Identification of Bcl2 as a stably expressed qPCR reference gene for human colon cancer cells treated with cottonseed-derived gossypol and bioactive extracts and bacteria-derived lipopolysaccharides

Authors: Cao, Heping; Sethumadhavan, Kandan

Submitted to: Molecules
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/1/2022
Publication Date: 11/4/2022
Citation: Cao, H., Sethumadhavan, K. 2022. Identification of Bcl2 as a stably expressed qPCR reference gene for human colon cancer cells treated with cottonseed-derived gossypol and bioactive extracts and bacteria-derived lipopolysaccharides. Molecules. 27(7560):1-19. https://doi.org/10.3390/molecules27217560.
DOI: https://doi.org/10.3390/molecules27217560

Interpretive Summary: Cottonseed contains many bioactive compounds. Cottonseed value might be increased by providing high-value bioactive polyphenols for improving nutrition and health. However, there was a lack of molecular evidence for cottonseed bioactivity in mammalian cells. One widely used method for evaluating the bioactivity of natural products is quantitative real-time-PCR (qPCR). Selection of stably expressed internal reference genes is a crucial task of qPCR assay for data analysis. The objective of this study was to select reference genes in human colon cancer cells (COLO 225) treated with cottonseed-derived gossypol and bioactive extracts along with bacterial endotoxin lipopolysaccharides (LPS). These results suggest that BCL2 is a preferable reference gene for qPCR assays in human colon cancer cells treated with cottonseed-derived gossypol and bioactive extracts as well as LPS. The result should facilitate studies designated to evaluate bioactivity of cottonseed materials and other natural products for nutrition and health uses.

Technical Abstract: Cottonseed contains many bioactive molecules including plant polyphenols. Cottonseed value might be increased by providing high-value bioactive polyphenols for improving nutrition and health. However, there was a lack of molecular evidence for cottonseed bioactivity in mammalian cells. One widely used method for evaluating the bioactivity of natural products is quantitative real-time-PCR (qPCR). Selection of stably expressed internal reference genes is a crucial task of qPCR assay for data analysis. The rational for reference gene selection is that the less of standard deviation of cycle of threshold (Cq) among the treatments indicates the more stable expression of the gene. The objective of this study was to select reference genes in human colon cancer cells (COLO 205) treated with cottonseed-derived gossypol and bioactive extracts along with bacterial endotoxin lipopolysaccharides (LPS). SYBR Green qPCR was used to analyze the mRNA levels of a wide-range of biomarkers involved in glucose transport, lipid biosynthesis, inflammatory response and cancer development. qPCR data (10,560 Cq values) were generated from 55 genes analyzed from 64 treatments with triplicate per treatment for each gene. The data showed that B-cell lymphoma 2 (Bcl2) mRNA was the most stable among the 55 mRNAs analyzed in the human colon cancer cells. Glyceraldehyde 3 phosphate dehydrogenase (Gapdh) and ribosome protein L32 (Rpl32) mRNAs were not good qPCR references for the colon cancer cells. These observations were consistent regardless of the treatment comparison between gossypol and LPS, glanded and glandless seed extracts, seed coat and kernel extracts, or treatment for 8 and 24 h. These results suggest that Bcl2 is a preferable reference gene for qPCR assays in human colon cancer cells treated with cottonseed-derived gossypol and bioactive extracts as well as LPS. The extensive qPCR results firmly support the conclusion that Bcl2 gene is stably expressed at the mRNA level in the human colon cancer cells regardless of the treatment, suggesting that Bcl2 gene expression is not regulated at the mRNA level but at the post-transcriptional level. These results should facilitate studies designated to evaluate bioactivity on gene expression regulation by cottonseed molecules and other natural and synthetic molecules for nutrition and health uses.