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CN111073981B - Application of circular RNA circEMB_003 as a marker of colorectal cancer - Google Patents

Application of circular RNA circEMB_003 as a marker of colorectal cancer Download PDF

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CN111073981B
CN111073981B CN202010053100.6A CN202010053100A CN111073981B CN 111073981 B CN111073981 B CN 111073981B CN 202010053100 A CN202010053100 A CN 202010053100A CN 111073981 B CN111073981 B CN 111073981B
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胡育菡
张哲莹
马帅
李健
钟加滕
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Abstract

本发明涉及一种环状RNA circEMB_003作为结直肠癌标志物的应用。该标志物的表达水平与结直肠癌肿瘤的大小(p<0.05)、TNM分期(p<0.05)及远处转移(p<0.05)呈负相关,而与病人的年龄、性别、肿瘤的分化没有相关性(p>0.05)。同时还通过RNA酶R消化和放线菌素D处理验证了circEMB_003的稳定性,能较好地反映结直肠肿瘤的发生发展,从而可用于监测结直肠肿瘤对治疗措施的反应等。该标志物的发现,为进一步研究结直肠癌的发病机理,探索结直肠癌的治疗靶点提供了新的实验理论基础和新的方向,并可应用于制备结直肠癌诊断和/或预后评估的试剂或试剂盒,丰富了结直肠癌的诊断和检测手段。

Figure 202010053100

The present invention relates to the application of a circular RNA circEMB_003 as a colorectal cancer marker. The expression level of this marker was negatively correlated with the size (p<0.05), TNM stage (p<0.05) and distant metastasis (p<0.05) of colorectal cancer tumors, and was correlated with the patient's age, gender, and tumor differentiation. There was no correlation (p>0.05). At the same time, the stability of circEMB_003 was verified by RNase R digestion and actinomycin D treatment, which can better reflect the occurrence and development of colorectal tumors, so that it can be used to monitor the response of colorectal tumors to therapeutic measures. The discovery of this marker provides a new experimental theoretical basis and a new direction for further research on the pathogenesis of colorectal cancer and exploration of therapeutic targets for colorectal cancer, and can be applied to the preparation of colorectal cancer diagnosis and/or prognosis evaluation The reagents or kits have enriched the diagnosis and detection methods of colorectal cancer.

Figure 202010053100

Description

环状RNA circEMB_003作为结直肠癌标志物的应用Application of circular RNA circEMB_003 as a marker for colorectal cancer

技术领域technical field

本发明涉及结直肠癌领域,特别是涉及一种环状RNA circEMB_003作为结直肠癌标志物的应用。The invention relates to the field of colorectal cancer, in particular to the application of a circular RNA circEMB_003 as a colorectal cancer marker.

背景技术Background technique

结直肠癌是一种常见的、严重危害人类健康的消化道恶性肿瘤。其发病率在全球各种恶性肿瘤中高居第三位,肿瘤相关死亡率高居第二位。随着肿瘤早期筛查工作的开展以及新的治疗方法的应用,结直肠癌的死亡率有所下降,但是其发病率却明显上升且日益年轻化。因此,深入研究结直肠癌的发生发展过程,寻找关键的分子标志物和药物靶点,对开发有效的治疗措施、提高结直肠癌患者的生存率具有重要的科学意义和临床价值。Colorectal cancer is a common digestive tract malignant tumor that seriously endangers human health. Its incidence ranks third among various malignant tumors in the world, and its tumor-related mortality ranks second. With the development of early tumor screening and the application of new treatment methods, the mortality rate of colorectal cancer has decreased, but its incidence has increased significantly and is getting younger and younger. Therefore, in-depth research on the occurrence and development of colorectal cancer and the search for key molecular markers and drug targets have important scientific significance and clinical value for developing effective treatment measures and improving the survival rate of colorectal cancer patients.

环状RNA(circRNA)是最近几年发现的一类在哺乳动物体内广泛存在的非编码RNA,其表达丰富,是由特殊的末端反向互补的前体mRNA经过可变剪切形成。与传统线性RNA(linear RNA)含5’和3’末端不同,circRNA的首尾相连形成闭合环状结构。circRNA的闭合环状结构使其比线性RNA更加稳定,能够耐受核酸外切酶的降解。同时,circRNA的表达具有组织及细胞类型的特异性,并且在血液、唾液、尿液或外泌体中也能检测到丰富的circRNA。circRNA的这些特征表明它具有作为疾病诊断标志物的潜能。现已在多种肿瘤中发现具有诊断和预后判断价值的circRNA。比如,Josh N.Vo等人通过外显子捕获RNA测序的方法检测了多种肿瘤中circRNA的表达情况,筛选到了能够作为前列腺癌潜在分子标志物的circRNA。Weng等研究发现circRNA ciRS-7在结直肠癌中表达水平增加且与病人的预后生存率相关。这些研究均表明circRNAs作为肿瘤早期标志物的筛查是一种有效的检测手段,有望成为新型的肿瘤分子治疗靶点。目前,与结直肠癌相关的环状RNA仍有很多未被发现,研究寻找这些环状RNA对结直肠癌的防治诊断具有重要的研究价值和临床价值。Circular RNAs (circRNAs) are a class of non-coding RNAs that are widely found in mammals in recent years. They are abundant in expression and are formed by alternative splicing of special reverse-complementary precursor mRNAs. Different from traditional linear RNA (linear RNA) which contains 5' and 3' ends, the end-to-end connection of circRNA forms a closed loop structure. The closed circular structure of circRNA makes it more stable than linear RNA and resistant to degradation by exonuclease. Meanwhile, the expression of circRNAs is tissue- and cell-type specific, and abundant circRNAs can also be detected in blood, saliva, urine or exosomes. These characteristics of circRNA suggest its potential as a disease diagnostic marker. CircRNAs with diagnostic and prognostic value have been found in a variety of tumors. For example, Josh N.Vo et al. detected the expression of circRNAs in various tumors by exon capture RNA sequencing, and screened circRNAs that can be used as potential molecular markers of prostate cancer. Weng et al. found that the expression level of circRNA ciRS-7 was increased in colorectal cancer and correlated with the prognosis and survival rate of patients. These studies all show that the screening of circRNAs as early tumor markers is an effective detection method, and it is expected to become a new tumor molecular therapeutic target. At present, there are still many circRNAs related to colorectal cancer that have not been discovered. It is of great research and clinical value to find these circRNAs for the prevention and diagnosis of colorectal cancer.

发明内容SUMMARY OF THE INVENTION

基于此,有必要提供一种环状RNA circEMB_003作为结直肠癌标志物的应用。Based on this, it is necessary to provide the application of a circular RNA circEMB_003 as a marker for colorectal cancer.

一种环状RNA circEMB_003的定量检测剂在制备用于结直肠癌诊断和/或预后评估的试剂盒中的应用,其特征在于,所述环状RNA circEMB_003具有如SEQ ID NO.1所示的核苷酸序列。The application of a quantitative detection agent of circular RNA circEMB_003 in the preparation of a kit for colorectal cancer diagnosis and/or prognosis evaluation, characterized in that the circular RNA circEMB_003 has the SEQ ID NO. Nucleotide sequence.

在其中一个实施例中,所述环状RNA circEMB_003的定量检测剂包括适用于如下至少一种方法的试剂:In one embodiment, the quantitative detection reagent for circular RNA circEMB_003 includes a reagent suitable for at least one of the following methods:

荧光染料法、数字PCR、共振光散射法、实时荧光定量PCR、测序或生物质谱法。Fluorescent dye method, digital PCR, resonance light scattering, real-time PCR, sequencing or biological mass spectrometry.

在其中一个实施例中,所述环状RNA circEMB_003的定量检测剂为能够特异性结合环状RNA circEMB_003或环状RNA circEMB_003对应的cDNA的探针或引物。In one embodiment, the quantitative detection agent for circular RNA circEMB_003 is a probe or primer that can specifically bind to circular RNA circEMB_003 or a cDNA corresponding to circular RNA circEMB_003.

在其中一个实施例中,所述探针或引物带有可检测的标记。In one embodiment, the probe or primer is detectably labeled.

在其中一个实施例中,所述标记为荧光标记物、化学发光探针或同位素标记物。In one embodiment, the label is a fluorescent label, a chemiluminescent probe or an isotopic label.

在其中一个实施例中,所述环状RNA circEMB_003的定量检测剂为环状RNAcircEMB_003的qRT-PCR引物,其上游引物如SEQ ID NO.2所示,下游引物如SEQ ID NO.3所示。In one embodiment, the quantitative detection agent of circular RNA circEMB_003 is the qRT-PCR primer of circular RNA circEMB_003, and the upstream primer is shown in SEQ ID NO.2, and the downstream primer is shown in SEQ ID NO.3.

在其中一个实施例中,所述试剂盒中还包括内参引物对。In one embodiment, the kit further includes an internal reference primer pair.

在其中一个实施例中,所述内参引物对包括18S rRNA扩增引物对和GAPDH扩增引物对中的一种或多种。In one embodiment, the internal reference primer pair includes one or more of a 18S rRNA amplification primer pair and a GAPDH amplification primer pair.

在其中一个实施例中,所述18S rRNA扩增引物对的核苷酸序列分别如SEQ IDNO.4和SEQ ID NO.5所示,所述GAPDH扩增引物对的核苷酸序列分别如SEQ ID NO.6和SEQID NO.7所示。In one embodiment, the nucleotide sequences of the 18S rRNA amplification primer pair are respectively shown in SEQ ID NO. 4 and SEQ ID NO. 5, and the nucleotide sequences of the GAPDH amplification primer pair are respectively shown in SEQ ID NO. ID NO.6 and SEQID NO.7.

在其中一个实施例中,所述试剂盒中还包括RNA提取试剂、PCR反应缓冲液、dNTPs以及DNA聚合酶中的至少一种。In one embodiment, the kit further includes at least one of RNA extraction reagents, PCR reaction buffer, dNTPs and DNA polymerase.

通过统计分析结直肠癌组织中上述结直肠癌分子标志物(circEMB_003)的相对表达量与结直肠癌患者临床病理参数之间的关系发现,circEMB_003的表达水平与结直肠癌肿瘤的大小(p<0.05)、TNM分期(p<0.05)及远处转移(p<0.05)呈负相关,而与病人的年龄、性别、肿瘤的分化没有相关性(p>0.05)。同时还通过RNA酶R消化和放线菌素D处理验证了circEMB_003的稳定性,能较好地反映结直肠肿瘤的发生发展,从而可用于监测结直肠肿瘤对治疗措施的反应等。另外,通过应用结直肠癌(CRC)组织及癌旁组织中circEMB_003的相对表达量或CRC患者和正常人外周血血浆中circEMB_003的相对表达量制作受试者工作特征曲线(ROC),评价其作为CRC诊断标志物的潜在价值。结果ROC曲线下面积(AUC)为0.768(95%可信区间为0.669~0.876),特异性为0.9275(95%可信区间为0.8389~0.9761)。ROC曲线下面积越接近1越具有诊断价值,这表明通过检测circEMB_003的表达可以高效地将结直肠癌从正常组织中鉴别出来,circEMB_003具备作为结直肠癌诊断标志物的潜在价值。该标志物的发现,为进一步研究结直肠癌的发病机理,探索结直肠癌的治疗靶点提供了新的实验理论基础和新的方向,并可应用于制备结直肠癌诊断和/或预后评估的试剂或试剂盒,丰富了结直肠癌的诊断和检测手段。Through statistical analysis of the relationship between the relative expression levels of the above colorectal cancer molecular markers (circEMB_003) in colorectal cancer tissues and the clinicopathological parameters of colorectal cancer patients, it was found that the expression level of circEMB_003 was associated with the size of colorectal cancer tumors (p< 0.05), TNM stage (p<0.05), and distant metastasis (p<0.05) were negatively correlated, but had no correlation with the patient's age, gender, and tumor differentiation (p>0.05). At the same time, the stability of circEMB_003 was verified by RNase R digestion and actinomycin D treatment, which can better reflect the occurrence and development of colorectal tumors, which can be used to monitor the response of colorectal tumors to therapeutic measures. In addition, by using the relative expression of circEMB_003 in colorectal cancer (CRC) tissues and adjacent tissues or the relative expression of circEMB_003 in peripheral blood plasma of CRC patients and normal people, a receiver operating characteristic curve (ROC) was prepared to evaluate its performance as a Potential value of diagnostic markers for CRC. Results The area under the ROC curve (AUC) was 0.768 (95% confidence interval, 0.669-0.876), and the specificity was 0.9275 (95% confidence interval, 0.8389-0.9761). The closer the area under the ROC curve is to 1, the more diagnostic value it is, which indicates that colorectal cancer can be efficiently differentiated from normal tissues by detecting the expression of circEMB_003, and circEMB_003 has potential value as a diagnostic marker for colorectal cancer. The discovery of this marker provides a new experimental theoretical basis and a new direction for further research on the pathogenesis of colorectal cancer and exploration of therapeutic targets for colorectal cancer, and can be applied to the preparation of colorectal cancer diagnosis and/or prognosis evaluation The reagents or kits have enriched the diagnosis and detection methods of colorectal cancer.

附图说明Description of drawings

图1为CircEMB_003形成的模式图;Fig. 1 is the pattern diagram that CircEMB_003 forms;

图2为CircEMB_003扩增片段的琼脂糖凝胶电泳图;Fig. 2 is the agarose gel electrophoresis figure of CircEMB_003 amplified fragment;

图3为CircEMB_003的Sanger测序结果图;Fig. 3 is the Sanger sequencing result graph of CircEMB_003;

图4为CircEMB_003的qRT-PCR扩增曲线;Fig. 4 is the qRT-PCR amplification curve of CircEMB_003;

图5为CircEMB_003的qRT-PCR溶解曲线;Fig. 5 is the qRT-PCR dissolution curve of CircEMB_003;

图6为CircEMB_003在43例配对结直肠癌组织与癌旁组织中表达的倍比关系图;Figure 6 is a graph showing the fold ratio of CircEMB_003 expression in 43 cases of matched colorectal cancer tissues and adjacent tissues;

图7为CircEMB_003在43例配对结直肠癌组织与癌旁组织中的平均表达水平图;Figure 7 is a graph showing the average expression level of CircEMB_003 in 43 matched colorectal cancer tissues and adjacent tissues;

图8为RNA酶R消化验证CircEMB_003的稳定性结果图,其中A为琼脂糖凝胶电泳图,B为qRT-PCR结果图;Figure 8 is a graph showing the stability results of RNase R digestion to verify CircEMB_003, wherein A is an agarose gel electrophoresis graph, and B is a qRT-PCR result graph;

图9为放线菌素D处理验证CircEMB_003的稳定性结果图;Figure 9 is a graph showing the stability results of Actinomycin D treatment to verify CircEMB_003;

图10为评估circEMB_003潜在诊断价值的受试者工作特征曲线;Figure 10 is the receiver operating characteristic curve for evaluating the potential diagnostic value of circEMB_003;

图11为FISH探针检测circEMB_003在结直肠癌组织中的表达结果图;Figure 11 shows the results of FISH probe detection of circEMB_003 expression in colorectal cancer tissue;

图12为CircEMB_003在7株结直肠癌细胞和正常肠上皮细胞NCM460中的表达结果图。Figure 12 is a graph showing the expression results of CircEMB_003 in 7 strains of colorectal cancer cells and normal intestinal epithelial cells NCM460.

具体实施方式Detailed ways

为了便于理解本发明,下面将对本发明进行更全面的描述,并给出了本发明的较佳实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。In order to facilitate understanding of the present invention, the present invention will be described more fully below, and preferred embodiments of the present invention will be given. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough and complete understanding of the present disclosure is provided.

除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

术语“环状RNA circEMB_003的定量检测剂”在本发明中不应仅仅理解为对环状RNA circEMB_003的检测剂,而应包括被本领域技术人员所知的其余可反映环状RNAcircEMB_003表达水平的检测试剂。例如可通过定量检测环状RNA circEMB_003反转录所得的cDNA,或对环状RNA circEMB_003的表达量进行间接检测。The term "quantitative detection agent for circular RNA circEMB_003" in the present invention should not only be understood as a detection agent for circular RNA circEMB_003, but should include other detections known to those skilled in the art that can reflect the expression level of circular RNA circEMB_003 reagents. For example, the cDNA obtained by reverse transcription of circular RNA circEMB_003 can be quantitatively detected, or the expression level of circular RNA circEMB_003 can be indirectly detected.

在本发明中,若无特别声明,“环状RNA circEMB_003”可被“标志物”、“标志物基因”、“生化标志物”、“结直肠癌肿瘤标志物”或“结直肠癌癌标志物”所替换。其具体指要用作分析患者实验样品的靶标的分子。In the present invention, unless otherwise stated, "circular RNA circEMB_003" can be used as a "marker", "marker gene", "biochemical marker", "colorectal cancer tumor marker" or "colorectal cancer cancer marker" thing" is replaced. It specifically refers to a molecule to be used as a target for the analysis of a patient's experimental sample.

在本发明中用作标志物的环状RNA circEMB_003预期包括其全长核糖核苷酸序列,或天然存在的变体,或全长序列及变体的片段,特别是可被检测并确定具体序列的片段,更优选为能够在结直肠组织中与其他RNA序列相区分的片段。优选地包含所述全长核糖核苷酸序列的至少7、8、9、10、11、12、15或20个连续的核糖核苷酸。The circular RNA circEMB_003 used as a marker in the present invention is expected to include its full-length ribonucleotide sequence, or a naturally-occurring variant, or a fragment of the full-length sequence and the variant, in particular, the specific sequence can be detected and determined , more preferably fragments that can be distinguished from other RNA sequences in colorectal tissue. Preferably at least 7, 8, 9, 10, 11, 12, 15 or 20 contiguous ribonucleotides of the full length ribonucleotide sequence are included.

本领域的技术人员可认识到,由细胞释放的核糖核苷酸或存在于胞外基质中的核糖核苷酸可能受到损害(例如,在炎症过程中),且可被降解或切割成这样的片段。如熟练的技术人员将明白的,mRNA或其片段也可以作为复合物的部分而存在。这样的复合物也可以用作本发明意义上的标志物。One skilled in the art will recognize that ribonucleotides released by cells or present in the extracellular matrix may be damaged (eg, during inflammation) and may be degraded or cleaved into such Fragment. As the skilled artisan will appreciate, mRNA or fragments thereof may also be present as part of a complex. Such complexes can also be used as markers in the sense of the present invention.

“天然存在的变体”应被理解为,高等动物的基因通常伴有高频率的多态性。也存在许多在剪接过程中产生含有相互不同的氨基酸序列的同种型的分子。与癌症相关疾病相关的具有与标志物基因的活性相似的活性的任何基因包括在标志物基因中,即使其由于多态性或为同种型而具有核苷酸序列差异。A "naturally occurring variant" is to be understood as a gene of a higher animal usually accompanied by a high frequency of polymorphisms. There are also many molecules that, during splicing, produce isoforms containing mutually different amino acid sequences. Any gene associated with a cancer-related disease that has an activity similar to that of the marker gene is included in the marker gene, even if it has nucleotide sequence differences due to polymorphism or being an isotype.

RNA的定量检测剂可选用本领域技术人员所公知的试剂,例如能够与该RNA杂交,且标记有荧光标记的核酸等;常见情况下RNA的检测剂可以选自RT-PCR的引物,以及用于扩增RT-PCR的产物——cDNA的引物。The quantitative detection reagent of RNA can be selected from reagents known to those skilled in the art, such as nucleic acid that can hybridize with the RNA and is labeled with fluorescent label, etc.; Primers for amplifying the product of RT-PCR - cDNA.

下面主要结合具体实施方式和附图对结直肠癌相关环状RNA基因、结直肠癌分子标志物及其应用作进一步详细的说明。The following describes the colorectal cancer-related circular RNA genes, colorectal cancer molecular markers and their applications in further detail mainly in conjunction with the specific embodiments and the accompanying drawings.

一、组织和细胞RNA的提取1. Extraction of tissue and cellular RNA

应用TRIzol试剂提取结直肠癌组织和细胞的总RNA。Total RNA was extracted from colorectal cancer tissues and cells using TRIzol reagent.

(1)研钵及所用剪刀等物品用锡箔纸包好,放置180℃烤箱内烤6~8小时以去除RNA酶;(1) Wrap the mortar and scissors with tin foil and bake in a 180°C oven for 6-8 hours to remove RNase;

(2)将研钵等物品从烤箱中取出冷却至室温,并用液氮预冷研钵;剪取约黄豆大小的组织块,放入研钵中小心研磨,边研磨边加液氮,直至将组织碾碎;(2) Take the mortar and other items out of the oven and cool to room temperature, and pre-cool the mortar with liquid nitrogen; cut tissue pieces about the size of soybeans, put them into the mortar and grind them carefully, and add liquid nitrogen while grinding, until the tissue crushing;

(3)组织被研磨成粉末状后,加入RNAisoTM Plus试剂(1mL/50mg~100mg)并进行匀浆处理,待到组织与RNAisoTM Plus混合物融合后,将研磨物转入无RNA酶的1.5mL离心管中;(3) After the tissue is ground into powder, add RNAiso TM Plus reagent (1mL/50mg~100mg) and perform homogenization treatment. After the tissue is fused with the RNAiso TM Plus mixture, transfer the ground material to 1.5 RNase-free 1.5 mL centrifuge tube;

(4)如果提取细胞的总RNA,则收集生长状态良好的细胞,胰酶消化收集至1.5mL离心管中,PBS洗3次,按每9.6cm2面积加入1mL RNAisoTM Plus,静置片刻后用枪头吹打至无细胞沉淀,室温静置5min;(4) If the total RNA of the cells is extracted, collect the cells in good growth state, digest them with trypsin and collect them into a 1.5 mL centrifuge tube, wash 3 times with PBS, add 1 mL of RNAiso TM Plus per 9.6 cm 2 area, and let stand for a while Pipette with a pipette tip until there is no cell precipitation, and let stand at room temperature for 5 min;

(5)每1mL TRIzol试剂加入200μL氯仿,加入氯仿后盖紧离心管,用力上下摇荡20秒钟,以保证水相和有机相能够充分接触,室温静置10分钟。将离心管置于低温离心机中离心,4℃,12000rpm,15分钟。离心后混合液出现分层,上面一层为基本无色的水相,内含有总RNA,下层为浅红色的酚-氯仿相;(5) Add 200 μL of chloroform per 1 mL of TRIzol reagent. After adding chloroform, cover the centrifuge tube tightly and shake vigorously up and down for 20 seconds to ensure that the aqueous phase and the organic phase can fully contact, and let stand for 10 minutes at room temperature. The centrifuge tube was centrifuged in a low temperature centrifuge at 4°C, 12000 rpm, for 15 minutes. After centrifugation, the mixed solution was stratified, the upper layer was a substantially colorless aqueous phase containing total RNA, and the lower layer was a light red phenol-chloroform phase;

(6)将上层水相转入一新离心管中,在离心管中加入与水相液体等体积的异丙醇,轻柔地上下颠倒使其充分混匀,室温静置10分钟,4℃,12000rpm离心15分钟。(6) Transfer the upper water phase into a new centrifuge tube, add isopropanol with the same volume as the water phase liquid in the centrifuge tube, gently invert it up and down to make it fully mixed, let stand for 10 minutes at room temperature, 4°C, Centrifuge at 12000 rpm for 15 minutes.

(7)小心弃去上清液,洗涤沉淀RNA,用1ml 75%乙醇(用高压灭菌后的0.1%的DEPC水配制)洗涤2次,再放置于低温离心机中离心,4℃,12000rpm离心5分钟,弃去上清液,室温干燥10分钟,加约20μL DEPC水溶解。(7) Carefully discard the supernatant, wash the precipitated RNA, wash twice with 1 ml of 75% ethanol (prepared with 0.1% DEPC water after autoclaving), and then centrifuge in a low temperature centrifuge at 4°C, 12000rpm Centrifuge for 5 minutes, discard the supernatant, dry at room temperature for 10 minutes, and add about 20 μL of DEPC water to dissolve.

(8)1%琼脂糖凝胶电泳检测总RNA的完整性,分光光度计检测RNA的浓度和纯度,并进行标记。(8) The integrity of total RNA was detected by 1% agarose gel electrophoresis, and the concentration and purity of RNA were detected by spectrophotometer, and labeled.

二、RNA逆转录2. RNA reverse transcription

(1)RNA逆转录按照PrimeScriptTM RT reagent Kit with gDNA Eraser(TaKaRa)说明书进行,首先去除RNA里可能污染的gDNA,按照下表配置反应体系:(1) RNA reverse transcription is carried out according to the instructions of PrimeScript TM RT reagent Kit with gDNA Eraser (TaKaRa), first remove the gDNA that may contaminate the RNA, and configure the reaction system according to the following table:

试剂reagent 体积(μL)Volume (μL) 5×gDNA Eraser Buffer5×gDNA Eraser Buffer 22 gDNA ErasergDNA Eraser 11 RNARNA 2(500ng/μL)2 (500ng/μL) RNase Free dH<sub>2</sub>ORNase Free dH<sub>2</sub>O up to 10up to 10

(2)去除gDNA程序:42℃,2min;(2) Procedure for removing gDNA: 42°C, 2min;

(3)按照下表在冰上加入如下试剂到已去除gDNA的前述反应体系内:(3) Add the following reagents on ice to the aforementioned reaction system from which gDNA has been removed according to the following table:

试剂reagent 体积(μL)Volume (μL) 5×PrimeScript Buffer5×PrimeScript Buffer 44 PrimeScript RT Enzyme Mix IPrimeScript RT Enzyme Mix I 11 Random 6mers(100μM)Random 6mers (100μM) 11 RNase Free dH<sub>2</sub>ORNase Free dH<sub>2</sub>O 44

(4)逆转录程序:37℃,15min;85℃,5sec;(4) Reverse transcription program: 37°C, 15min; 85°C, 5sec;

(5)逆转录产物用无菌超纯水稀释5~10倍,-20℃保存尽快使用或者直接进行PCR及qRT-PCR反应。(5) Dilute the reverse transcription product 5-10 times with sterile ultrapure water, store it at -20°C and use it as soon as possible or directly perform PCR and qRT-PCR reactions.

三、Hsa_circEMB_003序列(以下为cDNA序列)3. Hsa_circEMB_003 sequence (the following is the cDNA sequence)

位置:chr5:49694940-49707217strand:-Location: chr5:49694940-49707217strand:-

长度:954bpLength: 954bp

RNA sequence(circBank):RNA sequence (circBank):

aacattctagtatgccagtagaaaaaaatatcactttagaaaggccttctaatgtaaatctcacatgccagttcacaacatctggggatttgaatgcagtaaatgtgacttggaaaaaagatggtgaacaacttgagaataattatcttgtcagtgcaacaggaagcaccttgtatacccaatacaggttcaccatcattaatagcaaacaaatgggaagttattcttgtttctttcgagaggaaaaggaacaaaggggaacatttaatttcaaagtccctgaacttcatgggaaaaacaagccattgatctcttacgtaggggattctactgtcttgacatgtaaatgtcaaaattgttttcctttaaattggacctggtacagtagtaatgggagtgtaaaggttcctgttggtgttcaaatgaataaatatgtgatcaatggaacatatgctaacgaaacaaagctgaagataacacaacttttggaggaagatggggaatcttactggtgccgtgcactattccaattaggcgagagtgaagaacacattgagcttgtggtgctgagctatttggtgcccctcaaaccatttcttgtaatagtggctgaggtgattcttttagtggccaccattctgctttgtgaaaagtacacacaaaagaaaaagaagcactcagatgaggggaaagaatttgagcagattgaacagctgaaatcagatgatagcaatggtatagaaaataatgtccccaggcatagaaaaaatgagtctctgggccagtgaatacaaaacatcatgtcgagaatcattggaagatatacagagttcgtatttcagctttgtttatccttcctgttaagagcctctgagtttttagttttaaaaggatgaaaagcttatgcaacatgctcagcaggagcttcatcaacgatatatgtcagatctaaagaacattctagtatgccagtagaaaaaaatatcactttagaaaggccttctaatgtaaatctcacatgccagttcacaacatctggggatttgaatgcagtaaatgtgacttggaaaaaagatggtgaacaacttgagaataattatcttgtcagtgcaacaggaagcaccttgtatacccaatacaggttcaccatcattaatagcaaacaaatgggaagttattcttgtttctttcgagaggaaaaggaacaaaggggaacatttaatttcaaagtccctgaacttcatgggaaaaacaagccattgatctcttacgtaggggattctactgtcttgacatgtaaatgtcaaaattgttttcctttaaattggacctggtacagtagtaatgggagtgtaaaggttcctgttggtgttcaaatgaataaatatgtgatcaatggaacatatgctaacgaaacaaagctgaagataacacaacttttggaggaagatggggaatcttactggtgccgtgcactattccaattaggcgagagtgaagaacacattgagcttgtggtgctgagctatttggtgcccctcaaaccatttcttgtaatagtggctgaggtgattcttttagtggccaccattctgctttgtgaaaagtacacacaaaagaaaaagaagcactcagatgaggggaaagaatttgagcagattgaacagctgaaatcagatgatagcaatggtatagaaaataatgtccccaggcatagaaaaaatgagtctctgggccagtgaatacaaaacatcatgtcgagaatcattggaagatatacagagttcgtatttcagctttgtttatccttcctgttaagagcctctgagtttttagttttaaaaggatgaaaagcttatgcaacatgctcagcaggagcttcatcaacgatatatgtcagatctaaag

四、引物设计4. Primer Design

EMB及circEMB_003引物均应用Oligo7进行设计。NCBI查询EMB mRNA序列,选取其保守区域设计引物。circEMB_003序列为EMB第3~9外显子序列,将其5’端一部分序列(约150bp)连到3’末端,形成的模式图如图1所示。将序列输入Oligo7中,设定引物长度18bp~23bp,扩增片段长度100bp~250bp,选取评分高、上下游引物Tm值接近,尽量无发夹结构、无二聚体、无错配的引物,circEMB_003上下游引物需跨剪切位点(即5’端和3’末端的接点)。GAPDH、18S rRNA引物序列为本实验室已验证有效的序列。所有引物序列均在NCBI上blast以初步排除与其他基因存在非特异结合扩增。引物由生工生物工程股份有限公司合成,粉末状引物先高速离心后用无核酶水稀释为100μM储存液,再用无菌水稀释为10μM工作液,并通过qRT-PCR观察溶解曲线挑选无非特异及二聚体的引物进行后续实验。具体引物序列如下表所示:Both EMB and circEMB_003 primers were designed with Oligo7. NCBI queried the EMB mRNA sequence, and selected its conserved region to design primers. The circEMB_003 sequence is the sequence of exons 3 to 9 of EMB, and a part of its 5'-end sequence (about 150 bp) is connected to the 3'-end, and the pattern formed is shown in Figure 1. Input the sequence into Oligo7, set the primer length to be 18bp to 23bp, the amplified fragment length to be 100bp to 250bp, and select the primers with high scores, close Tm values of upstream and downstream primers, and try to have no hairpin structure, no dimers, and no mismatches. The upstream and downstream primers of circEMB_003 need to span the cleavage site (ie, the junction between the 5' and 3' ends). The sequences of GAPDH and 18S rRNA primers are the valid sequences verified by the laboratory. All primer sequences were blasted on NCBI to preliminarily rule out non-specific binding to other genes. The primers were synthesized by Sangon Bioengineering Co., Ltd. The powdered primers were first centrifuged at high speed and then diluted with nuclease-free water to 100 μM stock solution, and then diluted with sterile water to 10 μM working solution, and the dissolution curve was observed by qRT-PCR. Specific and dimer primers were used for subsequent experiments. The specific primer sequences are shown in the table below:

Figure BDA0002371900100000081
Figure BDA0002371900100000081

五、聚合酶链式反应(polymer chain reaction,PCR)Five, polymerase chain reaction (polymer chain reaction, PCR)

(1)根据PrimeSTAR GXL DNAPolymerase(Takara,R050Q)试剂盒说明书进行,首先在冰上配置如下反应体系:(1) According to the instructions of the PrimeSTAR GXL DNAPolymerase (Takara, R050Q) kit, first configure the following reaction system on ice:

试剂reagent 体积(μL)Volume (μL) 5×PrimeSTAR GXL BufferPrimeSTAR GXL Buffer 1010 dNTP Mixture(2.5mM each)dNTP Mixture (2.5mM each) 44 Forward PrimerForward Primer 1.51.5 Reverse PrimerReverse Primer 1.51.5 cDNAor DNAcDNA or DNA 11 PrimeSTAR GXL DNA PolymerasePrimeSTAR GXL DNA Polymerase 11 灭菌蒸馏水Sterilized distilled water up to 50up to 50

(2)PCR反应程序:98℃,10sec变性;55℃or 60℃,15sec退火(引物Tm值小于55℃设为55℃,大于55℃设为60℃);68℃,18sec延伸(按产物1kb设为1min计算);35个循环;(2) PCR reaction program: 98°C, 10sec denaturation; 55°C or 60°C, 15sec annealing (primer Tm value less than 55°C set to 55°C, greater than 55°C set to 60°C); 68°C, 18sec extension (according to the product 1kb is set as 1min calculation); 35 cycles;

(3)PCR产物结束后放于4℃保存,并尽快进行琼脂糖凝胶电泳。(3) Store the PCR product at 4°C and perform agarose gel electrophoresis as soon as possible.

六、琼脂糖凝胶电泳及Sanger测序6. Agarose gel electrophoresis and Sanger sequencing

根据产物片段长度,选择配置2%的琼脂糖凝胶,即2g琼脂粉加入到100mL1×TAE中,微波反复加热至完全溶解,溶液澄清,注意避免过度沸腾导致的液体蒸发而影响终浓度,冷却至60℃左右,加入10μL GelStain,摇匀后倒入已插好梳子的配胶槽内,室温约凝固40min,拔掉梳子,将胶放入电泳槽内,注意上样孔在负极侧,将1×TAE倒入槽内没过胶面约1mm,每孔上样10μL已加入loading buffer的PCR产物,5μL DNAmarker加于最左侧孔内,100V电压,电泳约25min~40min,电泳结束后在凝胶成像系统内采集图像,如图2所示,证实了CircEMB_003的反向剪切序列。然后用干净刀片切胶送测序,Sanger测序由北京睿博兴科生物技术有限公司完成,结果如图3所示,进一步证实了circEMB_003的反向剪切序列。According to the length of the product fragment, choose and configure 2% agarose gel, that is, add 2g agar powder to 100mL 1×TAE, microwave repeatedly to dissolve completely, the solution is clear, pay attention to avoid the liquid evaporation caused by excessive boiling and affect the final concentration, cool down When the temperature reaches about 60°C, add 10 μL of GelStain, shake well, pour it into the glue dispensing tank with the comb inserted, and solidify for about 40 minutes at room temperature. Pull out the comb and put the gel into the electrophoresis tank. Note that the sample hole is on the negative side. 1×TAE was poured into the tank for about 1mm below the glue surface, 10μL of PCR products added with loading buffer were loaded into each well, 5μL of DNA marker was added to the leftmost well, and the electrophoresis was performed at 100V for about 25min-40min. Images acquired within the gel imaging system, as shown in Figure 2, confirmed the reverse shearing sequence of CircEMB_003. Then, the gel was cut with a clean blade and sent for sequencing. Sanger sequencing was completed by Beijing Ruibo Xingke Biotechnology Co., Ltd. The results are shown in Figure 3, which further confirmed the reverse shearing sequence of circEMB_003.

七、荧光定量RT-PCR(qRT-PCR)7. Fluorescence quantitative RT-PCR (qRT-PCR)

(1)根据SybrGreen qPCR Master mix(DBI)说明书进行,避光条件下在冰上配置qRT-PCR反应体系如下:(1) According to the instructions of SybrGreen qPCR Master mix (DBI), configure the qRT-PCR reaction system on ice under dark conditions as follows:

试剂reagent 体积(μL)Volume (μL) SybrGreen qPCR master mixSybrGreen qPCR master mix 1010 Forward primer(10μM)Forward primer (10μM) 0.50.5 Reverse primer(10μM)Reverse primer (10μM) 0.50.5 cDNAcDNA 22 ddH<sub>2</sub>O(灭菌超纯水)ddH<sub>2</sub>O (sterile ultrapure water) 77 TotalTotal 2020

(2)每个样本加3个复孔,充分混匀后瞬时离心,八连管放入ABI7500荧光定量PCR仪中;(2) Add 3 duplicate wells to each sample, mix well and centrifuge briefly, and put the eight tubes into the ABI7500 fluorescence quantitative PCR instrument;

(3)qRT-PCR反应程序:95℃,预变性1min;95℃,变性15sec;60℃,退火及延伸34sec;40个循环;(3) qRT-PCR reaction program: 95°C, pre-denaturation for 1 min; 95°C, denaturation for 15sec; 60°C, annealing and extension for 34sec; 40 cycles;

(4)数据分析:用2-△△Ct方法进行数据整理分析,将各样品的Ct值(Ct值指所测反应管内的荧光信号达到所设定的阈值时总共经历的循环次数)减去内参的Ct值(△Ct=Ct目的基因-Ct内参)即△Ct值,放线菌素D处理实验用18S rRNA为内参,其余以GAPDH为内参,△△Ct=△Ct实验组-△Ct对照组,以公式2-△△Ct计算相对表达量。实验均重复3次,计算3次平均值及标准差。(4) Data analysis: use the 2- △△Ct method to organize and analyze the data, and subtract the Ct value of each sample (Ct value refers to the total number of cycles experienced when the fluorescence signal in the measured reaction tube reaches the set threshold) The Ct value of the internal reference (△Ct=Ct target gene-Ct internal reference) is the △Ct value, 18S rRNA was used as the internal reference in the actinomycin D treatment experiment, and GAPDH was used as the internal reference for the rest, △△Ct=△Ct experimental group-△Ct In the control group, the relative expression was calculated by formula 2 -ΔΔCt . The experiments were repeated three times, and the mean and standard deviation of the three times were calculated.

扩增曲线如图4所示,显示扩增效率高,且具有特异性。溶解曲线如图5所示,显示为单峰,说明无非特异性扩增及引物二聚体。CircEMB_003在43例配对结直肠癌组织与癌旁组织中表达的倍比关系图如图6所示(T/N,N代表正常结直肠组织,T代表结直肠癌组织),结果显示circEMB_003在结直肠癌组织表达下调。CircEMB_003在43例配对结直肠癌组织与癌旁组织中的平均表达水平如图7所示,Normal代表正常结直肠组织,Tumor代表结直肠癌组织,△Ct值越高则circEMB_003的表达水平越低,结果表明circEMB_003在结直肠癌组织的表达水平显著低于癌旁组织。The amplification curve is shown in Figure 4, showing that the amplification efficiency is high and specific. The melting curve is shown in Figure 5, showing a single peak, indicating the absence of non-specific amplification and primer dimers. The fold ratio of CircEMB_003 expression in 43 paired colorectal cancer tissues and adjacent tissues is shown in Figure 6 (T/N, N represents normal colorectal tissue, T represents colorectal cancer tissue). Down-regulated expression in rectal cancer tissues. The average expression levels of CircEMB_003 in 43 paired colorectal cancer tissues and adjacent tissues are shown in Figure 7. Normal represents normal colorectal tissue, and Tumor represents colorectal cancer tissue. The higher the △Ct value, the lower the expression level of circEMB_003. , the results showed that the expression level of circEMB_003 in colorectal cancer tissues was significantly lower than that in adjacent tissues.

八、RNA酶R消化验证circEMB_003稳定性8. RNase R digestion to verify the stability of circEMB_003

提取结直肠癌癌旁组织RNA,并分成1μg×4份,1份不加RNA酶R,另3份各加入0.05μL RNA酶R和RNA酶R buffer 0.7μL,加无核酸酶的灭菌水补至7μL,消化反应在PCR仪内进行,37℃分别消化15min、30min,消化完成后,与未消化的RNA一起分别在每管内加入gDNAbuffer 2μL和gDNA Eraser 1μL,42℃,2min去除RNA内可能会污染的gDNA,按照PrimeScriptTM RT reagent Kit with gDNAEraser(TaKaRa)说明书进行逆转录,逆转录成的cDNA用灭菌水稀释4倍,取2μL进行PCR及qRT-PCR,circEMB_003、EMB及GAPDH mRNA水平均与未消化前的RNA进行比较。RNA from adjacent tissues of colorectal cancer was extracted and divided into 1 μg×4 portions, one without RNase R, the other 3 with 0.05 μL RNase R and 0.7 μL RNase R buffer, and nuclease-free sterile water. Make up to 7 μL, the digestion reaction is carried out in a PCR machine, and digest at 37°C for 15min and 30min respectively. After the digestion is completed, add 2μL of gDNAbuffer and 1μL of gDNA Eraser to each tube together with the undigested RNA, and 42°C for 2min to remove possible RNA Contaminated gDNA was reverse transcribed according to the instructions of PrimeScript TM RT reagent Kit with gDNAEraser (TaKaRa), the reverse transcribed cDNA was diluted 4 times with sterilized water, and 2 μL was taken for PCR and qRT-PCR, circEMB_003, EMB and GAPDH mRNA levels Both were compared with the undigested RNA.

结果如图8所示,分别应用RNA酶R消化处理(+)结直肠细胞SW480的RNA15min、30min,0为未加RNA酶R(-)的RNA,应用(图8A)PCR扩增琼脂糖电泳及(图8B)qRT-PCR检测circEMB_003、EMB及GAPDH mRNA水平(*,p<0.05;**,p<0.01)。结果表明,与线性RNA相比,circEMB_003的确能够耐受RNA酶R的消化作用。The results are shown in Figure 8. The RNA of (+) colorectal cell SW480 was digested with RNase R for 15 min and 30 min, respectively, and 0 was the RNA without RNase R (-), and PCR amplification was performed by agarose electrophoresis (Figure 8A). And (Fig. 8B) qRT-PCR detection of circEMB_003, EMB and GAPDH mRNA levels (*, p<0.05; **, p<0.01). The results showed that, compared with linear RNA, circEMB_003 was indeed able to tolerate RNase R digestion.

九、放线菌素D处理细胞验证circEMB_003稳定性9. Actinomycin D treatment of cells to verify the stability of circEMB_003

(1)放线菌素D粉末(1mg)离心后在超净台内用100μL DMSO充分溶解(浓度10μg/μL),4℃保存,并尽快使用;(1) After centrifugation, actinomycin D powder (1 mg) was fully dissolved in 100 μL DMSO (concentration 10 μg/μL) in a clean bench, stored at 4°C, and used as soon as possible;

(2)SW480细胞铺于6孔板内,24h后,实验组加入放线菌素D(使终浓度为100ng/mL),对照组加入相同剂量的DMSO,每组设3个复孔;(2) SW480 cells were plated in 6-well plates, and after 24 hours, the experimental group was added with actinomycin D (to make the final concentration 100ng/mL), and the control group was added with the same dose of DMSO, with 3 duplicate wells in each group;

(3)分别在0h、12h、24h胰酶消化收集细胞提取RNA,定量后取500ng进行逆转录及后续qRT-PCR反应。(3) At 0h, 12h, and 24h, the cells were digested with trypsin to collect the RNA extracted from the cells, and 500 ng was taken for reverse transcription and subsequent qRT-PCR reaction after quantification.

结果如图9所示,应用放线菌素D抑制结直肠癌细胞SW480新生RNA合成验证circEMB_003的稳定性,qRT-PCR结果显示应用放线菌素D后12h,EMB已降解60%以上(p<0.0001),而circEMB_003未见明显降解(p>0.05),说明circEMB_003比线性RNA EMB稳定。The results are shown in Figure 9. The application of actinomycin D inhibited the nascent RNA synthesis of colorectal cancer cells SW480 to verify the stability of circEMB_003. The qRT-PCR results showed that 12h after the application of actinomycin D, EMB had been degraded by more than 60% (p <0.0001), while circEMB_003 was not significantly degraded (p>0.05), indicating that circEMB_003 is more stable than linear RNA EMB.

十、受试者工作特征曲线(ROC)评估circEMB_003潜在的诊断价值10. Receiver operating characteristic curve (ROC) to evaluate the potential diagnostic value of circEMB_003

应用CRC组织及癌旁组织中circEMB_003的相对表达量制作受试者工作特征曲线(ROC),评价其作为CRC诊断标志物的潜在价值。结果如图10所示,ROC曲线下面积(AUC)为0.768(95%可信区间为0.669~0.876),特异性为0.9275(95%可信区间为0.8389~0.9761)。ROC曲线下面积越接近1越具有诊断价值,这表明通过检测circEMB_003的表达可以高效地将结直肠癌从正常组织中鉴别出来,circEMB_003具备作为结直肠癌诊断标记物的潜在价值。The relative expression of circEMB_003 in CRC tissues and adjacent tissues was used to generate receiver operating characteristic (ROC) curves to evaluate its potential value as a diagnostic marker for CRC. The results are shown in Figure 10. The area under the ROC curve (AUC) was 0.768 (95% confidence interval, 0.669-0.876), and the specificity was 0.9275 (95% confidence interval, 0.8389-0.9761). The closer the area under the ROC curve is to 1, the more diagnostic value it is, which indicates that colorectal cancer can be efficiently identified from normal tissues by detecting the expression of circEMB_003, and circEMB_003 has potential value as a diagnostic marker for colorectal cancer.

十一、CircEMB_003在结直肠癌组织中的表达与患者临床病理特征的关系11. The relationship between the expression of CircEMB_003 in colorectal cancer tissues and the clinicopathological characteristics of patients

我们统计分析了结直肠癌组织中circEMB_003的相对表达量与结直肠癌患者临床病理参数之间的关系。结果如表1所示,circEMB_003的表达水平与结直肠癌肿瘤的大小(p<0.05)、TNM分期(p<0.05)及远处转移(p<0.05)呈负相关,而与病人的年龄、性别、肿瘤的分化没有相关性(p>0.05)。We statistically analyzed the relationship between the relative expression of circEMB_003 in colorectal cancer tissues and the clinicopathological parameters of colorectal cancer patients. The results are shown in Table 1. The expression level of circEMB_003 was negatively correlated with the tumor size (p<0.05), TNM stage (p<0.05) and distant metastasis (p<0.05) of colorectal cancer, and was negatively correlated with the patient's age, There was no correlation between gender and tumor differentiation (p>0.05).

表1CircEMB_003的表达水平和结直肠癌患者临床病理特征之间的关系Table 1 The relationship between the expression level of CircEMB_003 and the clinicopathological characteristics of colorectal cancer patients

Figure BDA0002371900100000111
Figure BDA0002371900100000111

Figure BDA0002371900100000121
Figure BDA0002371900100000121

a依据平均年龄分组。aGrouped by mean age.

b肿瘤直径依据平均值分组。bTumor diameters are grouped by mean.

*p<0.5。*p<0.5.

十二、荧光原位分子杂交(Fluorescence In situ hybridization,FISH)12. Fluorescence In situ hybridization (FISH)

将30例大肠癌标本的石蜡切片经脱石蜡、梯度脱水后,将载玻片浸入0.2N的HCl内5min,洗涤后用40μg/mL的蛋白酶K在37℃条件下消化10min,PBS洗涤后用乙醇梯度脱水:70%、80%、90%、无水乙醇浸泡。从乙醇中取出的组织迅速滴加1×杂交buffer,室温放置5min,轻轻倾去buffer,并加入由1×杂交buffer稀释的绿色荧光标记的circEMB_003探针(50nM),盖上盖玻片56℃处理1小时。SSC梯度洗杂,5×SSC 56℃5min,1×SSC 56℃5min×2次,0.2×SSC 56℃5min×2次,0.2×SSC常温5min,PBS冲洗5min×3次。滴加DAPI工作液(1:1000),室温孵育10min,PBS冲洗5min×3次。滴加适量抗荧光衰减封片剂封片,然后使用荧光显微镜进行观察、照相,结果如图11所示,表明circEMB_003在结直肠癌组织中表达下调。After deparaffinization and gradient dehydration of paraffin sections of 30 colorectal cancer specimens, the slides were immersed in 0.2N HCl for 5 min, washed with 40 μg/mL proteinase K at 37 °C for 10 min, and washed with PBS with Ethanol gradient dehydration: 70%, 80%, 90%, absolute ethanol soaking. The tissue taken out of ethanol was quickly added dropwise with 1× hybridization buffer, placed at room temperature for 5 min, the buffer was gently poured, and the green fluorescent labeled circEMB_003 probe (50 nM) diluted with 1× hybridization buffer was added, and covered with a coverslip 56 ℃ for 1 hour. SSC gradient washing, 5×SSC at 56°C for 5min, 1×SSC at 56°C for 5min×2 times, 0.2×SSC at 56°C for 5min×2 times, 0.2×SSC at room temperature for 5min, and PBS for 5min×3 times. DAPI working solution (1:1000) was added dropwise, incubated at room temperature for 10 min, and washed with PBS for 5 min × 3 times. An appropriate amount of anti-fluorescence decay mounting medium was added dropwise to mount the slides, and then observed and photographed using a fluorescence microscope. The results are shown in Figure 11, indicating that the expression of circEMB_003 is down-regulated in colorectal cancer tissues.

十三、CircEMB_003在结直肠癌细胞株中的表达13. Expression of CircEMB_003 in colorectal cancer cell lines

应用qRT-PCR技术分别检测circEMB_003在7种结直肠癌细胞株SW620、HCT116、RKO、SW480、LoVo、Caco-2、HCT8及正常结肠上皮细胞株NCM460中的表达,结果如图12所示,结果表明与NCM460相比,结直肠癌细胞中circEMB_003的表达水平显著下调。The expression of circEMB_003 in seven colorectal cancer cell lines SW620, HCT116, RKO, SW480, LoVo, Caco-2, HCT8 and normal colon epithelial cell line NCM460 was detected by qRT-PCR. The results are shown in Figure 12. showed that the expression level of circEMB_003 was significantly down-regulated in colorectal cancer cells compared with NCM460.

以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

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Claims (10)

1.环状RNA circEMB_003的定量检测剂在制备用于结直肠癌诊断的试剂盒中的应用,其特征在于,所述环状RNA circEMB_003的核苷酸序列如SEQ ID NO.1所示。1. The application of a quantitative detection agent of circular RNA circEMB_003 in the preparation of a kit for colorectal cancer diagnosis, wherein the nucleotide sequence of the circular RNA circEMB_003 is shown in SEQ ID NO.1. 2.根据权利要求1所述的应用,其特征在于,所述环状RNA circEMB_003的定量检测剂包括适用于如下至少一种方法的试剂:2. application according to claim 1, is characterized in that, the quantitative detection reagent of described circular RNA circEMB_003 comprises the reagent that is applicable to following at least one method: 数字PCR、共振光散射法、实时荧光定量PCR、测序或生物质谱法。Digital PCR, resonance light scattering, real-time PCR, sequencing or biological mass spectrometry. 3.根据权利要求1所述的应用,其特征在于,所述环状RNA circEMB_003的定量检测剂为能够特异性结合环状RNA circEMB_003或环状RNA circEMB_003对应的cDNA的探针或引物。3 . The application according to claim 1 , wherein the quantitative detection agent for circular RNA circEMB_003 is a probe or primer that can specifically bind to circular RNA circEMB_003 or cDNA corresponding to circular RNA circEMB_003. 4 . 4.根据权利要求3所述的应用,其特征在于,所述探针或引物带有可检测的标记。4. The use according to claim 3, wherein the probe or primer has a detectable label. 5.根据权利要求4所述的应用,其特征在于,所述标记为荧光标记物或同位素标记物。5. The application according to claim 4, wherein the label is a fluorescent label or an isotope label. 6.根据权利要求1所述的应用,其特征在于,所述环状RNA circEMB_003的定量检测剂为环状RNA circEMB_003的qRT-PCR引物,其上游引物如SEQ ID NO.2所示,下游引物如SEQID NO.3所示。6. The application according to claim 1, wherein the quantitative detection agent of the circular RNA circEMB_003 is the qRT-PCR primer of the circular RNA circEMB_003, and the upstream primer is shown in SEQ ID NO. As shown in SEQID NO.3. 7.根据权利要求1~6任一项所述的应用,其特征在于,所述试剂盒中还包括内参引物对。7. The application according to any one of claims 1 to 6, wherein the test kit also includes an internal reference primer pair. 8.根据权利要求7所述的应用,其特征在于,所述内参引物对包括18S rRNA扩增引物对和GAPDH扩增引物对中的一种或多种。8. The application according to claim 7, wherein the pair of internal reference primers comprises one or more of a pair of 18S rRNA amplification primers and a pair of GAPDH amplification primers. 9.根据权利要求8所述的应用,其特征在于,所述18S rRNA扩增引物对的核苷酸序列分别如SEQ ID NO.4和SEQ ID NO.5所示,所述GAPDH扩增引物对的核苷酸序列分别如SEQ IDNO.6和SEQ ID NO.7所示。9. The application according to claim 8, wherein the nucleotide sequences of the 18S rRNA amplification primer pair are respectively shown in SEQ ID NO.4 and SEQ ID NO.5, and the GAPDH amplification primer The nucleotide sequences of the pairs are shown in SEQ ID NO. 6 and SEQ ID NO. 7, respectively. 10.根据权利要求7所述的应用,其特征在于,所述试剂盒中还包括RNA提取试剂、PCR反应缓冲液、dNTPs以及DNA聚合酶中的至少一种。10 . The application according to claim 7 , wherein the kit further comprises at least one of RNA extraction reagents, PCR reaction buffer, dNTPs and DNA polymerase. 11 .
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