WO2023115601A1 - 一种用于结直肠癌预后的miRNA检测试剂盒 - Google Patents

一种用于结直肠癌预后的miRNA检测试剂盒 Download PDF

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WO2023115601A1
WO2023115601A1 PCT/CN2021/141650 CN2021141650W WO2023115601A1 WO 2023115601 A1 WO2023115601 A1 WO 2023115601A1 CN 2021141650 W CN2021141650 W CN 2021141650W WO 2023115601 A1 WO2023115601 A1 WO 2023115601A1
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mir
hsa
colorectal cancer
patients
mirnas
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张力图
刘海洲
宁淑芳
利基林
张皓嘉
韦文娥
陈伊
黄宝玥
王亚龙
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广西百谷生物科技有限公司
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

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  • the invention relates to a miRNA detection kit for the prognosis of colorectal cancer, which belongs to the technical field of medicine.
  • Colorectal cancer is one of the common digestive tract cancers, including colon cancer and rectal cancer.
  • the onset of colorectal cancer is insidious, there are often no obvious clinical symptoms in the early stage, and the disease develops slowly.
  • Most colorectal cancer patients are already in the advanced stage of tumor when they are diagnosed, and the best time for treatment has been lost, resulting in a five-year survival rate of less than 10%. 20%, the prognosis is poor.
  • the occurrence of liver metastasis in colorectal cancer patients is not only related to the abnormal expression or loss of proto-oncogenes and tumor suppressor genes, but also closely related to "epigenetics", such as acetylation, methylation and non-coding RNA regulation. Studies have confirmed that miRNAs are widely involved in the regulation of colorectal cancer liver metastasis, and some scholars call such miRNAs "metasta-miR".
  • miRNA is an RNA molecule of about 18-25 nucleotides widely present in eukaryotes, which can regulate the expression of other genes. miRNAs come from some RNAs (non-coding RNAs) that are transcribed from DNA but cannot be further translated into proteins. miRNA inhibits post-transcriptional gene expression by specifically binding to target messenger ribonucleic acid (mRNA), and plays an important role in regulating gene expression, cell cycle, and the timing of organism development. In animals, a single microRNA can often regulate dozens of genes. Alterations and dysfunctions of microRNAs play important roles in carcinogenesis, metastasis, and recurrence by regulating biological and pathological processes. In recent years, several studies have identified metastatic miRNAs in tumor tissues, which suggests that some miRNAs are tissue- and disease-specific. However, whether the miRNAs signature in plasma can be used as a diagnostic marker for colorectal cancer is still worthy of investigation.
  • the purpose of the present invention is to overcome the deficiencies of the prior art and provide a miRNA detection kit for the prognosis of colorectal cancer.
  • a miRNA detection kit for the prognosis of colorectal cancer including any one or two or more of miR-96, miR-99b and miR-155.
  • the miRNA detection kit for the prognosis of colorectal cancer of the present invention can quickly and effectively evaluate the prognosis, improve the accuracy, and provide therapeutic targets for clinical applications such as gene therapy and drug quality.
  • the miRNA detection kit for the prognosis of colorectal cancer of the present invention uses miRNAs related to the prognosis of colorectal cancer as non-invasive markers for the prognosis of colorectal cancer, providing an important theoretical basis for its clinical application.
  • the miRNA detection kit for the prognosis of colorectal cancer of the present invention belongs to non-invasive examination, which greatly facilitates the use of medical personnel and reduces the suffering of patients.
  • the miRNA detection kit for the prognosis of colorectal cancer of the present invention is to detect plasma, which is relatively easy to obtain.
  • the miRNA in the plasma reflects the pathological and physiological conditions of the body as a whole, and the detection results have accurate and detailed guiding significance .
  • the present invention can also be improved as follows.
  • sequence of the miR-96 is shown in SEQ ID NO.1; the sequence of the miR-99b is shown in SEQ ID NO.2; the sequence of the miR-155 is shown in SEQ ID NO.3.
  • the kit detects the expression levels of miR-96, miR-99b and miR-155 in biological samples by quantitative PCR technology.
  • the biological sample is selected from fresh tissues, fresh cells, fresh blood, fresh body fluids, formalin-fixed tissues, formalin-fixed cells, paraffin-embedded tissues and paraffin-embedded any type of cell.
  • the biological sample is plasma.
  • probes, gene chips or PCR primers with detection specificity for miR-96, miR-99b and miR-155 are also included.
  • Fig. 1 is technical method and data analysis flow chart of the present invention
  • Fig. 2 is the differential miRNA clustering analysis expression spectrum of liver cancer tissue and its adjacent normal liver tissue in Example 1 of the present invention, in the figure, C1 and C2 represent liver cancer tissue, N1 and N2 represent adjacent liver tissue;
  • Example 3 is a gel electrophoresis diagram of total RNA in plasma in Example 1 of the present invention.
  • from left to right 1-3 are colorectal cancer patients, and 4-6 are healthy people;
  • Fig. 4 is the amplification and melting curve figure of real-time fluorescent quantitative PCR of miRNAs in plasma according to the present invention.
  • the abscissa starts from 0, and the marked numerical value interval is 2; the vertical coordinate starts from 0, and the marked numerical value interval 10000;
  • Figure 5 is a graph showing the expression level of plasma miRNA in patients with colorectal cancer without distant metastasis and liver metastasis and healthy people according to the present invention; wherein, * is P ⁇ 0.05; ** is P ⁇ 0.01; *** is P ⁇ 0.001;
  • Fig. 6 is the ROC curve analysis miRNA liver metastases identification efficiency of the present invention.
  • FIG. 7 is a graph showing the relationship between miR-96 and miR-155 and the overall survival of colorectal cancer patients with liver metastases analyzed by the Kaplan-Meier method of the present invention.
  • Fig. 8 is the gel electrophoresis figure of total RNA in the tissue of the present invention.
  • Fig. 9 is the amplification and melting curve diagram of miRNAs real-time fluorescent quantitative PCR in the tissue of the present invention.
  • Figure 10 is a graph showing the expression levels of miRNA in formalin-fixed paraffin-embedded tissues of colorectal cancer patients without distant metastasis and patients with liver metastasis according to the present invention
  • Fig. 11 is a graph showing the expression of miRNA in colon cancer tissue and corresponding liver metastases tissue of patients with colorectal cancer liver metastases according to the present invention
  • Figure 12 is the ROC curve of the analysis of the diagnostic performance of miRNA among different groups of colorectal cancer patients according to the present invention.
  • Fig. 13 is a graph showing the relationship between miR-99b and overall survival of patients with colorectal cancer liver metastases analyzed by the Kaplan-Meier method of the present invention.
  • Example 1 Bioinformatics analysis and screening of miRNAs associated with colorectal cancer metastasis
  • the search keywords of this study in the GEO database are “Colorectal cancer”, “Colon cancer”, “rectal cancer”, “Colonic carcinoma”, “Metastasis”, “Hepatic metastasis”, “liver metastasis”, “MicroRNAs”.
  • Google Scholar and Baidu Academic were searched twice at different time points, and the chip set was included after 2010, and there was no language restriction. The chipsets included are shown in Table 1.
  • the miRNA chip data selected in this study came from 2 pairs of liver cancer tissues and adjacent normal liver tissues of liver cancer patients in the previous research of our research group.
  • the tissues were all from the specimen bank of the Cancer Hospital affiliated to Guangxi Medical University. All patients were informed about participating in the scientific research Agreed, all specimens were the first surgical resection samples of primary lesions, and none had received chemotherapy, radiotherapy, etc.
  • the excised tissue samples were immediately stored at -80°C after being snap-frozen in liquid nitrogen.
  • the chip was scanned by Agilent chip scanner to obtain hybridization pictures.
  • the Agilent Feature Extraction (v10.7) software was used to analyze the hybridization pictures and obtain the data, and the Agilent GeneSpring software was used to perform normalization and differential analysis on the data.
  • microRNA differential microRNAs
  • miRNA differential microRNAs
  • five genes including miR-96, miR-99b, miR-155, let-7a and let-7b were selected.
  • miR-96, miR-99b, miR-155, let-7a and let-7b were selected.
  • RNA data After the data is acquired, first we combine the original RNA data and the annotation files of the related chips, and group them, and then use the limma package of the R language to perform standardization and expression difference analysis. If the literature to which the chip belongs contains clinical data, we will The information was renamed, and the differential gene and clinical information were compared, and the chip set with complete information was found to be included in the study.
  • the selected data set is GSE72199.
  • the microarray samples come from the primary colorectal cancer and liver metastases respectively.
  • the standardized expression data of 28 cases of colorectal cancer primary tumors and 8 cases of liver metastases control samples are used for analysis and screening.
  • the expressions of up-regulated and down-regulated miRNAs are shown in Table 2. Due to the relatively large amount of differentially expressed miRNA data, we then narrowed down the scope of the study based on previous research and literature analysis.
  • hsa-miR-199a-3p 0.730114 raised 0.037959 hsa-let-7i 0.685427 raised 0.005026 hsa-miR-497 0.625685 raised 0.098753 hsa-miR-381 0.609518 raised 0.044039 hsa-miR-1274a 0.597932 raised 0.070462 hsa-miR-337-5p 0.568264 raised 0.06821 hsa-miR-154 0.554891 raised 0.052757 hsa-miR-1274b 0.543264 raised 0.026175 hsa-miR-132 0.535788 raised 0.087145 hsa-miR-3195 0.530642 raised 0.044603 hsa-miR-127-3p 0.48599 raised 0.09463 hsa-miR-409-3p 0.480208 raised 0.082304 hsa-miR-22 0.465164 raised 0.09
  • hsa-miR-181d -0.89785 lower down 0.005231 hsa-miR-7 -0.92823 lower down 0.060777 hsa-miR-20a* -0.93143 lower down 0.028441 hsa-miR-3189 -0.94779 lower down 0.07004 hsa-miR-183 -0.95729 lower down 0.046876 hsv1-miR-H8 -0.96162 lower down 0.094062 hsa-miR-203 -0.97528 lower down 0.059516 hsa-miR-432 -1.01261 lower down 0.022848 hsa-miR-195* -1.03305 lower down 0.024495 ebv-miR-BART4 -1.0841 lower down 0.024985 hsa-miR-1180 -1.12151 lower down 0.074813 hsa-miR-1290 -1.17811 lower down
  • liver cancer tissue C
  • paracancerous liver tissue N
  • the results of the association search between miR-96 and colorectal cancer found that it was closely related to keywords such as liver cancer, tumor metastasis, survival and prognosis; the joint search of miR-99b with tumor and colorectal cancer found no association with other keywords ;
  • the joint search of miR-155 and tumor found that it is closely related to keywords such as cell migration, tumor stage, survival and TP53; Repair, intraperitoneal chemotherapy, and keywords such as TP53, MYC, and ras family genes are closely related;
  • the results of an association search between let-7b and colorectal cancer found that it is related to tumor expression regulation, tumor metastasis, panitumumab, survival and prognosis, Key words such as KRAS gene are closely related.
  • Example 2 Expression of miRNAs associated with liver metastasis in plasma of patients with colorectal cancer and their diagnostic and prognostic value
  • Inclusion criteria for patients with liver metastases from colorectal cancer all patients had not received comprehensive treatment such as chemotherapy, radiotherapy, and targeted therapy at the time of collection, and the collected plasma was all preoperative plasma; no distant metastasis and/or recurrence at other locations were combined , not suffering from other benign diseases at the same time.
  • Inclusion criteria for patients with colorectal cancer without distant metastasis no distant metastasis and/or recurrence, and no other benign diseases at the same time.
  • a PCR reaction system was prepared according to the instructions of the small RNA polymerase chain reaction dye kit, and the primers were all from Tiangen. Each primer was replicated in 3 replicate wells, and each experiment was replicated 3 times.
  • the upstream primers are primers designed by Tiangen Company, and the downstream primers are general primers designed by Capgemini Company.
  • the reaction conditions were set according to the requirements of the kit: pre-denaturation at 95°C for 15 min; denaturation at 94°C for 15 s; annealing and extension at 55°C for 30 s; 40 cycles, and the melting curve was drawn at 70°C.
  • the reaction system is shown in Table 8.
  • the primer sequences are shown in Table 9.
  • SPSS 20.0, GraphPad Prism5.0 or Medcalc12.2.1 were used for data analysis. Normally distributed data were expressed as mean ⁇ standard deviation; t-test, one-way analysis of variance or non-parametric tests were used to evaluate the differences in the expression of miRNAs between different groups; receiver operating curve (ROC) and binary linear regression were used to evaluate The combined diagnostic efficacy of miRNAs; the prognostic value of each miRNA was assessed by univariate and multivariate survival analysis; the target genes of miRNAs and the signaling pathways involved were analyzed by bioinformatics methods. A P value less than 0.05 was considered statistically significant.
  • the OD value of 260/280 of the total RNA of each sample was detected by the trace nucleic acid analyzer, and the OD value of 260/280 was between 1.8-2.0; after the agarose gel electrophoresis experiment, the bands of 28S, 18S and 5S of each sample were clear, and the brightness of the 28S band was greater than that of 18S and 5S, so the quality of the extracted RNA is reliable and complete, and can be used for further research, as shown in Figure 3.
  • the amplification curves of the internal reference and the target miRNA both showed an S-shaped, smooth shape, with obvious amplification and plateau periods, indicating that the amplification results were ideal; the internal reference and the target miRNA fluorescence quantitative PCR
  • the melting curves of all the samples are narrow single-peak curves, and the melting point peaks are all at 76°C-80°C, without obvious double peaks, indicating that the specificity of the amplified product is good. As shown in Figure 4.
  • miR-96, miR-99b, miR-15 and Let-7b are related to tumor T stage, and the difference is statistically significant (P ⁇ 0.05); these 5 miRNAs were not observed in our study There was no significant difference in the relationship with other clinicopathological parameters (P>0.05).
  • ROC receiver operating curve
  • AUC area under the curve
  • the expression level of CEA was also detected, trying to compare the diagnostic efficacy of our five candidate miRNAs and CEA.
  • the results of ROC curve analysis showed that the area under the curve of plasma miR-96 was 0.937 (0.866-0.978), the area under the curve of plasma miR-99b was 0.929 (0.855-0.973), and the area under the curve of plasma miR-155 was 0.806 ( 0.710-0.882), the area under the curve of plasma let-7a is 0.843 (0.751-0.911), the area under the curve of plasma let-7b is 0.782 (0.682-0.862), the area under the curve of CEA is 0.693 (0.587-0.786), Among the six plasma detection indicators, miR-96, miR-99b and let-7a had the highest single diagnostic value in diagnosing patients without distant metastasis and liver metastasis, while the diagnostic efficiency of CEA was lower than that of the other 5 in this study. indicators.
  • Example 3 Expression of liver metastasis-related miRNAs in paraffin-embedded tissues of colorectal cancer patients and their diagnostic and prognostic value
  • Inclusion criteria for patients with liver metastases from colorectal cancer all patients had not received comprehensive treatment such as chemotherapy, radiotherapy, and targeted therapy at the time of collection, and the collected tumor tissues were all preoperative tissues; no distant metastasis from other locations and/or Recurrence without other benign diseases.
  • Inclusion criteria for patients with colorectal cancer without distant metastasis no distant metastasis and/or recurrence, and no other benign diseases at the same time.
  • the tumor tissue was immediately placed in neutral formalin solution with a volume fraction of 4% for internal fixation, and then made into a paraffin-embedded (FFPE) specimen after being processed, dehydrated, transparent, dipped in wax, and embedded.
  • FFPE paraffin-embedded
  • RNA extraction reagent in a new 1.5mL collection tube, add 14-30 ⁇ L enzyme-free water directly to the membrane of the kit, cover the lid carefully, centrifuge at full speed for 1min, and collect the eluate .
  • the total RNA collected in the 1.5mL centrifuge tube is the extracted total RNA.
  • the method for measuring the expression level of miRNA in FFPE tissue in this part is the same as step 2.4 in Example 2.
  • SPSS 20.0, GraphPad Prism5.0 or Medcalc12.2.1 were used for data analysis. Normally distributed data were expressed as mean ⁇ standard deviation (Mean ⁇ SD); t test, one-way analysis of variance or non-parametric test were used to evaluate the differences in the expression of miRNAs between different groups; receiver operating curve (ROC) and Binary linear regression was used to assess the diagnostic performance of miRNA combinations; univariate and multivariate survival analyzes were used to assess the prognostic value of individual miRNAs. A P value less than 0.05 was considered statistically significant.
  • the OD value of 260/280 of the total RNA of each sample was detected by the micro-nucleic acid analyzer, and the OD value of 260/280 was between 1.8-2.0; after the agarose gel electrophoresis experiment, the bands of 28S, 18S, and 5S of each sample were clear, and the brightness of the 28S band was greater than that of 18S and 5S, so the quality of the extracted RNA is reliable and complete, and can be used for further research, see Figure 8.
  • the amplification curves of the internal reference and the target miRNA both showed an S-shaped, smooth shape, with obvious amplification and plateau periods, indicating that the amplification results were ideal; the internal reference and the target miRNA fluorescence quantitative PCR
  • the melting curves are all a narrow single-peak curve, and the melting point peaks are all between 76-80°C, without obvious double peaks, indicating that the specificity of the amplified product is good. See Figure 9.
  • Table 15 The relationship between miRNA expression in colorectal cancer tissues and clinicopathological parameters in patients with colorectal cancer
  • ROC receiver operating curve
  • AUC area under the curve

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Abstract

一种用于结直肠癌预后的miRNA检测试剂盒，属于医药技术领域。该用于结直肠癌预后的miRNA检测试剂盒包括miR-96、miR-99b和miR-155中的任意一种或两种以上。该试剂盒可以快速有效地做到预后评估，提高了精确度，为基因治疗、药物质量的临床应用提供了治疗靶点。

Description

一种用于结直肠癌预后的miRNA检测试剂盒 技术领域
本发明涉及一种用于结直肠癌预后的miRNA检测试剂盒，属于医药技术领域。
背景技术
结直肠癌(Colorectal Cancer，简称CRC)是常见的消化道癌症之一，包括结肠癌和直肠癌，是由于结肠粘膜上皮在环境和遗传等多种致癌因素作用下发生的恶性癌变。结直肠癌起病隐匿，早期常无明显的临床症状，而且病情发展较慢，大部分结直肠癌患者确诊时已处于肿瘤进展期，失去了最佳的治疗时机，导致五年生存率不到20％，预后较差。结直肠癌患者肝转移的发生除了与原癌基因及抑癌基因表达异常或丢失有关，也与“表观遗传学”，如乙酰化、甲基化和非编码RNA调控紧密相关。有研究证实，在结直肠癌的肝转移发生进展过程中，miRNA广泛参与其调控，有学者称这类miRNA为“metasta-miR"。
微小RNA(microRNA，简称miRNA)是真核生物中广泛存在的一种长约18-25个核苷酸的RNA分子，可调节其他基因的表达。miRNA来自一些从DNA转录而来，但无法进一步转译成蛋白质的RNA(属于非编码RNA)。miRNA通过与靶信使核糖核酸(mRNA)特异结合，从而抑制转录后基因表达,在调控基因表达、细胞周期、生物体发育时序等方面起重要作用。在动物中，一个微小RNA通常可以调控数十个基因。微小RNA的改变和功能障碍通过调节生物学和病理过程在癌变、转移和复发过程中发挥重要作用。近年来，一些研究证实了肿瘤组织中的转移性miRNA，它表明一些miRNA具有组织和疾病特异性。但是，血浆中的miRNAs特征是否可以作为结直肠癌的诊断标志物仍然值得研究。
鉴于此，有必要提供一种用于结直肠癌预后的miRNA检测试剂盒，以解决现有技术的不足。
发明内容
本发明的目的是克服现有技术的不足，提供一种用于结直肠癌预后的miRNA检测试剂盒。
本发明解决上述技术问题的技术方案如下：一种用于结直肠癌预后的miRNA检测试剂盒，包括miR-96、miR-99b和miR-155中的任意一种或两种以上。
本发明的用于结直肠癌预后的miRNA检测试剂盒的有益效果是：
1、本发明的用于结直肠癌预后的miRNA检测试剂盒，可以快速有效地做到预后评估，提高了精确度，为基因治疗、药物质量等临床应用提供了治疗靶点。
2、本发明的用于结直肠癌预后的miRNA检测试剂盒，采用与结直肠癌预后相关的miRNA可作为结直肠癌预后的无创标志物，为其临床应用提供了重要的理论基础。
2、本发明的用于结直肠癌预后的miRNA检测试剂盒，与传统的筛检方法相比，属于无创检查，极大地方便了医疗人员的使用，减轻了患者的痛苦。
3、本发明的用于结直肠癌预后的miRNA检测试剂盒，是检测血浆，较易获得，血浆中的miRNA反映的是机体整体的病理、生理情况，其检测结果具有精确而详细的指导意义。
在上述技术方案的基础上，本发明还可以做如下改进。
进一步，所述miR-96的序列如SEQ ID NO.1所示；所述miR-99b的序列如SEQ ID NO.2所示；所述miR-155的序列如SEQ ID NO.3所示。
进一步，所述试剂盒是通过定量PCR技术检测生物样品中miR-96、miR-99b和miR-155的表达量。
进一步，所述生物样品选自新鲜的组织、新鲜的细胞、新鲜的血液、新鲜的体液、福尔马林固定的组织、福尔马林固定的细胞、石蜡包埋的组织和石蜡包埋的细胞中的任意一种。
更进一步，所述生物样品为血浆。
进一步，还包括对miR-96、miR-99b和miR-155具有检测特异性的探针、基因芯片或PCR引物。
附图说明
图1为本发明的技术方法和数据分析流程框图；
图2为本发明的实施例1中，肝癌组织及其癌旁正常肝组织差异性miRNA聚类分析表达谱图， 图中，C1和C2代表肝癌组织，N1和N2代表癌旁肝组织；
图3为本发明的实施例1中，血浆中总RNA凝胶电泳图，图中，从左至右1-3为结直肠癌患者，4-6为健康人；
图4为本发明所述的血浆中miRNAs实时荧光定量PCR的扩增和熔解曲线图；图中，横坐标由0开始，所标数值大小间隔2；纵坐标由0开始，所标数值大小间隔10000；
图5为本发明所述的结直肠癌患者无远处转移和肝转移患者、健康人血浆miRNA的表达水平图；其中，*为P＜0.05；**为P＜0.01；***为P＜0.001；
图6为本发明所述的ROC曲线分析miRNA的肝转移鉴别效能；
图7为本发明所述的Kaplan–Meier法分析miR-96和miR-155与结直肠癌肝转移患者总体生存期的关系图；
图8为本发明所述的组织中总RNA凝胶电泳图；
图9为本发明所述的组织中miRNAs实时荧光定量PCR的扩增和熔解曲线图；
图10为本发明所述的结直肠癌患者无远处转移患者和肝转移患者福尔马林固定石蜡包埋组织中miRNA的表达水平图；
图11为本发明所述的结直肠癌肝转移患者肠癌组织和相应肝转移瘤组织中miRNA的表达情况图；
图12为本发明所述的分析miRNA在结直肠癌患者不同组间的诊断效能的ROC曲线；
图13为本发明所述的Kaplan–Meier法分析miR-99b和结直肠癌肝转移患者总体生存期的关系图。
具体实施方式
以下结合具体附图对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。
实施例1：结直肠癌转移相关miRNA的生物信息学分析和筛选
材料与方法
1.技术方法和数据分析流程，如图1所示。
2.miRNA及基因表达数据的获取
本研究在GEO数据库中的检索关键词为“Colorectal cancer”，“Colon cancer”，“rectal cancer”，“Colonic carcinoma”，“Metastasis”，“Hepatic metastasis”，“liver metastasis”，“MicroRNAs”。并补充检索了Google Scholar和百度学术，在不同时间点检索两次，纳入芯片集在2010年之后，没有语言限制。纳入芯片集见表1。
表1 结直肠癌转移相关的芯片集
Figure PCTCN2021141650-appb-000001
注：*指芯片集类型为安捷伦品牌的miRNA芯片。
3.肝癌组织及其癌旁正常肝组织miRNA芯片数据处理
本研究选取的miRNA芯片数据来自于本课题组前期研究中肝癌患者的2对肝癌组织及癌旁正常肝组织，组织均源自广西医科大学附属肿瘤医院标本库，所有患者对参与科学研究均知情同意，所有标本均为原发病灶的首次手术切除样本，均未接受过化疗，放疗等。切除的组织标本经液氮 速冻后立即予以-80℃保存。
miRNA芯片实验数据提取及分析，芯片通过Agilent芯片扫描仪进行扫描，获得杂交图片。使用Agilent Feature Extraction(v10.7)软件分析杂交图片并获取数据，使用Agilent GeneSpring软件对数据进行归一化和差异分析。
4.PUBMED文献检索
为了综合评价以上差异微小RNA(microRNA，miRNA)对结直肠癌转移/肝转移的诊断及预后价值，选出miR-96、miR-99b、miR-155、let-7a和let-7b五个基因做详细的文献检索表格并列出文献中涉及miRNA的差异表达倍数。
5.统计方法
5.1主要统计软件
R-3.3.3，网址：https://www.r-project.org/
Fun rich网址：http://www.funrich.org/
GraphPad Prism 7网址：http://www.graphpad.com/
5.2芯片数据的标准化处理
数据获取后，首先我们将RNA原始数据和相关芯片的注释文件相结合，并进行分组，然后运用R语言的limma包进行标准化和表达差异分析，如果芯片所属文献中包含临床数据，我们将对其中信息重命名，并将差异基因和临床信息进行比对，找到完整信息的芯片集纳入研究。
筛选差异表达的miRNA，我们使用目前国内外研究都普遍使用的SAM方法，由斯坦福大学开发，并被广泛使用，是一种以t检验为基础的筛选差异基因的算法。本研究的筛选条件为P<0.1。即miRNA在转移灶和原发灶组织表达差异有统计学意义。本课题组前期研究的肝癌miRNA芯片数据的差异化筛选同上。
结果：
1.GEO数据集中差异表达的miRNA
1.1结直肠癌原发灶/转移灶组织相关的差异表达miRNA
入选的数据集为GSE72199，芯片标本分别来自结直肠癌原发灶和肝转移灶，应用28例结直肠癌原发灶和8例肝转移灶对照样本的标准化表达数据，进行分析筛选，根据是否有统计学意义及其差异倍数，符合条件的差异表达的miRNA共有83个，其中在转移灶组织中上调的33个，下调的50个，上调、下调的miRNA表达情况见表2。由于差异表达的miRNA数据相对较多，后续我们结合前期研究和文献分析再缩小研究范围。
表2 GSE72199中人结肠癌组织相对其肝转移组织差异表达的miRNA
基因 改变倍数 改变 P值
hsa-miR-551b 1.312296 上调 0.007907
hsa-miR-936 1.274284 上调 0.059048
hsa-miR-3174 1.119544 上调 0.032167
hsa-miR-146a 1.020621 上调 0.011194
hsa-miR-1285 0.945843 上调 0.057714
hsa-miR-149* 0.913007 上调 0.092106
hsa-miR-142-5p 0.899993 上调 0.021792
hsa-miR-223 0.887985 上调 0.072544
hsa-miR-513b 0.837228 上调 0.052481
hsa-miR-10b 0.754512 上调 0.050489
jcv-miR-J1-5p 0.750322 上调 0.095771
hsa-miR-494 0.731083 上调 0.089403
hsa-miR-125a-3p 0.730299 上调 0.061953
hsa-miR-199a-3p 0.730114 上调 0.037959
hsa-let-7i 0.685427 上调 0.005026
hsa-miR-497 0.625685 上调 0.098753
hsa-miR-381 0.609518 上调 0.044039
hsa-miR-1274a 0.597932 上调 0.070462
hsa-miR-337-5p 0.568264 上调 0.06821
hsa-miR-154 0.554891 上调 0.052757
hsa-miR-1274b 0.543264 上调 0.026175
hsa-miR-132 0.535788 上调 0.087145
hsa-miR-3195 0.530642 上调 0.044603
hsa-miR-127-3p 0.48599 上调 0.09463
hsa-miR-409-3p 0.480208 上调 0.082304
hsa-miR-22 0.465164 上调 0.096708
hsa-miR-4286 0.440413 上调 0.04343
hsa-miR-212 0.436136 上调 0.009051
hsa-miR-550a 0.396222 上调 0.012495
hsa-miR-939 0.391443 上调 0.080472
hsa-miR-3614-5p 0.300615 上调 0.035585
hsa-miR-33b* 0.289984 上调 0.079895
hsa-miR-664 -0.31863 下调 0.097762
hsa-miR-505 -0.38737 下调 0.08828
hsa-miR-128 -0.40766 下调 0.087887
hsa-miR-320c -0.43551 下调 0.096361
hsa-miR-148b -0.51217 下调 0.028773
hsa-miR-98 -0.51274 下调 0.098368
hsa-miR-200c -0.52989 下调 0.059143
hsa-miR-7-1* -0.58893 下调 0.06773
hsv1-miR-H1* -0.62097 下调 0.066866
hsa-miR-200a -0.63855 下调 0.091408
hsa-miR-29a* -0.69557 下调 0.026654
hsa-miR-96 -0.70432 下调 0.091916
hsa-miR-429 -0.74911 下调 0.067866
hsa-miR-192* -0.76874 下调 0.086049
hsa-miR-106b* -0.8446 下调 0.009767
hsa-miR-210 -0.84698 下调 0.049274
hsa-miR-598 -0.87103 下调 0.099125
hsa-miR-191 -0.89405 下调 0.078352
hsa-miR-181d -0.89785 下调 0.005231
hsa-miR-7 -0.92823 下调 0.060777
hsa-miR-20a* -0.93143 下调 0.028441
hsa-miR-3189 -0.94779 下调 0.07004
hsa-miR-183 -0.95729 下调 0.046876
hsv1-miR-H8 -0.96162 下调 0.094062
hsa-miR-203 -0.97528 下调 0.059516
hsa-miR-432 -1.01261 下调 0.022848
hsa-miR-195* -1.03305 下调 0.024495
ebv-miR-BART4 -1.0841 下调 0.024985
hsa-miR-1180 -1.12151 下调 0.074813
hsa-miR-1290 -1.17811 下调 0.035088
hsa-miR-449b* -1.19808 下调 0.000898
hsa-miR-375 -1.20615 下调 0.049515
hsa-miR-181c* -1.22109 下调 0.012019
ebv-miR-BART12 -1.24266 下调 0.044115
hsa-miR-3148 -1.25342 下调 0.089289
hsa-miR-1228* -1.30343 下调 0.014994
hsa-miR-19b-1* -1.33026 下调 0.044891
hsa-miR-1247 -1.33521 下调 0.030141
hsa-miR-595 -1.37846 下调 0.05597
hsa-miR-744* -1.38283 下调 0.026076
ebv-miR-BART10 -1.38421 下调 0.055649
hsa-miR-885-3p -1.4416 下调 0.043622
hsa-miR-3130-5p -1.46468 下调 0.004952
hsa-miR-3692* -1.46658 下调 0.092048
hsa-miR-1258 -1.54516 下调 0.042972
hsa-miR-514b-3p -1.83916 下调 0.013102
hsa-miR-205* -1.86067 下调 0.062458
hsa-miR-885-5p -2.00695 下调 0.002815
hsa-miR-122* -2.82073 下调 0.006442
hsa-miR-122 -4.33787 下调 0.004942
2.前期研究中肝癌组织与相匹配癌旁正常组织中差异表达的miRNA
利用miRNA芯片技术，对原始数据进行处理后，我们得到了33个差异性表达的miRNA，其中上调的miRNA有21个，下调的miRNA有12个，结果见表3。
表3 人肝癌组织相对其正常肝组织差异性表达miRNA
基因 基因库编号 改变 改变倍数
hsa-miR-127-3p MIMAT0000446 上调 27.01
hsa-miR-135a-3p MIMAT0004595 上调 25.25
hsa-miR-151a-3p MIMAT0000757 上调 2.05
hsa-miR-155-5p MIMAT0000646 上调 2.64
hsa-miR-339-3p MIMAT0004702 上调 6.25
hsa-miR-376a-3p MIMAT0000729 上调 33.43
hsa-miR-409-3p MIMAT0001639 上调 13.71
hsa-miR-410 MIMAT0002171 上调 10.15
hsa-miR-4291 MIMAT0016922 上调 2.13
hsa-miR-432-5p MIMAT0002814 上调 11.32
hsa-miR-4478 MIMAT0019006 上调 8.98
hsa-miR-4673 MIMAT0019755 上调 10.56
hsa-miR-500a-3p MIMAT0002871 上调 80.42
hsa-miR-503-5p MIMAT0002874 上调 3.24
hsa-miR-532-3p MIMAT0004780 上调 10.29
hsa-miR-542-5p MIMAT0003340 上调 2.48
hsa-miR-551b-3p MIMAT0003233 上调 53.53
hsa-miR-652-3p MIMAT0003322 上调 2.48
hsa-miR-654-3p MIMAT0004814 上调 10.57
hsa-miR-6717-5p MIMAT0025846 上调 2.19
hsa-miR-96-5p MIMAT0000095 上调 11.12
hsa-miR-122-3p MIMAT0004590 下调 4.97
hsa-miR-193b-5p MIMAT0004767 下调 2.15
hsa-miR-378a-3p MIMAT0000732 下调 3.67
hsa-miR-378d MIMAT0018926 下调 123.55
hsa-miR-378i MIMAT0019074 下调 3.79
hsa-miR-4270 MIMAT0016900 下调 3.01
hsa-miR-4286 MIMAT0016916 下调 6.45
hsa-miR-4508 MIMAT0019045 下调 2.13
hsa-miR-4649-3p MIMAT0019712 下调 2.15
hsa-miR-4701-5p MIMAT0019798 下调 2.31
hsa-miR-5585-3p MIMAT0022286 下调 2.13
hsa-miR-99a-5p MIMAT0000097 下调 4.45
对差异性的miRNA进行聚类分析，结果显示肝癌组织(C)及其癌旁肝组织(N)有明显不同的表达谱，见图2。
3.差异表达miRNA与肿瘤相关关键词的文本挖掘与文献检索
在pubmed数据库中，我们在差异表达的miRNA中，将miR-96、miR-99b、miR-155、let-7a、let-7b与“Neoplasms(disease)”、“Colorectal Neoplasms(alias COLORECTAL CANCER)”关键词共同检索。miR-96与结直肠癌关联检索的结果发现其与肝癌、肿瘤转移、生存及预后等关键词有密切关联；miR-99b与肿瘤、结直肠癌联合检索皆没有发现其与其他关键词有关联；miR-155 与肿瘤联合检索发现其与细胞迁移、肿瘤分期、生存和TP53等关键词有密切关联；let-7a与结直肠癌关联检索结果发现其与肿瘤的表达调控，甲基化，DNA修复，腹腔内化疗及TP53、MYC、ras家族基因等关键词密切相关；let-7b与结直肠癌关联检索的结果发现其与肿瘤的表达调控，肿瘤转移、帕尼单抗、生存及预后、KRAS基因等关键词密切相关。
利用文本挖掘的上述5个miRNA，符合我们进一步研究的入组要求。我们对其中一些重点文献进行了检索，其结果见表4。其中，研究普遍认为miR-96、miR-155、let-7a、let-7b四个基因参与了肿瘤的转移，并且被认为是潜在的预后标志物；其中miR-96、let-7a、let-7b的表达被认为与TP53，ras家族基因，KRAS基因相关；由于miR-99b在GEO数据库和前期研究中均发现为高表达，但是其在肿瘤和结直肠癌中的报道相对较少，为了探究其在结直肠癌中的潜在作用机制和价值，因此，我们将miR-99b纳入我们分析研究的指标中。
表4 文献中miR-96、miR-99b、miR-155、let-7a和let-7b表达情况一览表
Figure PCTCN2021141650-appb-000002
Figure PCTCN2021141650-appb-000003
注：*在癌组织/细胞/循环血中的表达。
综上所述，通过生物信息学方法，我们分析和筛选得到了在结直肠癌中与转移相关的5个 miRNA，分别是miR-96、miR-99b、miR-155、let-7a和let-7b，各自的序列见表5。它们可能在结直肠癌的发生、发展或转移中有重要作用，针对此推测可进行进一步分子生物学实验。
表5 miR-96、miR-99b、miR-155、let-7a和let-7b的序列
基因名称 序列
hsa-miR-96-5p uuuggcacuagcacauuuuugcu(SEQ ID NO.1)
hsa-miR-99b-5p cacccguagaaccgaccuugcg(SEQ ID NO.2)
hsa-miR-155-5p uuaaugcuaaucgugauaggggu(SEQ ID NO.3)
hsa-let-7a-5p ugagguaguagguuguauaguu(SEQ ID NO.4)
hsa-let-7b-5p ugagguaguagguugugugguu(SEQ ID NO.5)
实施例2：结直肠癌患者血浆中肝转移相关miRNA的表达及其诊断和预后价值
1.实验材料
病例选择和标本收集
本研究已通过广西医科大学附属肿瘤医院医学伦理委员会的批准，每位受试者均已签署知情同意书同意使用标本及临床资料。收集2008年12月30日至2011年12月30日期间在广西医科大学附属肿瘤医院就诊的CRC患者90例，同时收集2016年9月1日至2016年9月30日体检的健康人20例。患者中位年龄为55.9岁(年龄范围20-84岁)。男性56例，女性34例；左半结肠24例，右半结肠24例，直肠42例；低分化肿瘤26例，中分化肿瘤53例，高分化肿瘤11例；肿瘤直径＜5cm的57例，肿瘤直径≥5cm的33例；T1+T2有11例，T3+T4有79例；N0有22例，N1+N2有68例；临床分期为I+Ⅱ的有20例，临床分期Ⅲ+Ⅳ的有70例；无远处转移患者41例，肝转移患者49例。通过UICC和AJCC进行临床分期评估。详见表6。
结直肠癌肝转移患者入组标准：收集时所有患者均未接受过化疗、放疗、靶向治疗等综合治疗，收集的血浆均为术前血浆；未合并其他位置的远处转移和/或复发，未同时患有其他良性疾病。
结直肠癌无远处转移患者入组标准：无远处转移和/或复发，未同时患有其他良性疾病。
表6 90例结直肠癌患者和20例健康受试者的临床病理基本特征
Figure PCTCN2021141650-appb-000004
2.实验方法
2.1健康人血浆标本采集及初步处理
2.1.1收集健康人外周血2mL，立即置于含EDTA真空采血管中，室温静置约30min；
2.1.2将室温静置的血浆标本，以3000rpm离心15min，室温，取1mL上清；
2.1.3将所得上清，以12000rpm在4℃中离心15min，取上清分装，每管200μL，贴好标签，置于-80℃待用。
2.2结直肠癌患者血浆标本收集及处理(同上)
2.3血浆总RNA的提取
2.3.1将200μL血浆样品从-80℃冰箱取出，于室温解冻；
2.3.2向已解冻好的血浆中加入5体积(1mL)的裂解液，用移液器捶打充分混匀。常温静置约5min；
2.3.3在上述混合液中加入200μL的氯仿，用涡旋器混匀15s，常温静置约5min。此处的充分混匀是后续逆转录和聚合酶链反应顺利进展的保证；
2.3.4使用低温(4℃)离心机12000rpm，离心15分钟；
2.3.5小心将上清(约600μL)转移至新的1.5mL的离心管中，加入1.5倍体积(约900μL)的无水乙醇，用移液器充分混匀；
2.3.6取700μL上述混合液加入离心柱中，4℃，以12000rpm离心1min，弃废液；
2.3.7重复以上步骤，将剩下的混合液加入离心柱中离心；
2.3.8在离心柱中加入700μL初次冲洗缓冲液，12000rpm，4℃，离心1min，弃废液；
2.3.9在离心柱中加入500μL二次冲洗缓冲液，12000rpm，4℃，离心1min，弃废液；
2.3.10配制体积分数为80％的焦炭酸二乙酯酒精，在每个离心柱中加入500μL焦炭酸二乙酯酒精，于低温离心机12000rpm离心5min，弃废液；取出离心柱，避免酒精残留；
2.3.11将离心柱放置于新的离心管中，揭开盖子，4℃，12000rpm，离心5min，甩干管内膜片，弃废液；
2.3.12将离心柱置于新的离心管中，加入14μL无酶水，常温静置5min；
2.3.13将离心柱以12000rpm，离心5min，待用。
2.4测定血浆中miRNA表达水平
2.4.1血浆总RNA的逆转录
取12μL上述总RNA，按照凯捷公司小RNA逆转录试剂盒进行逆转录。整个配制过程在4℃中进行，37℃中反应60min，进行Poly(A)加尾和逆转录反应，95℃中5分钟灭活酶小RNA逆转录体系。反应体系见表7：
表7 反应体系
试剂名称 剂量
5x小RNA逆转录缓冲液 4μL
10x小RNA逆转录核酸体系 2μL
小RNA逆转录体系 2μL
总RNA 12μL
反应总体积 20μL
在得到的20μL逆转录产物中加入200μL无酶水，将所得产物cDNA进行浓度和纯度检测后置于-20℃冰箱保存备用。保存于-80℃冰箱中待用。
2.4.2荧光定量PCR
以逆转录后的cDNA为模板，按照小RNA聚合酶链反应染料法试剂盒的说明书配制PCR反应体系，引物均来自天根。每条引物重复3个复孔，每次试验重复3次。上游引物为天根公司设计的引物，下游引物为凯捷公司设计的通用引物。反应的条件按照试剂盒的要求设置：预变性95℃中15min；变性94℃中15s；退火延伸55℃中30s；40次循环，在70℃时绘制熔解曲线。反应体系见表8。引物序列见表9。
表8 反应体系
试剂名称 剂量
2x聚合酶链反应主要反应体系 10μL
10x上游引物，F链 2μL
下游引物，R链 2μL
无酶水 4μL
cDNA 2μL
反应总体积 20μL
表9 引物序列
基因名称 序列
hsa-miR-16-5p uagcagcacguaaauauuggcg(SEQ ID NO.6)
hsa-miR-96-5p uuuggcacuagcacauuuuugcu(SEQ ID NO.7)
hsa-miR-99b-5p cacccguagaaccgaccuugcg(SEQ ID NO.8)
hsa-miR-155-5p uuaaugcuaaucgugauaggggu(SEQ ID NO.9)
hsa-let-7a-5p ugagguaguagguuguauaguu(SEQ ID NO.10)
hsa-let-7b-5p ugagguaguagguugugugguu(SEQ ID NO.11)
我们以miR-16为标准计算各个miRNA的差异表达水平，每个miRNA的表达水平采用2-△△CT方法进行计算，其中-△△CT＝(CT实验组-CT标准参照)-(CT对照组-CT标准参照)，分析miRNA在结直肠癌患者和健康对照人群中的差异表达情况。
2.5生存分析
收集2008年12月30日至2011年12月30日期间在广西医科大学附属肿瘤医院就诊的结直肠癌患者，采用电话和走访的形式定期随访，以死亡为终点事件，截止至末次随访时未发生终点事件或失访都归入截尾数据。随访截止时间为2016年9月，中位随访时间为47.8个月。
2.6统计分析
采用SPSS 20.0、GraphPad Prism5.0或者Medcalc12.2.1进行数据分析。正态分布资料采用均数±标准差表示；采用t检验、单因素方差分析或者非参数检验评估不同组之间各个的miRNA表达差异；采用受试者工作曲线(ROC)和二元线性回归评估miRNA的联合诊断效能；采用单因素和多因素生存分析评估各个miRNA的预后价值；通过生物信息学方法分析miRNA的靶基因及其涉及的信号通路。P值小于0.05视为有统计学意义。
结果：
1结直肠癌患者血浆miRNAs表达水平和临床病理参数的关系
1.1提取的总RNA质量鉴定及PCR扩增和熔解曲线图
经微量核酸测定仪检测，各样本总RNA的260/280的OD值在1.8-2.0间；经琼脂糖凝胶电泳实验，各样本28S、18S和5S的条带清晰，28S条带亮度大于18S和5S，故提取的RNA质量可靠完整，可用于下一步研究，如图3所示。
本研究中，内参和目的miRNA的荧光定量PCR的扩增曲线均呈现出S型，走形光滑，有明显的扩增期和平台期，表示扩增结果理想；内参和目的miRNA的荧光定量PCR的熔解曲线均为一狭窄的单峰曲线，熔点峰均在76℃-80℃，无明显双峰，表明扩增产物特异性好。如图4所示。
1.2血浆miRNAs表达水平和临床病理参数的关系
我们检测了90例CRC患者和20例健康受试者的5个miRNA的表达水平，其和临床特征的关系如下表10。由10表可知，本研究中，血浆miR-96、miR-155和let-7a在肝转移组表达上调，而miR-99b和let-7b在肝转移组表达下调，差异有统计学意义(P＜0.05)。此外，本研究中5个miRNA的表达与肿瘤的N分期及临床分期有关，差异有统计学意义(P＜0.05)；miR-96、miR-99b和miR-155与肿瘤直径有关，差异有统计学意义(P＜0.05)；miR-96、miR-99b、miR-15和Let-7b与肿瘤T分期有关，差异有统计学意义(P＜0.05)；我们的研究未观察到此5个miRNA与其它临床病理参数的关系，差异无统计学意义(P＞0.05)。
表10 结直肠癌患者miRNA的表达情况及与临床病理特征的关系(均数±标准差)
Figure PCTCN2021141650-appb-000005
Figure PCTCN2021141650-appb-000006
注：*单因素方差分析；**秩和检验。
2结直肠癌患者和健康受试者血浆miRNAs表达水平
为了探索本研究中5个miRNA在CRC患者和健康受试者血浆之间是否有差异表达，我们采用实时荧光定量PCR(qRT-PCR)来检测这5个miRNA在血浆的表达水平。如表11所示，统计分析表明，miR-96、miR-155和let-7a在CRC患者血浆中高表达，而在健康受试者中呈现低表达；miR-99b和let-7b在CRC患者血浆中低表达，在健康受试者血浆中呈现高表达，差异均有统计学意义(P＜0.05)。
表11 结直肠患者和健康人血浆miRNA的表达水平
miRNA CRC(n＝90,M±S) 健康人(n＝20,M±S) 倍数 t P
miR-96 4.765±1.076 3.568±0.662 1.335 6.43 ＜0.001
miR-99b 0.529±0.3017 1.055±0.370 -1.994 -6.75 ＜0.001
miR-155 2.791±1.189 1.277±1.142 2.186 5.32 0.001
let-7a 2.685±0.966 1.098±0.540 2.445 10.06 ＜0.001
let-7b 0.470±0.347 1.236±1.034 -2.630 -3.27 0.004
为了进一步寻找上述5个差异表达的血浆miRNA与肝转移的关系，我们将90例CRC患者分为无远处转移组(临床分期I-Ⅲ)和肝转移组(临床分期Ⅳ)，比较各个血浆miRNA的表达水平是否有差异。统计分析表明，血浆miR-96、miR-155和let-7a在肝转移组中高表达，在无远处转移组中低表达，差异有统计学意义(P＜0.05)；而血浆miR-99b和let-7b在肝转移组中低表达，在无远处转移组中高表达，差异有统计学意义(P＜0.05)，如图5所示。
3结直肠癌患者血浆中miRNA的诊断效能分析
为了检测上述差异表达的血浆miRNA在检出肝转移方面是否具有诊断价值，我们采用受试者工作曲线(ROC)和二元线性回归模型进行分析各个miRNA在各组之间的诊断效能，见表12。首先，我们分析了上述5个差异表达的血浆miRNA在CRC患者和健康受试者之间的诊断效能，ROC和曲线下面积(AUC)表明本研究中5个血浆miRNA均有较好的诊断效能，miR-96、miR-99b、miR-155、let-7a和let-7b的曲线下面积分别为AUC＝0.823(CI:0.738-0.889)，0.86(CI:0.781-0.919)，0.858(CI:0.778-0.917)，0.908(CI:0.837-0.954)，0.869(CI:0.792-0.926)，差异均有统计学意义。
表12 结直肠癌患者血浆中miRNA的诊断效能分析
Figure PCTCN2021141650-appb-000007
在上述研究的基础上，我们进一步分析上述具有较优诊断价值的miRNA在鉴别和诊断无远处 转移(临床分期I－Ⅲ)和肝转移(临床分期Ⅳ)患者上的效能，如表12。
本研究同时检测了CEA的表达水平，试图比较我们候选的5个miRNA和CEA的诊断效能。ROC曲线分析的结果表明，血浆miR-96的曲线下面积为0.937(0.866-0.978)，血浆miR-99b的曲线下面积为0.929(0.855-0.973)，血浆miR-155的曲线下面积为0.806(0.710-0.882)，血浆let-7a的曲线下面积为0.843(0.751-0.911)，血浆let-7b的曲线下面积为0.782(0.682-0.862)，CEA的曲线下面积为0.693(0.587-0.786)，在这6个血浆检测指标中，miR-96、miR-99b和let-7a在诊断无远处转移患者和肝转移患者中的单独诊断价值最高，而CEA的诊断效能低于本研究其他的5个指标。
接着，我们通过二元线性回归模型分析了miRNA的联合诊断效能，miR-96，miR-99b和let-7a的联合诊断效能：曲线下面积为0.971(CI:0.911-0.995)，最佳截点值为0.565，敏感性为91.84％，特异性为80.49％。由此，我们的结果表明，血浆miR-96、miR-99b和let-7a三个指标的联合诊断的价值优于CEA及其它指标，见图15-图16。研究结果表明，血浆中miR-96、miR-99b和let-7a组合可能作为结直肠癌患者，特别是结直肠癌肝转移患者新型非侵入性诊断和监测的生物标志物，可能作为结直肠癌治疗的分子靶点。
4生存分析
为了评估本研究中5个血浆中miRNA和结直肠癌肝转移患者预后的关系，我们将49例肝转移患者根据各个miRNA表达水平的中位数将患者分为高表达组和低表达组，通过Kaplan–Meier法、Log-Rank检验和Cox多因素回归模型分析血浆miRNA的表达和总体生存期的关系。单因素分析结果表明高表达的miR-96，miR-155患者总体生存时间较低表达患者短(log-rank检验示P分别为0.031，0.047)；而高表达的miR-99b患者总体生存时间较低表达的患者长(log-rank检验示P分别为0.025)，见表13。然而，未发现其它miRNA及临床病理参数，例如CEA表达水平、年龄、性别和肿瘤直径等与总体生存时间的差异有统计学意义(P＞0.05)。多因素Cox回归模型示血浆中miR-96、miR-155的表达是结直肠癌肝转移患者预后的独立危险因素，见图7。表明血浆中miR-96和miR-155对结直肠癌肝转移患者具有预后评估价值。
实施例3：结直肠癌患者石蜡包埋组织中肝转移相关miRNA的表达及其诊断和预后价值
表13 结直肠癌患者总体生存时间的单因素和多因素分析
Figure PCTCN2021141650-appb-000008
1.实验材料
病例选择和标本收集
本研究已通过广西医科大学附属肿瘤医院医学伦理委员会的批准，每位受试者均已签署知情同意书同意使用标本及临床资料。收集于2008年12月30日至2011年12月30日期间在广西医科大学附属肿瘤医院就诊的结直肠癌患者58例。患者中位年龄为57岁(波动范围为33-89岁)。男性28例，女性30例；左半结肠13例，右半结肠23例，直肠22例；低分化肿瘤15例，中分化肿瘤26例，高分化肿瘤17例；肿瘤直径＜5cm的35例，肿瘤直径≥5cm的23例；T1+T2有27例，T3+T4有31例；N0有20例，N1+N2有38例；临床分期为I+II的有14例，临床分期III+IV的有44例；无远处转移患者25例，肝转移患者33例。通过UICC和AJCC进行临床分期评估。详见表14。
结直肠癌肝转移患者入组标准：收集时所有患者均未接受过化疗、放疗、靶向治疗等综合治疗，收集的肿瘤组织均为术前组织；未合并其他位置的远处转移和/或复发，未同时患有其他良性疾病。
结直肠癌无远处转移患者入组标准：无远处转移和/或复发，未同时患有其他良性疾病。
表14 结直肠癌患者的临床特征
Figure PCTCN2021141650-appb-000009
注：*P＜0.05
2实验方法
2.1患者手术标本的处理
肿瘤组织经手术切取后，立马放置于体积分数为4％的中性福尔马林溶液内固定，经取材、脱水、透明、浸蜡和包埋等处理后制成石蜡包埋(FFPE)的组织，切片经HE染色病理确诊，然后长期保存。所有无远处转移患者均留取肠癌组织和肠癌旁组织的蜡块；所有肝转移患者均留取肠癌组织、肠癌旁组织及相匹配的肝转移瘤组织的蜡块。
2.2石蜡包埋组织中总RNA的提取
2.2.1用手术刀削去石蜡层；
2.2.2切成5-20μm厚的片状(如果样本已经暴露在空气中，将头2-3层弃掉)；
2.2.3去石蜡操作：
2.2.3.1加1mL二甲苯、二戊烯或聚丙烯，涡旋震荡10s，全速离心2min；
2.2.3.2小心用枪头吸取上清，吸取上清的时候不要碰到沉淀物；
2.2.3.3在沉淀中加入1mL乙醇(体积分数为96-100％)，涡旋混匀，全速离心2min(乙醇的作用是将样本中的二甲苯去除)；
2.2.3.4小心用枪头吸取上清，吸取上清时不要碰到沉淀物，并用小枪头小心吸掉残留的庚烷/甲醇；
2.2.3.5开盖在室温下孵育(15-25℃，最高不要超过37℃)10min，或者孵育至所有残液挥发；
2.2.3.6加入150μL或240μL(切片数量大于2时)石蜡标本处理缓冲液，涡旋混匀；
2.2.3.7加入10μL蛋白酶K，用枪头温和混匀(反复吸打)；
2.2.3.在56℃下孵育15min，80℃下孵育15min。
2.2.4冰上孵育3min后，20,000×g(13,500rpm)离心15min；
2.2.5将上清转移至一个新的离心管，注意不要碰到沉淀物；
2.2.6加入约1/10样本体积的基因组脱氧核糖核酸去除缓冲液(约16μL或25μL，取决于样本量)，以及10μL的脱氧核糖核酸消化酶。正反倒置离心管混匀液体。简单离心以收集管壁上的残液；
2.2.7室温下孵育15min；
2.2.8加入320μL或500μL(样本大于2个切片时)红细胞缓冲液，彻底混合溶解产物；
2.2.9加入1120μL或1750μL乙醇(100％)至样本中，用枪头混匀，立即进行下一步；
2.2.10将700μL的样本(包括可能存在的沉淀)转移至微量核糖核酸抽提试剂盒上(将微量核糖核酸抽提试剂放置在2mL的收集管里，收集管试剂盒中已提供)。盖上收集管的盖子，在大于8,000×g(10,000rpm)条件下离心15s，丢弃收集管中的收集液；
2.2.11重复上面的步骤，直到之前的样本都处理完毕；
2.2.12加入500μL膜结合核糖核酸冲洗液至微量核糖核酸抽提试剂盒。小心盖上收集管的盖子，大于8,000×g(10,000rpm)条件下离心15s，丢弃收集管中的收集液；
2.2.13加入500μL膜结合核糖核酸冲洗液至微量核糖核酸抽提试剂盒。盖上收集管的盖子，大于8,000×g(10,000rpm)条件下离心2min，丢弃收集管；
2.2.14将微量核糖核酸抽提试剂放在一个新的2mL的收集管里，打开盖子，全速离心5min，丢掉收集管；
2.2.15将微量核糖核酸抽提试剂放在一个新的1.5mL的收集管里，直接加14-30μL无酶水至试剂盒的膜上，小心盖上盖子，全速离心1min，收集洗脱液。1.5mL离心管内收集到的即为提取的总RNA。
2.3组织总RNA的逆转录及实时荧光定量PCR
本部分FFPE组织中miRNA的表达水平测定方法同实施例2的步骤2.4。我们以U6为标准参照来归一化各个miRNA的表达。
2.4生存分析
收集于2008年12月30日至2011年12月30日期间在广西医科大学附属肿瘤医院就诊的结直肠癌患者，采用电话和走访的形式定期随访，以死亡为终点事件，截止至末次随访时未发生终点事件或失访都归入截尾数据。随访截止时间为2016年9月，中位随访时间为53.8月。
2.5统计分析
采用SPSS 20.0、GraphPad Prism5.0或者Medcalc12.2.1进行数据分析。正态分布资料采用均数±标准差(Mean±SD)表示；采用t检验、单因素方差分析或者非参数检验评估不同组之间各个的miRNA表达差异；采用受试者工作曲线(ROC)和二元线性回归评估miRNA组合的诊断效能；采用单因素和多因素生存分析评估各个miRNA的预后价值。P值小于0.05视为有统计学意义。
结果：
1结直肠癌患者组织中miRNAs表达水平和临床病理参数的关系
1.1提取的总RNA质量鉴定及PCR扩增和熔解曲线图
经微量核酸测定仪检测，各样本总RNA的260/280的OD值在1.8-2.0间；经琼脂糖凝胶电泳实验，各样本28S、18S、5S的条带清晰，28S条带亮度大于18S和5S，故提取的RNA质量可 靠完整，可用于下一步研究，见图8。
本研究中，内参和目的miRNA的荧光定量PCR的扩增曲线均呈现出S型，走形光滑，有明显的扩增期和平台期，表示扩增结果理想；内参和目的miRNA的荧光定量PCR的熔解曲线均为一狭窄的单峰曲线，熔点峰均在76-80℃之间，无明显双峰，表明扩增产物特异性好。见图9。
1.2福尔马林固定石蜡包埋组织中miRNAs表达水平和临床病理参数的关系
我们检测了25例结直肠癌无远处转移患者和33例结直肠癌肝转移患者的5个miRNA的表达水平，其和临床特征的关系如下表15。由表3-2可知，本研究中5个miRNA的表达与肿瘤的T分期或肝转移等临床参数有关，差异有统计学意义(P＜0.05)。
表15 结直肠癌患者肠癌组织miRNA的表达与临床病理参数的关系
Figure PCTCN2021141650-appb-000010
Figure PCTCN2021141650-appb-000011
注：*为非参数检验。
2结直肠癌患者癌组织和癌旁组织中miRNAs表达水平
为了探索本研究中5个miRNA在结直肠癌患者癌组织和癌旁组织之间是否有差异表达，我们采用实时荧光定量PCR(qRT-PCR)来检测这5个miRNA在组织的表达水平。如表16所示，统计分析表明，miR-96、miR-155和let-7a在CRC患者组织中高表达，而在癌旁组织中呈现低表达；miR-99b和let-7b在结直肠癌患者组织中低表达，在癌旁组织中呈现高表达，差异均有统计学意义(P＜0.05)，与血浆中的表达趋势一致。
表16 结直肠癌患者组织中miRNAs表达水平
Figure PCTCN2021141650-appb-000012
为了进一步寻找上述5个差异表达的miRNA与肝转移的关系，我们将58例结直肠癌患者分为无远处转移组(临床分期I-Ⅲ，n＝25)和肝转移组(临床分期Ⅳ，n＝33)，比较两组中miRNA的表达水平是否有差异。统计分析表明，福尔马林固定石蜡包埋组织中miR-96、miR-155和let-7a在肝转移组中高表达，在无远处转移组中低表达，差异有统计学意义(P＜0.05)；而miR-99b在肝转移组中低表达，在无远处转移组中高表达，差异有统计学意义(P＜0.05)，未发现let-7b在两组之间差异表达，见图10。
3结直肠癌肝转移患者肠癌组织和相匹配肝转移瘤中miRNA的表达
为了进一步分析这5个miRNA在结直肠癌肝转移患者中的表达情况，我们对33例有肝转移瘤的肠癌患者进行了详细的研究。我们通过配对t检验比较了每位患者的肠癌原发组织和相匹配的肝转移灶组织中这5个miRNA的表达情况，见图11。我们发现，与相匹配肝转移灶相比，miR-96和miR-155在肠癌原发灶中表达上调，而miR-99b表达下调，差异有统计学意义(P为0.027，＜0.001，0.032)，然而我们未发现let-7a和let-7b在肝转移患者肠癌和肝转移瘤组织中存在差异表达(P＝0.098,0.131)。
4结直肠癌患者FFPE组织中miRNA的诊断效能分析
为了检测上述差异表达的组织miRNA在检出肝转移方面是否具有诊断价值，我们采用受试者工作曲线(ROC)和二元线性回归模型进行分析各个miRNA在各组之间的诊断效能，见表3-4和图12。首先，我们分析了上述5个差异表达的血浆miRNA在结直肠癌组织和癌旁正常组织之间的诊断效能，ROC和曲线下面积(AUC)表明本研究中5个miRNA均有较好的诊断效能，miR-96、miR-99b、miR-155、let-7a和let-7b的ROC曲线如图12所示，差异均有统计学意义(P<0.0001)。
接着，我们比较了无远处转移患者和肝转移患者的肠癌组织中各个miRNA的诊断效能，发现miR-99b、miR-155和let-7a具有较优的诊断价值，差异均有统计学意义(P<0.0001)，而miR-96和let-7b的诊断价值较低(P＞0.05)；将具有统计学意义的3个指标miR-99b、miR-155和let-7a联合，评价其联合诊断效能，发现AUC＝0.954(0.864-0.992)均高于这3个指标的单独诊断效能及其它miRNAs的联合诊断效能，见图12。
5生存分析
为了评估本研究中5个miRNA和结直肠癌肝转移患者预后的关系，我们将33例肝转移患者根据各个miRNA表达水平的中位数将患分为高表达组和低表达组，通过Kaplan–Meier法和Cox多因素回归模型分析FFPE组织中miRNA的表达和总体生存期的关系，如表17。单因素分析结果表明miR-99b，let-7b和N分期与患者总体生存时间有关，P＜0.05)，未发现其它miRNA及临床病理参数，例如年龄、性别和肿瘤直径等与总体生存时间的差异有统计学意义(P＞0.05)。多因素Cox回归模型示miR-99b、N分期是结直肠癌肝转移患者预后的独立危险因素，见图13。
表17 结直肠癌肝转移患者总体生存时间的单因素和多因素分析
Figure PCTCN2021141650-appb-000013
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (6)

  1. 一种用于结直肠癌预后的miRNA检测试剂盒，其特征在于，包括miR-96、miR-99b和miR-155中的任意一种或两种以上。
  2. 根据权利要求1所述的用于结直肠癌预后的miRNA检测试剂盒，其特征在于，所述miR-96的序列如SEQ ID NO.1所示；所述miR-99b的序列如SEQ ID NO.2所示；所述miR-155的序列如SEQ ID NO.3所示。
  3. 根据权利要求1所述的用于结直肠癌预后的miRNA检测试剂盒，其特征在于，所述试剂盒是通过定量PCR技术检测生物样品中miR-96、miR-99b和miR-155的表达量。
  4. 根据权利要求3所述的用于结直肠癌预后的miRNA检测试剂盒，其特征在于，所述生物样品选自新鲜的组织、新鲜的细胞、新鲜的血液、新鲜的体液、福尔马林固定的组织、福尔马林固定的细胞、石蜡包埋的组织和石蜡包埋的细胞中的任意一种。
  5. 根据权利要求4所述的用于结直肠癌预后的miRNA检测试剂盒，其特征在于，所述生物样品为血浆。
  6. 根据权利要求1所述的用于结直肠癌预后的miRNA检测试剂盒，其特征在于，还包括对miR-96、miR-99b和miR-155具有检测特异性的探针、基因芯片或PCR引物。
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