The Mechanism Of UNC5BICD Reducing The Tolerance Of Bladder Cancer Cells To Mitochondrial Apoptosis

Introduction:Bladder cancer is one of the most common cancers in the world,and is listed as one of the top ten causes of cancer-related death.In China,bladder cancer ranks first in the incidence rate and mortality of malignant tumors of the urinary system.In recent years,biological therapy targeting the activation of tumor suppressor genes and related signal pathways has gradually shown unique advantages for patients with advanced bladder cancer.UNC5H（Uncoordinated-5 Homologue）family,which is highly expressed in the nervous system,is a candidate gene family of cancer that has received extensive attention in recent years.UNC5B is the most deeply studied member of its family.Many previous studies have confirmed its“dependent receptor”characteristics,that is,when its ligand,Netrin-1,exists,it can help it can help the survival,differentiation and migration of nerve cells;When Netrin-1 is absent,UNC5B is in a monomer state,and the intracellular region will be cut into intracellular domain,which will activate the downstream apoptosis-promoting pathway,leading to neuronal apoptosis.Because UNC5B has this characteristic and is low expressed in many types of cancer,including bladder cancer,researchers have been studying it as a tumor suppressor gene.This study explored the anti-tumor effect of UNC5BICD in bladder cancer and its specific molecular mechanism from multiple perspectives,such as in vivo and in vitro experiments,to provide a theoretical basis for using it as a therapeutic target for advanced bladder cancer in the future.Methods:1.Detection the relationship between UNC5B、UNC5BICD and the mitochondrial apoptosis of bladder cancer cells with In vivo and in vitro experiments.In vitro experiment:transfect lentivirus containing UNC5B and UNC5BICD into bladder cancer cell lines,T24 and UMUC-3,to construct bladder cancer cell lines,that stably overexpressing UNC5B and UNC5BICD.The expression of UNC5B and UNC5BICD in each cell lines that transfected with lentivirus was detected by q RT-PCR and Western Blot（WB）test.The tolerance of bladder cancer cell lines to H₂O₂-mediated mitochondrial apoptosis after overexpression of UNC5B and UNC5BICD was compared by using TMRE kit to detect mitochondrial membrane potential,WB to detect the activity of mitochondrial apoptosis pathway,and flow cytometry to detect the proportion of apoptotic cells.In vivo experiment:Conduct subcutaneous tumorigenesis experiment in nude mice,subcutaneously inoculate 4-week-old BALB/c nude mice with normal UMUC-3 cells and UMUC-3 cells that stably overexpressing UNC5B and UNC5BICD,and observe the growth of xenograft tumors.On the 7^th and 14^th day after inoculation,cisplatin（5mg/kg）was injected into the abdominal cavity of mice in the experimental group,and the corresponding volume of normal saline was injected into the abdominal cavity of mice in the control group.The mice were killed 4 weeks after inoculation,and the xenograft tumor was isolated.The weight of xenograft tumor was weighed and recorded to compare the difference of effect of cisplatin on bladder cancer cell lines after overexpression of UNC5B and UNC5BICD.2.Exploration of the mechanism of UNC5B and UNC5BICD affecting mitochondrial apoptosis of bladder cancer cells with in vitro experiments.In vitro experiment:The expression level of P53 and the change of its acetylation level in the nuclei of bladder cancer cell lines overexpressing UNC5B and UNC5BICD were detected by WB.Furthermore,the expression level of P53 deacetylase Sirt1 and the change of its subcellular distribution in bladder cancer cell lines overexpressing UNC5B and UNC5BICD were detected by WB and Immunofluorescence（IF）.3.In vitro recovery experiment 1 explored the relationship between the subcellular distribution of Sirt1 and the effect of UNC5B and UNC5BICD.In vitro experiment:the plasmid carrying CRM1 sh RNA（CRM1 can transport Sirt1 from nucleus to cytoplasm）was transfected into bladder cancer cells stably overexpressing UNC5B and UNC5BICD,and the expression of CRM1 in each cell lines after transfection of plasmid was detected by q RT-PCR and WB.The changes of P53 expression and its acetylation level in bladder cancer cell lines overexpressing UNC5B and UNC5BICD after CRM1 sh RNA transfection were detected by WB.The expression level of Sirt1 and the change of its subcellular distribution in the above cell lines were further detected by WB and IF.The tolerance of these cell lines to H₂O₂-mediated mitochondrial apoptosis was compared by using TMRE kit to detect mitochondrial membrane potential,WB to detect the activity of mitochondrial apoptosis pathway,and flow cytometry to detect the proportion of apoptotic cells.4.In vitro recovery experiment 2 explored whether the subcellular distribution pattern of Sirt1 changed under different expression levels of UNC5B and UNC5BICD.In vitro experiment:the plasmid carrying UNC5B sh RNA and UNC5BICD sh RNA was transfected into bladder cancer cells stably overexpressing UNC5B and UNC5BICD,and the expression of UNC5B and UNC5BICD in each cell line after transfection was detected by q RT-PCR and WB.The changes of P53 expression and its acetylation level in bladder cancer cell lines overexpressing UNC5B and UNC5BICD after transfection of sh UNC5B and sh UNC5BICD were detected by WB.The expression level of Sirt1 and the change of its subcellular distribution in the above cell lines were further detected by WB and IF.The tolerance of these cell lines to H₂O₂-mediated mitochondrial apoptosis was compared by using TMRE kit to detect mitochondrial membrane potential,WB to detect the activity of mitochondrial apoptosis pathway,and flow cytometry to detect the proportion of apoptotic cells.5.Exploration of the relationship between UNC5B,UNC5BICD and Sirt1 in the expression level and location,as well as the direct interaction between them with in vitro experiment.In vitro experiment:IF was used to quantitatively analyze the relationship between the specific expression of Sirt1 in the nucleus and cytoplasm and the expression of UNC5B and UNC5BICD in bladder cancer cell lines with different expressions of UNC5B and UNC5BICD,and draw regression curves.The distribution of UNC5B,UNC5BICD and Sirt1 in cells was analyzed by IF co localization.The direct interaction of UNC5B,UNC5BICD and Sirt1 in the cytoplasm was detected by co immunoprecipitation（Co-IP）.Results:1.UNC5B and UNC5BICD can reduce the tolerance of bladder cancer cell lines to mitochondrial apoptosis.In vitro experiments showed that the tolerance of bladder cancer cell lines overexpressing UNC5B and UNC5BICD to H₂O₂-mediated mitochondrial apoptosis was significantly lower than that of normal bladder cancer cell lines.In vivo experiments have proved that overexpression of UNC5B and UNC5BICD can significantly enhance the efficacy of cisplatin on xenografts of bladder cancer cell lines.2.Overexpression of UNC5B and UNC5BICD can change the distribution of Sirt1subcellular in bladder cancer cell lines,leading to the increase of P53 acetylation level in the nucleus.In vitro experiments showed that after overexpression of UNC5B and UNC5BICD,the subcellular distribution pattern of Sirt1 in bladder cancer cell lines changed（the expression level of Sit1 did not change significantly）,from mainly distributed in the nucleus to mainly distributed in the cytoplasm,thus leading to an increase in the level of P53 acetylation,the main target of Sirt1 in the nucleus（the expression level of P53 did not change significantly）.3.The change of the subcellular distribution of Sirt1 is the key factor for UNC5B and UNC5BICD to play its role,and the subcellular distribution pattern of Sirt1 will change with the change of the expression level of UNC5B and UNC5BICD.In vitro recovery experiment 1 proved that after CRM1 sh RNA was transfected into bladder cancer cell lines stably overexpressing UNC5B and UNC5BICD,Sirt1 returns to the nucleus（Sirt1 expression level did not change significantly）,while P53 acetylation level in the nucleus was significantly reduced（P53 expression level did not change significantly）,and the tolerance of each cell lines to H₂O₂-mediated mitochondrial apoptosis was significantly enhanced.It shows that the change of Sirt1 subcellular distribution plays a key role in the effect of UNC5B and UNC5BICD.In vitro recovery experiment 2 proved that after UNC5B sh RNA and UNC5BICD sh RNA were transfected into bladder cancer cell lines stably overexpressing UNC5B and UNC5BICD,the distribution pattern of Sirt1 changed to the nucleus-cytoplasm balance pattern（the expression level of Sirt1 did not change significantly）,and the level of P53acetylation in the nucleus also decreased partially（the expression level of P53 did not change significantly）,and the tolerance of each cell line to H₂O₂-mediated mitochondrial apoptosis enhanced significantly.It further confirmed the anti-tumor effect of overexpression of UNC5B and UNC5BICD in bladder cancer cells,and also showed that the distribution pattern of Sirt1 subcellular will change with the change of the expression of UNC5B and UNC5BICD.4.UNC5BICD can combine with Sirt1 in the cytoplasm.In vitro experiments showed that the expression of UNC5B and UNC5BICD was positively correlated with the expression of Sirt1 in the cytoplasm and negatively correlated with the expression of Sirt1 in the nucleus,and that UNC5B,UNC5BICD and Sirt1 were co-located in the cytoplasm.Co-IP proved that they could combine with each other in the cytoplasm.Conclusions:In bladder cancer cell lines,overexpression of UNC5BICD can play the same anti-tumor role as UNC5B.UNC5BICD can delay the entry of Sirt1 into the nucleus by combining with Sirt1,shuttling between nucleus and cytoplasm,in the cytoplasm,so that the subcellular distribution of Sirt1 changes（from the main distribution in the nucleus to the main distribution in the plasma）,leading to the increase of P53 acetylation level in the nucleus,activation of P53 activity in the nucleus and its downstream mitochondrial apoptosis pathway,thus reducing the tolerance of bladder cancer cells to mitochondrial apoptosis.