Study On The Regulation Mechanism Of E2F1 And Multi-target Combined Interference In Prostate Cancer Cells Based On Regulatory Network

Chapter 1 Key genes and pathways controlled by E2F1 in humancastration-resistant prostate cancer cellsCastration-resistant prostate cancer(CRPC)is an enormous challenge for prostate cancer treatment.E2 F transcription factor 1(E2F1)is an essential factor in CRPC,the primary purpose of this study was to identify of genes and pathways controlled by E2F1 and affected CRPC cellular behavior.Vitro assays were used to evaluate behaviors of CRPC cells(PC3 and DU145 with knock-down of E2F1).Cells’ expression was quantified by RNA sequencing(RNA-seq).The gene co-expression were performed by GeneMANIA database,and the correlation were analyzed by GEPIA database.Altered pathways of differentially expressed genes(DEGs)were revealed by functional annotation.Module analysis and hub genes were constructed by the STRING database and filtered by the Cytoscape software,respectively.Some of DEGs were validated by real-time quantitative PCR(RT-qPCR).Knock-down of E2F1 significantly inhibited proliferation and accelerated apoptosis in PC3 cells but not in DU145 cells,reduced the invasion and migration of both cells.Down-regulation of E2F1 brought 1811 DEGs into PC3 cells,27 DEGs into DU145 cells,10 overlapped DEGs such as TMOD2 and AIF1 L of two cell lines were obtained.These 10 overlapped DEGs were divided into 2 parts in gene co-expression network analysis,TMOD2 and KREMEN2 were coexpression genes of E2F1.6 overlapped DEGs were positively correlated with transcription factor E2F1.Moreover,10 overlapped DEGs were significantly enriched in actin filament organization pathways.The DEGs of PC3 and DU145 group were mainly associated with various pathways.5 overlapped DEGs and 3 hub genes of PC3 groups were validated by RT-qPCR.The current study suggests that E2F1 plays a critical role in regulating the actin filament organization by change the expression level of several genes such as TMOD2 and AIF1 L in CRPC,which extend the understanding of cellular responses affected by E2F1 in CRPC.Chapter 2 Study on the cytological function and regulatory mechanism of E2F1 knock-out in prostate cancer cellsThrough the pre-established regulatory network,it was found that E2F1 may be regulated by multiple upstream genes and also regulate the expression of multiple genes.However,the genes regulated by E2F1 in prostate cancer(PCa)cells are not clear,and only E2F1 is regulating the next gene.Therefore,we sought to find out the role of E2F1 in knocking out the cytological function,important pathways of PCa and regulating important genes.PC3 cells knocked out of E2F1 was constructed by CRISPR/Cas9 technology.The knockout effect of E2F1 in cells was detected by Western blotting.Flow cytometry,CCK8,and transwell were used to detect apoptosis,proliferation,migration,and invasion of cells.Combined with RNA-seq results,the Ch IP-seq results of E2F1 in prostate cancer cells were analyzed to find genes positively and negatively regulated by E2F1.These genes were verified by RT-q PCR.The PC3 cell line knocked out of E2F1 was successfully constructed using CRISPR/Cas9 technology.The proliferation of PC3 cells after knockout of E2F1 was significantly decreased(P<0.01),and had no significant effect on the apoptosis level of PC3 cells(P>0.05).The invasive ability of PC3 cells after knockout of E2F1 was significantly decreased(P<0.01),the migration ability of PC3 cells after knocking out E2F1 was extremely significantly reduced(P<0.001).TMOD2,AIF1 L,CFH,IL1RL1,EVA1 C,WIPF3,NXPH4,KREMEN2 and PI3 can be combined with E2F1.It was confirmed that E2F1 can regulate the cytoskeleton by regulating TMOD2 and AIF1 L involved in cell actin tissue.RT-q PCR confirmed that knockdown of E2F1 in PC3 cells can significantly down-regulate TMOD2 and AIF1 L,and up-regulate the expression of CFH,IL1RL1 and EVA1 C.After knocking out E2F1,the expression levels of CFH,IL1RL1 and EVA1 C increased,indicating that E2F1 can negatively regulate the next factor in PCa.On the one hand,it was demonstrated that E2F1 can be involved in the regulation of cellular actin by regulating TMOD2 and AIF1 L to affect the cytoskeleton,which in turn affects cytological function.On the other hand,it compensates for this gap in regulation and regulation of E2F1 in the reported literature,and improves the molecular regulatory network of E2F1 in prostate cancer.Chapter 3 Combined multi-target interference of key targets based on regulatory networkCastration-resistant Prostate Cancer(CRPC)occurs in patients with Prostate Cancer after further Castration and/or anti-androgen drug treatment for a period of time.In our previous studies,we had constructed CRPC regulatory network through text mining and study on the negative feedback loop study,and found that if we control E2F1,MEK and NF-κB targets at the same time,the expression of Androgen Receptor(AR)will be effectively controlled which will provide meaningful combined therapeutic targets for the treatment of CRPC.Stable CRPC cell lines PC3,DU145 and androgen-dependent LNCa P were constructed with sh RNA technology.Western blot and immunofluorescence were used to detect the inhibitory effect of MEK and NF-κB.Flow cytometry,CCK8 and transwell were used to detect the effects of the combined interference of the three targets on apoptosis,proliferation,migration and invasion of the three cell lines.Fluorescence quantitative PCR(RT-q PCR)was used to detect the effect of combined interference of three targets on the expression of tumor behavior-related genes(MMP2,VEGFA,CDH1 and BCL-2).Differentially expressed genes(DEGs)of CRPC cells were analyzed,and functional annotation pathway enrichment analysis of differentially expressed genes was conducted to find out which important pathways changed after combined interference with three targets.PPI network was constructed and analyzed to find the major modules that changed.Acquisition of the hub gene to understand what key genes changed after the combined interference.Combined interference on the three targets of PC3,DU145 and LNCa P cells showed that the proliferation of the three cell lines had a significant inhibitory effect(P < 0.001),and both the action time and the effect time were better than the inhibitory effect of the clinical drug abiraterone on the corresponding cells in the positive control group for prostate cancer.Combined interference with the three targets could significantly reduce the invasion of PC3(P < 0.001)and DU145(P < 0.05)cells,and the effect was stronger than that of the positive control group abiraterone.Combined interference with three targets significantly reduced the migration ability of PC3(P < 0.001),DU145(P < 0.05)and LNCa P(P < 0.001)cells,and the effect was stronger than that of the positive control group abiraterone.For PC3,DU145 and LNCa P cells,the combined interference of the three targets significantly enhanced the apoptosis after 96 h after the inhibitor action(P < 0.001),and the effect was stronger than that of the positive control group abiraterone.The expression level of MMP2 gene was up-regulated in PC3 cells after combined interference with three targets,and significantly down-regulated in DU145 cells after combined interference with three targets.The expression levels of three genes,VEGFA,BCL-2 and CDH1,were significantly downregulated in PC3 and DU145,and were all lower than the expression levels of corresponding genes after interference by abiraterone in the positive control group.After combined interference with E2F1,MEK and NF-κB targets,745 differentially expressed genes were found in PC3 cells of CRPC cells,and a total of 316 up-regulated genes and 429 down-regulated genes were identified.After combined interference with E2F1,MEK and NF-κB,three targets of PC3 cells,the five most important pathways were changed,including cytokine-mediated signaling,viral defense response,chromosome coagulation,chemotaxis and cell division.The effects of the combined interference of the three targets on cells mainly include four main cluster-like pathways: multiple pathways affecting chromosomes,multiple pathways related to chemotaxis,multiple pathways related to interferon,and some pathways related to cell behavior.Through PPI network construction,it was found that the central genes could be divided into 17 clusters including 6 major MCODEs.Twenty central genes including IL6,BIRC5,CDH1,PLK1,CDK1 and CDC20 were found.BIRC5 and CDC20 are significantly different in the expression and survival analysis of normal prostate and prostate cancer tissues,which is expected to be a potential indicator of prostate cancer prognosis.The combined interference of E2F1,MEK and NF-κB targets inhibited the proliferation,invasion and migration of CRPC cells(PC3 and DU145),and even the proliferation,invasion and migration of androgen-dependent LNCa P cells,promoted their apoptosis,and significantly down-regulated multiple tumor behavior related genes,which will provide meaningful combined therapeutic targets for the treatment of CRPC.We elucidated the molecular mechanism of the combined interference of three targets at the transcriptome level.After the combined interference,multiple important pathways and a number of key genes were changed,which resulting in changes in multiple aspects of cellular function in CRPC cells.