Peng Xinjian, McCormick David L
Life Sciences Group, IIT Research Institute, Chicago, IL 60616, USA.
Oncol Rep. 2016 Aug;36(2):1076-84. doi: 10.3892/or.2016.4883. Epub 2016 Jun 16.
Oral squamous cell carcinomas (OSCCs) induced in F344 rats by 4-nitroquinoline-1-oxide (4-NQO) demonstrate considerable phenotypic similarity to human oral cancers and the model has been widely used for carcinogenesis and chemoprevention studies. Molecular characterization of this model needs reliable reference genes (RGs) to avoid false- positive and -negative results for proper interpretation of gene expression data between tumor and adjacent normal tissues. Microarray analysis of 11 pairs of OSCC and site-matched phenotypically normal oral tissues from 4-NQO-treated rats identified 10 stably expressed genes in OSCC compared to adjacent normal tissues (p>0.5, CV<15%) that could serve as potential RGs in this model. The commonly used 27 RGs in the rat were also analyzed based on microarray data and most of them were found unsuitable for RGs in this model. Traditional RGs such as ACTB and GAPDH were significantly altered in OSCC compared to adjacent normal tissues (p<0.01, n=11); however, the Hsp90ab1 was ranked as the best RG candidate and the combination of Hsp90ab1 and HPRT1 was identified by NormFinder to be a superior reference for gene normalization among the commonly used RGs. This result was also validated by RT-PCR based on the selected top RG candidate pool. These data suggest that there are no common RGs suitable for different models and RG(s) should be identified before gene expression analysis. We successfully identified Hsp90ab1 as a stable RG in 4-NQO-induced OSCC compared to adjacent normal tissues in F344 rats. The combination of two stably expressed genes may be a better option for gene normalization in tissue samples.
4-硝基喹啉-1-氧化物（4-NQO）诱导F344大鼠产生的口腔鳞状细胞癌（OSCC）与人类口腔癌表现出相当大的表型相似性，该模型已广泛用于致癌作用和化学预防研究。此模型的分子特征分析需要可靠的内参基因（RG），以避免在正确解释肿瘤组织和相邻正常组织之间的基因表达数据时出现假阳性和假阴性结果。对11对来自4-NQO处理大鼠的OSCC组织和部位匹配的表型正常口腔组织进行微阵列分析，结果显示与相邻正常组织相比，OSCC中有10个基因稳定表达（p>0.5，CV<15%），这些基因可作为该模型潜在的内参基因。还基于微阵列数据对大鼠中常用的27个内参基因进行了分析，发现其中大多数不适用于该模型的内参基因。与相邻正常组织相比，传统的内参基因如ACTB和GAPDH在OSCC中发生了显著变化（p<0.01，n=11）；然而，Hsp90ab1被列为最佳内参基因候选，通过NormFinder软件分析，在常用的内参基因中，Hsp90ab1和HPRT1的组合被确定为基因标准化的最佳内参。基于所选的顶级内参基因候选库，通过逆转录-聚合酶链反应（RT-PCR）也验证了这一结果。这些数据表明，没有适用于不同模型的通用内参基因，在进行基因表达分析之前应确定合适的内参基因。我们成功地鉴定出，与F344大鼠相邻正常组织相比，Hsp90ab1是4-NQO诱导的OSCC中的一个稳定内参基因。两个稳定表达基因的组合可能是组织样本中基因标准化的更好选择。
