KAT2A-mediated CTBP1 Succinylation Promotes Prostate Cancer Progression By Blocking Its Inhibition Of CDH1 Transcriptional Activit

Background:Prostate cancer is a common malignant disease of the elderly,mostly seen in the male reproductive system and urinary system.According to the statistical data of China’s National Cancer Center in 2020,the incidence rate of male prostate cancer in China is 10.13/100000.It is the sixth male tumor incidence rate in China.This study takes prostate cancer glycolysis and protein succinylation modification as the starting point,and explores and analyzes the mechanism of KAT2A mediated CTBP1 succinylation modification regulating CDH1 expression and inhibiting prostate cancer glycolysis and metastasis through cellular and animal experiments.It elucidates its protein targets and upstream and downstream signaling pathways,providing new research ideas and experimental data for the development of new drugs and treatment technologies for prostate cancer.Method:1.1 Effect of CTBP1 on migration,invasion and glycolysis of prostate cancer cells(1)Cultured prostate cancer cells,the expression level of CTBP1 in normal cells and prostate cancer cells was detected by qRT-PCR and WB.(2)Transfected sh-CTBP1 knocks down the expression level of CTBP1 in prostate cancer cells,and its transfection efficiency was detected by qRT-PCR.After successful transfection,cell function analysis was performed.1.2 The regulatory effect of CTBP1 on CDH1 expression(1).qRT PCR detection of differentially expressed genes in prostate cancer cells after CTBP1 knockout.(2).After knocking down the expression of CTBP1,qRT PCR assay and WB detection were used to analyze the specific expression of CDH1.(3).Double Luciferase report test was used to detect the binding relationship between CTBP1 and CDH1.(4).The interaction between CDH1 and SP1 was detected by immuno coprecipitation assay and immunofluorescence staining.(5).Double Luciferase report and ChiP qPCR were used to detect the transcriptional regulation of SP1 on CDH 1.1.3 KAT2A regulates succinylation modification of CTBP1(1).WB detection of succinylation levels in prostate cancer cells.(2).WB detection of the expression level of CTBP1 in normal cells and prostate cancer cells after overexpression of KAT2A.(3).Transfect KAT2A overexpression plasmid into prostate cancer cells,overexpress KAT2A,and detect transfection efficiency by qRT PCR.(4).In prostate cancer cells overexpressing KAT2A,qRT PCR and WB were used to detect the expression level of CTBP1.(5).Western blot detection of ubisuccinylation modified proteins and succinylation modified CTBP1 in prostate cancer cells.(6).IP western blot was used to detect the overexpression of KAT2A and the succinylation of CTBP1 with K46,K280,K348 and K354 mutations.(7).The transcriptional activity of CDH1 after CTBP1 mutation at K36 and K280 sites was detected by double Luciferase report assay.1.4 CTBP1 affects the migration,invasion and glycolysis of prostate cancer cells by regulating KAT2A.CTBP1 overexpression and KAT2A knockdown in prostate cancer for functional analysis.1.5 CTBP1 affects prostate cancer tumor growth in mice with metabolic syndrome through KAT2A.(1).Mice were fed a high-fat diet for 16 weeks,and their body weight,cholesterol,and total testosterone levels were measured to confirm the occurrence of metabolic syndrome like diseases.(2).Establish a prostate cancer cell transplantation tumor model overexpressing CTBP1 and knocking down KAT2A in high-fat diet mice.After obtaining the tumor,detect tumor related indicators.Results:(1)CTBP1 is significantly upregulated in PCa tissues and cell lines.CCK-8 assay showed that knocking down CTBP1 inhibited the survival ability of DuCaP cells.Transwell analysis showed that CTBP1 silencing significantly inhibited the migration and invasion of DuCaP cells.Knocking down CTBP1 inhibited glucose consumption and lactate production in DuCaP cells.The results of the ECAR curve indicate that the glycolytic ability of the sh-CTBP1 group is lower than that of the control group.(2)Knockout of CTBP1 promotes mRNA and protein expression of CDH1.Silencing of CTBP1 enhances the transcriptional activity of CDH1.The CO-IP results indicate that CTBP1 can bind positively to SP1,but cannot bind positively to ATF3,KLF6,or NFE2.The immunofluorescence staining of CTBP1 and SP1 further confirmed the interaction between CTBP1 and SP1.SP1 can bind to the promoter region of CDH1(1501-2000bp)and regulate its transcriptional activity.(3)Overexpression of KAT2A enhances succinylation in DuCaP cells.Subsequently,we established CTBP1 mutants at sites K46,K280,K348,and K354,respectively.The results showed that the K46 and K280 mutants significantly reduced the succinylation level of CTBP1.In addition,we established CTBP1 mutants at the K46 and K280 loci.It was found that both K46 and K280 mutants had lower succinylation levels than the individual K46 or K280 mutants.However,we found that the CTBP1 mutant had no effect on the expression of CTBP1.Luciferase assay showed that CTBP1 knockout promoted the transcriptional activity of CDH1.The transcriptional activity of the K46 and K280 mutant groups was lower than that of the control group.In addition,cells transfected with mutants at the K46 and K280 sites exhibited transcriptional activity similar to that of the control group.(4)CTBP1 promotes the vitality,migration,invasion,and glycolysis of DuCaP cells.The silencing of KAT2A partially reversed the effect of CTBP1 on DuCaP cell phenotype.(5)Overexpression of CTBP1 promotes tumor weight,volume,and KI67 expression;The silencing of KAT2A partially reversed the effect of CTBP1 and inhibited its succinylation.Conclusion:We found that KAT2A-mediated succinylation of CDH1 can inhibit the transcriptional activity of CTBP1 to CDH1,thus inhibiting the glycolysis and metastasis of prostate cancer.