Taraxasterol Acetate Regulates The Occurrence And Development Of Colon Cancer By Activating Autophagy To Mediated RNF31/p53 Signal Axis

Background: Colon cancer is the third most common cancer worldwide,and its resistance rate of chemotherapy is up to 50%.Studies have shown that traditional Chinese medicine can reduce chemotherapy resistance.Taraxasterol acetate(TA)is the main component of traditional Chinese medicine dandelion.At present,its mechanism in colon cancer is not clear.E3 ligase RNF31 was originally cloned from breast cancer cells,and its m RNA was found to be highly expressed in cancer cells.Studies have shown that RNF31 is involved in the tumorigenesis of breast cancer,prostate cancer,and adrenocortical cancer,and a variety of E3 ligases were considered as the action targets of traditional Chinese medicine.But so far,it is unclear whether TA can act on RNF31 and whether RNF31 plays a role in the occurrence and development of colon cancer.Methods: This study was completed by using protease immunoblotting,RT-PCR,immunohistochemistry,immunofluorescence,immune co-precipitation,immunofluorescence,and other experimental methods.The data analysis was mainly completed by SPSS 17.0 software(SPSS Inc,Chicago,USA)and graphpad software.The values were expressed by mean ± standard deviation.P<0.05 means that the difference is statistically significant.Results: We examined the expression of RNF31 in CRC tissue samples via immunohistochemistry and elucidated the function of RNF31 in CRC cells by constructing a cell model with RNF31 depletion.A cycloheximide(CHX)-chase analysis and immunofluorescence assays were conducted to demonstrate that TA can promote RNF31 degradation by activating autophagy.We used the Pharm Mapper website to predict targets of TA and identified RNF31.CHX-chase experiments showed that TA could facilitate RNF31 degradation,which was inhibited by the administration of chloroquine.Immunofluorescence assays showed that RNF31 protein was colocalized with LC3I/II and p62,suggesting that TA promoted RNF31 degradation by activating autophagy.We also found that CRC patients with RNF31 overexpression had poorer survival than those with low RNF31 expression.The results of the CHXchase experiment showed that depletion of RNF31 alleviated p53 degradation,which was inhibited by MG132.A series of co-immunoprecipitation(Co-IP)assays revealed that RNF31 interacts with p53 and promotes p53 ubiquitination and degradation.A CoIP assay performed with a truncated RNF31 plasmid showed that the PUB domain interacts with p53.Moreover,the PUB domain is the key structure in the induction of p53 ubiquitination.Conclusion: Our findings reveal a key role of RNF31 in CRC cell growth and indicate a mechanism through which TA inhibits cell growth.