CN106148495A - The application in colorectal cancer biomarker of a kind of circular rna - Google Patents

Abstract

Translated fromChinese

本发明提供了一种环状RNA(Circular RNA\circRNA)(CPSF6-circRNA)在结直肠癌生物标志物中的应用，这条环状RNA(CPSF6-circRNA)长11434bp，位于人类的第12条染色体上，CPSF6是该circRNA的覆盖基因。本发明可用于鉴别可能患有结直肠癌的对象或结直肠癌的病理分型，再或者确定先前经鉴别的对象的手术预后的方法。本发明制备了筛查环状RNA(CPSF6-circRNA)的引物。所述方法包括检测来自所述对象的癌症和癌旁组织样品中的环状RNA(CPSF6-circRNA)的量，其中较高量的CPSF6-circRNA与所述对象结直肠癌预后不良的可能性增加相关。具有制备临床结直肠癌生物标志物的应用前景。The present invention provides the application of a circular RNA (CPSF6-circRNA) in colorectal cancer biomarkers. This circular RNA (CPSF6-circRNA) is 11434bp long and is located in the 12th human On the chromosome, CPSF6 is the covering gene of this circRNA. The present invention is useful in methods of identifying subjects likely to have colorectal cancer or pathological types of colorectal cancer, or determining surgical prognosis in previously identified subjects. The present invention prepares primers for screening circular RNA (CPSF6-circRNA). The method comprises detecting the amount of circular RNA (CPSF6-circRNA) in cancer and paracancerous tissue samples from the subject, wherein a higher amount of CPSF6-circRNA is associated with an increased likelihood of a poor prognosis for colorectal cancer in the subject relevant. It has the application prospect of preparing clinical colorectal cancer biomarkers.

Description

Translated fromChinese

一种环状RNA在结直肠癌生物标志物中的应用Application of a circular RNA in colorectal cancer biomarkers

技术领域technical field

本发明属于天然核糖核苷酸的用途，涉及环状核糖核苷酸(CPSF6-circRNA)的用途，尤其涉及CPSF6-circRNA结直肠癌患者的新的生物标记中的用途，用于鉴别结直肠癌及更不良预后的对象。The present invention belongs to the use of natural ribonucleotides, relates to the use of circular ribonucleotides (CPSF6-circRNA), in particular to the use of CPSF6-circRNA in new biomarkers for colorectal cancer patients, and is used to identify colorectal cancer and those with worse prognosis.

背景技术Background technique

结直肠癌又称大肠癌，是一种常见的恶性肿瘤。结肠癌在早期并无明显症状，直到发病中晚期发现。提高结直肠癌的早期诊断率对于及时治疗和改善预后至关重要。如通过在结直肠癌中出现的CPSF6-circRNA所证明的，已知在结直肠癌组织中有异常的CPSF6-circRNA表达。考虑到CPSF6-circRNA表达量在结直肠癌中可升高或降低，因此提供了疾病进展的病理学指征。Colorectal cancer, also known as colorectal cancer, is a common malignant tumor. Colon cancer has no obvious symptoms in the early stage until it is discovered in the middle and late stages of the disease. Improving the early diagnosis rate of colorectal cancer is crucial for timely treatment and improved prognosis. There is known to be aberrant CPSF6-circRNA expression in colorectal cancer tissues, as evidenced by the emergence of CPSF6-circRNA in colorectal cancer. Considering that the expression of CPSF6-circRNA can be increased or decreased in colorectal cancer, it provides a pathological indication of disease progression.

circRNA广泛存在于各种生物细胞中，具有结构稳定、丰度高和组织特异性表达等特征。是基因组DNA转录表达过程中，环化成环形RNA，的一类非编码RNA。研究表明，一些circRNA作为竞争性内源RNA(ceRNA)来发挥基因表达调控的作用。circRNA利用其microRNA(miRNA)应答元件结合miRNA，以阻断miRNA对其靶标基因表达的抑制作用，从而调控其他相关mRNA的表达水平。circRNA在基因表达调控中重要作用的发现提示circRNA在药物开发和疾病诊治中具有良好的应用前景。circRNAs widely exist in various biological cells and have the characteristics of stable structure, high abundance and tissue-specific expression. It is a type of non-coding RNA that is circularized into circular RNA during the transcription and expression of genomic DNA. Studies have shown that some circRNAs function as competitive endogenous RNAs (ceRNAs) to regulate gene expression. CircRNA uses its microRNA (miRNA) response element to bind miRNA to block the inhibitory effect of miRNA on the expression of its target genes, thereby regulating the expression level of other related mRNAs. The discovery of the important role of circRNA in the regulation of gene expression suggests that circRNA has a good application prospect in drug development and disease diagnosis and treatment.

circRNA作为一种调控性RNA(Regulatory RNA)近几年成为肿瘤研究的热点领域。circRNAs其表达具备组织特异性。circRNA中既有一部分作为竞争性内源RNA(ceRNA)来发挥基因表达调控的作用。Thomas B.Hansen等人通过对人AGO蛋白和miR-7的分析研究，证明了circRNA作为高效的microRNA海绵的作用。circRNAs在肿瘤和正常组织中的表达水平差别十分显著，甚至能达到数百甚至数千倍，这种显著的差异使得其在成为候选标志物上具有明显优势。As a kind of regulatory RNA (Regulatory RNA), circRNA has become a hot spot in tumor research in recent years. The expression of circRNAs has tissue specificity. Some of circRNAs function as competitive endogenous RNAs (ceRNAs) to regulate gene expression. Thomas B. Hansen et al. proved the role of circRNA as an efficient microRNA sponge through the analysis of human AGO protein and miR-7. The expression levels of circRNAs in tumors and normal tissues differ significantly, even reaching hundreds or even thousands of times. This significant difference makes them have obvious advantages in being candidate markers.

发明内容Contents of the invention

本发明的目的是提供环状RNA(Circular RNA\circRNA)CPSF6-circRNA在结直肠癌生物标志物中的应用，英文名CPSF6-circRNA，CPSF6-circRNA长11434bp，位于人类的第12条染色体上69644908到69656342，CPSF6-circRNA的碱基序列结构，CPSF6是该circRNA的覆盖基因。The purpose of the present invention is to provide the application of circular RNA (Circular RNA\circRNA) CPSF6-circRNA in colorectal cancer biomarkers, the English name is CPSF6-circRNA, CPSF6-circRNA is 11434bp long, located on the 12th chromosome of human beings 69644908 To 69656342, the base sequence structure of CPSF6-circRNA, CPSF6 is the covering gene of this circRNA.

本发明提供了鉴别可能患有结直肠癌的对象或结直肠癌的分期，再或者对先前经鉴别患有结直肠癌的对象的预后进行确定的方法及其引物序列结构、PCR产物识别序列。The present invention provides a method for identifying a subject who may have colorectal cancer or the stage of colorectal cancer, or determining the prognosis of a subject previously identified as having colorectal cancer, as well as its primer sequence structure and PCR product recognition sequence.

所述方法包括检测来自所述对象的样品中的CPSF6-circRNA量，其中较高量的CPSF6-circRNA与所述对象患有结直肠癌的可能性增加或者结直肠癌预后不佳的可能性增加相关。The method comprises detecting the amount of CPSF6-circRNA in a sample from the subject, wherein a higher amount of CPSF6-circRNA is associated with an increased likelihood that the subject has colorectal cancer or an increased likelihood that the colorectal cancer has a poor prognosis relevant.

本发明的有益效果有：1)提出了CPSF6-circRNA的用途；2)本发明的研究表明异常的量的CPSF6-circRNA与所述对象患结直肠癌的可能性增加或者可能提示结直肠癌预后不佳。3)circRNA具有结构稳定、丰度高和组织特异性表达等特征。且circRNA在结直肠癌病人的癌组织和癌旁组织中表达量存在差异，因此CPSF6-circRNA具有成为结直肠癌生物标志物的应用前景。The beneficial effects of the present invention include: 1) the use of CPSF6-circRNA is proposed; 2) the research of the present invention shows that the abnormal amount of CPSF6-circRNA is associated with an increased possibility of the subject suffering from colorectal cancer or may indicate the prognosis of colorectal cancer bad. 3) circRNA has the characteristics of stable structure, high abundance and tissue-specific expression. Moreover, there are differences in the expression of circRNA in cancer tissues and adjacent tissues of colorectal cancer patients, so CPSF6-circRNA has the prospect of becoming a biomarker of colorectal cancer.

附图说明Description of drawings

图1为CPSF6-circRNA的PCR识别序列与PCR产物测序结果的对比图。Figure 1 is a comparison chart of the PCR recognition sequence of CPSF6-circRNA and the sequencing results of PCR products.

图2为使用CPSF6-circRNA筛查引物对病人组织样品表达量筛查的结果图。Figure 2 is a graph showing the results of screening the expression level of patient tissue samples using CPSF6-circRNA screening primers.

图3为CPSF6-circRNA的简易结构及引物位置图。Figure 3 is a simplified structure and primer location map of CPSF6-circRNA.

具体实施方式detailed description

下面结合具体实验步骤对本发明进行进一步描述。The present invention will be further described below in conjunction with specific experimental steps.

1)实验材料：1) Experimental materials:

临床样品：16例结直肠癌病人的癌组织和癌旁组织样品由301医院所提供。Clinical samples: 16 colorectal cancer patients' cancer tissue and paracancerous tissue samples were provided by 301 Hospital.

试剂：CPSF6-circRNA筛查引物由生工生物工程(上海)股份有限公司北京引物合成部合成；Trizol(货号15596-018)购自Invitrogen；氯仿(货号20100927)购自北京化工，异丙醇(货号1205031)购自西陇化工，乙醇(货号101860)购自北化经销；Random primers(货号C1181)，M-MLV(货号M1705)，Ribonuclease Inhibitor(货号2511)，Nuclease-FreeWater(货号2511)，dNTP mix(货号P1195)均购自Promega；Premix Ex TaqTM II(货号DRR081A)购自Takara；Mineral oil(货号D7295)购自Sigma。Reagents: CPSF6-circRNA screening primers were synthesized by the Beijing Primer Synthesis Department of Sangon Bioengineering (Shanghai) Co., Ltd.; Trizol (product number 15596-018) was purchased from Invitrogen; chloroform (product number 20100927) was purchased from Beijing Chemical Industry, isopropanol ( Product No. 1205031) was purchased from Xilong Chemical Industry; ethanol (Product No. 101860) was purchased from Beihua Distribution; Random primers (Product No. C1181), M-MLV (Product No. M1705), Ribonuclease Inhibitor (Cat. No. 2511), Nuclease-FreeWater (Cat. No. 2511), dNTP mix (Cat. No. P1195) were purchased from Promega; Premix Ex TaqTM II (Cat. No. DRR081A) was purchased from Takara; Mineral oil (Cat. No. D7295) was purchased from Sigma.

2)实验方法：2) Experimental method:

A、结直肠癌癌变组织/癌旁组织RNA提取A. RNA extraction from colorectal cancer tissues/paracancerous tissues

称取1克组织材料，液氮研磨后，转移至1.5毫升RNase-free的离心管中，混匀后室温静置5分钟左右。加入200微升的氯仿，混匀。11000转每分钟离心10分钟。取上清加入200ul的氯仿抽提，11000转每分钟离心10分钟。取上清加入500微升的异丙醇，-20℃沉淀10分钟。11000转每分钟离心15分钟，将沉淀转移至1.5毫升RNase-free离心管中，预冷的75％乙醇洗涤2遍，晾干，RNase-free水溶解(根据情况适当溶解，一般是20-35微升)。Nanodrop测算所得粗提液的OD值以及浓度。Weigh 1 gram of tissue material, grind it with liquid nitrogen, transfer it to a 1.5 ml RNase-free centrifuge tube, mix well, and let stand at room temperature for about 5 minutes. Add 200 µl of chloroform and mix well. Centrifuge at 11,000 rpm for 10 minutes. Take the supernatant and add 200ul of chloroform for extraction, and centrifuge at 11000 rpm for 10 minutes. Take the supernatant and add 500 microliters of isopropanol, and precipitate at -20°C for 10 minutes. Centrifuge at 11,000 rpm for 15 minutes, transfer the precipitate to a 1.5 ml RNase-free centrifuge tube, wash twice with pre-cooled 75% ethanol, dry in the air, and dissolve in RNase-free water (appropriately dissolved according to the situation, generally 20-35 µl). Nanodrop measured and calculated the OD value and concentration of the obtained crude extract.

B、表达量筛查B. Expression screening

1.cDNA合成1. cDNA synthesis

1-2微克RNA到14微升RNase-free水中，再加1微升random primer轻柔混匀后加入0.2mlPCR管中。PCR仪70℃孵育5分钟。快速放在冰上2分钟。Add 1-2 micrograms of RNA to 14 microliters of RNase-free water, add 1 microliter of random primer, mix gently and add to a 0.2ml PCR tube. Incubate at 70°C for 5 minutes in a PCR instrument. Place quickly on ice for 2 min.

配置反应混合物：Configure the reaction mixture:

42℃孵育1小时，70℃孵育15分钟，4℃保温。Incubate at 42°C for 1 hour, at 70°C for 15 minutes, and incubate at 4°C.

2.Real-time PCR2. Real-time PCR

cDNA中加入50ul的ddH₂O。Add 50ul of ddH₂ O to the cDNA.

在冰上制备QPCR混合物：Prepare the qPCR mix on ice:

每管中加入10微升矿物油。离心机1000转每分钟离心1分钟。将样品放置入QPCR仪中运行，程序如下：Add 10 µl of mineral oil to each tube. Centrifuge at 1000 rpm for 1 minute. Place the sample into the QPCR instrument to run, the procedure is as follows:

3)实验结果：3) Experimental results:

参见图1和图2，图1为CPSF6-circRNA的PCR识别序列与PCR产物测序结果的对比图。实验结果表明CPSF6-circRNA的PCR识别序列与PCR产物测序结果比对成功，CPSF6-circRNA在结直肠癌病人的癌组织和癌旁组织中表达。图2为使用CPSF6-circRNA筛查引物对病人组织样品表达量筛查的结果图。实验结果表明CPSF6-circRNA在结直肠癌病人的癌组织和癌旁组织中的表达差异较大，癌组织相对于癌旁组织下调存在着从0.1905349倍到0.9904351倍的变化其中6/11的病人下调倍数在0.5以下；上调存在1.599753倍到18.333660倍的表达量变化，其中4/5的病人上调倍数在5倍以内，1/5的病人上调在10倍以上。如此大的表达差异给了CPSF6-circRNA作为结直肠癌生物标志物的应用前景。See Figure 1 and Figure 2, Figure 1 is a comparison chart of the PCR recognition sequence of CPSF6-circRNA and the sequencing results of PCR products. The experimental results showed that the PCR recognition sequence of CPSF6-circRNA was successfully compared with the PCR product sequencing results, and CPSF6-circRNA was expressed in cancer tissues and adjacent tissues of colorectal cancer patients. Figure 2 is a graph showing the results of screening the expression level of patient tissue samples using CPSF6-circRNA screening primers. The experimental results show that the expression of CPSF6-circRNA in colorectal cancer patients' cancer tissue and para-cancerous tissue is quite different, and the down-regulation of cancer tissue relative to para-cancerous tissue has a change from 0.1905349 times to 0.9904351 times, of which 6/11 patients are down-regulated The multiple is below 0.5; the up-regulation has a change in expression level from 1.599753 times to 18.333660 times, of which 4/5 patients have an up-regulation factor within 5 times, and 1/5 patients have an up-regulation rate of more than 10 times. Such a large expression difference gave CPSF6-circRNA a promising application as a biomarker for colorectal cancer.

Claims (9)

1. circular rna (Circular RNA circRNA) CPSF6-circRNA is in preparation colorectal cancer biomarkerApplication, it is characterised in that the long 11434bp of described CPSF6-circRNA, is positioned on the 12nd article of chromosome of the mankind 69644908To 69656342, the base sequence of described CPSF6-circRNA is as shown in SEQ ID NO:1.

Applying the most as claimed in claim 1, it includes detecting in the sample of the object that may suffer from colorectal cancerCPSF6-circRNA measures, and wherein the CPSF6-circRNA of higher amount and described object suffer from the probability increasing of colorectal cancerAdd or probability that colorectal cancer prognosis is the best increases relevant.

Applying the most as claimed in claim 2, after treatment of colorectal cancer, the CPSF6-circRNA of reduced levels represents that treatment acts asWith.

Applying the most as claimed in claim 2 or claim 3, the amount of described CPSF6-circRNA is cancerous tissue and cancer beside organism in sampleAbsolute magnitude.

Applying the most as claimed in claim 2 or claim 3, the amount of wherein said CPSF6-circRNA is from the blood serum sample from objectMiddle determine.

Applying the most as claimed in claim 2 or claim 3, the amount of wherein said CPSF6-circRNA is from the plasma sample from objectMiddle determine.

7. the primer detecting colorectal cancer biomarker, it is characterised in that described primer is used for identifying CPSF6-circRNA,The long 11434bp of described CPSF6-circRNA, is positioned on the 12nd article of chromosome of the mankind 69644908 to 69656342,The base sequence of described CPSF6-circRNA is as shown in SEQ ID NO:1.

8. detection primer as claimed in claim 7, it is for obtaining the CPSF6-circRNA expression of cancerous tissue and cancer beside organism,By the risk that the two contrast suffers from colorectal cancer in denoted object is increased or prognosis mala.

9. the test kit detecting colorectal cancer biomarker, it is characterised in that include for identifying CPSF6-circRNA'sThe combination of primer, probe, primer or probe combinations, the long 11434bp of described CPSF6-circRNA, be positioned at the 12nd of the mankind theOn bar chromosome 69644908 to 69656342, the base sequence of described CPSF6-circRNA such as SEQ ID NO:1 instituteShow.