CN114657249A - 长非编码rnalinc01963作为肺癌肿瘤标志物及治疗靶点 - Google Patents
长非编码rnalinc01963作为肺癌肿瘤标志物及治疗靶点 Download PDFInfo
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Abstract
本发明属于生物医药领域，具体属于分子生物学技术领域，尤其涉及长非编码RNA LINC01963作为肺癌肿瘤标志物及治疗靶点的应用。本发明提供了长非编码RNA LINC01963作为肺癌肿瘤标志物及治疗靶点，长非编码RNA LINC01963在肺癌患者中低表达，其表达与患者***转移和临床分期显著相关，作为肺癌的诊断标志物具有较高的准确性，LINC01963过表达抑制了肺癌细胞增殖、迁移、侵袭和管形成，促进了肺癌细胞的凋亡，因此可以成为肺癌基因治疗的重要靶点。
Description
技术领域
本发明属于生物医药领域，具体属于分子生物学技术领域，尤其涉及长非编码RNALINC01963作为肺癌肿瘤标志物及治疗靶点的应用。
背景技术
肺癌被认为是全球男性癌症相关死亡率的第一大原因，女性死亡率的第四大原因。2018年全球癌症统计数据显示，肺癌新发病例2093876例，死亡1761007例，占新发癌症病例的11.6%和癌症相关死亡病例的18.4%。近年来，尽管在化疗、放疗、手术、免疫治疗和靶向治疗等方面取得了很大的努力，但预后不良伴有耐药和复发的情况仍屡见不鲜。由于65%的肺癌病例在初次诊断时被归类为中晚期，导致患者5年生存率只有4-17%。因此，提高肺癌的早期诊断率和改进肺癌的治疗手段是目前亟待解决的问题。
长链非编码RNA（lncRNA），长度超过200个核苷酸，是一组缺乏编码蛋白质能力的RNA，过去被认为是无用的“暗物质”或“转录噪声”。然而，越来越多的证据逐渐揭示了lncRNA 在包括生理和病理在内的多个生物过程中的重要作用。值得注意的是，lncRNA 的异常表达在包括 肺癌在内的多种恶性肿瘤的发展中发挥重要作用，并且是肺癌诊断和预后的有潜力的生物标志物。LINC01963是一种新出现的lncRNA，被认为是口腔和口咽鳞状细胞癌的潜在预后指标。然而，LINC01963在肺癌中的作用至今仍不清楚。
发明内容
本发明涉及一种肺癌的诊断标志物，所述诊断标志物为长非编码RNA LINC01963。
进一步的，所述LINC01963在肺癌中低表达，所述低表达的范围是正常细胞的0.2-0.8倍。
本发明还提供了一种用于诊断肺癌的试剂盒，所述试剂盒包括检测LINC01963表达水平的试剂。
进一步的，所述试剂为特异性扩增LINC01963的引物。
进一步的，所述特异性扩增LINC01963的引物序列为： LINC01963，F：5’-CCCGGTGTAGGGTAAATGCA-3’ and R：5’-ATTGGCCACTCCCGGATTTT-3’；GAPDH，F：5’-CTGGGCTACACTGAGCACC-3’ and R：5’-AAGTGGTCGTTGAGGGCAATG-3’。
本发明还提供了一种治疗肺癌的药物组合物，所述药物组合物含有LINC01963。
进一步的，所述药物组合物为含有LINC01963的质粒。
本发明的主要有益效果在于：
1、本发明提供了长非编码RNA LINC01963作为肺癌肿瘤标志物及治疗靶点；
2、长非编码RNA LINC01963在肺癌患者中低表达，低表达范围是正常细胞的0.2-0.8倍，其表达与患者***转移和临床分期显著相关，作为肺癌的诊断标志物具有较高的准确性；
3、LINC01963过表达抑制了肺癌细胞增殖、迁移、侵袭和管形成，促进了肺癌细胞的凋亡，因此可以成为肺癌基因治疗的重要靶点。
附图说明
图1：LINC01963在肺癌中低表达并且可以充当肺癌的诊断生物标志物；
图2：LINC01963的表达对肺癌细胞的克隆形成和迁移的影响；
图3：LINC01963的表达对肺癌细胞的侵袭和凋亡的影响；
图4：LINC01963的表达对肺癌细胞的血管生成的影响附图标记。
具体实施方式
以下将结合本发明的附图，对本发明实施例中的技术方案进行清楚、完整的描述和讨论，显然，这里所描述的仅仅是本发明的一部分实例，并不是全部的实例，基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明的保护范围。
为了便于对本发明实施例的理解，下面将结合附图以具体实施例为例作进一步的解释说明，且各个实施例不构成对本发明实施例的限定。
本发明的实施例1，LINC01963是肺癌的潜在诊断生物标志物。
（1）、LINC01963在肺癌患者中低表达：
来自癌症基因组图谱 (TGCA) 数据库的数据通过基因表达谱交互分析 (GEPIA;http://gepia.cancer-pku.cn/)，以显示正常组织 (n = 59) 和肺腺癌组织(n = 483)之间LINC01963的差异表达。结果如图1A所示，LINC01963在肺腺癌中的表达显著低于正常对照。
肺癌组织和癌旁组织取自 2019 年 5 月至 2021 年 7 月确诊的 80 名患者。血清样本来自上述肺癌患者和 80 名健康志愿者。所有患者均签署知情同意书，并同意将其组织用于临床研究。 临床试验方案经第一作者医院伦理委员会（20190430095）审核通过。用匀浆器研磨组织，然后使用 Trizol 试剂从中提取总 RNA。 使用 High-Capacity cDNA反转录试剂盒(4368813, 赛默飞, 美国) 合成 cDNA。 然后通过 PowerUp™ SYBR™Green Master Mix（A25743，赛默飞，美国）进行实时定量 PCR。条件如下：95°C 预变性 2分钟，然后是 95°C 15 秒和 60°C 1 分钟，共40 个循环。 引物序列见表1。GAPDH为内源对照，数据采用2-ΔΔCT法定量。结果如图1B所示，LINC01963在肺癌组织中的表达水平显著低于癌旁组织中的表达水平。
按照实施案例1（1）中的方法对收集的肺癌患者和健康志愿者的血清样本进行QRT-PCR，结果如图1C所示，LINC01963在83.75%（80个配对中的67个）肺癌患者血清中下调。
（2）、LINC01963与临床病理特征的关系
通过分析LINC01963的表达和患者的临床病理特征的关系（表2），得到LINC01963表达与年龄（p = 0.621）、吸烟（p = 0.094）、分化状态（p = 0.217）和分级（p = 0.078）无显着相关，而与***转移（p = 0.003）和 临床分期 (p = 0.005) 显著相关。
绘制ROC曲线以评估血清LINC01963的诊断价值，结果如图1D所示，LINC01963的ROC曲线下面积为0.8156（95% CI 0.7492-0.8821），表明LINC01963是一个很好的肺癌诊断标志物。
表1、引物序列
表2、LINC01963的表达和患者的临床病理特征的关系
实施案例2：LINC01963的表达对肺癌细胞生物学行为的影响
（1）、细胞转染效率的测定：为了调节肺癌细胞的LINC01963水平，将短发夹RNA（shLINC01963; 5'-CTCTCCCTACACCAGAATTTA-3'）和LINC01963过表达质粒通过Lipo 3000转染到 H1299 和 A549细胞中。使用空载体作为阴性对照。胰蛋白酶消化细胞后，将细胞以2×105 个细胞/孔接种在 24 孔板中并培养至 70-90% 融合。用Opti-MEM培养基（25 µL×2） 稀释Lipo 3000 (0.75 µL) 和样品 (1 µg)，然后将 2 µL P3000 试剂加入稀释样品中。接着将样品-试剂混合物与稀释的Lipo 3000 (1:1) 混合，并在室温下培养 10分钟。最后，将复合物（50 µL/孔）加入细胞中，并在 37℃下培养 24小时。然后按照实施案例1（1）中的方法进行qRT-PCR。结果如图2A-B所示，shLINC01963成功抑制了H1299和A549细胞中LINC01963的表达，而LINC01963过表达质粒显著提高了H1299和A549细胞中LINC01963的表达。
（2）、LINC01963的表达对肺癌细胞增殖的影响：按照实施案例2（1）中转染的细胞以每孔 1×103 个细胞的密度接种在 6 孔板中，并每两天更换一次的培养基，直到形成集落。 随后弃去培养基，细胞用4%多聚甲醛（1 mL/孔）在室温下固定 20 分钟后用磷酸盐缓冲盐水（PBS）洗涤细胞两次。然后将 200 μL 结晶紫染色液添加到每个孔中对细胞进行染色。结果如图2C-D所示，shLINC01963促进了H1299和A549细胞的增殖，而LINC01963过表达质粒显著抑制了H1299和A549细胞的增殖。
（3）、LINC01963的表达对肺癌细胞迁移的影响：按照实施案例2（1）中转染的细胞以每孔 5×105 个的密度接种在 6 孔板中，并孵育至 80%-100% 汇合。 使用移液器吸头在细胞单层上划一条直线，然后用 PBS 洗涤细胞以去除细胞碎片。将6孔板在无血清培养基中培养 48 小时。 通过生物显微镜（放大倍数：×100）记录0小时和48小时的照片，并通过Image J分析迁移能力。结果如图2E-F所示，shLINC01963促进了H1299和A549细胞的迁移，而LINC01963过表达质粒显著抑制了H1299和A549细胞的迁移。
（4）、LINC01963的表达对肺癌细胞侵袭的影响：按照实施案例2（1）中转染的细胞用PBS 清洗后以 4×105/ml 悬浮于无血清培养基中，然后将细胞（200 μL）接种在预涂基质胶的***物中。下室的培养基补充有 10% FBS 作为化学引诱剂。在37℃培养48小时后，用棉签清楚***物内层细胞，***物底部的细胞用4%多聚甲醛固定30分钟，然后用结晶紫染色30分钟。 在生物显微镜（放大倍数：×250）下观察和计数细胞。结果如图3A-B所示，shLINC01963促进了H1299和A549细胞的侵袭，而LINC01963过表达质粒显著抑制了H1299和A549细胞的侵袭。
（5）、LINC01963的表达对肺癌细胞凋亡的影响：按照实施案例2（1）中转染的细胞用PBS洗涤后，将细胞以2×105个细胞/mL 的浓度重悬于1×结合缓冲液(195μL) 中。然后将Annexin V-FITC(5μL)加入细胞中并在暗室中孵育15分钟。 接下来，用 1×结合缓冲液(200μL)冲洗细胞并将细胞以1000rpm离心5分钟。去除上清液后，将细胞悬浮在1× 结合液(190 μL) 中并与 10 μL PI进行孵育。 之后上机，使用流式细胞仪分析细胞凋亡。结果如图3C-D所示，shLINC01963抑制了H1299和A549细胞的凋亡，而LINC01963过表达质粒显著促进了H1299和A549细胞的凋亡。
（6）、LINC01963的表达对肺癌细胞血管形成的影响：按照实施案例2（1）中转染的细胞在无血清培养基中培养 24小时，然后收集、离心和过滤以获得肿瘤条件培养基。将基质胶预先在 4℃下解冻过夜，并铺在预冷的 24孔板中，然后在 37℃下孵育 30 分钟。将人脐静脉血管内皮细胞饥饿两小时然后悬浮在肿瘤条件培养基中并加入到24孔板，在37℃、5% CO2条件下培养 6小时。用显微镜拍摄图像（放大倍数：×100）。结果如图4A-B所示，shLINC01963促进了H1299和A549细胞的血管生成，而LINC01963过表达质粒显著抑制了H1299和A549细胞的血管生成。
最后应说明的是：以上所述实施例，仅为本发明的具体实施方式，用以说明本发明技术方案，而非对其限制，本发明的保护范围并不局限于此，尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，其依然可以对前述实施例所记载的技术方案进行修改或可轻易想到变化，或者对其中部分技术特征进行等同替换；而这些修改、变化或者替换，并不使相应技术方案的本质脱离本发明实施例技术方案的精神和范围，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应所述以权利要求的保护范围为准。
Claims (7)
1.一种肺癌的诊断标志物，其特征在于：所述诊断标志物为长非编码RNA LINC01963。
2.根据权利要求1所述的一种肺癌的诊断标志物，其特征在于：所述LINC01963在肺癌中低表达，所述低表达的范围是正常细胞的0.2-0.8倍。
3.一种用于诊断肺癌的试剂盒，其特征在于：所述试剂盒包括检测LINC01963表达水平的试剂。
4.根据权利要求3所述的一种用于诊断肺癌的试剂盒，其特征在于：所述试剂为特异性扩增LINC01963的引物。
5.根据权利要求4所述的一种用于诊断肺癌的试剂盒，其特征在于，所述特异性扩增LINC01963的引物序列为：
LINC01963，F：5’-CCCGGTGTAGGGTAAATGCA-3’ and R：5’-ATTGGCCACTCCCGGATTTT-3’；
GAPDH，F：5’-CTGGGCTACACTGAGCACC-3’ and R：5’-AAGTGGTCGTTGAGGGCAATG-3’。
6.一种治疗肺癌的药物组合物，其特征在于：所述药物组合物含有LINC01963。
7.根据权利要求6所述的一种治疗肺癌的药物组合物，其特征在于：所述药物组合物为含有LINC01963的质粒。
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