CN110714072B - A cancer prognosis assessment kit based on detection of LncRNA - Google Patents

Abstract

Translated fromChinese

本发明公开了一种基于检测LncRNA的癌症预后评估试剂盒，试剂盒包括可检测RP11‑783K16.13、RP11‑727F15.11、PRKAG2‑AS1、AC013460.1、CRNDE表达水平的检测试剂；本发明提供了可高效预测癌症的预后的试剂盒。

The invention discloses a cancer prognosis assessment kit based on the detection of LncRNA. The kit includes detection reagents capable of detecting the expression levels of RP11‑783K16.13, RP11‑727F15.11, PRKAG2‑AS1, AC013460.1, and CRNDE; A kit capable of efficiently predicting the prognosis of cancer is provided.

Description

Translated fromChinese

一种基于检测LncRNA的癌症预后评估试剂盒A cancer prognosis assessment kit based on detection of LncRNA

技术领域Technical Field

本发明涉及医药卫生领域，尤其涉及一种基于检测LncRNA的癌症预后评估试剂盒。The present invention relates to the field of medicine and health, and in particular to a cancer prognosis assessment kit based on detecting LncRNA.

背景技术Background Art

前列腺癌是指发生在前列腺的上皮性恶性肿瘤。2012年我国肿瘤登记地区前列腺癌发病率为9.92/10万，列男性恶性肿瘤发病率的第6位。发病年龄在55岁前处于较低水平，55岁后逐渐升高，发病率随着年龄的增长而增长，高峰年龄是70～80岁。家族遗传型前列腺癌患者发病年龄稍早，年龄≤55岁的患者占43％。Prostate cancer refers to an epithelial malignant tumor that occurs in the prostate gland. In 2012, the incidence of prostate cancer in my country's tumor registration areas was 9.92/100,000, ranking sixth among male malignant tumors. The age of onset is relatively low before the age of 55, and gradually increases after the age of 55. The incidence rate increases with age, with the peak age being 70 to 80 years old. Patients with familial hereditary prostate cancer develop at a slightly earlier age, with 43% of patients aged ≤55 years old.

前列腺癌根治术是治疗局限性前列腺癌的重要手段之一。但是，前列腺癌根治术后，仍有相当多的患者会出现生化复发。当患者在发生生化复发后，需要明确是否已经发生临床复发，以及是局部复发，区域淋巴结转移还是远处转移。在临床工作中，前列腺癌根治术后生化复发的评估常常是令人头疼的，从而导致其治疗也是缺乏精准的。Radical prostatectomy is one of the important means of treating localized prostate cancer. However, after radical prostatectomy, a considerable number of patients will still experience biochemical recurrence. When a patient has a biochemical recurrence, it is necessary to clarify whether a clinical recurrence has occurred, and whether it is a local recurrence, regional lymph node metastasis, or distant metastasis. In clinical work, the evaluation of biochemical recurrence after radical prostatectomy is often a headache, which leads to a lack of precision in its treatment.

长链非编码RNA(Long non-coding RNA,LncRNA)是长度大于200个核苷酸的非编码RNA。研究表明,LncRNA在剂量补偿效应、表观遗传调控、细胞周期调控和细胞分化调控等众多生命活动中发挥重要作用，成为遗传学研究热点。研究发现,长链非编码RNA的表达或功能异常与人类疾病的发生密切相关,其中就包括癌症、退行性神经疾病在内的多种严重危害人类健康的重大疾病,具体表现为长链非编码RNA在序列和空间结构上的异常、表达水平的异常、与结合蛋白相互作用的异常等。Long non-coding RNA (LncRNA) is a non-coding RNA with a length greater than 200 nucleotides. Studies have shown that LncRNA plays an important role in many life activities such as dosage compensation effect, epigenetic regulation, cell cycle regulation and cell differentiation regulation, and has become a hot topic in genetic research. Studies have found that the expression or functional abnormality of long non-coding RNA is closely related to the occurrence of human diseases, including cancer, degenerative neurological diseases and many other major diseases that seriously endanger human health, which are specifically manifested in the abnormality of long non-coding RNA in sequence and spatial structure, abnormal expression level, abnormal interaction with binding proteins, etc.

现有研究已经发现了大量的与前列腺癌相关基因，2018年发表于癌症研究和临床肿瘤学杂志(Cancer Research and Clinical Oncology)的一篇论文，Validation of a10-gene molecular signature for predicting biochemical recurrence andclinical metastasis in localized prostate cancer，公开了FRZB,LEF1,SDCBP,WNT2,ING3,ANK3,MEIS2,ANXA4,PLA2G7和CHD5共10个基因的表达用于预测前列腺癌生化复发的模型。该论文还公开了采用该模型预测前列腺癌的结果：AUC值为0.65，HR值为0.24，95％CI：0.09-0.59。Existing studies have discovered a large number of genes related to prostate cancer. A paper published in Cancer Research and Clinical Oncology in 2018, Validation of a 10-gene molecular signature for predicting biochemical recurrence and clinical metastasis in localized prostate cancer, disclosed a model for predicting biochemical recurrence of prostate cancer using the expression of 10 genes, including FRZB, LEF1, SDCBP, WNT2, ING3, ANK3, MEIS2, ANXA4, PLA2G7 and CHD5. The paper also disclosed the results of using the model to predict prostate cancer: AUC value was 0.65, HR value was 0.24, 95% CI: 0.09-0.59.

虽然现有技术公开了大量的与前列腺癌相关基因，但是前列腺癌相关基因在实际应用中用于评估前列腺癌却受限。现有模型的预测效果仍有待增强，需要从大量的前列腺癌基因中建立能够更加精准预测前列腺癌复发风险的模型。Although the prior art has disclosed a large number of genes related to prostate cancer, the practical application of prostate cancer-related genes in assessing prostate cancer is limited. The prediction effect of the existing model still needs to be enhanced, and a model that can more accurately predict the risk of prostate cancer recurrence needs to be established from a large number of prostate cancer genes.

发明内容Summary of the invention

针对上述技术问题，本发明提供了一种基于检测LncRNA的癌症预后评估试剂盒。In view of the above technical problems, the present invention provides a cancer prognosis assessment kit based on the detection of LncRNA.

本发明的一个方面，公开了一种基于检测LncRNA的癌症预后评估试剂盒，所述试剂盒包括可检测RP11-783K16.13、RP11-727F15.11、PRKAG2-AS1、AC013460.1、CRNDE表达水平的检测试剂。In one aspect of the present invention, a cancer prognosis assessment kit based on detecting LncRNA is disclosed, wherein the kit comprises a detection reagent capable of detecting the expression levels of RP11-783K16.13, RP11-727F15.11, PRKAG2-AS1, AC013460.1, and CRNDE.

优选地，所述检测试剂为多核苷酸引物或探针。Preferably, the detection reagent is a polynucleotide primer or a probe.

优选地，多核苷酸引物的序列如SEQ ID NO:1-10所示。Preferably, the sequences of the polynucleotide primers are shown in SEQ ID NOs: 1-10.

优选地，其中所述癌症选自前列腺癌，优选的，所述试剂盒用于前列腺癌根治术后预后的评估。Preferably, the cancer is selected from prostate cancer. Preferably, the kit is used for evaluating the prognosis after radical prostatectomy.

优选地，本发明的癌症预后方法包括以下步骤：Preferably, the cancer prognosis method of the present invention comprises the following steps:

a)检测样本中RP11-783K16.13、RP11-727F15.11、PRKAG2-AS1、AC013460.1、CRNDE的mRNA表达水平；a) Detect the mRNA expression levels of RP11-783K16.13, RP11-727F15.11, PRKAG2-AS1, AC013460.1, and CRNDE in the samples;

b)根据步骤a)获得的表达水平数据评估患者的癌症复发风险。b) assessing the patient's risk of cancer recurrence based on the expression level data obtained in step a).

优选地，步骤b)中通过以下方法评估患者的癌症复发风险：Preferably, in step b), the patient's risk of cancer recurrence is assessed by the following method:

风险值＝(0.0766×RP11-783K16.13表达水平)+(0.2443×RP11-727F15.11表达水平)+(0.0042×PRKAG2-AS1表达水平)+(2.8117×AC013460.1表达水平)+(0.0162×CRNDE表达水平)，其中所述的表达水平为步骤a)中检测获得的mRNA表达值。Risk value=(0.0766×RP11-783K16.13 expression level)+(0.2443×RP11-727F15.11 expression level)+(0.0042×PRKAG2-AS1 expression level)+(2.8117×AC013460.1 expression level)+(0.0162×CRNDE expression level), wherein the expression level is the mRNA expression value obtained by detection in step a).

优选的，所述检测mRNA表达水平的方法包括：Affymetrix/Illumina的芯片检测、全转录组鸟枪法测序、RT-PCR。Preferably, the method for detecting mRNA expression level includes: Affymetrix/Illumina chip detection, whole transcriptome shotgun sequencing, and RT-PCR.

本发明的另一个方面，检测RP11-783K16.13、RP11-727F15.11、PRKAG2-AS1、AC013460.1、CRNDE表达水平的检测试剂在制备用于癌症预后的产品中的用途。Another aspect of the present invention is the use of a detection reagent for detecting the expression levels of RP11-783K16.13, RP11-727F15.11, PRKAG2-AS1, AC013460.1, and CRNDE in the preparation of a product for cancer prognosis.

优选地，本发明可用于前列腺癌生化复发的早期预测，优选的，本发明可用于前列腺癌根治术后预后的评估。Preferably, the present invention can be used for early prediction of biochemical recurrence of prostate cancer. Preferably, the present invention can be used for evaluation of prognosis after radical prostatectomy.

优选地，所述癌症预后方法包括以下步骤：Preferably, the cancer prognosis method comprises the following steps:

a)检测样本中RP11-783K16.13、RP11-727F15.11、PRKAG2-AS1、AC013460.1、CRNDE的mRNA表达水平；a) Detect the mRNA expression levels of RP11-783K16.13, RP11-727F15.11, PRKAG2-AS1, AC013460.1, and CRNDE in the samples;

b)根据步骤a)获得的表达水平数据评估患者的癌症复发风险。b) assessing the patient's risk of cancer recurrence based on the expression level data obtained in step a).

优选地，步骤b)中通过以下方法评估患者的癌症复发风险：Preferably, in step b), the patient's risk of cancer recurrence is assessed by the following method:

风险值＝(0.0766×RP11-783K16.13表达水平)+(0.2443×RP11-727F15.11表达水平)+(0.0042×PRKAG2-AS1表达水平)+(2.8117×AC013460.1表达水平)+(0.0162×CRNDE表达水平)，其中所述的表达水平为步骤a)中检测获得的mRNA表达值。Risk value=(0.0766×RP11-783K16.13 expression level)+(0.2443×RP11-727F15.11 expression level)+(0.0042×PRKAG2-AS1 expression level)+(2.8117×AC013460.1 expression level)+(0.0162×CRNDE expression level), wherein the expression level is the mRNA expression value obtained by detection in step a).

优选的，所述检测mRNA表达量的方法包括：Affymetrix/Illumina的芯片检测、全转录组鸟枪法测序、RT-PCR。Preferably, the method for detecting the mRNA expression level includes: Affymetrix/Illumina chip detection, whole transcriptome shotgun sequencing, and RT-PCR.

与现有技术相比，本发明的技术方案具有以下优点：本发明的试剂盒和方法能够准确预测癌症预后后果，尤其是对于前列腺癌生化复发的诊断具有较高的预测价值和重要的基础及临床价值和广阔的应用前景。Compared with the prior art, the technical solution of the present invention has the following advantages: the kit and method of the present invention can accurately predict the prognosis of cancer, especially for the diagnosis of biochemical recurrence of prostate cancer, it has high predictive value and important basic and clinical value and broad application prospects.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

图1是本发明实施例的5个LncRNA为基础的基因决策模型；FIG1 is a gene decision model based on 5 LncRNAs according to an embodiment of the present invention;

图2是本发明实施例的343例患者ROC曲线图；FIG2 is a ROC curve diagram of 343 patients in an embodiment of the present invention;

图3是本发明实施例的343例患者生化复发曲线图；FIG3 is a biochemical recurrence curve diagram of 343 patients in an embodiment of the present invention;

图4是本发明实施例的114例患者ROC曲线图；FIG4 is a ROC curve diagram of 114 patients in an embodiment of the present invention;

图5是本发明实施例的114例患者生化复发曲线图。FIG5 is a biochemical recurrence curve diagram of 114 patients according to an embodiment of the present invention.

具体实施方式DETAILED DESCRIPTION

以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention.

术语“癌症”和“癌”是指或者描述哺乳动物中通常以异常或失控的细胞生长为特征的生理状态。癌症和癌症病理可以伴随着例如转移、干扰邻近细胞的正常机能、以异常水平释放细胞因子或其他分泌产物、抑制或加重炎性或免疫应答、瘤形成、癌前病变、恶性肿瘤、周围或远距离组织或器官如淋巴结的浸润等。尤其包括的是前列腺癌。The terms "cancer" and "carcinoma" refer to or describe the physiological state in mammals that is generally characterized by abnormal or uncontrolled cell growth. Cancer and cancer pathology can be accompanied by, for example, metastasis, interference with the normal function of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immune responses, neoplasia, precancerous lesions, malignancies, infiltration of surrounding or distant tissues or organs such as lymph nodes, etc. Particularly included is prostate cancer.

术语“预后”指对医学结果(medical outcome)，例如不良或良好结果的预测(例如长期存活的可能性)；消极的预后或不良结果包括复发、疾病进展(例如肿瘤生长或转移或耐药性)或死亡的预测。积极的预后或良好结果包括疾病好转(例如无疾状态)、改善(例如肿瘤消退)或稳定的预测。The term "prognosis" refers to a medical outcome, such as a prediction of a poor or good outcome (e.g., the likelihood of long-term survival); a negative prognosis or poor outcome includes a prediction of recurrence, disease progression (e.g., tumor growth or metastasis or drug resistance), or death. A positive prognosis or good outcome includes a prediction of disease improvement (e.g., disease-free state), improvement (e.g., tumor regression), or stability.

本发明所述的RP11-783K16.13同The Cancer Genome Atlas(TCGA)数据库中的RNA-seq数据中的RP11-783K16.13。The RP11-783K16.13 described in the present invention is the same as the RP11-783K16.13 in the RNA-seq data in The Cancer Genome Atlas (TCGA) database.

本发明所述的RP11-727F15.11同The Cancer Genome Atlas(TCGA)数据库中的RNA-seq数据中的RP11-727F15.11。The RP11-727F15.11 described in the present invention is the same as the RP11-727F15.11 in the RNA-seq data in The Cancer Genome Atlas (TCGA) database.

本发明所述的PRKAG2-AS1同The Cancer Genome Atlas(TCGA)数据库中的RNA-seq数据中的PRKAG2-AS1。The PRKAG2-AS1 described in the present invention is the same as the PRKAG2-AS1 in the RNA-seq data in The Cancer Genome Atlas (TCGA) database.

本发明所述的AC013460.1同The Cancer Genome Atlas(TCGA)数据库中的RNA-seq数据中的AC013460.1。The AC013460.1 described in the present invention is the same as the AC013460.1 in the RNA-seq data in The Cancer Genome Atlas (TCGA) database.

实施例1前列腺癌复发和/或存活后果或预后的评估模型的建立Example 1 Establishment of a model for assessing prostate cancer recurrence and/or survival outcome or prognosis

通过从The Cancer Genome Atlas(TCGA)数据库检索并通过倾向性匹配分析，筛选了343例前列腺癌患者作为发现集，从其中提取了相应的RNA-seq数据，应用Lasso Cox回归模型分析预测这些患者生化复发密切相关的LncRNA。最终确定和构建了以5个LncRNA为基础的基因决策模型(见图1)。并根据决策模型的风险，采用以下评分公式，得出风险值，将患者分为前列腺癌术后生化复发高危组和低危组。By searching from The Cancer Genome Atlas (TCGA) database and using propensity matching analysis, 343 prostate cancer patients were screened as the discovery set, from which the corresponding RNA-seq data were extracted, and the Lasso Cox regression model was used to analyze and predict the LncRNAs closely related to biochemical recurrence in these patients. Finally, a gene decision model based on 5 LncRNAs was determined and constructed (see Figure 1). According to the risk of the decision model, the following scoring formula was used to obtain the risk value, and the patients were divided into high-risk and low-risk groups for biochemical recurrence after prostate cancer surgery.

风险值＝(0.0766×RP11-783K16.13表达水平)+(0.2443×RP11-727F15.11表达水平)+(0.0042×PRKAG2-AS1表达水平)+(2.8117×AC013460.1表达水平)+(0.0162×CRNDE表达水平)。Risk value = (0.0766 × RP11-783K16.13 expression level) + (0.2443 × RP11-727F15.11 expression level) + (0.0042 × PRKAG2-AS1 expression level) + (2.8117 × AC013460.1 expression level) + (0.0162 × CRNDE expression level).

决策LncRNA表达值代入模型中计算出风险值，风险值大于0.55判定为前列腺癌生化复发高危组，风险值小于0.55判定为前列腺癌生化复发低危组。The decision-making LncRNA expression value was substituted into the model to calculate the risk value. The risk value greater than 0.55 was determined to be a high-risk group for biochemical recurrence of prostate cancer, and the risk value less than 0.55 was determined to be a low-risk group for biochemical recurrence of prostate cancer.

将343例前列腺癌患者的风险值分别计算出，结合患者的临床资料，绘制ROC曲线，见图2，可见两年内，前列腺癌患者的早期生化复发诊断AUC值为0.722,推衍至五年生化复发的AUC值为0.704。并根据risk score分为的前列腺癌术后生化复发高危组和低危组，绘制生化复发曲线，见图3，可见两组术后生化复发存在着显著差异，HR＝0.44,95％CI：0.27-0.72，C-index值为0.63。The risk values of 343 prostate cancer patients were calculated and combined with the clinical data of the patients to draw the ROC curve, as shown in Figure 2. It can be seen that within two years, the AUC value for early biochemical recurrence diagnosis of prostate cancer patients is 0.722, and the AUC value for five-year biochemical recurrence is 0.704. The high-risk group and low-risk group for postoperative biochemical recurrence of prostate cancer were divided according to the risk score, and the biochemical recurrence curve was drawn, as shown in Figure 3. It can be seen that there is a significant difference in postoperative biochemical recurrence between the two groups, HR = 0.44, 95% CI: 0.27-0.72, and C-index value is 0.63.

实施例2用于癌症预后评估的试剂盒Example 2 Kit for cancer prognosis assessment

本实施例中用于癌症预后的试剂盒包括：The kit for cancer prognosis in this embodiment includes:

总RNA提取试剂Trizol；Total RNA extraction reagent Trizol;

氯仿(三氯甲烷)；Chloroform (chloroform);

异戊醇；Isoamyl alcohol;

无水乙醇；Anhydrous ethanol;

DEPC水(DD1005)；DEPC water (DD1005);

焦磷酸乙二酯(DEPC)；Diethylene glycol pyrophosphate (DEPC);

抗RNA酶液(RNaseZap)；Anti-RNase solution (RNaseZap);

反转录试剂盒；Reverse transcription kit;

iQ SYBR Green Supermix；iQ SYBR Green Supermix;

序列为SEQ ID NO:1-10的多核苷酸引物。The sequences of the polynucleotide primers are SEQ ID NOs: 1-10.

利用试剂盒对各LncRNA进行实时定量PCR(qRT-PCR)检测后，初始数据结果以Ct值(cycle threshold)表示，即每个反应体系内的荧光信号达到设定的阈值所需要的循环数。每一样品的Ct值与该样品起始拷贝数的对数存在线性关系，起始拷贝数越多，则Ct值越小。采用ΔΔCT法计算各LncRNA表达水平，并进行标准化处理。After using the kit to perform real-time quantitative PCR (qRT-PCR) detection on each LncRNA, the initial data results are expressed as Ct values (cycle threshold), that is, the number of cycles required for the fluorescence signal in each reaction system to reach the set threshold. There is a linear relationship between the Ct value of each sample and the logarithm of the starting copy number of the sample. The more the starting copy number, the smaller the Ct value. The ΔΔCT method was used to calculate the expression level of each LncRNA and perform standardization.

根据上述步骤获得的各LncRNA表达水平，代入以下前列腺癌生化复发预测模型计算前列腺癌生化复发的风险值，风险值大于0.55判定为前列腺癌生化复发高危组，风险值小于0.55判定为前列腺癌生化复发低危组。According to the expression levels of each LncRNA obtained in the above steps, the following prostate cancer biochemical recurrence prediction model was substituted to calculate the risk value of prostate cancer biochemical recurrence. A risk value greater than 0.55 was determined to be a high-risk group for prostate cancer biochemical recurrence, and a risk value less than 0.55 was determined to be a low-risk group for prostate cancer biochemical recurrence.

风险值＝(0.0766×RP11-783K16.13表达水平)+(0.2443×RP11-727F15.11表达水平)+(0.0042×PRKAG2-AS1表达水平)+(2.8117×AC013460.1表达水平)+(0.0162×CRNDE表达水平)。Risk value = (0.0766 × RP11-783K16.13 expression level) + (0.2443 × RP11-727F15.11 expression level) + (0.0042 × PRKAG2-AS1 expression level) + (2.8117 × AC013460.1 expression level) + (0.0162 × CRNDE expression level).

实施例3样本RNA的制备和预处理Example 3 Preparation and pretreatment of sample RNA

(1)组织RNA提取实验用枪头、镊子在DEPC水中浸泡过夜后高温高压灭菌，预冷4℃离心机，实验台、移液枪、手套等用抗RNA酶液(RNase Zap)擦拭。(1) For RNA extraction from tissues, pipette tips and tweezers were soaked in DEPC water overnight and then sterilized by high temperature and high pressure. The centrifuge was precooled to 4°C. The laboratory bench, pipette, gloves, etc. were wiped with anti-RNase solution (RNase Zap).

(2)组织匀浆取50～100mg组织样品，用无菌手术刀片稍切碎后置于1.5mL EP管中，加入500μL Trizol试剂，用电动组织研磨器充分匀浆，然后补充加入500μL Trizol试剂。(2) Tissue homogenization: Take 50-100 mg of tissue sample, mince it slightly with a sterile surgical blade, and place it in a 1.5 mL EP tube. Add 500 μL of Trizol reagent, homogenize it thoroughly with an electric tissue grinder, and then add another 500 μL of Trizol reagent.

(3)每1mL Trizol试剂匀浆样品中加入0.2mL氯仿，盖紧EP管盖。剧烈振荡15秒，室温放置3min。4℃12,000rpm离心15min(提前预冷离心机)。离心后混合体系将分为上层的无色水相，中层的蛋白质和下层的红色酚氯仿相。DNA溶于氯仿分布于下层，RNA溶于水相分布于上层。水相的体积为匀浆时加入Trizol试剂体积的60％左右。(3) Add 0.2 mL of chloroform to every 1 mL of Trizol reagent homogenate sample and cover the EP tube tightly. Shake vigorously for 15 seconds and leave at room temperature for 3 minutes. Centrifuge at 12,000 rpm at 4°C for 15 minutes (pre-cool the centrifuge in advance). After centrifugation, the mixed system will be divided into a colorless aqueous phase on the upper layer, a protein phase in the middle layer, and a red phenol-chloroform phase on the lower layer. DNA is dissolved in chloroform and distributed in the lower layer, while RNA is dissolved in the aqueous phase and distributed in the upper layer. The volume of the aqueous phase is about 60% of the volume of Trizol reagent added during homogenization.

(4)RNA沉淀，将上层水相转移至新的EP管中。每1mL Trizol试剂匀浆样品中加入0.5mL异丙醇。混匀后室温放置10min，4℃下12,000rpm离心10min。离心后将在管壁底部和侧壁上可见白色沉淀物。(4) RNA precipitation, transfer the upper aqueous phase to a new EP tube. Add 0.5 mL of isopropanol to each 1 mL of Trizol reagent homogenate sample. After mixing, place at room temperature for 10 minutes and centrifuge at 12,000 rpm for 10 minutes at 4°C. After centrifugation, white precipitates will be visible on the bottom and side walls of the tube.

(5)RNA清洗，轻轻弃去上清，向每1mL Trizol试剂匀浆样品中加入1mL 75％的乙醇，清洗RNA沉淀。振荡，4℃下10,000rpm离心5min。(5) RNA cleaning: Gently discard the supernatant, add 1 mL of 75% ethanol to each 1 mL of Trizol reagent homogenate sample to clean the RNA precipitate. Oscillate and centrifuge at 10,000 rpm for 5 min at 4°C.

(6)RNA溶解，轻弃乙醇溶液，空气中干燥RNA沉淀约5～10min。注意切勿完全干燥RNA沉淀，否则将大大降低RNA的可溶性。溶解RNA时，加入适量无RNase的DEPC水，用移液枪反复吹打，确保RNA充分溶解后，将RNA溶液保存于-80℃冰箱。(6) RNA is dissolved, the ethanol solution is discarded, and the RNA precipitate is dried in air for about 5 to 10 minutes. Be careful not to completely dry the RNA precipitate, otherwise the solubility of the RNA will be greatly reduced. When dissolving RNA, add an appropriate amount of RNase-free DEPC water, and repeatedly blow with a pipette to ensure that the RNA is fully dissolved, and then store the RNA solution in a -80℃ refrigerator.

(7)RNA浓度测定和质控，使用

ND-2000紫外分光光度计测定RNA溶液的浓度和纯度。(7) RNA concentration determination and quality control, use

The concentration and purity of RNA solution were determined by ND-2000 UV spectrophotometer.

1)测量前先用溶解RNA用的DEPC水调零；1) Before measurement, adjust the zero value with DEPC water used to dissolve RNA;

2)用移液枪吸取2μL RNA样品滴加至测量基座的表面；2) Use a pipette to draw 2 μL of RNA sample and drop it onto the surface of the measurement base;

3)轻轻合上基座后，液滴会自动在上下基座之间形成液柱，完成测定后电脑中即显示RNA溶液的各种参数包括RNA浓度和纯度等。A260/A280的比值是一种常用的评估RNA纯度的参数，一般认为其比值范围1.8～2.1表示RNA纯度较好。3) After gently closing the base, the droplet will automatically form a liquid column between the upper and lower bases. After the measurement is completed, the computer will display various parameters of the RNA solution, including RNA concentration and purity. The ratio of A260/A280 is a commonly used parameter for evaluating RNA purity. It is generally believed that a ratio range of 1.8 to 2.1 indicates good RNA purity.

4)一次检测完成后，用擦镜纸轻轻擦去上下基座表面液体，即可进行下一个样品的检测4) After one test is completed, use lens cleaning paper to gently wipe off the liquid on the upper and lower base surfaces, and then proceed to test the next sample.

ii)琼脂糖凝胶电泳ii) Agarose gel electrophoresis

1)制胶：称取1g琼脂糖加入100mL 1×TAE buffer中，置于微波炉加热至沸腾，灌制凝胶板，待胶凝后取下梳子，将凝胶板放入电泳槽内，加入适量的1×TAE buffer至液面完全覆盖胶面。1) Gel preparation: Weigh 1g agarose and add it to 100mL 1×TAE buffer. Heat it in a microwave oven until it boils. Make a gel plate. After gelling, remove the comb and place the gel plate in an electrophoresis tank. Add an appropriate amount of 1×TAE buffer until the liquid surface completely covers the gel surface.

2)准备RNA样品：取3μg RNA，加3倍体积的甲醛上样染液，再加EB于甲醛上样染液中至EB终浓度为10ug/mL，将体系加热至70℃孵育5min使样品变性。2) Prepare RNA samples: Take 3 μg RNA, add 3 volumes of formaldehyde loading solution, and then add EB to the formaldehyde loading solution to a final EB concentration of 10 ug/mL. Heat the system to 70°C and incubate for 5 minutes to denature the sample.

3)电泳：上样后，5～6V/cm电压下电泳，至溴酚蓝指示剂进入胶内至少2～3cm。3) Electrophoresis: After loading the sample, run electrophoresis at 5-6 V/cm until the bromophenol blue indicator has penetrated at least 2-3 cm into the gel.

4)紫外透射光下观察结果：经过变性RNA电泳后，在凝胶成像系统上可见28SrRNA、18S rRNA和5S rRNA三条带。观察到28S rRNA条带的强度约为18S rRNA的2倍，而且5SrRNA条带较弱，说明总RNA未发生明显降解。4) Results observed under UV transmission light: After denaturing RNA electrophoresis, three bands of 28S rRNA, 18S rRNA and 5S rRNA were visible on the gel imaging system. The intensity of the 28S rRNA band was about twice that of the 18S rRNA, and the 5S rRNA band was weak, indicating that the total RNA was not significantly degraded.

实施例4决策LncRNA检测Example 4 Decision-making LncRNA Detection

(1)采用全式金公司的

1st Strand cDNA Synthesis SuperMix进行第一链cDNA的合成，反应体系的配制在冰上进行，反应体系如下：(1) Adopting the full-style gold company

1st Strand cDNA Synthesis SuperMix was used to synthesize the first strand cDNA. The reaction system was prepared on ice. The reaction system was as follows:

(2)采用Bio-Rad公司的iQ SYBR Green Supermix进行实时定量PCR反应。一般引物终浓度为0.2μM可以得到较好的结果，反应性能不佳时，可在0.1～1.0μM范围内调整引物浓度。(2) Real-time quantitative PCR reaction was performed using iQ SYBR Green Supermix from Bio-Rad. Generally, a final primer concentration of 0.2 μM can yield good results. When the reaction performance is poor, the primer concentration can be adjusted within the range of 0.1 to 1.0 μM.

(3)反应体系(3) Reaction system

PCR引物分别为：RP11-783K16.13的扩增引物为：SEQ ID NO:1、SEQ ID NO:2；The PCR primers were: RP11-783K16.13 amplification primers were: SEQ ID NO: 1, SEQ ID NO: 2;

RP11-727F15.11的扩增引物为：SEQ ID NO:3、SEQ ID NO:4；The amplification primers for RP11-727F15.11 are: SEQ ID NO: 3, SEQ ID NO: 4;

PRKAG2-AS1的扩增引物为：SEQ ID NO:5、SEQ ID NO:6；The amplification primers for PRKAG2-AS1 are: SEQ ID NO: 5, SEQ ID NO: 6;

AC013460.1的扩增引物为：SEQ ID NO:7、SEQ ID NO:8；The amplification primers for AC013460.1 are: SEQ ID NO: 7, SEQ ID NO: 8;

CRNDE的扩增引物为：SEQ ID NO:9、SEQ ID NO:10。The amplification primers of CRNDE are: SEQ ID NO:9, SEQ ID NO:10.

(4)反应程序(4) Reaction procedure

(5)对各LncRNA的表达进行定量(5) Quantification of the expression of each LncRNA

实时定量PCR反应结束后对Real time PCR扩增曲线及熔解曲线进行数据分析处理，初始数据结果以Ct值(cycle threshold)表示，即每个反应体系内的荧光信号达到设定的阈值所需要的循环数。每一样品的Ct值与该样品起始拷贝数的对数存在线性关系，起始拷贝数越多，则Ct值越小。采用ΔΔCT法计算各LncRNA表达水平。After the real-time quantitative PCR reaction, the real time PCR amplification curve and melting curve were analyzed and processed. The initial data results were expressed as Ct values (cycle threshold), that is, the number of cycles required for the fluorescence signal in each reaction system to reach the set threshold. There is a linear relationship between the Ct value of each sample and the logarithm of the starting copy number of the sample. The more the starting copy number, the smaller the Ct value. The ΔΔCT method was used to calculate the expression level of each LncRNA.

实施例5前列腺癌生化复发模型与前列腺癌生化复发的相关性分析Example 5 Correlation analysis between prostate cancer biochemical recurrence model and prostate cancer biochemical recurrence

采用本发明的试剂盒进行预测，对114例验证集的前列腺癌患进行了验证，通过上述实施例的方法，将验证人群风险值计算出并分为高危组和低危组，绘制ROC曲线及生化复发曲线，见图4和图5，结果显示在验证人群中本发明的方法对前列腺癌术后患者生化复发有很强的预测价值，两年及五年的生化复发ACU值分别为0.680和0.702，HR＝0.22,95％CI：0.09-0.56，C-index值为0.65。The test kit of the present invention was used for prediction, and 114 prostate cancer patients in the validation set were verified. The risk value of the validation population was calculated and divided into a high-risk group and a low-risk group by the method of the above embodiment. ROC curve and biochemical recurrence curve were drawn, as shown in Figures 4 and 5. The results showed that in the validation population, the method of the present invention had a strong predictive value for biochemical recurrence in patients after prostate cancer surgery, and the two-year and five-year biochemical recurrence ACU values were 0.680 and 0.702, respectively, HR=0.22, 95%CI: 0.09-0.56, and the C-index value was 0.65.

以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

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<110> 复旦大学附属肿瘤医院<110> Fudan University Cancer Hospital

<120> 一种基于检测LncRNA的癌症预后评估试剂盒<120> A cancer prognosis assessment kit based on detection of LncRNA

<141> 2018-06-20<141> 2018-06-20

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Claims (6)

1. A prostate cancer prognosis evaluation kit based on detection of LncRNA, characterized in that the kit comprises detection reagents capable of detecting expression levels of RP11-783K16.13, RP11-727F15.11, PRKAG2-AS1, AC013460.1, CRNDE.

2. The kit of claim 1, wherein the detection reagent comprises a polynucleotide primer or probe.

3. The kit of claim 2, wherein the polynucleotide primer has a sequence as set forth in SEQ ID NO. 1-10.

4. The kit of claim 1, wherein the determination of the prognosis of prostate cancer comprises the steps of:

a) Detecting the expression level of RP11-783K16.13, RP11-727F15.11, PRKAG2-AS1, AC013460.1, CRNDE of the sample;

b) Assessing the risk of cancer recurrence in the patient based on the expression level data obtained in step a);

assessing the risk of cancer recurrence in the patient in step b) by:

risk value = (0.0766×rp11-783K16.13 expression level) + (0.2443 ×rp11-727F15.11 expression level) + (0.0042×prkag2-AS1 expression level) + (2.8117 ×ac013460.1 expression level) + (0.0162×crnde expression level).

5. Use of a detection reagent for detecting expression levels of RP11-783K16.13, RP11-727F15.11, PRKAG2-AS1, AC013460.1, CRNDE in the preparation of a product for prognosis of prostate cancer.

6. The use of claim 5, wherein the determination of the prognosis of prostate cancer comprises the steps of:

a) Detecting the expression level of RP11-783K16.13, RP11-727F15.11, PRKAG2-AS1, AC013460.1, CRNDE of the sample;

b) Assessing the risk of cancer recurrence in the patient based on the expression level data obtained in step a);

assessing the risk of cancer recurrence in the patient in step b) by:

risk value = (0.0766×rp11-783K16.13 expression level) + (0.2443 ×rp11-727F15.11 expression level) + (0.0042×prkag2-AS1 expression level) + (2.8117 ×ac013460.1 expression level) + (0.0162×crnde expression level).

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