The Regulation And Mechanism Of ALDH6A1 On Bladder Cancer Cells

Objective: To explore the key role and mechanism of aldehyde dehydrogenase ALDH6A1 in regulating bladder tumor proliferation and cisplatin resistance.Research content and methods: ALDH6A1,a member of the ALDHs family,catalyzes the irreversible oxidative decarboxylation of malonate and methylmalonate semialdehyde to acetyl-Co A and propionyl-Co A,and plays an important role in valine and pyrimidine catabolic pathway.In this study,We searched GEO database,and extracted m RNA and protein from clinical bladder caner samples,to detect the expression level of ALDH6A1 in non-muscle-invasive and muscle-invasive bladder tumors by RT-PCR and Western blot.We also analyzed the correlation between the ALDH6A1 expression level and the overall survival rate of patients.Secondly,in our in vitro experiments,we constructed sh RNA lentivirus to transfect bladder cancer cell lines to obtain ALDH6A1 knockdown cell lines,and Ch IP-PCR result found that the orphan nuclear receptor HNF4 a can bind to the promoter region of ALDH6A1 gene and participate in regulation expression of ALDH6A1.Then MTT assay,flow cytometry,Seahorse,small interfering RNA and HNF4 a agonist were used to compare the proliferation ability,clony formation,cisplatin sensitivity,cell cycle and glycolysis levels among wild-type,knock-down,and up-regulated ALDH6A1 bladder tumor cells.Finally,a mouse subcutaneous tumor model was established,cisplatin and HNF4 a agonists were administered respectively,and the subcutaneous tumor size of the mice in each group was dynamically monitored.Results: Both GEO database and clinical samples found that the expression of ALDH6A1 in bladder tumors was significantly lower than normal adjacent tissues.The lower expression of ALDH6A1 was correlated with higher the malignancy of the tumor and the overall survival rate of the patient was worse.In vitro results showed that the gene and protein expressions of ALDH6A1 and HNF4 a were lower in bladder tumor cells than normal epithelial cell,they also decreased in bladder tumor cells after cisplatin intervention.Ch IP-PCR result showed the orphan nuclear receptor HNF4 a is involved in regulating ALDH6A1 in bladder tumor cells.Bladder tumor cells with low expression of ALDH6A1 have higher cell proliferation rate,clone formation ability and glycolysis rate.After cisplatin intervention,bladder tumor cells with low expression of ALDH6A1 were unsensitive to cisplatin and had lower levels of total reactive oxygen species produced in tumor cells.After using the agonists of HNF4 a to up-regulate HNF4 a in bladder tumor cells,both in vitro and in vivo results showed that both HNF4 a and ALDH6A1 were upregulated in bladder tumor cells,the proliferation rate of tumor cells was significantly reduced,and improved treatment effect of cisplatin on tumor cells obviously.Conclusion: Decreased ALDH6A1 in bladder tumor cells can affect the normal function of the mitochondrial aerobic respiratory chain,ultimately leading to a decrease in the production of reactive oxygen species in mitochondria and a compensatory increase in the level of aerobic glycolysis.Using HNF4 a agonist to upregulate ALDH6A1 in bladder tumor cells can reduce the proliferation rate of bladder tumor cells and improve the sensitivity of tumor cells to cisplatin.In clinical practice,detecting the expression of ALDH6A1 in bladder cancer can provide new clues for the diagnosis and prognosis of bladder cancer,find a new way for targeted therapy of bladder cancer,and provide a new idea for future research on bladder cancer.