Identification of mRNAs and lincRNAs associated with lung cancer progression using next-generation RNA sequencing from laser micro-dissected archival FFPE tissue specimens.
Identification of mRNAs and lincRNAs associated with lung cancer progression using next-generation RNA sequencing from laser micro-dissected archival FFPE tissue specimens.
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DOI：
10.1016/j.lungcan.2014.03.020
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发表时间：
2014-07
期刊：
影响因子：
--
通讯作者：
Thompson CL
中科院分区：
文献类型：
--
作者：
Morton ML;Bai X;Merry CR;Linden PA;Khalil AM;Leidner RS;Thompson CL
原位腺癌（AIS）是正常肺组织进展为浸润性腺癌的中间步骤。然而，由于在获得用于下游分析的新鲜临床样品方面的挑战，这种进展的分子机制仍有待充分阐明。福尔马林固定和石蜡包埋（FFPE）是一种广泛用于长期储存的组织保存系统。直到最近，FFPE工作的挑战排除了使用新的RNA测序技术（RNA-seq），这将有助于澄清癌症进展的关键途径。此外，包括激光捕获显微切割在内的分离技术提供了选择组织病理学不同组织的能力，使研究人员能够研究紧密并列的细胞和组织类型之间的转录变异。利用这些技术和新的比对工具，我们检查了来自6名患者的正常、AIS和浸润性腺癌样本中长基因间非编码RNA（lincRNA）和mRNA的差异表达，以确定肺癌进展的可能标志物。从这18个样本中提取和测序的RNA平均每个样本产生1.98亿个读数。在比对和过滤后，唯一比对的读数代表总读数的平均35%。当比较正常与AIS或AIS与浸润性腺癌时，我们检测到许多lincRNA和mRNA的差异表达。其中，5个lincRNA和31个mRNA从正常到AIS一直上调或下调，并且更多地是浸润性癌。我们通过RT-qPCR验证了两种mRNA和一种lincRNA的上调作为原理证明。我们的研究结果表明，不仅mRNA，而且lincRNA在浸润性腺癌的进展中的潜在作用。我们预计，这些发现将为未来FFPE候选RNA的实验研究奠定基础，以确定其在肺癌中的功能作用。
Adenocarcinoma in situ (AIS) is an intermediate step in the progression of normal lung tissue to invasive adenocarcinoma. However, molecular mechanisms underlying this progression remain to be fully elucidated due to challenges in obtaining fresh clinical samples for downstream analyses. Formalin fixation and paraffin embedding (FFPE) is a tissue preservation system widely used for long-term storage. Until recently, challenges in working with FFPE precluded using new RNA sequencing technologies (RNA-seq), which would help clarify key pathways in cancer progression. Also, isolation techniques including laser-capture micro-dissection provide the ability to select histopathologically distinct tissues, allowing researchers to study transcriptional variations between tightly juxtaposed cell and tissue types. Utilizing these technologies and new alignment tools we examined differential expression of long intergenic non-coding RNAs (lincRNAs) and mRNAs across normal, AIS and invasive adenocarcinoma samples from six patients to identify possible markers of lung cancer progression. RNA extracted and sequenced from these 18 samples generated an average of 198 million reads per sample. After alignment and filtering, uniquely aligned reads represented an average 35% of the total reads. We detected differential expression of a number of lincRNAs and mRNAs when comparing normal to AIS, or AIS to invasive adenocarcinoma. Of these, 5 lincRNAs and 31 mRNAs were consistently up- or down-regulated from normal to AIS and more so to invasive carcinoma. We validated the up-regulation of two mRNAs and one lincRNA by RT-qPCR as proof of principle. Our findings indicate a potential role of not only mRNAs, but also lincRNAs in the progression to invasive adenocarcinoma. We anticipate that these findings will lay the groundwork for future experimental studies of candidate RNAs from FFPE to identify their functional roles in lung cancer.
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