目的 建立一种催化发夹自组装（CHA）联合成簇间隔的短回文重复序列及其关联蛋白12a（CRISPR-Cas12a）的传感技术，用于外泌体源性微小RNA-21（miR-21）的检测并分析其性能。 方法 选取2023年9—10月在陆军军医大学第一附属医院确诊为乳腺癌的患者8例为乳腺癌组；选取同期健康体检者8名为健康对照组。采用试剂盒提取血浆外泌体及其miR-21。针对miR-21序列设计DNA发夹和CRISPR RNA序列。通过聚丙烯酰胺凝胶电泳和荧光分光光度计验证检测技术可行性。进一步优化发夹浓度、CHA反应时间、Cas12a蛋白浓度和Cas12a蛋白反应时间。在此基础上，检测不同浓度（0、0.1、0.5、1.0、2.5、5.0、7.5、10.0 nmol/L）的miR-21，收集荧光强度采用一元线性回归方法做线性分析以评价方法学的灵敏度，同时检测不同类型miRNA（miR-31、miR-26a、miR-192、miR-25-3p）及空白对照以评价方法学特异性。采用病例对照研究，用该检测技术和逆转录PCR方法检测乳腺癌组和健康对照组血浆外泌体源性miR-21的相对表达水平以评价其临床样本检测能力。 结果 应用CHA联合CRISPR-Cas12a技术建立了外泌体源性miR-21的检测方法。该方法检测miR-21的浓度与荧光强度呈良好的线性关系（相关系数为0.966 7），线性检测范围为0.1~10.0 nmol/L，检测限为87.81 pmol/L。miR-21的检测荧光强度为450.27±23.96，高于miR-31、miR-26a、miR-192、miR-25-3p和空白对照组（分别为98.89±7.35、98.12±2.07、98.93±2.45、96.66±2.45、82.93±3.54），差异均有统计学意义（P均<0.001）。逆转录PCR结果显示，乳腺癌组血浆外泌体源性miR-21相对表达水平高于健康对照组（1.83±0.27比0.93±0.12，P<0.001）；CHA联合 CRISPR-Cas12a检测技术结果显示，乳腺癌组血浆外泌体源性miR-21相对表达水平高于健康对照组（1.94±0.21比0.98±0.08，P<0.001）；CHA联合 CRISPR-Cas12a检测技术与逆转录PCR检测的乳腺癌组和健康对照组的血浆外泌体源性miR-21相对表达水平比较，差异无统计学意义（P均>0.05）。 结论 建立了CHA联合CRISPR-Cas12a检测外泌体源性miR-21的高灵敏度与高特异性的传感技术，其检测血浆外泌体源性miR-21的结果与逆转录PCR结果一致，可用于乳腺癌患者筛查。 Objective To establish a sensing technology of catalytic hairpin self-assembly (CHA) combining with clustered interspaced short palindromic repeats with associated protein 12a (CRISPR-Cas12a) for the detection of exosomal microRNA-21 (miR-21), and to analyze the performance. Methods Eight patients diagnosed as breast cancer in the First Affiliated Hospital of the Army Military Medical University from September to October 2023 were selected as the breast cancer group 8 healthy individuals who underwent physical examinations during the same period were selected as the healthy control group. Plasma exosomes and their miR-21 were extracted using the kit. DNA hairpins and CRISPR RNA sequences were designed for miR-21 sequences. The feasibility of detection technology was validated using polyacrylamide gel electrophoresis and fluorescence spectrophotometer. Hairpins concentration, CHA reaction time, Cas12a protein concentration and Cas12a protein reaction time were further optimized. On this basis, miR-21 was detected at different concentrations (0, 0.1, 0.5, 1.0, 2.5, 5.0, 7.5, 10.0 nmol/L), and fluorescence intensity was collected for unary linear regression analysis to evaluate methodological sensitivity meanwhile, different types of miRNAs (miR-31, miR-26a, miR-192, miR-25-3p) and blank controls were detected to evaluate methodological specificity. A case-control study was conducted to detect the relative expression level of plasma exosomal miR-21 in breast cancer group and healthy control group using this detection technology and reverse transcription PCR (RT-PCR) to evaluate the detection ability of clinical samples. Results A detection method for exosomal miR-21 was established using CHA combining with CRISPR-Cas12a. The concentration of miR-21 detected by this method showed a good linear relationship with fluorescence intensity (the linear correlation coefficient 0.966 7), and the linear detection range was 0.1-10.0 nmol/L, and the detection limit was 87.81 pmol/L. The fluorescence intensity of miR-21 was 450.27±23.96 which was higher than that of miR-31, miR-26a, miR-192, miR-25-3p, and the blank group (98.89±7.35, 98.12±2.07, 98.93±2.45, 96.66±2.45, 82.93±3.54, respectively), with statistical significance (P<0.001). The results of RT-PCR showed that the relative expression levels of plasma exosomal miR-21 in the breast cancer group were higher than that in healthy control group (1.83±0.27 vs 0.93±0.12,P<0.001) CHA combining with CRISPR-Cas12a detection technology showed that the relative expression levels of plasma exosomal miR-21 in breast cancer group were higher than that in healthy control group (1.94±0.21 vs 0.98±0.08,P<0.001) There was no significant difference in the relative expression levels of plasma exosomal miR-21 between CHA combining with CRISPR-Cas12a detection technology and reverse transcription PCR in breast cancer group and healthy control group (P>0.05). Conclusion In this study, a highly sensitive and specific sensing technology of CHA combining with CRISPR-Cas12a for exosomal miR-21 was established. The results of detecting plasma exosomal miR-21 were consistent with the results of reverse transcription PCR, which can be used for screening of breast cancer patients.
Application of catalytic hairpin self-assembly combining with CRISPR-Cas12a sensing technology in exosomal microRNA-21
Objective To establish a sensing technology of catalytic hairpin self-assembly (CHA) combining with clustered interspaced short palindromic repeats with associated protein 12a (CRISPR-Cas12a) for the detection of exosomal microRNA-21 (miR-21), and to analyze the performance. Methods Eight patients diagnosed as breast cancer in the First Affiliated Hospital of the Army Military Medical University from September to October 2023 were selected as the breast cancer group 8 healthy individuals who underwent physical examinations during the same period were selected as the healthy control group. Plasma exosomes and their miR-21 were extracted using the kit. DNA hairpins and CRISPR RNA sequences were designed for miR-21 sequences. The feasibility of detection technology was validated using polyacrylamide gel electrophoresis and fluorescence spectrophotometer. Hairpins concentration, CHA reaction time, Cas12a protein concentration and Cas12a protein reaction time were further optimized. On this basis, miR-21 was detected at different concentrations (0, 0.1, 0.5, 1.0, 2.5, 5.0, 7.5, 10.0 nmol/L), and fluorescence intensity was collected for unary linear regression analysis to evaluate methodological sensitivity meanwhile, different types of miRNAs (miR-31, miR-26a, miR-192, miR-25-3p) and blank controls were detected to evaluate methodological specificity. A case-control study was conducted to detect the relative expression level of plasma exosomal miR-21 in breast cancer group and healthy control group using this detection technology and reverse transcription PCR (RT-PCR) to evaluate the detection ability of clinical samples. Results A detection method for exosomal miR-21 was established using CHA combining with CRISPR-Cas12a. The concentration of miR-21 detected by this method showed a good linear relationship with fluorescence intensity (the linear correlation coefficient 0.966 7), and the linear detection range was 0.1-10.0 nmol/L, and the detection limit was 87.81 pmol/L. The fluorescence intensity of miR-21 was 450.27±23.96 which was higher than that of miR-31, miR-26a, miR-192, miR-25-3p, and the blank group (98.89±7.35, 98.12±2.07, 98.93±2.45, 96.66±2.45, 82.93±3.54, respectively), with statistical significance (P<0.001). The results of RT-PCR showed that the relative expression levels of plasma exosomal miR-21 in the breast cancer group were higher than that in healthy control group (1.83±0.27 vs 0.93±0.12,P<0.001) CHA combining with CRISPR-Cas12a detection technology showed that the relative expression levels of plasma exosomal miR-21 in breast cancer group were higher than that in healthy control group (1.94±0.21 vs 0.98±0.08,P<0.001) There was no significant difference in the relative expression levels of plasma exosomal miR-21 between CHA combining with CRISPR-Cas12a detection technology and reverse transcription PCR in breast cancer group and healthy control group (P>0.05). Conclusion In this study, a highly sensitive and specific sensing technology of CHA combining with CRISPR-Cas12a for exosomal miR-21 was established. The results of detecting plasma exosomal miR-21 were consistent with the results of reverse transcription PCR, which can be used for screening of breast cancer patients.
Breast cancerExosomeMicroRNACatalytic hairpin self-assembly
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