| Objective: Bladder cancer is the 10 th most common cancer in the world,with more than 500,000 new cases and 200,000 deaths estimated in 2018.More male patients are found than female ones.If patients aged 35 or above with bladder irritation or gross hematuria take cystoscopy,test to measure renal function,and upper urinary tract imaging at an early stage,the grading of malignant tumor can be identified.Early diagnosis and treatment are the key to improving the prognosis of the disease.Transurethral resection of the urethra can further verify the diagnosis,determine the tumor stage and initial treatment plan.The treatments of non-muscle invasive bladder cancer(NMIBC)are mainly through transurethral resection,intravesical BCG or intravesical chemotherapy.Because of its rapid progression and high recurrence rate,muscle invasive bladder cancer(MIBC)is usually treated with radical cystectomy and neoadjuvant chemotherapy.The use of immunosuppressants encourages us to fight against metastatic urothelial cancer.The development of new drugs to treat bladder cancer has never stopped.Ferroptosis is a newly discovered type of cell death characterized by iron-dependent lipid peroxidation accumulation.New evidence suggests that ferroptosis could be induced in the treatment of cancer,particularly in the treatment of malignant tumors that are resistant to drugs.Can ferroptosis inducer produce unexpected therapeutic effects in the treatment of bladder cancer? What are the pathways by which it induces cell death? What genes are involved in this process? We hope that we can find new ways to treat bladder cancer through our study.Methods: 1.Genes that are associated with oxygen metabolism were selected by bioinformatics.2.The gene MCT4(SLC16A3)related to BCa was screened by analysis of prognosis.3.Knock-down of MCT4(SLC16A3)cell line in BCa cell line was created.4.The knock-down efficiency of MCT4(SLC16A3)gene was determined by RT-PCR.5.The regulation of gene knock-down on transcriptome level of bladder cancer 5637 cell line by RNA-seq technique was analyzed.6.Western Blot was used to detect the knock-down efficiency of target gene protein levels and the change in the expression of ferroptosis-related protein,AMPK pathway-related protein and autophagy-related protein after MCT4(SLC16A3)knock-down.7.The changes of clonogenic ability and proliferation ability of cells in knock-down cell lines were detected by plate cloning experiment.8.Apoptosis and reactive oxygen species(DCFH-DA staining)production were detected by Flow cytometry(FC).9.The lipid metabolism disorder of the knock-down cells was detected by MDA test.10.ROS activity(DCFH-DA staining),autophagy levels(Ad Plus-m Cherry-GFP-LC3 B reporting system),and lipid peroxidation specific oxidation(Liperfluo staining)were observed by fluorescence microscope.l PO assay was performed to detect the changes of lipid-reactive oxygen species in knock-down cell lines treated with ferroptosis inhibitor.11.The ultrastructure of erastin-treated cells in the knock-down group and those in the control group were observed under transmission electron microscope(TEM).12.tumorigenicity testing was conducted to test whether the tumorigenicity of the 5637 cell line in nude mice was affected.13.The activity of caspase-3 and caspase-7 was measured by fluorescent labeling before and after gene knockout with RSL3 and CQ respectively.Results: 1.RNA-seq analysis revealed that the genes of 5637 bladder carcinoma cells with MCT4(SLC16A3)knockdown were mainly enriched in ferroptosis,oxidative stress and autophagy-related pathways.2.Transfection of si MCT4(SLC16A3)to 5637 cells and T24 cells to knock down MCT4(SLC16A3)in vitro was effective in inhibiting the proliferation and clonality of 5637 cells,but less effective in T24 cells.Besides,5637 cells with MCT4(SLC16A3)knock-down showed decreased tumorigenicity and proliferative ability in nude mice.3.In 5637 cells with MCT4(SLC16A3)knockdown,expression levels of the key proteins associated with ferroptosis were down-regulated.After inducing with Erastin,more obvious signs of ferroptosis and apoptosis were observed in the 5637 cell group with MCT4(SLC16A3)knockdown under a transmission electron microscope(TEM).When another inducer RSL3 was used,the level of reactive oxygen species(ROS)in 5637 cell group with MCT4(SLC16A3)knockdown was increased under a fluorescence microscope.MDA test and LPO test showed that intracellular lipid peroxidation(LPO)also increased(lipid metabolism disorder),and LPO test showed that such increase could be inhibited by fer-1,a ferroptosis inhibitor.4.After MCT4(SLC16A3)knock-down,the expression levels of the key proteins of the AMPK pathway were significantly inhibited.After both RSL3 and AMPK agonist A-769662 were used to treat 5637 cells with MCT4(SLC16A3)knock-down,it was found that AMPK agonists reversed the increase of ROS and LPO induced by such knock-down under a fluorescence microscope.5.RSL3 was used to treat 5637 cells with MCT4(SLC16A3)knock-down.Flow cytometry showed that knock-down of MCT4(SLC16A3)induced apoptosis.The pure enzyme activities of Caspase-3 and Caspase-7,which represent the degree of apoptosis,were measured by fluorescence microplate reader before and after MCT4(SLC16A3)knock-down of 5637 cells treated with RSL3,and such activities were found to increase.6.The report of Ad Plus-m Cherry-GFP-LC3 B showed that the autophagy of 5637 cells was inhibited after MCT4(SLC16A3)knock-down.Flow cytometry showed that MCT4(SLC16A3)knockdown could increase the level of ROS and apoptosis induced by the autophagy inhibitor CQ.Conclusion: MCT4(SLC16A3)knock-down can inhibit the proliferation of 5637 bladder carcinoma cells and affect their tumorigenicity in nude mice.MCT4(SLC16A3)knock-down induces ferroptosis of 5637 cells through AMPK pathway.RSL3 causes the death of 5637 and T24 cells after knock-down in two ways: promoting ferroptosis and inducting apoptosis.This study also found that MCT4(SLC16A3)knock-down could inhibit autophagy,and it can also increase ROS level caused by inhibition and make cells more apoptotic. |
