CN107937527B - Glioma diagnostic markers circ1:43920404|43920928 and its application - Google Patents

Abstract

The invention belongs to the technical field of biology, and discloses a glioma diagnosis marker circ1:43920404|43920928 and application thereof. The content of circ1:43920404|43920928 in the serum exosomes of the glioma patients is analyzed by a fluorescent quantitative PCR technology, and the expression level of circ1:43920404|43920928 in the serum exosomes of the glioma patients is obviously reduced compared with that of a normal control group (p is less than 0.0001), and ROC curve analysis shows that the expression level of circ1:43920404|43920928 in the serum exosomes of the glioma patients has higher diagnostic value on glioma, wherein AUC is 0.877, p is 0.003, and the sensitivity and the specificity are 85.7% and 100% respectively.

Description

Translated fromChinese

胶质瘤诊断标志物circ1:43920404|43920928及应用Glioma diagnostic markers circ1:43920404|43920928 and its application

技术领域technical field

本发明属于生物技术领域，涉及一种用于胶质瘤诊断的血清外泌体circRNA标志物、以及检测该标志物的试剂用于制备胶质瘤诊断制剂的应用、还有试剂盒。The invention belongs to the field of biotechnology, and relates to a serum exosome circRNA marker for glioma diagnosis, an application of a reagent for detecting the marker for preparing a glioma diagnostic preparation, and a kit.

背景技术Background technique

胶质瘤是颅内发病率最高的恶性肿瘤，占颅内肿瘤的40.49％，虽然临床上采用手术、化疗结合的方法进行治疗，但由于其浸润性，对化疗药物低敏感性等原因，经常导致患者术后复发，严重威胁人类生命健康。而且一旦确诊，大部分都为胶质瘤中晚期，术后生存率不高。另外，在WHO3-4级即高级别脑胶质瘤患者中，预后较差，平均生存寿命不超过五年。因此，寻找胶质瘤诊断标志物对患者进行诊断分析，尽早确诊病情，并相应地选择合理的后续治疗方案，提高生存率，是神经科学领域亟待解决的研究任务。Glioma is the malignant tumor with the highest incidence of intracranial tumors, accounting for 40.49% of intracranial tumors. Although the combination of surgery and chemotherapy is used for clinical treatment, due to its invasiveness and low sensitivity to chemotherapy drugs, it is often It leads to postoperative recurrence of patients, which seriously threatens human life and health. And once diagnosed, most of them are in the middle and late stages of glioma, and the postoperative survival rate is not high. In addition, in patients with WHO grades 3-4 or high-grade gliomas, the prognosis is poor, with an average lifespan of less than five years. Therefore, it is an urgent research task to be solved in the field of neuroscience to search for glioma diagnostic markers to diagnose and analyze patients, diagnose the disease as soon as possible, and select a reasonable follow-up treatment plan accordingly to improve the survival rate.

circRNA是一类广泛且多样地存在于哺乳动物细胞中、具有调控基因表达作用的内源性非编码RNA分子，具有共价闭合的环形结构，广泛存在于各种细胞中，也是继microRNA(miRNA)后RNA家族的最新研究热点。近年来，随着深度测序技术的广泛应用和生物物理和信息学技术的快速发展，人们发现人类许多外显子的转录本可被非线性地反向剪接或通过基因重排而形成circRNA，且它们在所有剪接转录本中占了相当大的比例。近年来逐渐发现外泌体中也含有大量的circRNA，可能发挥重要作用。目前，由于circRNA具有丰富性、稳定性、高保守性和时空特异性等特点，在肿瘤诊断标志物方面正发挥越来越大的作用。CircRNAs are a class of endogenous non-coding RNA molecules that are widely and diversely present in mammalian cells and can regulate gene expression. They have a covalently closed ring structure and are widely present in various cells. ) of the latest research hotspots in the post-RNA family. In recent years, with the wide application of deep sequencing technology and the rapid development of biophysical and informatics technologies, it has been found that transcripts from many human exons can be non-linearly backspliced or rearranged to form circRNAs, and They make up a considerable proportion of all spliced transcripts. In recent years, it has been gradually discovered that exosomes also contain a large number of circRNAs, which may play an important role. Currently, circRNAs are playing an increasingly important role in tumor diagnostic markers due to their abundance, stability, high conservation, and spatiotemporal specificity.

发明内容SUMMARY OF THE INVENTION

本发明的第一个目的是：提供一种用于胶质瘤诊断的血清外泌体circRNA标志物。The first objective of the present invention is to provide a serum exosomal circRNA marker for glioma diagnosis.

主要内容包括：一种用于胶质瘤诊断的血清外泌体circRNA标志物circ1:43920404|43920928，其序列如SEQ NO:1所示。The main contents include: a serum exosomal circRNA marker circ1:43920404|43920928 for the diagnosis of glioma, the sequence of which is shown in SEQ NO:1.

本发明的第二个目的是，提供检测所述的circRNA标志物在血清外泌体中表达量的试剂在制备胶质瘤诊断制剂中的应用。The second object of the present invention is to provide the application of a reagent for detecting the expression of the circRNA marker in serum exosomes in the preparation of a glioma diagnostic preparation.

本发明的第三个目的是，提供一种胶质瘤诊断试剂盒，其能够测定血清外泌体中的circ1:43920404|43920928的含量。The third object of the present invention is to provide a glioma diagnostic kit, which can measure the content of circ1:43920404|43920928 in serum exosomes.

所述的胶质瘤诊断试剂盒含有检测circ1:43920404|43920928含量的PCR引物。优选引物的序列如SEQ NO:2和3所示。The glioma diagnostic kit contains PCR primers for detecting the content of circ1:43920404|43920928. The sequences of preferred primers are shown in SEQ NO: 2 and 3.

所述的胶质瘤诊断试剂盒，除circ1:43920404|43920928的引物外，还含有从血清中提取外泌体、由外泌体中提取RNA并进行逆转录及荧光定量PCR的所有试剂。包括：(1)提取血清外泌体所需试剂：Total Exosome Isolation Reagent(from serum)，可由Invitrogen公司购得，货号4478360；(2)提取外泌体RNA所需试剂：Trizol试剂、三氯甲烷、异丙醇、75％乙醇、无酶水；(3)逆转录所需试剂：随机引物(Random Primer)、无酶水、5×逆转录缓冲液、三磷酸碱基脱氧核苷酸、RNA酶抑制剂、MMLV逆转录酶；(4)荧光定量PCR所需试剂：circ1:43920404|43920928上下游引物、GAPDH内参上下游引物、SYBR染料、无酶水。The glioma diagnostic kit, in addition to the primers of circ1:43920404|43920928, also contains all the reagents for extracting exosomes from serum, extracting RNA from exosomes, and performing reverse transcription and fluorescence quantitative PCR. Including: (1) Reagents required for extracting serum exosomes: Total Exosome Isolation Reagent (from serum), which can be purchased from Invitrogen, Cat. No. 4478360; (2) Reagents required for extracting exosome RNA: Trizol reagent, chloroform , isopropanol, 75% ethanol, enzyme-free water; (3) Reagents required for reverse transcription: random primer (Random Primer), enzyme-free water, 5× reverse transcription buffer, triphosphate base deoxynucleotide, RNA Enzyme inhibitor, MMLV reverse transcriptase; (4) Reagents required for fluorescence quantitative PCR: circ1:43920404|43920928 upstream and downstream primers, GAPDH internal reference upstream and downstream primers, SYBR dye, enzyme-free water.

本发明的有益效果在于：首次发现circ1:43920404|43920928这种血清外泌体中的环状RNA，并发现其对胶质瘤具有较高的诊断价值；通过血清circRNA标志物和诊断试剂盒的研制和应用，可以使得胶质瘤的诊断更加方便易行，为临床医生快速准确掌握患者病情，为提高临床治疗效果奠定基础，并为发现具有潜在治疗价值的新型小分子药物靶标提供帮助。The beneficial effects of the present invention are as follows: circ1:43920404|43920928, a circular RNA in serum exosomes, is discovered for the first time, and it is found to have high diagnostic value for glioma; through the detection of serum circRNA markers and diagnostic kits The development and application can make the diagnosis of glioma more convenient and feasible, provide clinicians with a quick and accurate grasp of the patient's condition, lay a foundation for improving the clinical treatment effect, and provide help for the discovery of new small-molecule drug targets with potential therapeutic value.

附图说明Description of drawings

图1为实时荧光定量PCR分析circ1:43920404|43920928在正常人血清外泌体与胶质瘤患者血清外泌体中的表达差异；Figure 1 shows the difference in the expression of circ1:43920404|43920928 between normal human serum exosomes and glioma patient serum exosomes by real-time quantitative PCR analysis;

图2为ROC曲线分析血清外泌体来源的circ1:43920404|43920928对胶质瘤诊断的特异性，灵敏性。Figure 2 shows the specificity and sensitivity of ROC curve analysis of serum exosome-derived circ1:43920404|43920928 for the diagnosis of glioma.

具体实施方式Detailed ways

以下结合实施例旨在进一步说明本发明，而非限制本发明。The following examples are intended to further illustrate the present invention, rather than limit the present invention.

一、研究对象1. Research objects

病例组为2016年1月至2017年6月在湖南省肿瘤医院收集的30例胶质瘤血清样本。对照组为同期进行社区疾病筛查的健康个体12例，按性别和年龄(±5岁)与病例进行频数匹配。用于研究的样本为同期收取，采样、分装、保存条件均一致。The case group consisted of 30 glioma serum samples collected in Hunan Cancer Hospital from January 2016 to June 2017. The control group consisted of 12 healthy individuals who were screened for community diseases during the same period, and frequency-matched with cases by gender and age (±5 years). The samples used for the study were collected at the same time, and the sampling, packaging and storage conditions were all the same.

二、研究方法2. Research methods

(1)血清外泌体的制备：取血清200μl于2000g常温离心30分钟，用微量移液器抽取上层清液至新的600μl离心管，加入40μl外泌体提取试剂(Total Exosome IsolationReagent(from serum)，货号4478360，Invitrogen公司)轻轻上下颠倒摇匀，4℃孵育45分钟。孵育结束后10000g常温离心10分钟，弃掉上清液，于沉淀中加入200μl Trizol(MRC公司)使沉淀重悬，将悬液移至新的1.5ml tube管，补Trizol至1ml。(1) Preparation of serum exosomes: Take 200 μl of serum and centrifuge at 2000g at room temperature for 30 minutes, extract the supernatant with a micropipette to a new 600 μl centrifuge tube, add 40 μl of exosome extraction reagent (Total Exosome Isolation Reagent (from serum ), Cat. No. 4478360, Invitrogen), shake upside down gently, and incubate at 4°C for 45 minutes. After the incubation, centrifuge at 10,000 g at room temperature for 10 minutes, discard the supernatant, add 200 μl Trizol (MRC) to the pellet to resuspend the pellet, transfer the suspension to a new 1.5 ml tube, and add Trizol to 1 ml.

(2)外泌体中RNA的抽提：将上述重悬液于冰上静止裂解15分钟。裂解结束后4℃，12000rpm离心10min，上清液移至新的tube管。加氯仿200μl于Tube中，用手震荡15-30s，冰上放置5min，4℃12000rpm离心15min；小心取上层水相入新tube中，加入预冷的异丙醇0.5ml混匀，冰上静置大于20min，4℃12000rpm离心10min；弃上清，加入75％DEPC水稀释的乙醇1ml混匀，4℃，7500rpm离心5min，尽量弃上清，室温干燥5-10min，加入无酶水10μl溶解RNA，-80℃保存。(2) Extraction of RNA from exosomes: The above resuspended solution was statically lysed on ice for 15 minutes. After lysis, centrifuge at 12,000 rpm for 10 min at 4°C, and transfer the supernatant to a new tube. Add 200 μl of chloroform to the tube, shake by hand for 15-30 s, place on ice for 5 min, and centrifuge at 12,000 rpm at 4°C for 15 min; carefully take the upper aqueous phase into a new tube, add 0.5 ml of pre-cooled isopropanol, mix well, and keep on ice Set aside for more than 20min, centrifuge at 12000rpm for 10min at 4°C; discard the supernatant, add 1ml of ethanol diluted with 75% DEPC water, mix well, centrifuge at 4°C, 7500rpm for 5min, discard the supernatant as much as possible, dry at room temperature for 5-10min, add 10μl of enzyme-free water to dissolve RNA, stored at -80°C.

(3)cDNA制备：按照逆转录试剂盒(Thermo公司)说明书进行逆转录反应。反应总体积为20μl(总RNA10μl，Random primer 1μl,无酶水1μl，5×Reaction Buffer 4μl，RI 1μl，RT1μl和10mM dNTP 2μl)。(3) cDNA preparation: The reverse transcription reaction was performed according to the instructions of the reverse transcription kit (Thermo Company). The total reaction volume was 20 μl (10 μl of total RNA, 1 μl of Random primer, 1 μl of enzyme-free water, 4 μl of 5×Reaction Buffer, 1 μl of RI, 1 μl of RT and 2 μl of 10 mM dNTP).

成分Element剂量/管dose/tube随机逆转录引物(1μM)Random reverse transcription primer (1μM)1μl1μlRNA样本RNA sample10μl10μl无酶水Enzyme-free waterTo 12μlTo 12μl

逆转录第一步条件：65℃5分钟Conditions for the first step of reverse transcription: 65°C for 5 minutes

成分Element剂量/管dose/tube5×逆转录缓冲液5x reverse transcription buffer4μl4μl三磷酸碱基脱氧核苷酸(10mM)Deoxynucleotide triphosphate (10mM)2μl2μlRNA酶抑制剂(40U/μl)RNase inhibitor (40U/μl)1μl1μlMMLV逆转录酶(200U/μl)MMLV reverse transcriptase (200U/μl)1μl1μl第一步逆转录产物first step reverse transcript12μl12μl总体积total capacity20μl20μl

逆转录第二步程序：25℃5分钟，42℃60分钟，70℃5分钟。The reverse transcription second step program: 25°C for 5 minutes, 42°C for 60 minutes, and 70°C for 5 minutes.

(4)实时荧光定量PCR：汉恒生物科技(上海)有限公司合成的circ1:43920404|43920928特异性引物(见序列表SEQ NO：2和3)进行实时定量PCR：先将逆转录产物稀释10倍，混匀。20μl反应体系如下：(4) Real-time quantitative PCR: circ1:43920404|43920928 specific primers synthesized by Hanheng Biotechnology (Shanghai) Co., Ltd. (see SEQ NOS: 2 and 3 in the sequence table) for real-time quantitative PCR: first dilute the reverse transcription product by 10 times, mix well. The 20 μl reaction system is as follows:

实时荧光定量PCR反应程序：95℃3分钟,40个循环，95℃10秒，60℃30秒。Real-time quantitative PCR reaction program: 95°C for 3 minutes, 40 cycles, 95°C for 10 seconds, and 60°C for 30 seconds.

(5)数据分析：采用2^-ΔΔCT表示胶质瘤血清外泌体的circ1:43920404|43920928相对于正常血清外泌体的表达倍数，其中△CT＝CT_样本–CT_内参，ΔΔCT＝ΔCT–ΔCT_正常。本实验数据采用相对定量的分析方法，GAPDH作为内参基因(引物序列见SEQ NO：4和5)ΔCT_正常为正常血清外泌体的ΔCT平均值，数据利用软件GraphPad Prism及SPSS 17.0进行分析。三、研究结果(5) Data analysis: 2^{-ΔΔCT was} used to represent the expression fold of circ1:43920404|43920928 of glioma serum exosomes relative to normal serum exosomes, where ΔCT=CT_sample –CT_{internal reference} , ΔΔCT=ΔCT–ΔCT_normal . The data of this experiment were analyzed by relative quantitative analysis method,_GAPDH was used as the internal reference gene (see SEQ NO: 4 and 5 for the primer sequence). 3. Research results

病例组血清外泌体circ1:43920404|43920928表达水平较对照组显著下调(p<0.0001)。具体数据如图1所示。The expression level of serum exosome circ1:43920404|43920928 in the case group was significantly lower than that in the control group (p<0.0001). The specific data is shown in Figure 1.

ROC曲线分析显示，circ1:43920404|43920928作为生物标记物对胶质瘤具有较高诊断价值(AUC＝0.877，p＝0.003，敏感度和特异度分别为85.7％和100％)。详细结果见图2。ROC curve analysis showed that circ1:43920404|43920928 as a biomarker has high diagnostic value for glioma (AUC=0.877, p=0.003, sensitivity and specificity were 85.7% and 100%, respectively). The detailed results are shown in Figure 2.

序列表sequence listing

<110> 中南大学湘雅医院<110> Central South University Xiangya Hospital

<120> 胶质瘤诊断标志物 circ1:43920404|43920928及应用<120> Glioma diagnostic markers circ1:43920404|43920928 and its application

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<213> 智人(Homo sapiens)<213> Homo sapiens

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<210> 3<210> 3

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<212> DNA<212> DNA

<213> 未知(Unknown)<213> Unknown

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<210> 4<210> 4

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<212> DNA<212> DNA

<213> 未知(Unknown)<213> Unknown

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<212> DNA<212> DNA

<213> 未知(Unknown)<213> Unknown

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catcacgcca cagtttcc 18catcacgcca cagtttcc 18

Claims (4)

Translated fromChinese

1.检测circ1:43920404|43920928在血清外泌体中表达量的试剂在制备胶质瘤诊断制剂中的应用circ1:43920404|43920928其序列如SEQ NO:1所示，胶质瘤患者血清外泌体中c i r c 1 : 43920404|43920928表达水平比正常人低。1. The application of reagents for detecting the expression of circ1:43920404|43920928 in serum exosomes in the preparation of glioma diagnostic preparations circ1:43920404|43920928 Its sequence is shown in SEQ NO:1, serum exosomes of glioma patients The expression level of c i r c 1 : 43920404|43920928 in the body was lower than that of normal people.2.根据权利要求1所述的应用，其特征在于，检测circ1:43920404|43920928在血清外泌体中表达量的试剂含有检测circ1:43920404| 43920928含量的PCR引物。2. The application according to claim 1, wherein the reagent for detecting the expression of circ1:43920404|43920928 in serum exosomes contains PCR primers for detecting the content of circ1:43920404|43920928.3.根据权利要求2所述的应用，其特征在于，引物的序列如SEQ NO:2和3 所示。3 . The application according to claim 2 , wherein the sequences of the primers are shown in SEQ NO: 2 and 3. 4 .4.根据权利要求1或2或3所述的应用，其特征在于，检测circ1:43920404|43920928在血清外泌体中表达量的试剂除含有检测circ1:43920404| 43920928的引物外，还含有从血清中提取外泌体、由外泌体中提取RNA并进行逆转录及荧光定量PCR的所有试剂。4. The application according to claim 1, 2 or 3, wherein the reagent for detecting the expression of circ1:43920404|43920928 in serum exosomes contains, in addition to the primers for detecting circ1:43920404|43920928, a primer from circ1:43920404|43920928. All reagents for extracting exosomes from serum, extracting RNA from exosomes and performing reverse transcription and real-time PCR.

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