短扩增子对结直肠癌转移石蜡组织LncRNA定量分析的可行性

摘要

目的 探讨短扩增子对结直肠癌转移石蜡包埋组织(以下称石蜡组织)长链非编码RNA(LncRNA)定量分析的可行性.方法 筛选与结直肠癌转移相关的4条LncRNA (LncRNA-Enst00000554679、LncRNA-Enst00000550851、LncRNA-Enst00000497498、LncRNA-Enst00000513016).收集结直肠癌石蜡组织55例份、结直肠癌冰冻组织40例份,每份标本包含结直肠癌组织、癌旁正常组织和转移淋巴结组织.采用实时荧光定量PCR法检测25例份冰冻组织中结直肠癌组织、癌旁正常组织、转移淋巴结组织4条长扩增子扩增后LncRNA表达.设计4条LncRNA的短扩增子引物,评估长、短扩增子在冰冻组织和石蜡组织中的扩增效率及测定系数(R2).分析冰冻组织中短扩增子和长扩增子的定量一致性以及冰冻组织和石蜡组织短扩增子的定量一致性.分别为4条LncRNA设计3对短扩增子引物,分析石蜡组织中3个短扩增子的定量一致性.结果 癌旁正常组织、结直肠癌组织、转移淋巴结组织4条LncRNA相对表达量逐渐升高(P均<0.05).短扩增子在冰冻组织和石蜡组织中均能达到最佳扩增效率,而长扩增子均未达到最佳扩增效率,短扩增子的R2明显高于长扩增子(P<0.05).在冰冻组织中短扩增子和长扩增子的定量一致性为62.5％;冰冻组织与石蜡组织短扩增子的定量一致性仅为25.0％.4条LncRNA中3个短扩增子扩增循环数变化趋势一致率为37.5％,2个短扩增子扩增循环数变化趋势一致率达到100％.结论 筛选的4条LncRNA在结直肠癌转移组织中表达升高;使用短扩增子对石蜡组织LncRNA进行定量分析是可行的.%Objective To explore the feasibility of quantitative analysis of long non-coding RNA (lncRNA) in FFPE tissues of metastatic colorectal cancer with short amplicons. Methods We selected 4 target lncRNAs associated with colorectal cancer metastasis (LncRNA-Enst00000554679, LncRNA-Enst00000550851, LncRNA-Enst00000497498, and LncRNA-Enst00000513016). Fifty-five cases of FFPE tissue specimens and 40 cases of frozen tissue specimens were collected, each specimen contained the colorectal cancer tissues, adjacent normal tissues, and lymph node metastasis tissues. The qRT-PCR was used to detect the expression of 4 LncRNAs after amplification of long amplicons in 25 samples of colorectal cancer tissues, adjacent normal tissues and metastatic lymph node tissues. Four lncRNA short amplicon primers were designed to assess the amplification efficiency and the assay coefficient (R2) of long and short amplicons in frozen and FFPE tissues. The quantitative concordance of short amplicons and long amplicons in the frozen tissues was analyzed, as well as the quantitative concordance of short amplicons in the frozen tissues and FFPE tissues. We separately designed three pairs of short amplicon primers for four lncRNAs, and analyzed the quantitative concordance of three short amplicons in the FFPE tissues. Results Four lncRNAs expressed significantly higher in the colorectal cancer tissues (all P＜0. 05). Short amplicons could achieve the best amplification efficiency in both frozen and FFPE tissues, while the long amplicons did not reach the best amplification efficiency. The R2 of the short amplicon was significantly higher than that of the long amplicon(P＜0. 05). The quantitative concordance of short and long amplicons in the frozen tissues was 62. 5％, and that of short amplicons in the frozen and FFPE tissues was only 25. 0％. The coincidence rate of the three short amplicons in the four LncRNAs was 37. 5％, while that of the two short amplicons was 100％. Conclusion Four lncRNAs are highly expressed in the metastatic colorectal cancer tissues, and the short amplicons can be used for lncRNA quantitative analysis in the FFPE tissues.