| Objective:In this study,we observed the effects of grape seed proanthocyanidins(GSPs)on the production of ROS in colon cancer cell lines SW480 and SW620,combined with transcriptome sequencing to screen out target genes regulated by GSPs and regulate ROS production.Further,we investigated the effects of target gene on biological characteristics of colon cancer cell lines SW480 and SW620,and explored the relevant regulatory mechanisms,which may provide effective molecular targets and new treatments for the diagnosis and treatment of colon cancer.Methods:The cells of SW480 and SW620 were treated with GSPs and the intracellular ROS production was detected by DCFH-DA staining.GSPs regulated genes were screened by transcriptome sequencing,and verified by fluorescence quantitative PCR and Western Blotting.With specific inhibitors of the target gene,the effect of the target gene on the biological characteristics of colon cancer cells was investigated,such as cell viability,cell cycle,cell apoptosis,cell migration and cell cloning formation.Further,we used software to predict the upstream regulatory transcription factors of target genes,and verified their regulation of target genes by siRNA interference after verifying that they are GSPs regulatory genes.Results:GSPs treatment of SW480 and SW620 cells resulted in increased intracellular ROS production compared with the control group.Combined with the transcriptome sequencing results of previous work,a large number of GSPs regulatory genes were screened.Among these candidate genes,FoxMl was inhibited by GSPs,and there was negative feedback on the expression of this gene and ROS production.After down-regulation by FoxMl specific inhibitors,the experimental group showed an increase in ROS production consistent with GSPs treatment compared to the control group.FoxM1 down-regulated by its specific inhibitor could change many characteristics of cell biology,such as:decreasing cell viability,arresting S phase of cell cycle,promoting cell apoptosis,suppressing cell migration and clone formation.Down-regulation of upstream transcription factor POU2F1 could cause the decrease of FoxM1 mRNA level.Conclusion:(1)FoxMl is a target gene regulated by GSPs,its expression is inhibited by GSPs,and exists a negative feedback with ROS production,down-regulation of FoxMl could affect cell characteristics of SW480 and SW620 cells;(2)GSPs inhibit the transcription of FoxMl by inhibiting the expression of POU2F1. |
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