CN113736887A - 长链非编码rna在制备食管鳞癌癌前病变预警检测试剂中的应用 - Google Patents
长链非编码rna在制备食管鳞癌癌前病变预警检测试剂中的应用 Download PDFInfo
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Abstract
本发明公开了长链非编码RNA在制备食管鳞癌癌前病变预警检测试剂或试剂盒中的应用，本发明在获取的血液样本中检测HUESCC‑LncRNA2表达量水平，提早筛查诊断，早发现早治疗，提高早诊率，降低ESCC发病率。检测方法更容易被受检者接受，待测者依从性高，便于推广。
Description
技术领域
本发明涉及肿瘤检测领域，更具体地说，它涉及一种长链非编码RNA HUESCC-LncRNA2在用于食管鳞癌癌前病变预警检测试剂或试剂盒及应用。
背景技术
食管癌（EC）是一种严重威胁人类健康的常见多发恶性肿瘤，全世界范围内，食管癌主要的病理类型——食管鳞癌（esophageal squamous cell carcinoma, ESCC）占 90%以上，我国EC患者也以鳞癌为主，且发病率仍呈上升趋势。近年来经过多学科综合治疗，食管癌的治疗效果有所改善，但患者的长期生存获益仍然十分有限，2015 年新英格兰医学杂志报道：国外 EAC 患者的5年生存率约为17%，而ESCC患者的总体5年生存率甚至更低。宿迁地区属于我国食管鳞癌高发地区，当地ESCC患者总体5年生存率仅10%左右，95%患者确诊时已属中晚期，丧失了早期根治性切除的手术时机，而早期诊断的患者5年生存率可高达90%以上，早期诊断是提高ESCC患者5年生存率最为有效的手段。
食管癌变进程一般经由正常食管粘膜发展为低级别上皮内瘤变再到高级别上皮内瘤变，再逐步进展为早期ESCC与中晚期ESCC。目前，食管癌的早期诊断研究主要集中在癌前病变，包括低级别瘤变和高级别瘤变。上皮内瘤变（ intraepithelialneoplasia ，IN）是指上皮性恶性肿瘤浸润前的肿瘤性改变，包括异型增生和原位癌。食管低级别上皮内瘤变（Low Grade intraepithelial neoplasia, LGIN）是指食管异型增生的细胞仅限于上皮层下2/3，相当于轻度和中度不典型增生。食管高级别上皮内瘤变（High Gradeintraepithelial neoplasia, HGIN）是指食管异型增生的细胞超过上皮全层的2/3，多形性显著，或异形细胞累及上皮全层，但仍未突破基底膜，相当于重度异型增生和原位癌。现今肿瘤诊断仍以病理诊断为金标准，但受限于活检取材部位及病理医师的诊断水平，且癌前病变和早期肿瘤病灶小故漏诊率高，因而病理诊断也有其局限性。
早诊早治是改善食管癌预后的有效途径。目前食管癌的早期诊筛主要依靠消化内镜，然而我国人口基数巨大，无法对适龄人口全部进行内镜检查，故目前需对所有成年人进行食管鳞癌危险评估，根据评估后的风险程度进行筛查，对于高危人群进一步行内镜检查。然而目前的风险评估手段暂无在食管早期癌变进程中即可预警肿瘤的分子生物学靶标。因此，寻找新的敏感性及特异性好的生物标志物具有十分重要的临床应用价值，是未来改善食管癌患者临床治疗效果及预后的方向。
发明内容
针对现有技术存在的不足，本发明的目的在于提供一种解决食管鳞癌癌前病变预警检测的试剂或试剂盒及应用，在获取的高危人群血液中检测HUESCC-LncRNA2表达量水平，提早筛查诊断，早发现早治疗，提高早诊率，降低ESCC发病率。
为实现上述目的，本发明提供了如下技术方案：
长链非编码RNA在制备食管鳞癌癌前病变预警检测试剂或试剂盒中的应用，在获取的血液样本中检测HUESCC-LncRNA2表达量水平，HUESCC-LncRNA2转录区域位于8号染色体反义链186,435,038-186,478,330bp，基因全长约为1164bp。
本发明进一步改进技术方案是，所述试剂盒包含检测对象样本中 HUESCC-LncRNA2表达量的试剂。
本发明更进一步改进技术方案是，所述试剂包括能够特异性扩增HUESCC-LncRNA2的引物，上游引物序列为５'－ＡＧＡＴＧＡＡＧＣＣＧＧＴＡＣＣＴＣＡＧＡＴ－３'
下游引物序列为５'－ＴＣＡＣＴＴＴＡＡＡＧＡＧＧＧＡＧＡＧＧＡＧ－３'。
本发明更进一步改进技术方案是，长链非编码RNA HUESCC-LncRNA2的检测方法具体步骤包括：
使用Trizol 试剂提取血液中总RNA；紫外分光光度计进行RNA 定量及纯度分析后，吸取1μg 总RNA 用A-MLV 反转录试剂盒在20μl 体系下进行反转录，Oligo(dT)15 作为引物，反转录得到的cDNA等量于RNA，用于基因mRNA 水平的定量检测；应用ABI 公司的7500型定量PCR 仪进行实时定量RT-PCR 检测，结果采用2-△ct 理论进行分析。
本发明另一种目的在于提供一种长链非编码RNA用于食管鳞癌癌前病变预警检测试剂或试剂盒，所述长链非编码RNA 是HUESCC-LncRNA2，其转录区域位于8号染色体反义链186,435,038-186,478,330bp，基因全长约为1164bp。
本发明有益效果：
一、从癌组织与癌旁组织中检测具有滞后性，无法实现癌变前早诊和预后评估，失去最佳诊治和干预期。本发明血液样本中HUESCC-LncRNA2作为ESCC患者诊断及临床分期的分子标志物，成为ESCC患者癌变前筛查的依据，有利于早发现早治疗，降低ESCC发病率，提高早诊率、提高疗效以及评估预后提供重要的工作基础。
二、血液作为检测样本更容易获取，更容易被受检者接受，待测者依从性高，便于推广。
附图说明
图1为在正常食管粘膜-低级别上皮内瘤变-高级别上皮内瘤变-浸润癌等组织样本中HUESCC-LNCRNA2的表达；
图2为在正常食管粘膜-低级别上皮内瘤变-高级别上皮内瘤变-浸润癌等血液样本HUESCC-LNCRNA2的表达；
图3为血液和组织中HUESCC-LNCRNA2的表达量呈线性关系；
图4为内镜黏膜下剥离术或切除术后患者血液中HUESCC-LNCRNA2的表达。
具体实施方式
为了使本发明所解决的技术问题、技术方案及有益效果更加清楚明白，以下结合附图与实施例，对本发明进行进一步详细说明。
(1).样本收集
在南京鼓楼医院集团宿迁市人民医院以及南京医科大学附属南京第一医院收集不同病变阶段的内镜和手术切除样本以及对照的周旁近似正常组织及血液样本，丰富样本库。并对样本进行病理诊断，建立病例资料和随访联系。
(2).Real-Time PCR扩大样本量验证癌变进程各期样本中差异表达的HUESCC-LncRNA2
使用Trizol 试剂（Invitrogen）提取样本总RNA。紫外分光光度计进行RNA 定量及纯度分析后，吸取1μg 总RNA 用A-MLV 反转录试剂盒在20μl 体系下进行反转录（Oligo(dT)15 作为引物）。反转录得到的cDNA（等量于RNA）用于基因mRNA 水平的定量检测。应用ABI 公司的7500 型定量PCR 仪（Applied Biosystem, Foster City, CA）进行实时定量RT-PCR 检测。结果采用2-△ct 理论进行分析。
(3).核酸探针原位杂交确定HUESCC-LncRNA2在癌变进程各期样本中差异表达及组织定位
1）冰冻切片与杂交前预处理 取出各期冰冻的食管样本，OCT包埋，-23 ℃平衡30min，固定，切片（10μm），平涂于有多聚赖氨酸(1mg／m1)的玻片上(玻片预先180 ℃干烤6小时)，冰冻保存（-70 ℃）；室温干燥10min，4％的多聚甲醛-PBS(pH 7.4)固定10min；活跃的DEPC-PBS洗2次， 5 min；0.2M的盐酸，10 min后，重复步骤；切片上滴加蛋白酶K(0.1μg／m1)，37 ℃孵育15 min，重复。
2）杂交 滴加预杂交液(约100 μl／玻片)，置于放有湿盒液(50％甲酰胺v／v；0.3 MNaCl；1Mm EDTA；10 mM Tris-Cl，pH 8.O)的湿盒中，55～58 ℃烘箱中预杂交2 h；甩掉预杂交液，地高辛标记的反义寡核苷酸HUESCC-LncRNA2探针(浓度1-2ng／μl) 70 ℃变性1O分钟，置冰上1 min，玻片上滴加预杂交液(约60μl／玻片)，覆盖parafilm膜，放湿盒中在48～58 ℃下杂交18-30 h。
3）杂交后处理及信号检测 取出玻片，去掉Parafilm膜，甩掉杂交液， 52 ℃预热5×SSC洗30 min;无DNA的RNA酶A(20μg／ml)溶液中37 ℃孵育30 min;分别依次用52 ℃预热的2 X SSC，1 X SSC和O.1 X SSC洗2次，每次30 min；、缓冲液平衡5min；在玻片上滴加碱性磷酸酶的抗地高辛抗体，室温反应2h；用缓冲液洗2次，每次15 min；在缓冲液中平衡5min．显色过夜；后EDTA洗15 min终止反应；95％乙醇，洗l h以除去非特异的背景；用蒸馏水洗15min，除去结晶体；脱水、透明，用中性树胶封片；充分干燥后在显微镜下观察、照相。
4).RNA免疫共沉淀—质谱检测HUESCC-LncRNA2是否绑定相关蛋白复合物
（1）利用生物素标记的UTP为原料，体外转录合成目的RNA
（2）将RNA与cell lysate混合后过链亲和素柱子
（3）洗脱亲和柱上
（4）收集柱子上的蛋白质，进行质谱检测
5). 检测并评估HUESCC-LncRNA2在食管鳞癌早诊中的临床应用价值
采用qRT-PCR在正常食管粘膜-低级别上皮内瘤变-高级别上皮内瘤变-浸润癌等组织中验证发现： HUESCC-LNCRNA2在食管鳞状上皮的癌变进程表达逐渐升高（见附图1），我们进一步检测了经病理证实为癌前病变的患者的血液样本中HUESCC-LNCRNA2的表达量，发现HUESCC-LNCRNA2在高级别上皮内瘤变患者的血液中的表达显著增高（见附图2），且血液和组织中HUESCC-LNCRNA2的表达量呈线性关系（见附图3），在内镜粘膜下剥离术（Endoscopic submucosal dissection，ESD）或内镜下黏膜切除术（endoscopic mucosalresection，EMR）后，有相当数量的患者血液中HUESCC-LNCRNA2的表达量显著下降（见附图4），可见血液中HUESCC-LncRNA2表达水平在食管鳞癌临床早诊的预警价值是显见的。
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.长链非编码RNA在制备食管鳞癌癌前病变预警检测试剂或试剂盒中的应用，其特征在于：在获取的血液样本中检测HUESCC-LncRNA2表达量水平，HUESCC-LncRNA2转录区域位于8号染色体反义链186,435,038-186,478,330bp，基因全长约为1164bp。
2.根据权利要求1所述的应用，其特征在于：所述试剂盒包含检测对象样本中 HUESCC-LncRNA2表达量的试剂。
3.根据权利要求1所述的应用，其特征在于：所述试剂包括能够特异性扩增HUESCC-LncRNA2的引物，
上游引物序列为５'－ＡＧＡＴＧＡＡＧＣＣＧＧＴＡＣＣＴＣＡＧＡＴ－３'
下游引物序列为５'－ＴＣＡＣＴＴＴＡＡＡＧＡＧＧＧＡＧＡＧＧＡＧ－３'。
4.根据权利要求1所述的应用，其特征在于：长链非编码RNA HUESCC-LncRNA2的检测方法具体步骤包括：
使用Trizol 试剂提取血液中总RNA；紫外分光光度计进行RNA 定量及纯度分析后，吸取1μg 总RNA 用A-MLV 反转录试剂盒在20μl 体系下进行反转录，Oligo(dT)15 作为引物，反转录得到的cDNA等量于RNA，用于基因mRNA 水平的定量检测；应用ABI 公司的7500 型定量PCR 仪进行实时定量RT-PCR 检测，结果采用2-△ct 理论进行分析。
5.一种长链非编码RNA用于食管鳞癌癌前病变预警检测试剂或试剂盒，所述长链非编码RNA 是HUESCC-LncRNA2，其转录区域位于8号染色体反义链186,435,038-186,478,330bp，基因全长约为1164bp。
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