Database-based Analysis Of FOXG1 Expression In Tumors And The Construction Of Human FOXG1 Eukaryotic Expression Vector

Research Background Cancer is one of the major diseases that threaten human life and health.Despite the rapid development of modern medical technology,the life quality and prognosis of many cancer patients are still poor.Therefore,it is urgent to find more molecular targets or biomarkers to improve the effects of diagnosis and treatment.Tumorigenesis is a complex process involving multiple genes,including oncogenes,and tumor suppressor genes.Recent studies have shown that some genes palying a key role in embryonic development,or cell differentiation have been identified to involve in tumorigenesis.FOXG1 is an highly conserved winged-helix transcriptional repressor,belonging to the forkhead box transcription factor family.FOXG1 is highly expressed in brain which plays a key role in neural progenitor cell differentiation and then participates in the development of telencephalon and cerebral cortex.Previous researches on FOXG1 function were mostly focused on brain and neurogenesis.Recently,some researches also suggested that FOXG1 was involved in tumorigenesis.It has been reported that the abnormal expression of FOXG1 in glioblastoma,breast cancer,ovarian cancer,liver cancer and colorectal cancer.But the functions of FOXG1 in other human cancers,and the mechanism by which FOXG1 involved in tumorigenesis remain unclear.In this study,we performed a database-based analysis of FOXG1 expression and prognostic values in human cancers,and then construct human FOXG1 eukaryotic expression vector,which provide experimental and theoretical basis for further study of FOXG1 in tumorigenesis.Objectives1.To analyze the expression and clinical significance of human FOXG1 gene in tumorsbased on database,explore its value as a new target for tumor diagnosis and prognosis,and provide ideas for subsequent basic research.2.To construct human FOXG1 eukaryotic expression vector,establish stable overexpressing of FOXG1 cell line,and provide the experimental basis for further researches.Methods1.We analyzed the m RNA expression differences of FOXG1 between cancers and normal tissues via Oncomine database,especially in glioblastoma,invasive breast cancer,colorectal cancer and lung cancer.We then we assessed the prognostic values by using Kaplan Meier Plotter and GEPIA databases.2.One step cloning method was used to construct the human FOXG1 eukaryotic expression vector.We firstly amplified human FOXG1 gene from p WPXLd-IRES puro-FOXG1 plasmid,and then cloned into Eco RI site of p EGFP-C1 vector.The recombinant plasmid p EGFP-C1-FOXG1 was verified by enzyme digestion and DNA sequencing,and then transient transfected into Hela cells by liposome.The p EGFP-C1-FOXG1 expression was detected via western blot.3.We carried out the lentiviral packaging in HEK 293 T cells.The virus concentrate was then infected A549 cells.Stable overexpression of FOXG1 cell lines were established by purinomycin screening.Results1.Oncomine database analysis showed that FOXG1 m RNA expression levels varied with tumor types.Compared with normal tissues,FOXG1 expression was significant lower in glioblastoma(P<0.05),but higher in breast cancer,colorectal cancer and small cell lung cancer(P<0.05).2.Survival analysis showed that recurrence free survival rate of rectal adenocarcinoma patients with low FOXG1 expression was higher than that with high FOXG1 expression(Log-rank P<0.05),which indicated a poor prognosis.There was no significant correlation between FOXG1 expression and survival prognosis in patients withglioblastoma,breast cancer,lung adenocarcinoma and lung squamous cell carcinoma(Log-rank P>0.05).3.Enzyme digestion and DNA sequencing confirmed that the human FOXG1 gene was correctly inserted into the Eco R I site of p EGFP-C1 vector.Western blot showed that the recombinant plasmid p EGFP-C1-FOXG1 could be successfully expressed in Hela cells.4.Lentivirus was successfully packaged in HEK 293 T cells.24 hours after lentivirus infection,the expression of green fluorescent protein was observed in A549 cells under inverted fluorescence microscope.Through purinomycin screening,we successfully estabolised the stable overexpression of FOXG1 cell lines,A549-FOXG1.Conclusions1.FOXG1 was significantly underexpressed in glioblastoma,and there was no significant correlation between FOXG1 expression level and survival rates in glioma patients.2.FOXG1 was significantly highly expressed in invasive breast cancer,and there was no correlation between FOXG1 expression level and survival rates in breast cancer patients.3.FOXG1 was significantly highly expressed in colorectal cancer,and high expression of FOXG1 in patients with rectal adenocarcinoma indicates poor prognosis.4.FOXG1 was highly expressed in small cell lung cance,but no significant differences was found in lung adenocarcinoma and lung squamous cell carcinoma.Survival analysis showed that there was no significant correlation between FOXG1 expression level and the survival rates of patients with lung adenocarcinoma and lung squamous cell carcinoma.5.Eukaryotic expression vector p EGFP-C1-FOXG1 was successfully constructed and expressed in Hela cells.6.We successfully established A549-FOXG1 and A549-NC stable cell lines via lentiviral infection.