CN118121620A - hsa_circ_0066187在制备脓毒症检测试剂盒和治疗药物中的应用 - Google Patents
hsa_circ_0066187在制备脓毒症检测试剂盒和治疗药物中的应用 Download PDFInfo
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Abstract
本发明涉及hsa_circ_0066187在制备脓毒症检测试剂盒和治疗药物中的应用，涉及生物医学技术领域。本发明发现hsa_circ_0066187在经脂多糖(LPS)刺激的人血管内皮细胞中高表达，敲低该circRNA能够降低炎症因子IL‑1β及IL‑6的表达，并提高紧密连接蛋白Occludin和ZO‑1的表达。hsa_circ_0066187通过调控miRNA‑185‑5p对下游RhoA/ROCK1信号通路活性进行调节，最终表现为hsa_circ_0066187在脓毒症患者及健康对照人群外周血中表达差异，其表达量与疾病严重程度及总体预后显著相关，能够作为脓毒症检测和治疗的关键靶点。
Description
技术领域
本发明涉及生物医药技术领域，尤其是指hsa_circ_0066187在制备脓毒症检测试剂盒和治疗药物中的应用。
背景技术
脓毒症指由细菌等病原微生物侵入机体引起的全身炎症反应综合征，是危重患者常见并发症，也是临床危重病患者死亡的重要原因。脓毒症的发病机制尚未明了，涉及到复杂的全身炎症网络效应、基因多态性、免疫功能障碍、凝血功能异常、组织损伤以及宿主对不同感染病原微生物及其毒素的异常反应等多个方面。由于脓毒症临床症状非特异性，给早期诊断和治疗带来困难。脓毒症与机体多器官功能障碍密切相关，该疾病治疗花费高，医疗资源消耗大，严重影响人类的生活质量，已经对人类健康造成巨大威胁。因此，如何对脓毒症进行及时的诊断，以及后续如何针对脓毒病患者进行高效的治疗，是当前亟待解决的问题。
发明内容
为解决上述技术问题，本发明发现了一个脓毒症检测与治疗的关键靶点hsa_circ_0066187，该环状RNA在经LPS刺激的人血管内皮细胞中高表达，敲低该circRNA表达能够降低炎症因子IL-1β及IL-6的表达，提高紧密连接蛋白Occludin和ZO-1的表达。hsa_circ_0066187对miRNA-185-5p存在调控作用，其通过miRNA-185-5p对下游RhoA/ROCK1信号通路活性进行调控，最终表现为hsa_circ_0066187在脓毒症患者及健康对照人群外周血中表达差异。
本发明的第一个目的是提供hsa_circ_0066187在制备脓毒症治疗药物中的应用。
进一步地，hsa_circ_0066187的核苷酸序列如SEQ ID NO.1所示。
进一步地，上述药物敲低hsa_circ_0066187的表达。
进一步地，使用siRNA敲低hsa_circ_0066187的表达，siRNA的序列如SEQ IDNO.2-5所示
进一步地，siRNA序列为CAAAUCAUAACUUGAGGCATT(SEQ ID NO.2)，UGCCUCAAGUUAUGAUUUGTT(SEQ ID NO.3)(siRNA-1)或CAUAACUUGAGGCAAACUATT(SEQ IDNO.4)，UAGUUUGCCUCAA GUUAUGTT(SEQ ID NO.5)(siRNA-2)。
本发明的第二个目的是提供hsa_circ_0066187在制备脓毒症检测试剂盒中的应用。
本发明的第三个目的是提供一种脓毒症的检测试剂盒，试剂盒检测样本中hsa_circ_0066187的表达量。
进一步地，上述样本为外周血样本。
进一步地，脓毒症患者外周血中hsa_circ_0066187的表达量上升。
进一步地，检测试剂盒中包括用于扩增hsa_circ_0066187的引物，引物序列如SEQID NO.6-7所示。
进一步地，用于扩增hsa_circ_0066187的引物为正向引物5’-CCGAAGAGCCTGCATTAGTA-3’(SEQ ID NO.6)；反向引物5’-CTTGCATAGT TTGCCTCAAG-3’(SEQID NO.7)。
进一步地，检测试剂盒中还包括用于PCR的其他组分。
本发明的第四个目的是提供扩增hsa_circ_0066187的引物在制备脓毒症检测试剂盒中的应用。
本发明的上述技术方案相比现有技术具有以下优点：
本发明提供了hsa_circ_0066187作为靶点在脓毒症检测和治疗中的应用，敲低hsa_circ_0066187降低炎症因子IL-1β及IL-6的表达，并提高紧密连接蛋白Occludin和ZO-1的表达。hsa_circ_0066187通过miRNA-185-5p对下游RhoA/ROCK1信号通路活性进行调控，最终表现为hsa_circ_0066187在脓毒症患者及健康对照人群外周血中表达差异，其表达量与疾病严重程度及总体预后显著相关，能够作为脓毒症检测和治疗的关键靶点，可以用于制备脓毒症检测试剂盒及治疗脓毒症的药物，为脓毒症患者的快速检测节约了时间，有广阔的应用前景。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明，其中
图1是本发明实施例1中hsa_circ_0066187在LPS刺激的血管内皮细胞中不同时间点的表达差异；
图2是本发明实施例2中对hsa_circ_0066187环状结构的验证；
图3是本发明实施例3中敲低hsa_circ_0066187降低炎症因子IL-1β及IL-6表达，提高紧密连接蛋白Occludin和ZO-1表达的实验结果图；
图4是本发明实施例4中hsa_circ_0066187对miRNA-185-5p存在调控作用的实验结果图；
图5是本发明实施例5中miRNA-185-5p表达增高可降低炎症因子IL-1β及IL-6表达，并提高紧密连接蛋白Occludin和ZO-1表达的实验结果图；
图6是本发明实施例6中hsa_circ_0066187通过miRNA-185-5p对下游RhoA/ROCK1信号通路活性进行调控的实验结果图；
图7是本发明实施例7中hsa_circ_0066187在脓毒症患者及健康对照人群外周血中表达差异的实验结果图；
图8是本发明实施例8中脓毒症患者外周血中hsa_circ_0066187表达量与疾病严重程度及总体预后显著相关的实验结果图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1：hsa_circ_0066187在LPS刺激的人血管内皮细胞中高表达
用LPS(10μg/mL)刺激人源血管内皮细胞(HMEC-1)，12小时后收集细胞并用Trizol提取总RNA，通过反转录及RT-CR(反应体系SYBR Green Master Mix 10μL，Forward primer0.5μL，Reverse primer 0.5μL，cDNA 2μL，DEPC水7μL，反应步骤：预变性：95℃，5min，1个循环；循环反应：95℃，10sec以及60℃，34sec，共40个循环；熔解曲线：95℃，15sec，60℃，60sec，以及95℃，15sec，1个循环)检测hsa_circ_0066187表达量(hsa_circ_0066187上游引物为5’-CCGAAGAGCCTGCATTAGTA-3’(SEQ ID NO.6)，下游引物为5’-CTTGCATAGTTTGCCTCAAG-3’(SEQ ID NO.7))。研究发现，与对照组相比，hsa_circ_0066187在LPS刺激的人血管内皮细胞中高表达，如图1所示。
实施例2：验证hsa_circ_0066187环状结构
RNase R酶消化试验提示，环形的hsa_circ_0066187相对于其线性的母基因Cacna1d更加耐受RNase R酶消化(将提取的总RNA分成两份，将10U RNase R加入到5μg总RNA中，于37.0℃孵育20min之后进行qRT-PCR实验，另一份不经RNase R处理直接进行qRT-PCR。RNase R消化体系：10×Reaction Buffer 2μL；RNA 5μg；RNase R 0.7μL；DEPC水16.5μL，实验结果)(如图2A)。我们接下来用Trizol提取细胞的RNA，反转录成cDNA，以及直接从细胞中提取gDNA(TIANamp Genomic DNA Kit血液/细胞/组织基因组DNA提取试剂盒)，通过设计circRNA的正向和反向引物(hsa_circ_0066187反向引物CCGAAGAGCCTGCATTAGTA，CTTGCATAGTTTGCCTCAAG；hsa_circ_0066187正向引物CTCCCAAAGAAAACGTCAGCA，AATGCAGGCTCTTCGGATGG)，用qRT-PCR进行扩增(反应体系同实施例1)。之后再进行1.5％琼脂糖凝胶电泳，确认该circRNA为环形结构。如图2B所示。
实施例3：敲低hsa_circ_0066187降低炎症因子表达，提高紧密连接蛋白表达
在HMEC-1细胞中转染siRNA(siRNA-1和siRNA-2各5μL与Lipofe ctamine2000 5μL混合后静置10分钟，然后将混合液加入六孔板中完成瞬时转染，siRNA-1序列：CAAAUCAUAACUUGAGGCATT(SEQ ID NO.2)；UGCCUCAAGUUAUGAUUUGTT(SEQ ID NO.3)和siRNA-2序列：CA UAACUUGAGGCAAACUATT(SEQ ID NO.4)；UAGUUUGCCUCAAGU UAUGTT(SEQ IDNO.5))干扰hsa_circ_0066187表达，发现siRNA-1及siRNA-2均可以显著降低该circRNA表达，其中以siRNA-2干扰效果最为明显(图3A)。用siRNA-2序列包装慢病毒(由吉凯基因设计)侵染HME C-1细胞，荧光显微镜下可见满视野绿色荧光(图3B)。qRT-PCR提示慢病毒侵染HMEC-1细胞后hsa_circ_0066187表达显著下降(图3C)。在LPS刺激细胞后shRNA-hsa_circ_0066187仍可以有效敲低该circRNA表达(图3D)。上述结果表明稳定敲低hsa_circ_0066187表达HMEC-1细胞株构建成功。Western blot结果提示敲低细胞中hsa_circ_0066187表达后可降低炎症因子IL-1β及IL-6表达，提高紧密连接蛋白Occludin和ZO-1表达(图3E)。抗体的来源及稀释倍数：IL-1β(1:1000,Abcam),IL-6(1:1000,Abcam),oc cludin(1:1000,Abcam),ZO-1(1:1000,Boster Biological Technology)。
实施例4：hsa_circ_0066187对miRNA-185-5p的调控作用
对hsa_circ_0066187下游的miRNA进行预测，发现miRNA-185-5p是其潜在靶基因(图4A)，miRNA-185-5p在LPS刺激的HMEC-1细胞中低表达(图4B)。在细胞中敲低hsa_circ_0066187表达，可提高miRNA-185-5p表达(图4C)，提示hsa_circ_0066187对miRNA-185-5p存在负性调节。FISH试验(图4D)证实hsa_circ_0066187与miRNA-185-5p共同定位于细胞质，双荧光素酶试验(图4E)证实hsa_circ_0066187与miRNA-185-5p之间存在结合位点，及RIP试验(图4F)证实hsa_circ_0066187在AGO2蛋白上高度富集，上述结果表明hsa_circ_0066187对miRNA-185-5p存在调控作用，类似于miRNA-185-5p“海绵”，发挥吸附作用。
实施例5：提高miRNA-185-5p表达降低炎症因子表达，并提高紧密连接蛋白表达
提高miRNA-185-5p表达(miRNA-185-5p mimics 5μL与Lipofectamine2000 5μL混合后静置10分钟，然后将混合液加入六孔板中完成瞬时转染)与敲低hsa_circ_0066187表达一样，均可以降低炎症因子IL-1β及IL-6表达，提高紧密连接蛋白Occludin和ZO-1表达(图5)。最终产生同样的生物学作用(即miRNA-185-5p有助于减轻脓毒症肺损伤)。miRNA-185-5p在其他领域的生物学功能已有报道，但在脓毒症及肺损伤中的作用和机制既往未有报道过(miRNA-185-5p mimics：UGGAGAGAAAGGCAGUUCCUGA(SEQ ID NO.8)，AGGAACUGCCUUUCUCUCCAUU(SEQ ID NO.9))。
实施例6：hsa_circ_0066187通过miRNA-185-5p对下游RhoA/ROCK1信号通路活性进行调控
miRNA-185-5p对下游RhoA/ROCK1信号通路存在负性调控作用(图6A)，且双荧光素酶报告基因实验提示miRNA-185-5p与下游RhoA之间存在结合(图6B，C)。hsa_circ_0066187对下游RhoA/ROCK1信号通路存在正性调控作用(图6D)。在稳定敲低hsa_circ_0066187表达的HMEC-1细胞中转染miRNA-185-5p inhibitor可逆转单纯敲低hsa_circ_0066187导致的RhoA及ROCK1表达下降(图6E)，且对RhoA/ROCK1信号通路下游IL-1β、IL-6、Ocludin及ZO-1产生同样的影响(图6F)。由此表明hsa_circ_0066187通过作用于miRNA-185-5p对下游RhoA/ROCK1信号通路活性进行调控(miRNA-185-5p inhibitor：5’-UCAGGAACUGCCUUUCUCUCCA-3’(SEQ ID NO.10))。
最终的信号通路就是：hsa_circ_0066187通过对miRNA-185-5p的海绵吸附作用，增强RhoA/ROCK1通路活性(既往研究已经明确证实RhoA/ROCK1通路活性越强，脓毒症肺损伤越严重)，加重肺损伤。因此敲低hsa_circ_0066187表达，则减弱了对miRNA-185-5p的吸附作用，因此抑制了RhoA/ROCK1通路活性，最终减轻脓毒症肺损伤。
实施例7：hsa_circ_0066187在脓毒症患者及健康对照人群外周血中表达差异
抽取脓毒症患者及健康对照人群外周血，用qRT-PCR检测外周全血中circRNA的表达量。hsa_circ_0066187脓毒症早期表达明显升高(图7)。
实施例8：脓毒症患者外周血中hsa_circ_0066187表达量与疾病严重程度及总体预后显著相关
hsa_circ_0066187在脓毒症合并ARDS患者中表达量显著高于非ARDS患者(图8A)。hsa_circ_0066187在28天住院死亡患者中表达量高于存活患者(图8B)。运用spearman相关性分析，分析该circRNA表达与患者病情严重程度及总体预后的相关性。结果表明，hsa_circ_0066187表达量与脓毒症ARDS患者氧合指数显著负相关(图8C)。hsa_circ_0066187表达量与脓毒症患者APACHE-Ⅱ及SOFA评分评显著正相关(图8D，E)。上述结果表明，脓毒症患者外周血hsa_circ_0066187表达量与疾病严重程度及总体预后显著相关。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.hsa_circ_0066187在制备脓毒症治疗药物中的应用。
2.根据权利要求1所述的应用，其特征在于：hsa_circ_0066187的核苷酸序列如SEQ IDNO.1所示。
3.根据权利要求1所述的应用，其特征在于：所述药物敲低hsa_circ_0066187的表达。
4.根据权利要求3所述的应用，其特征在于：所述药物为siRNA，所述siRNA的序列如SEQID NO.2-5所示。
5.hsa_circ_0066187在制备脓毒检测试剂盒中的应用。
6.一种脓毒症的检测试剂盒，其特征在于：所述试剂盒检测样本中hsa_circ_0066187的表达量。
7.根据权利要求6所述的检测试剂盒，其特征在于：所述样本为外周血样本。
8.根据权利要求6所述的检测试剂盒，其特征在于：所述检测试剂盒中包括用于扩增hsa_circ_0066187的引物。
9.根据权利要求6所述的检测试剂盒，其特征在于：所述检测试剂盒中还包括用于PCR的其他组分。
10.扩增hsa_circ_0066187的引物在制备脓毒症检测试剂盒中的应用。
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