Effect And Mechanism Of POU6F1 On Proliferation And Metastasis Of Lung Adenocarcinoma

Part Ⅰ.Abnormal expression and prognosis of POU6F1 and the biological influence of POU6F1 on lung adenocarcinoma cellsObjectives:To screen and investigate the essential transcription factors(TF s)for regulating lung adenocarcinoma(LUAD)progression and the effects on the proliferation and invasion of LUAD cells.Methods:A public LUAD dataset was used to analyze the significantly expressed TFs that were associated with death,tumor stage,and metastasis using The Cancer Genome Atlas(TCGA).The Kaplan-Meier survival analysis was used to analyze the relationship between gene expression levels and clinicopathological features and survival prognosis.Western blot assay was used to determine gene expression levels in LUAD tissues and adjacent tissues.Stably overexpressing LUAD lines were established by antibiotic stress puromycin and Western blot and real-time quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR)assays were used to validate expression level.The effects on the proliferation and invasion of cells were detected by in vitro assays,including Cell Counting Kit-8(CCK8),colony formation,soft agar colony formation,and Transwell assays.Subcutaneous and caudal vein tumor models were performed to determine the growth and metastasis capacity of A549 cells.Results:Four TFs,including caudal type homeobox transcription factor 4(CDX4),conerod homeobox(CRX),heat shock transcription factor family,X linked 1(HSFX1),and Pou domain,class 6,transcription factor 1(POU6F1)were identified and associated with death,tumor stage,and metastasis in LUAD.No significant difference in the expression of HSFX1,CDX4,and CRX between LUAD tissues and normal tissues.Kaplan-Meier analysis indicated that no significant survival difference in the overall survival was observed between the high-expression and low-expression groups of HSFX1,CDX4,and CRX.On the contrary,POU6F1 was significantly downregulated within LUAD tissues,and patients with upregulation of POU6F1 had a significantly better prognosis.The in vitro assays revealed that overexpressing POU6F1 of A549 and NCI-H1299 cells decreased viability and invasion.The subcutaneous model showed that the growth of tumors,weight,proliferation index Ki-67,and CD31-positive intratumoral microvessels in stably overexpressing POU6F1 A549 cells was decreased.In tail vein metastasis,the number of pulmonary metastasis was reduced.Conclusions:POU6F1 was downregulated and downregulation of POU6F1 predicted poor prognosis in LUAD patients.POU6F1 repressed the proliferation,invasion,and metastasis of LUAD.Part Ⅱ.POU6F1 regulated HIF1A signaling pathway in lung adenocarcinoma cellsObjectives:To reveal the mechanism via which POU6F1 suppressed tumorigenesis in LUAD cells.Methods:RNA-seq assay in A549 cells upon POU6F1 overexpression was used to screen differentially expressed genes(DEGs).Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway category and Gene Ontology(GO)analysis of DEGs were performed to determine the cellular process.Western blot and qRT-PCR were used to validate the expression levels.Dual-luciferase assays and chromatin immunoprecipitation assays indicated were conducted to detect the effect of gene promoter activity and enrichment level.Results:RNA-seq results revealed 1269 upregulated and 1467 downregulated genes.KEGG pathway and GO analysis indicated that DEGs were involved in various cellular biological processes,such as cell growth and death,and cell adhesion.DEGs participated in several growths-and invasion-related pathways,such as the HIF-1 signaling pathway and PI3K-AKT signaling pathway.Western blot and qRT-PCR assays showed that POU6F1 affected the expression levels of ENO1,PDK1,and PRKCB.The dual-luciferase assay revealed that overexpressing POU6F1 decreased HIF1A activity.And forced POU6F1 expression decreased ENO1 and PDK1 activity and enhanced PRKCB activity in A549 and NCI-H1299 cells.But,HEK293T cells displayed dropped PDK1 activity and increased ENO1 and PRKCB activity.The chromatin immunoprecipitation and qRT-PCR assays revealed that overexpression of POU6F1 enhanced the enrichment of POU6F1 on the promoter regions of ENO1,PDK1,and PRKCB in A549 cells.Conclusions:POU6F1 inhibited the transcriptional activity of HIF1A and was directly bound to the promoter regions of ENO1,PDK1,and PRKCB,affecting their expression levels.Part Ⅲ.POU6F1 interacted with RORA and affected the activity and stability of RORA in lung adenocarcinoma cellsObjectives:To explore proteins that interact with transcription factor POU6F1 in LUAD cells.Methods:Immunofluorescence and Western blot were used to determine the localization of POU6F1 in LUAD cells.Immunoprecipitation(IP),mass spectrometry(MS),and biomolecular fluorescent complementation(BiFC)were used to detect the interaction of POU6F1 and retinoid-related orphan receptor alpha(RORA).Expression levels of POU6F1 or RORA were examined in LUAD cells transfected with ectopic expression or knockdown of RORA or POU6F1 using Western blot and qRT-PCR assays.The proteasome inhibitor MG132 was used to investigate the effect of POU6F1 on RORA stability.A dual luciferase assay was used to detect the influence of POU6F1 on the RORA promoter activity.Results:POU6F1 could bind to RORA in the nucleus of cells revealed by immunofluorescence and MS.BiFC assay was further performed for direct visualization of their interaction.qRT-PCR and Western blot showed that POU6F1 affected the expression of RORA,but ectopic expression or knockdown of RORA did not affect the expression level of POU6F1.Furthermore,the decreased expression of RORA induced by the knockdown of POU6F1 could be reversed by treatment with MG132 in LUAD cells.Dual luciferase assay showed that ectopic expression of POU6F1 increased the activity of the RORA promoter in LUAD cells.Conclusions:POU6F1 interacted with RORA and affected the stability and activity of RORA in LUAD cells.Part Ⅴ.POU6F1 coordinated with RORA to inhibit lung adenocarcinoma progression Objectives:To further examine the functional interplay between POU6F1 and RORA in the regulation of LUAD progression.Methods:Western blot and qRT-PCR assays were performed to detect the expression levels of genes in cancer cells transfected with knockdown of POU6F1 and overexpression of RORA.Dual luciferase assay was conducted to reveal the activity of gene promoter.Soft agar colony formation and Transwell invasion assays were used to observe the proliferation and invasive ability of LUAD cells.The expression levels and survival of genes were evaluated using Kaplan-Meier in LUAD patients.Results:Western blot and qRT-PCR assays showed that forced expression of RORA suppressed the increased expression of HIF1A,ENO1,or PDK1 and reduction of PRKCB in LUAD cells induced by impaired POU6F1 expression.In the dual luciferase assay,the knockdown of POU6F1 increased the promoter activity of ENO1 and PDK1 and decreased PRKCB activity,which was prevented by the ectopic expression of RORA in LUAD cells.Soft agar colony formation and Transwell invasion assays indicated that the decreased POU6F1 caused an increase in growth and invasiveness,which were decreased by the forced expression of RORA in LUAD cells.Mining public datasets indicated that ENO1 and PDK1 expression were highly expressed in LUAD tissues and patients with higher ENO1 and PDK1 expression were linked with poor outcomes.At the same time,PRKCB was reduced in tumor tissues and downregulated PRKCB expression was indicative of a low survival possibility in LUAD patients.Conclusions:POU6F1 coordinated with RORA to inhibit LUAD progression.