CN114457161A - lncRNA AC145207.5在结直肠癌诊断、治疗和提高药物敏感性中的应用 - Google Patents
lncRNA AC145207.5在结直肠癌诊断、治疗和提高药物敏感性中的应用 Download PDFInfo
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Abstract
本发明公开了lncRNA AC145207.5在结直肠癌诊断、治疗和提高药物敏感性中的应用。本发明首次发现敲减lncRNA AC145207.5后，结直肠癌细胞的增殖能力、克隆形成能力、迁移能力、侵袭能力均产生了显著下降；同时能够提高结直肠癌细胞对伊立替康的敏感性。上述发现能够用于结直肠癌诊断、治疗、提高靶向药物敏感性等产品的制备中，具有较大的应用前景。
Description
技术领域
本发明属于生物技术领域，具体涉及lncRNA AC145207.5在结直肠癌诊断、治疗和提高药物敏感性中的应用。
背景技术
结直肠癌(Colorectal cancer，CRC)是消化道中最常见的恶性肿瘤之一。近年来，结直肠癌的发病率及死亡率逐渐升高，正在成为日益严重的公共卫生问题。本发明专利依托单位为江西省癌症中心，于2018年至今承担国家城市癌症早诊早治项目(结直肠癌、上消化道癌、肺癌、乳腺癌、肝癌等肿瘤)，本研究团队负责结直肠癌等相关肿瘤的筛查，在结直肠癌早发现、早诊断和早治疗、降低死亡率等方面，累积了大量筛查经验。
目前，生物标志物在结直肠癌患者的检测和治疗中起着重要作用。通过寻找新的生物标志物可以增加筛查的风险分层，这些标志物单独或作为现有测试的补充物可能会识别出疾病的易感性或早期阶段。深入了解结直肠癌的发病机制，明确分子标志物在结直肠癌诊断、转移、耐药、生存预后中的作用，可能为为结直肠癌个体化精准治疗奠定理论基础，同时可能为结直肠癌的化疗疗效的预测提供新的生物标志物。
发明内容
本发明的目的是解决现有技术的不足，提供一种与结直肠癌相关的生物标志物，具体技术方案如下：
本发明的第一个目的在于提供lncRNAAC145207.5(其序列如下所记载：ENSG00000263731；https://www.ncbi.nlm.nih.gov/nuccore/AC145207.2)或其抑制剂在制备治疗结直肠癌药物中的应用。申请人发现敲减lncRNA AC145207.5后，结直肠癌细胞的增殖能力、克隆形成能力、迁移能力、侵袭能力均产生了显著下降。
优选地，所述抑制剂为siRNA1、siRNA2或siRNA3；所述siRNA1的序列如SEQ ID NO：1(CCCTTTGTCATCCATAAAT)所示；所述siRNA2的序列如SEQ ID NO：2(TCTGTTTCACTTCCAAGTT)所示；所述siRNA3的序列如SEQ ID NO：3(GACCTACGATTTCTTTCTT)所示。
本发明的第二个目的在于提供检测lncRNAAC145207.5的试剂在制备结直肠癌诊断试剂或试剂盒中的应用。
优选地，所述检测lncRNAAC145207.5的试剂包括引物对，所述引物对的上游引物的序列如SEQ ID NO：4(5′-TTAATTTCAGCATCCACTGGTG-3’)所示，下游引物的序列如SEQ IDNO：5(5′-GAAGGGACTGCTCTTTCTTAGAGA-3′)所示。
本发明的第三个目的在于提供lncRNAAC145207.5在制备提高结直肠癌细胞对伊立替康的敏感性的产品中的应用。
本发明的有益效果为：本发明首次发现敲减lncRNA AC145207.5后，结直肠癌细胞的增殖能力、克隆形成能力、迁移能力、侵袭能力均产生了显著下降；同时能够提高结直肠癌细胞对伊立替康的敏感性。上述发现能够用于结直肠癌诊断、治疗、提高靶向药物敏感性等产品的制备中，具有较大的应用前景。
附图说明
图1所示为lncRNA AC145207.5的表达情况结果；
图2所示为siRNA1、siRNA2、siRNA3的敲减结果；
图3所示为MTS实验检测细胞增殖结果；
图4所示为克隆形成实验结果；
图5所示为划痕实验结果；
图6所示为Transwell实验结果；
图7所示为MTS检测敲减lncRNAAC145207.5对结直肠癌细胞伊立替康药物敏感性的影响结果；
图8所示为流式细胞仪检测敲减lncRNAAC145207.5对结直肠癌伊立替康药物敏感性影响结果；
图9所示为荧光原位杂交实验结果；
图10所示为Western blot实验结果。
具体实施方式
以下将结合实施例和附图对本发明的构思及产生的技术效果进行清楚、完整的描述，以充分地理解本发明的目的、方案和效果。
实施例1：
一、临床样本验证
方法：采用RT-qPCR法对在我院收集的28例结直肠癌组织及28例正常结直肠组织检测lncRNA AC145207.5的表达情况。
本实施例公开的一种lncRNA AC145207.5诊断检测方法，收集结直肠癌患者肿瘤组织或正常结直肠组织，按照说明书操作步骤进行提取总RNA，浓度为150-1000ng/μL，将提好的总RNA反转录为cDNA，并将剩余的RNA保存于-80℃。
(1)逆转录反应如下：
①体系1(去除基因组DNA)
反应条件：42℃，2min，4℃，+∞。
②体系2(逆转录)
将反应结束后的体系1直接加入体系2，混匀，反应条件为37℃，15min；85℃，5s；4℃，+∞。
(2)逆转录产物的操作
用于RT-qPCR分析，将以上cDNA混匀，吸取2μL配制反应体系如下：
反应条件采用两步法PCR标准扩增程序：
第一步：变性 95℃30s；
第二步：PCR反应 95℃5s，60℃30s；40个循环；
第三步：溶解曲线 95℃10s，65℃5s。
引物浓度调整为10μM，以GAPDH为内参，按照2-ΔCt法计算相对表达量。
其中检测lncRNA AC145207.5基因水平拷贝数的引物序列为：
F:5′-TTAATTTCAGCATCCACTGGTG-3’(SEQ ID NO：4)
R:5′-GAAGGGACTGCTCTTTCTTAGAGA-3′(SEQ ID NO：5)
GAPDH基因水平拷贝数的引物序列为：
F:5′-CCCATCACCATCTTCCAGGAG-3′
R:5′-GTTGTCATGGATGACCTTGGC-3′
(3)结果
结果如图1所示，由图1可知，发现lncRNA AC145207.5在胶质瘤中的表达显著高于正常脑组织。
此外，通过提取TCGA-GTEx-COADREAD数据库中共计742例结直肠癌及正常相关数据进行分析，包括tumor＝383，Normal＝359，发现lncRNA AC145207.5在结直肠癌组织中的表达显著高于正常对照组，这与本课题组前期收集的样本分析结果一致；在预测Normal和Tumor结局上，变量lncRNA AC145207.5的预测能力有一定准确性(AUC＝0.826，CI＝0.797-0.855)，即lncRNA AC145207.5有可能成为结直肠癌诊断的标志物。(备注:ROC曲线下的面积值在0.5和1之间。AUC越接近于1，说明诊断效果越好。AUC在0.5～0.7时有较低准确性，AUC在0.7～0.9时有一定准确性，AUC在0.9以上时有较高准确性)。
二、检测lncRNA AC145207.5敲减效率
(1)siRNA转染
①IncRNA AC145207.5siRNA序列
siRNA1-AC145207.5 1：CCCTTTGTCATCCATAAAT(SEQ ID NO：1)
siRNA2-AC145207.5 2：TCTGTTTCACTTCCAAGTT(SEQ ID NO：2)
siRNA3-AC145207.5 3:GACCTACGATTTCTTTCTT(SEQ ID NO：3)
②操作步骤
转染前一天取结直肠癌细胞HCT116、SW480种于6孔板中，待细胞生至长融合度达60％时进行转染。弃去旧的细胞培养液，PBS清洗两遍，每孔加入1.5mL含10％FBS的完全培养基。配制转染体系：取4个洁净无酶的1.5mL EP管，各加入250μL Opti-MEM，再分别加入10μL IncRNA AC145207.5siRNA1、siRNA2、siRNA3，NC片段，轻轻混匀后静置5min；另取4个1.5mL EP管中各加入250μL Opti-MEM，再分别加入5μL lipo-RNA iMAX，轻轻混匀后静置5min。将两个体系轻轻混匀，静置20min后分别加入各孔中，放入细胞培养箱继续培养48h。
(2)RNA的提取
转染48h后，从细胞培养箱取出种有HCT116、SW480细胞的6孔板。弃去旧的细胞培养液，PBS清洗两遍，每孔加入1mL Trizo。用1mL枪头反复吸打，室温静置5min。1mL trizol试剂加入200uL的氯仿，震荡混匀15s后室温放置2-3min。4℃，12000g/min，离心15min，样品分为三层，底层为黄色有机相，上层为无色水相和一个中间层，RNA主要存在于上层水相中。收集上层水相至一个新的无RNase的1.5mL离心管中。在离心管中加入和水相等体积的异丙醇，缓慢上下颠倒混匀，室温静置5-10min。4℃，12000g/min，离心10min，RNA沉淀在管底或者管壁。弃上清，加入500uL预冷的75％乙醇，充分吹打混匀，洗涤沉淀。4℃，12000g/min，离心5min。弃去上清，于超净台中静置15-30min。根据提取的RNA浓度加入适量的DEPC水(RNase Free)溶解RNA(一般加入20uL DEPC水)。
(3)RNA浓度的测定和稀释
控制样品总RNA浓度在200-1000ng/uL之间(浓度过高可以用DEPC水稀释)。
(4)RT-qPCR法检测lncRNA AC1455207.5的相对表达，并计算3个siRNA的敲减效率。
(5)结果：结果如图2所示，由图2可知，3个siRNA都有较好的敲减效率，选取敲除效率最好的siRNA1和siRNA3进行后续实验。
三、MTS实验检测细胞增殖
转染前一天取结直肠癌细胞HCT116、SW480种于96孔板中，每孔2×103个细胞，根据以上操作步骤转染，继续培养24h、48h、72h、96h，分别于各时间点检测细胞增殖情况。具体操作如下，按MTS：DMEM＝1:9比例配制工作液，混匀后每孔加入100μL工作液。置于细胞培养箱37℃孵育30min，酶标仪于490nm波长处检测吸光度。
结果如图3所示，由图3可知，与NC组对比，敲减lncRNA AC145207.5后细胞增殖能力显著下降。
四、克隆形成实验
将NC，siRNA1，siRNA3转染48h后的HCT116、SW480细胞胰酶消化后，完全培养基重悬成细胞悬液，并计数。于6孔板培养板中各实验组接种1,000个细胞/孔。继续培养到14天或绝大多数单个克隆中细胞数大于50为止，中途每隔3天进行换液并观察细胞状态。克隆完成后，在显微镜下对细胞进行拍照，然后PBS洗涤1次，每孔加入1mL 4％多聚甲醛固定30-60min，PBS洗涤1次。每孔加入结晶紫染液1mL，染细胞30min。PBS洗涤细胞数次，晾干，相机拍照。
其结果如图4所示，由图4可知，与NC组对比，敲减lncRNAAC145207.5可明显抑制细胞克隆形成能力，克隆数目降低。
五、划痕实验
转染前一天取结直肠癌细胞HCT116、SW480种于6孔板中，待细胞生至长融合度达60％时进行转染。转染后继续培养，待细胞融合度达90％，用marker笔在6孔板背后画横线(用直尺比着)，每孔至少穿过5条线，每条线均匀且平行。用200uL枪头(灭菌)，垂直孔板背后的黑线划痕，使划痕与标记线相交。划线完成后，使用无菌PBS洗细胞2-3次，去除划下的细胞，使留下的间隙肉眼即清晰可见，然后更换新鲜低血清(<2％)的培养基，在显微镜下观察并拍照。将细胞放入37℃，5％CO2培养箱中培养。然后在24小时后取出细胞，在显微镜下观察并拍照。
其结果如图5所示，由图5可知，与NC组对比敲减lncRNA AC145207.5可明显抑制结直肠癌细胞迁移能力。
六、Transwell实验
转染48h后消化细胞，制备无血清细胞悬浮液，计数，加入细胞悬液100uL，接种细胞2*105/孔。24孔板(下室)加入600uL含10％胎牛血清培养基，将小室置于24孔板中，上室加100uL无血清培养基悬浮的细胞。37℃培养24h-48h倒扣小室于吸水纸上，弃去小室内部培养基，用PBS冲洗小室。小室置于4％多聚甲醛((24孔板，1mL))中，固定15-20min，PBS冲洗小室。小室置于结晶紫染液(24孔板，1mL)中，染色30min，PBS冲洗小室用棉拭子轻轻擦去小室内部非转移细胞，空气晾干。显微镜下拍照。200X照片计数，进行数据分析，比较实验组、对照组转移，侵袭能力差异：计算各组转移，侵袭细胞数、标准差、T-Test分析得到的p值，判断是否有统计学差异。
其结果如图6所示，由图6可知，与NC组对比，敲减lncRNA AC145207.5可明显抑制细胞迁移和侵袭能力。
七、MTS检测敲减lncRNAAC145207.5对结直肠癌细胞伊立替康药物敏感性的影响
通过MTS实验，检测lncRNA AC145207.5对HCT116、SW480细胞伊立替康药物敏感性的影响。将lncRNA AC1455207.5干扰6h后，分别加入含有伊立替康浓度为0、2、4、8、16μM的培养基，继续培养72小时并检测每组细胞生存率。
其结果如图7所示，由图7可知，当将lncRNA AC145207.5敲减后提高了结直肠癌细胞对伊立替康的药物敏感性，细胞生存率显著下调。
八、流式细胞仪检测敲减lncRNAAC145207.5对结直肠癌伊立替康药物敏感性影响
将HCT116、SW480细胞干扰6h后，加入含伊立替康终浓度为8μM培养基继续培养72h。胰酶消化并收集细胞，转移至1.5ml EP管中，3000rpm离心10min，弃去上清；PBS洗涤后，加入195μL结合液重悬细胞，依次加入5μL Annexin V-FITC及10μL碘化丙啶后，轻轻混匀，室温避光染色20min，流式细胞仪检测细胞凋亡情况。
其结果如图8所示，由图8可知，lncRNA AC145207.5敲减后细胞凋亡数目显著增加，即结直肠癌细胞对伊立替康的药物敏感性显著增加。
九、荧光原位杂交实验(FISH实验)
1.细胞培养
将细胞爬片置于24孔板孔底，培养适量细胞(～6 104/孔)。实验前使细胞融合度达到60％-70％。
2.细胞固定与通透
a.【1 PBS】清洗细胞5min；
b.【4％多聚甲醛】室温固定10min；
c.【1 PBS】清洗细胞5min，共3次；
d.每孔加入1mL预冷的【通透液】，4℃静置5min；
e.弃去【通透液】后，加入【1 PBS】清洗细胞5min，3次。
3.探针检测
a.每孔加入200uL【预杂交液】，37℃封闭30min；
b.预杂交同时，将【杂交液】在37℃中预热；
c.避光条件下，把2.5uL 20uM【lncRNA FISH Probe Mix储存液】或【内参FISHProbe Mix储存液】加入到100μL【杂交液】中；
d.弃去每孔细胞中的【预杂交液】，加入100uL含有探针的【探针杂交液】，避光，37℃杂交过夜；
e.避光，42℃，【杂交洗液I】清洗每孔细胞3次，每次5min，以降低背景信号；
f.避光，42℃，【杂交洗液II】清洗细胞1次；
g.避光，42℃，【杂交洗液III】清洗细胞1次；
h.避光，【1 PBS】清洗细胞，室温5min。
4.DNA染色
a.避光，【DAPI染色液】染色10min；
b.避光，【1 PBS】清洗细胞3次，每次5min。
5.封片
避光条件下，从孔中小心取出细胞爬片，用封片剂将其固定于载玱片上，进行荧光检测。
6.结果
其结果如图9所示，由图9可知，lncRNA AC145207.5在结直肠癌细胞HCT116、SW480的细胞质和细胞核中都有分布，大部分分布在细胞核中。
十、Western blot实验
其结果如图10所示，由图10可知，lncRNA AC145207.5调控结直肠癌细胞HCT116、SW480增殖，迁移侵袭，凋亡相关蛋白，介导结直肠癌细胞的增殖，迁移侵袭和凋亡。敲减lncRNA AC145207.5，抑制结直肠癌细胞增殖，迁移和侵袭，促进凋亡。
以上所述，只是本发明的较佳实施例而已，本发明并不局限于上述实施方式，只要其以相同的手段达到本发明的技术效果，都应属于本发明的保护范围。在本发明的保护范围内其技术方案和/或实施方式可以有各种不同的修改和变化。
SEQUENCE LISTING
<110> 江西省肿瘤医院（江西省第二人民医院、江西省癌症中心）
<120> lncRNA AC145207.5在结直肠癌诊断、治疗和提高药物敏感性中的应用
<130> 2022
<160> 5
<170> PatentIn version 3.5
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<212> DNA
<213> 人工合成
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<210> 5
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Claims (5)
1.lncRNA AC145207.5或其抑制剂在制备治疗结直肠癌药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述抑制剂为siRNA1、siRNA2或siRNA3；所述siRNA1的序列如SEQ ID NO：1所示；所述siRNA2的序列如SEQ ID NO：2所示；所述siRNA3的序列如SEQ ID NO：3所示。
3.检测lncRNA AC145207.5的试剂在制备结直肠癌诊断试剂或试剂盒中的应用。
4.根据权利要求3所述的应用，其特征在于，所述检测lncRNA AC145207.5的试剂包括引物对，所述引物对的上游引物的序列如SEQ ID NO：4所示，下游引物的序列如SEQ ID NO：5所示。
5.lncRNA AC145207.5在制备提高结直肠癌细胞对伊立替康的敏感性的产品中的应用。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20210277395A1 (en) * | 2020-01-26 | 2021-09-09 | Washington University | Methods and compositions for modulating lncrnas and methods of treatment based on lncrna expression |
| CN113832227A (zh) * | 2021-09-17 | 2021-12-24 | 中山大学孙逸仙纪念医院 | 一种肝细胞肝癌患者预后预测模型的构建及应用 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113832227A (zh) * | 2021-09-17 | 2021-12-24 | 中山大学孙逸仙纪念医院 | 一种肝细胞肝癌患者预后预测模型的构建及应用 |
Non-Patent Citations (2)
| Title |
|---|
| XIGANG XIA等: "Identification of Glycolysis- Related lncRNAs and the Novel lncRNA WAC-AS1 Promotes Glycolysis and Tumor Progression in Hepatocellular Carcinoma", FRONTIERS IN ONCOLOGY, vol. 11, pages 1 - 16 * |
| 董涛，等: "lncRNA MALAT1对结直肠癌SW480细胞增殖、侵袭、迁移及凋亡的影响", 解剖科学进展, vol. 26, no. 05, pages 591 - 594 * |
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| CN114990214B (zh) * | 2022-05-26 | 2023-09-19 | 中南大学湘雅医院 | lncRNA分子在诊断肿瘤化疗耐药性试剂中的应用及检测试剂盒 |
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