Abstract
Numerous evidences indicate that aspirin usage causes a significant reduction in colorectal cancer. However, the molecular mechanisms about aspirin preventing colon cancer are largely unknown. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a most frequently used method to identify the target molecules regulated by certain compound. However, this method needs stable internal reference genes to analyze the expression change of the targets. In this study, the transcriptional stabilities of several traditional reference genes were evaluated in colon cancer cells treated with aspirin, and also, the suitable internal reference genes were screened by using a microarray and were further identified by using the geNorm and NormFinder softwares, and then were validated in more cell lines and xenografts. We have showed that three traditional internal reference genes, β-actin, GAPDH and α-tubulin, are not suitable for studying gene transcription in colon cancer cells treated with aspirin, and we have identified and validated TMEM208 and PQLC2 as the ideal internal reference genes for detecting the molecular targets of aspirin in colon cancer in vitro and in vivo. This study reveals stable internal reference genes for studying the target genes of aspirin in colon cancer, which will contribute to identify the molecular mechanism behind aspirin preventing colon cancer.
Highlights
Aspirin, one of the most frequently used nonsteroidal anti-inflammatory drugs (NSAIDs), has been demonstrated to possess an attractive effect on cancer chemoprevention [1,2,3].The anti-cancer effect of aspirin was first identified in animal models in 1970s [4]
We have showed that three traditional internal reference genes, β-actin, GAPDH and α-tubulin, are not suitable for studying gene transcription in colon cancer cells treated with aspirin, and we have identified and validated TMEM208 and PQLC2 as the ideal internal reference genes for detecting the molecular targets of aspirin in colon cancer in vitro and in vivo
The results showed that expression of GAPDH decreased in three cell lines treated with aspirin (Figure 1D), whereas the expression of β-actin in HCT116 and SW620 cells treated with aspirin significantly increased, but decreased in aspirin treated DLD1 cells (Figure 1E)
Summary
One of the most frequently used nonsteroidal anti-inflammatory drugs (NSAIDs), has been demonstrated to possess an attractive effect on cancer chemoprevention [1,2,3].The anti-cancer effect of aspirin was first identified in animal models in 1970s [4]. Identification of the target molecules of aspirin is an attractive topic in the cancer chemoprevention field. One of the most frequently used strategies to identify the target molecules regulated by a certain compound is to determine the altered gene expression after treatment with that compound. There are evidences suggesting that the transcript levels of some internal reference genes may be changed under certain conditions, such as drug treatment, serum stimulation and hypoxia [22, 23]
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