阅读说明：本技术 hsa_circ_0008961作为痛风诊断标志物的应用 (Application of hsa _ circ _0008961 as gout diagnosis marker ) 是由 青玉凤 张全波 戴菲 黄玉琴 郑建雄 唐乙萍 董曾荣 周闻君 于 2020-12-10 设计创作，主要内容包括：本发明具体涉及hsa-circ-0008961作为痛风诊断标志物的应用。circRNA在痛风的发生发展过程中具有重要的生理作用,为了进一步明确circRNA在痛风中的调节机制,开发具有痛风诊断意义的circRNAs。本发明通过基因芯片技术筛选分析了痛风患者和健康对照者外周血单个核细胞中差异表达的circRNA,并通过实时荧光定量PCR验证了芯片结果,表明hsa-circ-0008961可作为痛风诊断的生物标志物,公开的相应试剂或试剂盒对痛风病人的诊断灵敏性高、特异性好、检测方便,具有良好的临床应用价值。(The invention particularly relates to application of hsa _ circ _0008961 as a gout diagnosis marker. The circRNA has important physiological action in the occurrence and development process of gout, and in order to further define the regulation mechanism of the circRNA in the gout, the circRNAs with gout diagnosis significance are developed. According to the invention, circRNA differentially expressed in peripheral blood mononuclear cells of gout patients and healthy contrast persons is screened and analyzed by a gene chip technology, and a chip result is verified by real-time fluorescent quantitative PCR (polymerase chain reaction), which indicates that hsa _ circ _0008961 can be used as a biomarker for gout diagnosis, and the disclosed corresponding reagent or kit has high diagnostic sensitivity, good specificity and convenient detection on gout patients, and has good clinical application value.)

1. Application of the circRNA marker for gout diagnosis in preparation of a gout diagnosis kit.

2. The use according to claim 1, wherein the circRNA marker is SEQ ID NO: hsa _ circ _0008961 shown in FIG. 1.

3. The use of claim 1, wherein the level of expression of hsa _ circ _0008961 in the sample is detected using a fluorescent quantitative PCR technique.

4. The marker for diagnosing gout according to claim 1, wherein hsa _ circ _0008961 is expressed in an increased amount in peripheral blood of a gout patient.

5. The marker of claim 2 or 3 or 4, wherein the primer pair of the circRNA marker is a specific primer for hsa _ circ _ 0008961.

6. The use of claim 5, wherein the specific primer for hsa _ circ _0008961 comprises the sequence as set forth in SEQ ID NO: 2 as shown in SEQ ID NO: 3, or a reverse primer as shown in the figure.

7. The test kit according to claim 1, further comprising enzymes and/or reagents required for reverse transcription and PCR reactions.

8. The blood test kit of claim 7, further comprising reverse transcriptase, buffer, dNTPs, MgC12, enucleated enzyme water, fluorescent dye, probe, and/or Taq enzyme.

9. Use according to claim 1, wherein the sample is peripheral blood.

Technical Field

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

Background

Gout is one of the common autoinflammatory diseases of human, and is characterized in that purine metabolic disorder and/or uric acid excretion disorder increase the uric acid level in the body, and monosodium urate crystals are formed and deposited in tissues, so that arthritis, soft tissue lumps (namely tophus), uric acid nephropathy and the like are caused. It is associated with a significant geographical distribution, a meta-analysis involving 17476 subjects showing a gout prevalence of 1.1% in the chinese population; the american epidemiological survey showed that the overall prevalence of gout was 3.9% during 2015-2016, and the overall affected population trended upward year by year.

The basis for the onset of gout is hyperuricemia, but not all hyperuricemia progresses to gout. In fact, gout occurs in only 10% of patients with hyperuricemia. Serum uric acid may also drop to normal levels during gout attacks. At present, gout diagnosis mostly depends on clinical manifestations, hematuria routine, blood uric acid determination, joint ultrasound, joint cavity puncture examination and the like, which have advantages, but are invasive examination, or lack of sensitivity or specificity, or have high technical requirements, need abundant clinical experience, and cannot meet clinical needs. Meanwhile, gout not only causes arthritis, but also can become an important risk factor for hypertension, hyperlipidemia, cardiovascular diseases and cerebrovascular diseases in long-term gout, so that the search for markers with high sensitivity and specificity for gout diagnosis and early treatment is necessary.

Circular RNA (circRNA) is a closed circular non-coding RNA, the main biological function of the circular RNA is to play a role of sponge by combining with micro RNA (miRNA), and in addition, the circular RNA can also regulate protein combination, participate in transcriptional regulation and encode protein polypeptide, and participate in the occurrence of various diseases. In existing studies, it was found that circRNA is differentially expressed between disease and normal populations and has the potential to be a biomarker. The inventor further excavates and integrates gout sequencing data through a microarray chip technology, constructs a bioinformatics method-miRNA interaction bioinformatics regulation network, respectively performs GO function analysis and Pathway analysis on source genes of the circRNA, searches key molecules participating in regulating gout, and discovers hsa _ circ _ 0008961.

Disclosure of Invention

Application of the circRNA marker for diagnosing primary gout in preparation of a primary gout diagnostic 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 technology.

The hsa _ circ _0008961 has increased expression in peripheral blood of patients with primary gout.

The primer pair of the circRNA marker is a specific primer aiming at hsa _ circ _ 0008961.

The specific primer aiming at hsa _ circ _0008961 comprises the nucleotide sequence shown in SEQ ID NO: 2 as shown in SEQ ID NO: 3, or a reverse primer as shown in the figure.

The kit may further comprise enzymes and/or reagents required for reverse transcription and PCR reactions.

The kit also comprises reverse transcriptase, dNTP, MgC12, buffer solution, fluorescent dye, enucleated enzyme water, a probe and/or Taq enzyme.

The sample was peripheral blood.

The application has the advantages that: the invention utilizes microarray chip technology to detect circRNA differentially expressed in peripheral blood mononuclear cells, and confirms that the circRNA is differentially expressed in primary gout patients and normal persons and can be used as a potential gout diagnosis marker, which is helpful for exploring pathogenesis of gout and providing a new direction for diagnosis thereof. Through the development and application of the blood biomarker and the diagnosis kit, only the blood of a patient is needed without any other tissue, and the sensitivity of detection is improved through amplifying the expression level of the circRNA of peripheral blood, so that the diagnosis and treatment of gout are more convenient and easier, the condition of the patient can be quickly and accurately mastered by a clinician, and a foundation is laid for the evaluation of clinical treatment effect.

Drawings

Figure 1 is a volcano plot analysis of differentially expressed circRNA.

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

FIG. 3 shows that has _ circ _0008961 is up-regulated in peripheral blood of gout patients by qRT-PCR detection.

FIG. 4 is the diagnosis performance of has _ circ _0008961 alone in peripheral blood.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. 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 technology analysis of circRNA differentially expressed between patients with primary gout and normal persons

1. Material

60 gout patients who are subjected to rheumatism and immunology clinics outpatient service in hospitals affiliated to the medical college of northchuanbei between 4 months in 2018 and 9 months in 2020 and are collected as experimental groups. The patients are divided into 30 cases of Acute Gout (AG) and Intermittent Gout (IG) according to whether the clinical symptoms exist. All patients meet the gout diagnosis standard established by ACR in 1977 or ACR/EULAR in 2015, and secondary gout patients caused by medicines, tumors, primary kidney diseases and the like are excluded. 30 cases of patients who had normal health examinations and had no history of gout and joint disease in the examination center affiliated to the medical college of north-chuan were collected and set as a Health Control (HC) group. There was no statistical significance in the differences in age and gender composition between the normal control group and the gout patient group included in the study.

2. Experimental methods

2.1 Total RNA extraction and quality detection

All subjects collected 4ml of fasting peripheral blood in the early morning using heparin anticoagulation tubes, separated PMBCs by Ficoll density gradient centrifugation, and extracted total RNA from PMBCs according to the instructions of Trizol kit (Invitrogen, USA). RNA integrity was assessed by standard denaturing agarose gel electrophoresis, using a NanoDrop ND-1000 spectrophotometer (Agilent, USA) to test RNA concentration and purity. After the sample is qualified, subpackaging and storing in a refrigerator at minus 80 ℃.

2.2 microarray chip technology analysis

And 3 cases of AG, IG and HC which are qualified in RNA quality identification and matched with ages are selected for gene chip analysis. Sample preparation and microarray hybridization were performed according to the standard protocol of Arraystar: 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 labeling kit (Arraystar, USA) protocol. The labeled cRNA was hybridized to an Arraystar Human circRNA Array v2(8X15K) chip (Arraystar, USA) containing a 13617 Human circRNA probe. After washing the slides, array scans were performed using an Agilent scanner G2505C (Agilent, USA). The images obtained from the scans were analyzed by Agilent feature extraction software (version 11.0.1.1), and quantile normalization and subsequent data processing were performed using the R language limma software package to screen for differentially expressed circRNAs with the criteria of Fold Change (FC) > 1.5 and P < 0.05. The analysis result shows that there are circrnas with differential expression among gout AG, IG and HC, and the above-mentioned differences are expressed by volcano chart (fig. 1) and hierarchical cluster chart (fig. 2). Wherein, compared with HC group, there are 93 and 14 circRNAs up-regulated and 23 and 27 circRNAs down-regulated in AG and IG groups, respectively; in contrast to the IG group, the AG group up-regulated and down-regulated expression of 86 circRNAs and 19 circRNAs.

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: 191 bp.

2. Reverse transcription

Total RNA samples were collected from 30 cases of AG, IG and HC each having no statistical difference in sex and age, and subjected to reverse transcription. Push buttonPhoto pimeScript^TMThe RT reagent Kit (Perfect Real Time) RR047A (Takara) instructions configure reverse transcription reaction system, take 2 u l total RNA for reverse transcription synthesis of cDNA. The method comprises the steps of firstly carrying out flash dissociation on each reagent, sequentially adding the reagents into an EP tube without RNase by using a pipette, fully mixing the reagents uniformly, placing the reagents in a circulator for incubation to invert 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 storing the reagents at 4 ℃.

RT-PCR verification of hsa _ circ _0008961 expression level

In thatqPCR was performed on a PCR instrument. SYBR Premix Ex Taq (Takara) real-time fluorescent quantitative kit is used for carrying out fluorescent quantitative PCR detection, and the reaction system is as follows: mu.l TB Green Premix Ex Tap II, 0.3. mu.l each of the upstream and downstream primers, 2. mu.l cDNA, ddH20 to 20. mu.l. The reaction conditions are as follows: the first step is as follows: 95 ℃ 30sec 1 cycle → cycle ℃ 5sec → 60 ℃ 34sec for a total of 40 cycles. The second step is that: 1 cycle consisting of 95 ℃ 5sec → 60 ℃ 60sec → 95 ℃ 15 sec. After the reaction is finished, a dissolution curve is drawn, beta-actin is used as an internal reference, and each sample is provided with a plurality of holes. Using relative quantification method with 2^-ΔΔc(t)As the relative expression quantity of the circRNA, the difference of the target gene expression among different samples is judged.

4. Statistical treatment

Statistical analysis is carried out by adopting statistical software SPSS 23.0 software, and continuous variables are expressed by adopting an average value +/-standard deviation (x +/-SD) and a Median (Median); two comparisons were performed using either the t-test or the Mann-Whitney U-test. The diagnostic efficacy of the biomarkers was evaluated by the receiver operating characteristic curve (ROC curve) and calculating the corresponding area under the curve (AUC), with P < 0.05 as the difference being statistically significant.

5. Verification result

Displaying the result; the RT-PCR amplification curve has good overall parallelism, which shows 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 raising phenomenon exists, the slope of the exponential phase of the curve is large, which shows that the amplification efficiency is higher; the dissolution curve of the sample amplification product is a single peak, which indicates that the amplification product is single and is specific amplification. The expression level of hsa _ circ _0008961 in peripheral blood mononuclear cells of gout patients is obviously higher than that of healthy control groups, and the difference is 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) between the gout patient group and HC group (FIG. 4), where AUC (95% IC) was 0.839(0.741-0.937), sensitivity was 85.2%, and specificity was 77.8% indicating that hsa _ circ _0008961 had good diagnostic value for gout.