The Mechanism Study Of Function And Molecule That CPSF4 Regulates COX-2 Expression In Lung Cancer

Objective To make clear if CPSF4, the gene with transcriptional function,could regulate COX-2, which is the most significant rate-limiting enzyme taking responsibility for making the conversion about arachidonic acid to prostaglandin endoperoxide 2(PGE2) synthesis,in lung cancer。If there could,clarifying the specific regulation mechanism at some certain degree。To clarify the clinical pathological co-relationship between the both genes and tumor stage tumor progression overall survival rates in clinical tumor patients.Furthermore,to confirm the conclusion got from in vitro and clinica l patients sample by experiments in vivo.Methods Making sure two kinds of lung cancer cell,H1299 and H460,To up-regulate CPSF4 stablely in H1299 by lentiviral transfection. To up-regulate CPSF4 transiently in H460 by plasmid mixing liposome. To knock down CPSF4 expression in lung cancer by specific CPSF4 si RNA mixing liposome.To detect the COX-2 expression variation at protein level after up-regulating or knocking down CPSF4 by western blot assay. To detect the COX-2 expression variation at m RNA level after up-regulating or knocking down CPSF4 by sounthern blot assay. To detect the celluar migration ability variation after up-regulating or knocking down CPSF4 by scratch assay. To detect the celluarinvasion ability variation after up-regulating or knocking down CPSF4 by transwell assay. To detect the celluar proliferation ability variation after up-regulating or knocking down CPSF4 by MTS assay. To detect the COX-2 promoter ability variation after up-regulating or knocking down CPSF4 by Dual- Luciferase Reporter Assay. To detect the NF-κB pathway proteins in cytoplasm and in nucleus variation after up-regulating or knocking down CPSF4 by western blot assay. To detect the NF-κB pathway proteins translocation after up-regulating or knocking down CPSF4 by laser confocal assay. To detect the binding quantity variation of NF-κB pathway proteins and CPSF4 itself to COX-2 promoter after up-regulating or knocking down CPSF4 by pulldown and C HIP assay. To detect if there has binding between CPSF4 and NF-κB pathway activator in lung cancer by IP assay. To detect if there has binding between CPSF4 and NF-κB pathway activator in lung cancer by laser confocal assay. Obtained clinical patients microarray sample, To detect if there has relationship between CPSF4 expression and COX-2 expression in clinical patient tumor tissue by statistical methods. To detect if there has relationship between both CPSF4 and COX-2 expression with some clinical indicator of clinical patients by statistical methods. To detect if there has relationship between CPSF4 expression and COX-2 expression in clinical patient tumor tissue by immunohistochemistry assay. To detect the tumor volume and tumor weight variation at m RNA after up-regulating or knocking down CPSF4 in vivo assay.Results In lung cancer cell H1299 and H460,1.CPSF4 expression has positively co-relationship with COX-2 expression at protein level.2.CPSF4 expression has positively co-relationship with COX-2 expression at m RNA level.3. CPSF4 expression has positively co-relationship with celluar migration ability.4.CPSF4 expression has positively co-relationship with celluar invasion ability.5.CPSF4 expression has positively co-relationship with celluar proliferation ability.6.CPSF4 expression has positively co-relationship with celluar proliferation ability.7.CPSF4 expression has positively co-relationship with NF-κB pathway activity.8.CPSF4 expression has positively co-relationship with NF-κB pathway activator protein in nuclues and CPSF4 itself binding to COX-2 promoter DNA. 9.There has the binding between CPSF4 andNF-κB pathway activator protein in lung cancer. In clinical patients microarray sample 10. there has relationship between CPSF4 expression and COX-2 expression in clinical patient tumor tissue.11.The both expression of CPSF4 and COX-2 has co-relationship with clinical patients tumor stage.12.The both expression of CPSF4 and COX-2 has negative co-relationship with clinical patients overall survival rate.The experiments in vivo,CPSF4 and COX-2 expression at the sametime have positive relationship with tumor volume and tumor weight.Conclusions In lung cancer cells.1.To overexpress(knock down) CPSF4 will up-regulate(down-regulate)COX-2 expression.2.CPSF4 regulate COX-2 through two wholely different patheway,one of them affecting COX-2 expression trought affecting NF-κB pathway activity 3.The second way is trought affecting the binding CPSF4 to COX-2 promoter,then changing COX-2 expression.