse transcriptase, dNTP, mgC12, buffer solution, fluorescent dye, nuclease water, a probe and/or Taq enzyme.

The sample is peripheral blood.

The application has the advantages that: the invention detects the differential expression of the circRNA in peripheral blood mononuclear cells by utilizing a microarray chip technology, and proves that the circRNA has differential expression in primary gout patients and normal people and can be used as a potential gout diagnosis marker, which is helpful for exploring the pathogenesis of gout and providing a new direction for diagnosis thereof. Through the development and application of the blood biomarker and the diagnostic kit, only the blood of a patient is needed without any other tissues, and the detection sensitivity is improved by amplifying the expression level of peripheral blood circRNA, so that the diagnosis and treatment of gout are more convenient and feasible, the condition of the patient is rapidly and accurately mastered by a clinician, and a foundation is laid for the evaluation of clinical treatment effects.

Drawings

FIG. 1 is a volcanic analysis of differentially expressed circRNA.

FIG. 2 is a hierarchical cluster map analysis of differentially expressed circRNA.

FIG. 3 shows qRT-PCR detection of up-regulation of his_circ_ 0008961 expression in peripheral blood of gout patients.

Fig. 4 shows the diagnostic efficacy of his_circ_ 0008961 alone in peripheral blood.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

EXAMPLE 1 microarray chip technique analysis of circRNA differentially expressed between primary gout patients and Normal humans

1. Material

60 gout patients belonging to the department of medical science of northern Sichuan of 4 months in 2018 to 9 months in the department of medical science of Rheumatoid immunology of department of hospitalization were collected as an experimental group. According to whether there are clinical symptoms, it is divided into 30 cases of Acute Gout (AG) group and Intermittent Gout (IG) group. All patients meet the gout diagnosis standard formulated by ACR in 1977 or ACR/EULAR in 2015, and the patients with secondary gout caused by medicine, tumor, kidney primary diseases and the like are excluded. 30 cases of healthy physical examination, normal and free of gout and joint history, were collected at the same time at the physical examination center of the affiliated hospital of the medical college of north of China and were set as a Healthy Control (HC) group. There was no statistical significance in age and sex composition differences between the normal control group and the gout patient group that were included in the study.

2. Experimental method

2.1 Total RNA extraction and quality detection

All subjects were collected with 4ml of early morning fasting peripheral blood using heparin anticoagulation tubes, PMBCs were isolated using Ficoll density gradient centrifugation, and total RNA was extracted from PMBCs according to Trizol kit (Invitrogen, usa) instructions. RNA integrity was assessed by standard denaturing agarose gel electrophoresis, and RNA concentration and purity were tested using a NanoDrop ND-1000 spectrophotometer (Agilent Corp., U.S.A.). And (5) after the sample is qualified, packaging and storing in a refrigerator at the temperature of-80 ℃.

2.2 microarray chip technology analysis

And 3 cases of AG, IG and HC which are qualified in RNA quality identification and are matched with each other in age are selected for gene chip analysis. Sample preparation and microarray hybridization were performed according to the standard protocol for Arraystar: that is, total RNA was digested with RNase R (Epicentre, USA) to remove linear RNA and enrich for circular RNA, which was amplified and transcribed into fluorescent cRNA according to the Arraystar Super RNA labelling kit (Arraystar, USA) procedure. The labeled cRNA was hybridized to a Arraystar Human circRNA Array v (8 x 15K) chip (Arraystar, usa) containing 13617 personal circRNA probes. After washing the slide, an array scan was performed with an Agilent scanner G2505C (Agilent company, usa). The scanned images were analyzed by Agilent feature extraction software (version 11.0.1.1), quantile normalization and subsequent data processing were performed using the R language limma software package, screening for differentially expressed circRNAs with Fold Change (FC) > 1.5 and P < 0.05 criteria. Analysis results show that the differential expression of the circRNA exists among gout AG, IG and HC, and the differential condition is expressed by adopting a volcanic diagram (figure 1) and a hierarchical clustering diagram (figure 2). Wherein, compared with HC group, AG group and IG group have 93 and 14 upregulated expressed circRNAs and 23 and 27 downregulated expressed circRNAs respectively; whereas the AG group up-regulated 86 circRNAs and down-regulated 19 circRNAs compared to the IG group.

Example 2 RT-PCR verification of relative expression level of hsa_circ_0008961 in peripheral blood mononuclear cells of gout patients

1. Primer design

hsa_circ_0008961 primer:

an upstream primer: 5'-CGGCTGCTCAACTCTGTGTG-3'

A downstream primer: 5'-TGTTCCTCCCCCTGCTCAGTC-3'

Target gene amplification length: 191bp.

2. Reverse transcription

30 cases of AG, IG and HC with no statistical difference in gender and age were taken and total RNA samples were reverse transcribed. According to PimeScript ^TM A reverse transcription reaction system was prepared according to the RT reagent Kit (Perfect Real Time) RR047A (Takara) protocol, and 2. Mu.l of total RNA was used for the reverse transcription to synthesize cDNA. Firstly, each reagent is instantaneously separated, the reagents are sequentially added into an EP tube of the RNase by using a pipettor, fully and uniformly mixed, and the mixture is placed into a circulator for incubation to reverse RNA into cDNA, wherein the reverse transcription condition of the first step is 42 ℃ for 2min, the reverse transcription condition of the second step is 42 ℃ for 15min, and finally the mixture is preserved at 4 ℃.

RT-PCR verification of hsa_circ_0008961 expression level

At the position of

qPCR was performed on a PCR instrument. Fluorescent quantitative PCR detection was performed using a SYBR Premix Ex Taq (TakaRa) real-time fluorescent quantitative kit, and the reaction system was: 10. Mu. l TB Green Premix Ex Tap II, 0.3. Mu.l each of the upstream and downstream primers, 2. Mu.l cDNA, ddH20 was supplemented to 20. Mu.l. The reaction conditions are as follows: the first step: 95℃30sec 1 cycles → 5sec of cycle → 34sec of cycle → 40 cycles total. And a second step of: a total of 1 cycle from 95℃for 5sec to 60sec to 95℃for 15 sec. After the reaction is finished, a dissolution curve is made, beta-actin is used as an internal reference, and each specimen is provided with a compound hole. By a relative quantitative method, 2 ^-ΔΔc(t) As the relative expression amount of the circRNA, the difference in expression of the target gene between the different samples was interpreted.

4. Statistical treatment

Statistical analysis was performed using the statistical software SPSS 23.0, and continuous variables were represented by mean ± standard deviation (x±sd) and Median (Median); the two sets of comparisons were either t-test or Mann-Whitney U test. Diagnostic efficacy of the biomarkers was assessed by subject working characteristics (receiver operating characteristic curve, ROC curve) and calculating the corresponding area under the curve (AUC), with a difference of P < 0.05 being statistically significant.

5. Verification result

Displaying results; the whole parallelism of the RT-PCR amplification curve is good, which indicates that the amplification efficiency of each reaction tube is similar, the inflection point of the amplification curve is clear, the limit is flat and no upward phenomenon exists, the slope of the exponential phase of the curve is large, and the amplification efficiency is higher; the sample amplification product dissolution curve is unimodal, which indicates that the amplification product is single and is specific amplification. hsa_circ_0008961 was expressed in peripheral blood mononuclear cells of gout patients significantly higher than that of healthy control group, and the difference was statistically significant (P < 0.001) (FIG. 3). ROC curve analysis showed that the expression level of hsa_circ_0008961 was statistically significant (p < 0.001) for the difference between gout patient and HC groups (fig. 4), with AUC (95% ic) of 0.839 (0.741-0.937), sensitivity of 85.2%, and specificity of 77.8% indicating that hsa_circ_0008961 has good diagnostic value for gout.

Claims

1. The application of a reagent for detecting the expression level of a circRNA marker for gout diagnosis in preparing a gout diagnosis kit is characterized in that the circRNA marker is SEQ ID NO: hsa_circ_0008961 shown in FIG. 1.

2. The use according to claim 1, wherein the expression level of hsa_circ_0008961 in the sample is detected using fluorescent quantitative PCR techniques.

3. The use according to claim 1, wherein hsa_circ_0008961 is expressed in peripheral blood of patients with gout.

4. The use according to claim 2 or 3, wherein the reagent for detecting expression level of the circRNA marker for gout diagnosis is a specific primer for hsa_circ_0008961.

5. The use according to claim 4, wherein the specific primer for hsa_circ_0008961 comprises the nucleotide sequence set forth in SEQ ID NO:2, and the upstream primer is shown as SEQ ID NO:3, and a downstream primer shown in 3.

6. The use according to claim 2, wherein the sample is peripheral blood.