Effects And Mechanisms Of PBRM1 On Aerobic Glycolysis And Proliferation In Clear Renal Cell Carcinoma Cells

Objective:Kidney cancer is a common tumor of the urinary system,accounting for3%-5% of all tumors,of which the most typical histomorphological type is cc RCC(clear renal cell carcinoma).The mutation rate of PBRM1(encoding PB1/BAF180protein)in clear renal cell carcinoma is as high as 41%.However,the function of PBRM1 and molecular mechanisms remain to be determined.In addition,aerobic glycolysis is a common phenomenon in many tumors,but whether the two are related is not clear.This study aims to investigate the function of PBRM1 and molecular mechanisms of PBRM1,and effects of PBRM1 on glycolysis in cc RCC cells,so as to provide theoretical basis for the diagnosis and treatment of renal cell carcinoma and other related cancers.Methods:786-O and SN12 C cell lines were cultured in complete RPMI1640 medium.(1)Small interfering RNAs were transfected into 786-O to knock down PBRM1 gene,followed by transcriptome sequencing analysis.(2)Stable knockdown cell lines were set up by using sh RNAs targeting PBRM1 gene,cell proliferation and migration capabilities were examined by using CCK-8,transwell and scratch assays.(3)After knocking down PBRM1 in cc RCC cells,q RT-PCR was used to detect the expression levels of glycoly-related genes under hypoxia or normoxia conditions,and the change of lactic acid content in normoxia and hypoxia states was measured with lactate dehydrogenase concentration detection kit.(4)Upon knockdown of PBRM1 in cc RCC cells,the expression levels of AKT-m TOR pathway-related proteins were examined by using Western Blot technology.(5)Upon knockdown of PBRM1 in cc RCC cells,the expression levels of HIF1α under hypoxia or normoxia conditions were detected by q RT-PCR and Western Blot.(6)786-O cells with stable knockdown of PBRM1 were injected subcutaneously into nude mice to examine the effect of PBRM1 on tumorformation rate.(7)COIP assay was performed to further validate the interaction between PB1 and Lamin A/C protein,which was observed in our previous study.Results:(1)Expression profiling of 786-O cells upon knockdown of PBRM1 showed characteristics changes and genes involved in glycolysis,cell cycle pathways were enriched.(2)The proliferation and migration capabilities of 786-O/SN12 C cells were enhanced upon knockdown of PBRM1.(3)The expression levels of glycoly-related genes in 786-O/SN12 C cells were up-regulated after PBRM1 knockdown under normoxia conditions,and the expression levels of glycoly-related genes were further increased under hypoxia conditions.Reduced PBRM1 expression and hypoxia conditions showed a synergistic effect on the production of cellular lactic acid.(4)After knockdown of PBRM1 in 786-O/SN12 C cells,the expression levels of Akt-m TOR pathway proteins(AKT,p-AKT,m TOR,p-MTOR)increased.(5)Reduced PBRM1 expression and hypoxia conditions exert a synergistic effect on HIF1α expression in SN12 C cells.(6)Upon knockdown of PBRM1 in 786-O cells,subcutaneous tumor bearing experiment showed that the growth rate of the tumor increased.(7)COIP results showed that PB1 and lamin A/C interacts with each other.Conclusion:(1)Our study demonstrates that PBRM1 does function as a tumor suppressor.(2)Knockdown of PBRM1 in 786-O/SN12 C cells activated the AKT-m TOR signaling pathway and increased aerobic glycolysis activity.(3)PBRM1 deficiency and hypoxia play a synergistic role in lactic acid production and HIF1α expression in SN12 C cells.(4)COIP assay confirmed that PB1 and Lamin A/C protein interacts with each other.