CN113249478A - Hsa_circ_0068464作为标志物在制备结直肠癌诊断及预后药物中的应用 - Google Patents
Hsa_circ_0068464作为标志物在制备结直肠癌诊断及预后药物中的应用 Download PDFInfo
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- CN113249478A CN113249478A CN202110552684.6A CN202110552684A CN113249478A CN 113249478 A CN113249478 A CN 113249478A CN 202110552684 A CN202110552684 A CN 202110552684A CN 113249478 A CN113249478 A CN 113249478A
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Abstract
本发明公开了Hsa_circ_0068464作为标志物在制备结直肠癌诊断及预后药物中的应用，具体涉及环状hsa_circ_0068464作为肿瘤标志物在制备结直肠癌诊断及预后药物中的应用。本发明通过qPCR检测RNA hsa_circ_0068464结果显示，Circ464在结直肠癌组织及细胞系中均高表达，生物信息学预测Circ464与miR‑383结合，提示Circ464可能在结直肠癌的发生发展中具有重要的作用，在癌症组血清中较健康及良性组均显著增高，且在转移组血清中的水平显著高于未转移组，提示Circ464可作为结直肠癌诊断及预后肿瘤标志物。
Description
【技术领域】
本发明涉及生物医学技术领域，涉及Hsa_circ_0068464作为标志物在制备结直肠癌诊断及预后药物中的应用，具体涉及环状hsa_circ_0068464作为肿瘤标志物在制备结直肠癌诊断及预后药物中的应用。
【背景技术】
结直肠癌是常见的消化道恶性肿瘤，每年全球约有120万例患者被确诊为结直肠癌，而有超过60万例患者直接或间接死于结直肠癌。在我国，结直肠癌发病率、死亡率在恶性肿瘤中均位居前5位，其中每年新发病例37.6万，死亡病例19.1万，已成为严重危害我国人民健康的疾病。遗传和表观遗传在直肠癌的发生过程中起到非常重要的作用。大量非编码RNA被证实参与到肿瘤的发生发展过程中。其中环状RNA作为一种新的内源性RNA参与了包括肿瘤在内的多种疾病的过程，研究其分子调控机制具有重要临床和科研意义。
环状RNA(circular RNA，circRNA)是一类在真核细胞中广泛存在的非编码RNA，具有结构稳定、丰度高及细胞或组织特异性表达等特征，可能通过多种作用方式参与基因表达调控。例如，有些circRNA富含微小RNA(miRNA)结合位点，可充当竞争性内源RNA(ceRNA)结合miRNA并阻断其对靶基因表达的抑制作用。最新研究表明，circRNA的表达及作用与多种疾病的发生发展、生物组织发育及细胞衰老等相关。circRNA在不同生物样本中的表达差异使其可能成为用于疾病诊断、组织发育鉴定等方面理想的生物标记物，其在疾病中作用方式的逐步阐明，使之具有成为有效治疗靶点的潜力。例如中国专利CN201711482488公开了一种用于预测中期结直肠癌预后及死亡风险的环状RNA分子标记物及其应用，所述环状RNA分子标记物包括circRNA13452，hsa_circ_0008039，hsa_circ_0079480和hsa_circ_0087391，通过检测上述四个环状RNA分子在中期结直肠癌患者组织中的表达水平，并通过相应的预测模型，达到预测患者预后及死亡风险的目的，提高了通过结直肠癌患者组织标本预测预后及死亡风险的准确性，对患者的进一步诊治方案和延长患者生存期具有重大意义。
已有研究表明结直肠癌肿瘤组织中存在着circRNA的异常表达，相关circRNA在结直肠癌中的作用也成为研究热点，circRNA对结直肠癌的影响主要体现在以下几个方面:
(1)circRNA作为ceRNA通过与miRNA相互作用调控结直肠癌发生发展、侵袭转移，如：hsa_circ_0020397能促进miR-138的靶基因TERT和PD-L1的表达从而调节结直肠癌细胞的生长、凋亡以及侵袭。hsa_circ_001569在结直肠癌组织中显著高表达，通过直接抑制miR-145而上调E2F5、BAG4以及FMNL2，从而影响肿瘤细胞的增殖以及侵袭能力，导致远端转移以及低分化直接影响结直肠癌患者诊断后生存率。同样地，hsa_circ_0000069在结直肠癌组织中显著高表达，且与患者TNM分期相关，敲减hsa_circ_0000069能显著抑制细胞增殖、迁移、侵袭以及诱导G0/G1期细胞周期阻滞。而另一种在结直肠癌中显著高表达的circRNA circHIPK3，可通过与miR-124结合并抑制miR-124的活性进而增加促癌基因IL-6以及DLX2表达，诱导肿瘤发生发展。因此，circHIPK3的高表达能逆转miR-124对促癌基因Il-6以及DLX2的抑制从而发挥促进结直肠癌增殖的作用。
(2)circRNA通过肿瘤信号通路参与调控结直肠癌发生发展、侵袭转移，结直肠癌中肿瘤细胞的异常生长涉及到与细胞增殖相关的多条信号通路的异常激活，circRNA通过直接调节细胞增殖信号通路相关的靶基因或对与细胞增殖信号通路密切相关的miRNA进行调控，而参与到结直肠癌的发生。已有报道多种circRNA参与EGFR/RAF1/MAPK信号通路的激活，对这些信号通路的激活涉及到对miR-7表达的抑制。circRNA CiRS-7可作为RNA海绵吸附miR-7而削弱其对EGFR/RAF1/MAPK信号通路的抑制，从而促进肿瘤细胞的生长。MRPS35_hsa_circ_001042可通过对miR-21、miR-148a、miR-148b、miR-152调节参与MAPK信号通路的激活。而circRNA CER则通过吸附miR-136实现对TGFβ、JNK、ERK通路的调节。circ-ITCH在结直肠癌组织中表达显著下调，而有研究报道circ-ITCH能增加ITCH表达水平，抑制Wnt/β-Catenin通路从而抑制癌细胞增殖。
(3)近期研究表明circRNA参与到了结直肠癌多个病理进程并有望成为结直肠癌的诊断和治疗的标志物及治疗靶点，hsa_circ_001988在结直肠癌组织中比健康标本中表达显著下调，可能在肿瘤沿神经浸润中起重要作用，而肿瘤沿神经浸润能预测结直肠癌预后和疗效，并与结直肠癌生存率和原位复发负相关。因此，hsa_circ_001988是一个可能的结直肠癌预后和治疗靶点。circ-BANP在癌组织中高表达，敲减circ-BANP能减弱结直肠癌细胞增殖，有望成为诊断和治疗的标志物。RNA-Seq结果及qRT-PCR结果显示circCCDC66在结肠癌和息肉中表达增高，且与不良预后相关。有研究验证了ciRS-7在结直肠癌细胞过表达能抑制miR-7并激活致癌基因EGFR以及RAR1，是一个有前景的预后生物标记物。另有研究证实结肠癌患者血浆外泌体内存在67种circRNA的缺失与257种不出现于正常个体的circRNA的异常表达。结肠血清外泌体中可检测到差异表达circRNA分子，具备诊断标记物潜能。
从研究者的几项研究结果来看，circRNAs非常有希望成为结肠癌治疗靶标和诊断标记物和靶点，作为临床诊断标记物在血液或体液中检测将更有意义，并为临床应用带来新的视角和方向。
【发明内容】
针对现有技术中结肠癌治疗靶标和诊断标记物和靶点研究的不足，提供了Hsa_circ_0068464作为标志物在制备结直肠癌诊断及预后药物中的应用，具体是环状RNA hsa_circ_0068464(简称Circ464)作为结直肠癌诊断及预后标志物的使用方法。本发明通过qPCR检测RNA hsa_circ_0068464结果显示，Circ464在结直肠癌组织及细胞系中均高表达，生物信息学预测Circ464与miR-383结合，提示Circ464可能在结直肠癌的发生发展中具有重要的作用，在癌症组血清中较健康及良性组均显著增高，且在转移组血清中的水平显著高于未转移组，提示Circ464可作为结直肠癌诊断及预后肿瘤标志物。
Hsa_circ_0068464作为标志物在制备结直肠癌诊断及预后药物中的应用，具体是环状RNA hsa_circ_0068464作为结直肠癌诊断及预后标志物的使用方法，包括如下步骤：
1)Circ464作为结直肠癌诊断标志物的鉴定：
(1)收集常见的恶性肿瘤患者的血清标本、收集癌症组患者年龄及性别匹配的各良性组患者血清及健康对照血清标本分别作为良性组及健康组；
(2)提取总RNA，逆转录获得cDNA，设计特异性PCR引物，通过qPCR检测Circ464在癌症组、良性组和健康组间的表达水平差异，分析Circ464的预警功能；
(3)分析(2)的实验结果，计算Circ464在每一种癌症诊断中的特异性及灵敏度，作为泛癌血清诊断标志物；
(4)分析肿瘤Ⅰ+Ⅱ分期血清中Circ464的表达水平，确定Circ464作为肿瘤的早期诊断；
2)Circ464作为结直肠癌转移及预后标志物的鉴定：
(1)收集不同临床等级的结直肠癌患者病例标本，提取总RNA，逆转录获得cDNA，设计特异性PCR引物，qPCR检测Circ464的表达水平；所述的特异性PCR引物是：
hsa_circ_0068464-F-2，CAGTTTCTGCTCTGCATGGT；
hsa_circ_0068464-R-2，CAGCCTGTGTAATGGTCAGTG；
(2)利用统计软件分析Circ464与临床患者病理分级、TNM分期及预后情况的相关性；
(3)通过对比不同病理分级及TNM分期间Circ464的表达差异，确定Circ464作为结直肠癌转移及预后标志物；
3)Circ464体内及体外生物学功能的鉴定：
(1)通过慢病毒感染的方法构建Circ464的稳定细胞系SW480和LS174-T过表达和沉默；
(2)通过MTT实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后增殖能力的改变；
(3)通过Transwell小室实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后运动迁移能力的改变；
(4)通过Matrigel基质胶实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后侵袭能力的改变；
(5)通过流式检测技术分别比较SW480和LS174-T细胞Circ464过表达和沉默后细胞周期分布的改变；
(6)通过Annexin-V/PI实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后细胞凋亡水平的改变；
(7)通过顺铂及5-氟尿嘧啶耐药实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后细胞耐药能力的改变；
(8)通过构建裸鼠皮下成瘤模型，分别比较LS174-T细胞Circ464过表达和沉默后肿瘤细胞体内成瘤能力的改变；
(9)通过构建尾静脉注射肺转移模型，分别比较LS174-T细胞Circ464过表达和沉默后肿瘤细胞体内转移能力的改变；
4)Circ464靶基因的挖掘及鉴定：
(1)分别构建靶基因3’-UTR-WT(Wild Type)及结合位点序列突变的3’-UTR-MT(Mutant Type)荧光素酶报告载体，与Circ464共转染细胞，检测荧光素酶活性；
(2)提取稳定过表达及沉默表达细胞(SW480和LS174-T)总RNA，逆转录获得cDNA，设计特异性PCR引物，qPCR检测靶基因在不同处理后的表达水平变化；所述的特异性PCR引物是：
hsa_circ_0068464-F-2，CAGTTTCTGCTCTGCATGGT；
hsa_circ_0068464-R-2，CAGCCTGTGTAATGGTCAGTG；
(3)提取稳定过表达及沉默表达细胞(SW480和LS174-T)总蛋白SDS-PAGE电泳蛋白，稳压转膜至PVDF膜，4℃过夜孵育一抗，37℃，1小时孵育二抗，ECL显色发光记录结果；
5)Circ464的作用结果：
(1)在TCGA数据库下载多种肿瘤的circRNAseq数据；首先，根据癌旁组织样本数小于3例不予纳入的标准排除了部分肿瘤；
(2)对剩余的肿瘤数据按照FDR＜0.05，FC＞2的标准分别进行差异circRNAs筛选[注FDR(False Discovery Rate)：错误发现率；FC(Fold Change)：差异倍数]；
(3)选择在结直肠癌中特异性高表达且未见功能报道的几个circRNAs进行深入研究；
(4)通过引物设计，采用qPCR检测所选结肠癌组织及相应的癌旁组织中这些circRNAs的表达水平；然而采用不同的FC标准，筛选出来特异性表达的circRNA；
(5)比较恶性肿瘤患者的血清标本和较健康及良性组表达水平是否增高、且在转移组结直肠癌血清中的表达水平显著高于未转移组(P＜0.05)，如果是，确定Circ464作为无创型泛癌诊断及预后标志物；
(6)功能缺失性研究：通过siRNA方法敲减hsa_circ_0068464的表达；然后通过CCK8、克隆形成、Transwell迁移侵袭小室、细胞周期及凋亡等试验方法检测hsa_circ_0068464对结肠癌细胞表型如增殖、侵袭、转移、凋亡的影响。
本发明中：
步骤1)中所述的常见的恶性肿瘤，是12种常见的恶性肿瘤，是指结直肠癌、肝细胞癌、胃癌、宫颈癌、子宫内膜癌、肺癌、食管癌、乳腺癌、膀胱癌、鼻咽癌癌、前列腺癌及卵巢癌。
步骤1)中所述的良性组及健康组，是排除高血脂、糖尿病、复合癌症及合并其他自身免疫性疾病患者的标本。
步骤3)中所述的通过慢病毒感染的方法构建Circ464的稳定细胞系SW480和LS174-T过表达和沉默，是根据前期的研究，通过瞬时转染结合细胞功能学实验，初步证实Circ464可在体外促进结肠癌细胞的侵袭转移能力。
步骤5)所述的选择在结直肠癌中特异性高表达且未见功能报道的几个circRNAs进行深入研究，是选择Circ389、Circ294、Circ273和Circ464进行深入研究。
通常而言，作为药物，均是在制备成制剂后才临床应用。本发明所述的药物，作为药物组合物可根据本领域公知的方法制备。可通过将本发明药物与一种或多种药学上可接受的固体或液体赋形剂和/或辅剂结合，制成适于人或动物使用的任何剂型。本发明药物在其药物组合物中的含量通常为0.1-95％(w/w)。
本发明药物或含有它的药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、静脉注射、肌肉注射、皮下注射、鼻腔、口腔粘膜、眼、肺和呼吸道、皮肤、阴道、直肠等。
给药剂型可以是液体剂型、固体剂型或半固体剂型。液体剂型可以是溶液剂(包括真溶液和胶体溶液)、乳剂(包括o/w型、w/o型和复乳)、混悬剂、注射剂(包括水针剂、粉针剂和输液)、滴眼剂、滴鼻剂、洗剂和搽剂等；固体剂型可以是片剂(包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片)、胶囊剂(包括硬胶囊、软胶囊、肠溶胶囊)、颗粒剂、散剂、微丸、滴丸、栓剂、膜剂、贴片、气(粉)雾剂、喷雾剂等；半固体剂型可以是软膏剂、凝胶剂、糊剂等。
本发明药物可以制成普通制剂、也可以制成缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。为了将本发明药物制成片剂，可以广泛使用本领域公知的各种赋形剂，包括稀释剂、黏合剂、润湿剂、崩解剂、润滑剂、助流剂。稀释剂可以是淀粉、糊精、蔗糖、葡萄糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙等；润湿剂可以是水、乙醇、异丙醇等；黏合剂可以是淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、微晶纤维素、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、丙烯酸树脂、卡波姆、聚乙烯吡咯烷酮、聚乙二醇等；崩解剂可以是干淀粉、微晶纤维素、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠、羧甲基淀粉钠、碳酸氢钠与枸橼酸、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠等；润滑剂和助流剂可以是滑石粉、二氧化硅、硬脂酸盐、酒石酸、液体石蜡、聚乙二醇等。
还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。
为了将给药单元制成胶囊剂，可以将有效成分本发明药物与稀释剂、助流剂混合，将混合物直接置于硬胶囊或软胶囊中。也可将有效成分本发明药物先与稀释剂、黏合剂、崩解剂制成颗粒或微丸，再置于硬胶囊或软胶囊中。用于制备本发明药物片剂的各稀释剂、黏合剂、润湿剂、崩解剂、助流剂品种也可用于制备本发明药物的胶囊剂。
为将本发明药物制成注射剂，可以用水、乙醇、异丙醇、丙二醇或它们的混合物作溶剂并加入适量本领域常用的增溶剂、助溶剂、pH调剂剂、渗透压调节剂。增溶剂或助溶剂可以是泊洛沙姆、卵磷脂、羟丙基-β-环糊精等；pH调剂剂可以是磷酸盐、醋酸盐、盐酸、氢氧化钠等；渗透压调节剂可以是氯化钠、甘露醇、葡萄糖、磷酸盐、醋酸盐等。如制备冻干粉针剂，还可加入甘露醇、葡萄糖等作为支撑剂。
此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂或其他添加剂。
和现有技术相比，本发明具有如下优点：
1、本发明所述的Hsa_circ_0068464作为标志物在制备结直肠癌诊断及预后药物中的应用，通过qPCR检测RNA hsa_circ_0068464结果显示，Circ464在结直肠癌组织及细胞系中均高表达，生物信息学预测Circ464与miR-383结合，提示Circ464可能在结直肠癌的发生发展中具有重要的作用，在癌症组血清中较健康及良性组均显著增高，且在转移组血清中的水平显著高于未转移组，提示Circ464可作为结直肠癌诊断及预后肿瘤标志物；功能实验显示，Circ464可促进结肠癌细胞转移及侵袭。
2、本发明所述的Hsa_circ_0068464作为标志物在制备结直肠癌诊断及预后药物中的应用将为结直肠癌筛查及预后提供新思路，并对阐明结直肠癌恶性行为的分子机制具有重要意义。并且可以根据肿瘤标志物Circ464来开发结直肠癌诊断及预后药物。
【附图说明】
图1是本发明实施例中Circ464在结直肠癌组织中高表达的图。
图2是本发明实施例中Circ464在结直肠癌、肝细胞癌、胃癌、宫颈癌及子宫内膜癌组织中高表达的图。
图3是本发明实施例中Circ464在结直肠癌、肝细胞癌、胃癌、宫颈癌及子宫内膜癌血清中高表达的图。
图4是本发明实施例中Circ464在转移组结直肠癌血清中显著高表达的图。
图5是本发明实施例中的Circ464相关细胞功能研究的图。
图6是本发明实施例中与Circ464可潜在结合的miRNAs及其靶基因网络的图。
【具体实施方式】
以下结合实施例对本发明的具体实施方式做进一步说明。
实施例：
Hsa_circ_0068464作为标志物在制备结直肠癌诊断及预后药物中的应用，包括如下步骤：
1)Circ464作为结直肠癌诊断标志物的鉴定：
(1)预实验的qPCR结果提示Circ464作为结直肠癌的血清标志物；
分别收集12种常见的恶性肿瘤，是指结直肠癌、肝细胞癌、胃癌、宫颈癌、子宫内膜癌、肺癌、食管癌、乳腺癌、膀胱癌、鼻咽癌癌、前列腺癌及卵巢癌的血清标本数量各120例，与癌症组患者年龄及性别匹配的各良性组患者血清(收集120例)及健康对照血清标本(收集120例)分别作为良性组及健康组(高血脂、糖尿病、复合癌症及合并其他自身免疫性疾病患者的标本均不纳入该研究)；
(2)提取总RNA，逆转录获得cDNA，设计特异性PCR引物，通过qPCR检测Circ464在癌症组、良性组和健康组间的表达水平差异，作为Circ464的预警功能；
(3)分析上述实验结果，计算Circ464在每一种癌症诊断中的特异性及灵敏度，作为泛癌血清诊断标志物；
(4)分析肿瘤Ⅰ+Ⅱ分期血清中Circ464的表达水平，确定Circ464在肿瘤早期诊断中的作用；
2)Circ464作为结直肠癌转移及预后标志物的鉴定：
(1)预实验的结果提示Circ464作为肿瘤转移及预后标志物；
收集不同临床等级的结直肠癌患者病例标本，提取总RNA，逆转录获得cDNA，设计特异性PCR引物，qPCR检测Circ464的表达水平；所述的特异性PCR引物是：
hsa_circ_0068464-F-2，CAGTTTCTGCTCTGCATGGT；
hsa_circ_0068464-R-2，CAGCCTGTGTAATGGTCAGTG；
(2)利用统计软件分析Circ464与临床患者病理分级、TNM分期及预后情况的相关性；
(3)通过对比不同病理分级及TNM分期间Circ464的表达差异，确定Circ464作为预后标志物；
3)Circ464体内及体外生物学功能的鉴定：
(1)预实验中，通过瞬时转染结合细胞功能学实验，确认Circ464在体外促进结肠癌细胞的侵袭转移能力；接下来通过慢病毒感染的方法构建Circ464的稳定细胞系SW480和LS174-T(过表达和沉默)；
(2)通过MTT实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后增殖能力的改变；
(3)通过Transwell小室实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后运动迁移能力的改变；
(4)通过Matrigel基质胶实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后侵袭能力的改变；
(5)通过流式检测技术分别比较SW480和LS174-T细胞Circ464过表达和沉默后细胞周期分布的改变；
(6)通过Annexin-V/PI实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后细胞凋亡水平的改变；
(7)通过顺铂及5-氟尿嘧啶耐药实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后细胞耐药能力的改变；
(8)通过构建裸鼠皮下成瘤模型，分别比较LS174-T细胞Circ464过表达和沉默后肿瘤细胞体内成瘤能力的改变；
(9)通过构建尾静脉注射肺转移模型，分别比较LS174-T细胞Circ464过表达和沉默后肿瘤细胞体内转移能力的改变；
4)Circ464靶基因的挖掘及鉴定：
首先利用在线生物信息学MiRanda Program分析预测，确定miR-383，miR-199a，miR-668-5p，miR-589-3p为hsa_circ_0068464结合的miRNAs，同时预测出他们各自的靶基因调控网络(参见附图3)；进一步在SW480和LS174-T细胞中沉默或过表达hsa_circ_0068464，Trizol抽提RNA，qRT-PCR检测4个靶miRNAs的表达水平，发现仅miR-383与hsa_circ_0068464的表达呈现负向相关的关系，其余3个miRNAs没有显著变化，这些结果确定hsa_circ_0068464靶向结合miR-383，miR-383的靶基因为CREPT等；
(1)分别构建靶基因3’-UTR-WT(Wild Type)及结合位点序列突变的3’-UTR-MT(Mutant Type)荧光素酶报告载体，与Circ464共转染细胞，检测荧光素酶活性；
(2)提取稳定过表达及沉默表达细胞(SW480和LS174-T)总RNA，逆转录获得cDNA，设计特异性PCR引物，qPCR检测靶基因在不同处理后的表达水平变化；所述的特异性PCR引物是：
hsa_circ_0068464-F-2，CAGTTTCTGCTCTGCATGGT；
hsa_circ_0068464-R-2，CAGCCTGTGTAATGGTCAGTG；
(3)提取稳定过表达及沉默表达细胞(SW480和LS174-T)总蛋白SDS-PAGE电泳蛋白，稳压转膜至PVDF膜，4℃过夜孵育一抗，37℃，1小时孵育二抗，ECL显色发光记录结果；
5)Circ464的作用结果：
预先在TCGA数据库下载27种肿瘤的circRNAseq数据；首先，根据癌旁组织样本数小于3例不予纳入的标准排除了10种肿瘤；
然后，对剩余的17种肿瘤数据(结直肠癌，肾上腺癌，胆管癌，膀胱尿路上皮癌，脑胶质瘤，乳腺癌，宫颈癌，食管癌，头颈部鳞状细胞癌，肾癌，肝细胞癌，肺癌，胰腺癌，前列腺癌，胃腺癌，甲状腺癌，子宫癌)按照FDR＜0.05，FC＞2的标准分别进行差异circRNAs筛选[注FDR(False Discovery Rate)：错误发现率；FC(Fold Change)：差异倍数]；
最后，选择在结直肠癌中特异性高表达且未见功能报道的4个circRNAs(Circ389，Circ294，Circ273，Circ464)进行深入研究；由于低表达的circRNAs不适合做临床血清标志物，因此不做重点研究；
通过引物设计，采用qPCR检测12例结肠癌组织及相应的癌旁组织中这些circRNAs的表达水平，发现Circ464与数据挖掘结果一致(附图1，NT：癌旁正常组织；CRC：结直肠癌；***P＜0.001)；
然而采用不同的FC标准，所筛选出来的特异性表达的circRNA也会有所不同，经计算，如果按照FDR＜0.05，FC＞1.5的标准，TCGA数据挖掘结果显示Circ464在四种肿瘤中均有特异性高表达(结直肠癌，乳腺癌，肝细胞癌，胃腺癌)，qPCR实验结果显示Circ464在结直肠癌组织中表达显著增高(附图2，NT：癌旁正常组织；CRC：结直肠癌；HCC：肝细胞癌；GC：胃癌；CC：宫颈癌；EC：子宫内膜癌；**P＜0.01；***P＜0.001)，两者结果基本吻合；
继续收集结直肠癌、肝细胞癌、胃癌、宫颈癌及子宫内膜癌血清标本各8例，检测发现，Circ464在癌症患者血清较健康及良性组表达水平均显著增高(附图3，HC：健康对照；BE：良性病变；CRC：结直肠癌；HCC：肝细胞癌；GC：胃癌；CC：宫颈癌；EC：子宫内膜癌；*P＜0.05；**P＜0.01)；且在转移组结直肠癌血清中的表达水平显著高于未转移组(P＜0.05)(附图4，CRC-NM：非转移结直肠癌；CRC-M：转移结直肠癌；*P＜0.05)，上述结果确认Circ464作为无创型泛癌诊断及预后标志物；
功能缺失性研究：通过siRNA方法敲减hsa_circ_0068464的表达；然后通过CCK8、克隆形成、Transwell迁移侵袭小室、细胞周期及凋亡等试验方法检测hsa_circ_0068464对结肠癌细胞表型如增殖、侵袭、转移、凋亡等的影响；实验结果显示：干扰hsa_circ_0068464的表达，可以明显抑制结肠癌细胞的增殖、侵袭、转移和细胞周期并促进细胞的凋亡，确定hsa_circ_0068464在促进结肠癌的发生发展中具有重要的作用(附图5，*P＜0.05；*P＜0.05；**P＜0.01)；
讨论：
在前期工作中，首先利用在线生物信息学MiRanda Program分析预测，发现miR-383，miR-199a，miR-668-5p，miR-589-3p为hsa_circ_0068464结合的miRNAs，同时确定它们各自的靶基因调控网络(参见附图3)；进一步在SW480和LS174-T细胞中沉默或过表达hsa_circ_0068464，Trizol抽提RNA，qRT-PCR检测4个靶miRNAs的表达水平，发现仅miR-383与hsa_circ_0068464的表达呈现负向相关的关系，其余3个miRNAs没有显著变化，这些结果确定hsa_circ_0068464靶向结合miR-383，miR-383的靶基因为CREPT等(附图6)。
上述说明是针对本发明较佳可行实施例的详细说明，但实施例并非用以限定本发明的专利申请范围，凡本发明所提示的技术精神下所完成的同等变化或修饰变更，均应属于本发明所涵盖专利范围。
序列表
<110> 柳州市工人医院
<120> hsa_circ_0068464作为标志物在制备结直肠癌诊断及预后药物中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cagtttctgc tctgcatggt 20
<210> 2
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cagcctgtgt aatggtcagt g 21
Claims (4)
1.Hsa_circ_0068464作为标志物在制备结直肠癌诊断及预后药物中的应用，其特征在于：具体是环状RNA hsa_circ_0068464作为结直肠癌诊断及预后标志物的使用方法，包括如下步骤：
1)Circ464作为结直肠癌诊断标志物的鉴定：
(1)收集常见的恶性肿瘤患者的血清标本、收集癌症组患者年龄及性别匹配的各良性组患者血清及健康对照血清标本分别作为良性组及健康组；
(2)提取总RNA，逆转录获得cDNA，设计特异性PCR引物，通过qPCR检测Circ464在癌症组、良性组和健康组间的表达水平差异，分析Circ464的预警功能；
(3)分析(2)的实验结果，计算Circ464在每一种癌症诊断中的特异性及灵敏度，作为泛癌血清诊断标志物；
(4)分析肿瘤Ⅰ+Ⅱ分期血清中Circ464的表达水平，确定Circ464作为肿瘤的早期诊断；
2)Circ464作为结直肠癌转移及预后标志物的鉴定：
(1)收集不同临床等级的结直肠癌患者病例标本，提取总RNA，逆转录获得cDNA，设计特异性PCR引物，qPCR检测Circ464的表达水平；所述的特异性PCR引物是：
hsa_circ_0068464-F-2，CAGTTTCTGCTCTGCATGGT；
hsa_circ_0068464-R-2，CAGCCTGTGTAATGGTCAGTG；
(2)利用统计软件分析Circ464与临床患者病理分级、TNM分期及预后情况的相关性；
(3)通过对比不同病理分级及TNM分期间Circ464的表达差异，确定Circ464作为结直肠癌转移及预后标志物；
3)Circ464体内及体外生物学功能的鉴定：
(1)通过慢病毒感染的方法构建Circ464的稳定细胞系SW480和LS174-T过表达和沉默；
(2)通过MTT实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后增殖能力的改变；
(3)通过Transwell小室实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后运动迁移能力的改变；
(4)通过Matrigel基质胶实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后侵袭能力的改变；
(5)通过流式检测技术分别比较SW480和LS174-T细胞Circ464过表达和沉默后细胞周期分布的改变；
(6)通过Annexin-V/PI实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后细胞凋亡水平的改变；
(7)通过顺铂及5-氟尿嘧啶耐药实验分别比较SW480和LS174-T细胞Circ464过表达和沉默后细胞耐药能力的改变；
(8)通过构建裸鼠皮下成瘤模型，分别比较LS174-T细胞Circ464过表达和沉默后肿瘤细胞体内成瘤能力的改变；
(9)通过构建尾静脉注射肺转移模型，分别比较LS174-T细胞Circ464过表达和沉默后肿瘤细胞体内转移能力的改变；
4)Circ464靶基因的挖掘及鉴定：
(1)分别构建靶基因3’-UTR-WT Wild Type及结合位点序列突变的3’-UTR-MT MutantType荧光素酶报告载体，与Circ464共转染细胞，检测荧光素酶活性；
(2)提取稳定过表达及沉默表达细胞SW480和LS174-T总RNA，逆转录获得cDNA，设计特异性PCR引物，qPCR检测靶基因在不同处理后的表达水平变化；所述的特异性PCR引物是：
hsa_circ_0068464-F-2，CAGTTTCTGCTCTGCATGGT；
hsa_circ_0068464-R-2，CAGCCTGTGTAATGGTCAGTG；
(3)提取稳定过表达及沉默表达细胞SW480和LS174-T总蛋白SDS-PAGE电泳蛋白，稳压转膜至PVDF膜，4℃过夜孵育一抗，37℃，1小时孵育二抗，ECL显色发光记录结果；
5)Circ464的作用结果：
(1)在TCGA数据库下载多种肿瘤的circRNAseq数据；首先，根据癌旁组织样本数小于3例不予纳入的标准排除了部分肿瘤；
(2)对剩余的肿瘤数据按照FDR＜0.05，FC＞2的标准分别进行差异circRNAs筛选；
(3)选择在结直肠癌中特异性高表达且未见功能报道的几个circRNAs进行深入研究；
(4)通过引物设计，采用qPCR检测所选结肠癌组织及相应的癌旁组织中这些circRNAs的表达水平；然而采用不同的FC标准，筛选出来特异性表达的circRNA；
(5)比较恶性肿瘤患者的血清标本和较健康及良性组表达水平是否增高、且在转移组结直肠癌血清中的表达水平显著高于未转移组P＜0.05，如果是，确定Circ464作为无创型泛癌诊断及预后标志物；
(6)功能缺失性研究：通过siRNA方法敲减hsa_circ_0068464的表达；然后通过CCK8、克隆形成、Transwell迁移侵袭小室、细胞周期及凋亡等试验方法检测hsa_circ_0068464对结肠癌细胞表型如增殖、侵袭、转移、凋亡的影响。
2.根据权利要求1所述的Hsa_circ_0068464作为标志物在制备结直肠癌诊断及预后药物中的应用，其特征在于：步骤1)中所述的常见的恶性肿瘤，是12种常见的恶性肿瘤，是指结直肠癌、肝细胞癌、胃癌、宫颈癌、子宫内膜癌、肺癌、食管癌、乳腺癌、膀胱癌、鼻咽癌癌、前列腺癌及卵巢癌。
3.根据权利要求1所述的Hsa_circ_0068464作为标志物在制备结直肠癌诊断及预后药物中的应用，其特征在于：步骤1)中所述的良性组及健康组，是排除高血脂、糖尿病、复合癌症及合并其他自身免疫性疾病患者的标本。
4.根据权利要求1所述的Hsa_circ_0068464作为标志物在制备结直肠癌诊断及预后药物中的应用，其特征在于：步骤5)所述的选择在结直肠癌中特异性高表达且未见功能报道的几个circRNAs进行深入研究，是选择Circ389、Circ294、Circ273和Circ464进行深入研究。
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| CN114350802A (zh) * | 2022-01-06 | 2022-04-15 | 南方医科大学珠江医院 | 用于肠癌患者预后预测及治疗的lncRNA |
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| CN108866195A (zh) * | 2018-08-17 | 2018-11-23 | 柳州市工人医院 | miR-3942-5p作为肿瘤标志物在制备无创型泛癌诊断及预后药物中的应用 |
| US20210079474A1 (en) * | 2018-04-25 | 2021-03-18 | Stc. Unm | Circular rnas for the diagnosis and treatment of brain disorders |
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| CN114350802A (zh) * | 2022-01-06 | 2022-04-15 | 南方医科大学珠江医院 | 用于肠癌患者预后预测及治疗的lncRNA |
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