| Objective: Bladder cancer is one of the most common malignant tumors of the urinary system.It is characterized by multiple foci,easy recurrence and easy progression.In China,the incidence of bladder cancer ranks second among male genitourinary system malignancies.Some patients with bladder cancer will be supplemented with systemic treatments such as radiotherapy and chemotherapy after radical cystectomy.However,patients with bladder cancer often develop drug resistance after a period of chemotherapy,which reduces the efficacy of treatment.Therefore,further study of the molecular mechanism and drug resistance is still need for the prevention,early diagnosis and treatment of bladder cancer.The RASSF8 gene is a member of the Ras-related domain family.RASSF8 involved in the regulation of cell signaling pathways,cell cycle,microtubule network and cell apoptosis.RASSF8 has been reported associated with many kinds of malignant tumors,including gastric cancer,colorectal cancer,cervical cancer,non-small cell lung cancer,ovarian cancer and osteosarcoma.However,there are few research reports on RASSF8 in bladder cancer.In this research,we explored RASSF8 expression pattern,the role of RASSF8 on tumor cell proliferation,invasion,apoptosis,especially drug resistance,and its potential molecular biological mechanisms in bladder cancer.Methods: 1.Bioinformatics Analysis: We download the expression data of RASSF8 m RNA for bladder cancer in the TCGA(The Cancer Genome Atlas)database,which include 414 cases of bladder tumor tissues and 19 cases of normal bladder epithelial tissues.The difference of RASSF8 expression between tumor tissues and healthy tissues was statistically analyzed by Prism 8 software.2.RASSF8 expression in bladder cancer tissues and cells: RASSF8 levels in the collected bladder cancer tissues and adjacent healthy tissues were analyzed by immunohistochemical method.RASSF8 protein and m RNA expression in the bladder epithelial cells and bladder cancer cell lines were detected by western blot and real-time fluorescent quantitative PCR.3.The effect of RASSF8 on proliferation,invasion,migration,cell cycle,apoptosis,mitochondrial function,oxidative stress and glucose metabolism in bladder cancer cells:The empty plasmid and RASSF8 plasmid were transfected in T24 cells.The non-targeting interference sequence and RASSF8 si RNA were transfected in J82 cells.Western blot and real-time fluorescent quantitative PCR methods were used to verify the transfection efficiency.T24-control,T24-RASSF8,J82-control,J82-RASSF8 si RNA cells were applied to CCK8 assays,colony formation assays,Transwell invasion assays,scratch assays,PI staining assays,Annexin V/PI double staining assays,JC-1 staining assays,ROS assays,2-NBDG staining assays,GOD staining assays and ATP assays to verify the changes of various biological behaviors including cell proliferation,invasion,migration,cell cycle,apoptosis,mitochondrial membrane potential,oxidative stress,glucose uptake,glucose consumption and ATP production.4.The molecular mechanism of RASSF8 affecting the biological behavior of bladder cancer cells: Total RNA from T24-control and T24-RASSF8 cells was extracted and then applied for high-throughput sequencing.The m RNA expression data was subjected to GSEA enrichment analysis to explore the influence of RASSF8 expression differences on the biological processes and signal pathways in bladder cancer cells.Total protein from T24-control,T24-RASSF8,J82-control,J82-RASSF8 si RNA cells was extracted and then applied to western blot assays.The interaction between proteins in bladder cancer cells is verified by Co-IP assays.Results: 1.Bioinformatics analysis results: According to the RASSF8 m RNA expression data from the TCGA database,the expression of RASSF8 in bladder cancer tissues was significantly down-regulated compared with healthy tissues(p<0.001).2.RASSF8 expression in bladder cancer tissues and cells: Immunohistochemistry results showed that RASSFF8 was highly expressed in bladder epithelial tissues,but was low or not expressed in bladder cancer tissues.RASSF8 low expression was significantly associated with advanced TNM stage and higher tumor grade in bladder cancer.Western blot results showed that the expression of RASSF8 was down-regulated in 7 cases(7/11)of bladder cancers.3.RASSF8 regulates the biological behaviors of bladder cancer cell including proliferation,invasion,migration,cell cycle,mitochondrial membrane potential,glucose metabolism,cell apoptosis and chemotherapy sensitivity: The growth rate decreased and the number of colonies decreased after RASSF8 overexpression in bladder cancer cells;on the contrary,proliferation rate increased and the number of colonies increased after RASSF8 depletion.Transwell invasion results showed that the number of invasive cells decreased after RASSF8 overexpression,while increased after RASSF8 depletion in bladder cancer cells.Scratch results showed that the migration distance of the cells was shortened after RASSF8 overexpression,while was increased after RASSF8 depletion in bladder cancer cells.PI staining assay results showed that the proportion of cells in G1 phase increased and the proportion of cells in S phase decreased after RASSF8overexpression;On the contrary,RASSF8 depletion led to a decrease in the proportion of cells in G1 phase and an increase in the proportion of cells in S phase.Annexin V/PI double staining assay results showed that cell apoptosis increased after RASSF8 overexpression while decreased after RASSF8 depletion in bladder cancer cells.In addition,RASSF8 overexpression could further increase cisplatin-induced apoptosis level,but RASSF8 depletion could decrease cisplatin-induced apoptosis level.JC-1staining assay results showed that the mitochondrial membrane potential decreased after RASSF8 overexpression,while was increased after RASSF8 depletion in bladder cancer cells.ROS assay results showed that ROS content was increased after RASSF8 overexpression and the oxidative stress response was enhanced;On the contrary,ROS content was decreased after RASSF8 depletion and the oxidative stress response was weakened.Glucose metabolism related assay results showed that RASSF8 overexpressing cells had reduced glucose uptake and consumption,and reduced ATP production;On the contrary,glucose uptake,glucose consumption,and ATP production increased after RASSF8 depletion in bladder cancer cells.4.RASSF8 regulates the YAP signaling and the expression of cyclin D1,Bcl-2,caspase3 in bladder cancer cells.RASSF8 inhibits the YAP/TEAD3 combination in bladder cancer cells: GSEA enrichment analysis results showed that the bladder cancer phenotype with RASSF8 overexpression was enriched in malignant tumor progression related pathways,cell apoptosis,DNA repair,mitosis,oxidative phosphorylation,reactive oxygen pathways,cellular chemical stress response and metabolism.Western blot results showed that the expression of cyclin D1,Bcl-2,caspase 3,YAP was decreased,and the expression of cleaved-caspase 3,p-Lats1/2,p-YAP was increased after RASSF8 overexpression.On the contrary,the expression of cyclin D1,Bcl-2,caspase 3,YAP was increased,and the expression of cleaved-caspase 3,p-Lats1/2,p-YAP was decreased after RASSF8 depletion in bladder cancer cells.Co-IP assay results showed that RASSF8 overexpression can inhibit the interaction between YAP and TEAD3 in bladder cancer cells.RASSF8 inhibits the binding of YAP-TEAD3,thereby increasing the sensitivity of bladder cancer cells to cisplatin.Conclusion:1.RASSF8 is down-regulated in bladder cancer.2.RASSF8 overexpression inhibits the proliferation,invasion,migration,and cell cycle transition of bladder cancer cells.RASSF8 overexpression induces oxidative stress and apoptosis,and enhances the sensitivity of cisplatin chemotherapy.3.RASSF8 overexpression accelerates the loss of mitochondrial membrane potential and inhibits glucose metabolism in bladder cancer cells.4.RASSF8 regulates the expression of cell cycle and apoptosis related proteins in bladder cancer cells,and enhances the cisplatin sensitivity through the YAP-TEAD3 signaling. |
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