Research On The Role And Mechanism Of GTF2E2 In The Progression Of Esophageal Squamous Cell Carcinoma And Early Postoperative Recurrence

PART Ⅰ The expression and clinical significance of GTF2E2 in esophageal squamous cell carcinoma and postoperative recurrent tissues,and its effect on the proliferation and metastasis of esophageal squamous cell carcinoma cellsObjective:To investigate the role of GTF2E2 in esophageal squamous cell carcinoma(ESCC)and postoperative recurrent tissues,and its correlation with clinicopathological parameters and prognosis,and to determine the effects of interfering with GTF2E2 expression on the proliferation,apoptosis and metastasis of ESCC cells.Methods:The expression of GTF2E2 in ESCC tissues and its relationship with prognosis were analyzed by bioinformatics methods.Immunohistochemistry assay(IHC)was applied to detect the expression of GTF2E2 in ESCC and adjacent tissues of tissue microarray.The correlation of GTF2E2 expression with clinicopathological parameters and survival outcomes was explored based on the tissue microarray data.IHC was performed to detect GTF2E2 expression in ESCC patients with early or late postoperative recurrence,and to analyze the relationship between GTF2E2 level and clinicopathological characteristics of ESCC patients with early or late postoperative recurrence.The mRNA and protein levels of GTF2E2 in human ESCC cell lines and in the normal esophageal epithelial cell line were detected by RT-PCR and Western blot.The levels of GTF2E2 in ESCC cells with knockdown or overexpression by transfection with lentiviral vectors were determined by RT-PCR and Western blot,respectively.CCK8,clony formation,EdU and flow cytometry assays were used to detect the effects of GTF2E2 on proliferation and apoptosis of ESCC cells.Transwell and wound-healing assays were employed to verify the effect of GTF2E2 on the migration and invasion abilities of ESCC cells.Immunofluorescence and Western blot were used to detect the relationship between GTF2E2 expression and the levels of molecular markers related to epithelial-mesenchymal transformation(EMT).The effect of GTF2E2 on the proliferation of ESCC cells was verified by the subcutaneous xenograft tumor model,and its role on distant metastasis and prognosis was determined by the tail vein metastasis model in nude mice.Results:The analysis of the TCGA database and tissue microarray IHC results showed that GTF2E2 expression was significantly higher in ESCC tissues than in adjacent normal tissues.Analysis of tissue microarray clinicopathological data found that the expression of GTF2E2 was closely related to the N stage and clinical stage of ESCC patients.The univariate Cox regression model revealed that N stage,clinical stage,and GTF2E2 expression were associated with the survival of ESCC patients.The multivariate analysis indicated that GTF2E2 expression was an independent prognostic factor in ESCC patients.The overall survival(OS)was significantly decreased in patients with high GTF2E2 expression.Moreover,the IHC results demonstrated that high GTF2E2 expression was positively correlated with early postoperative recurrence.By analyzing the clinicopathological data of postoperative recurrent patients,it was found that the time of postoperative recurrence was closely related to the gender and GTF2E2 score.The results of RT-PCR and Western blot showed that GTF2E2 was successfully knocked down in Eca-109 and KYSE-150 cells and GTF2E2 was overexpressed in TE-1 cells.CCK8,colony formation,EdU,and flow cytometry assays showed that ESCC cells with higher GTF2E2 expression exhibited stronger proliferation ability and reduced apoptosis compared with counterpart cells of lower GTF2E2 expression.Transwell and wound-healing assays indicated that overexpression of GTF2E2 promoted and knockdown of GTF2E2 inhibited migration and invasion in ESCC cells.Furthermore,the western blot results suggested that high GTF2E2 level upregulated the level of mesenchymal markers and downregulated the level of epithelial marker in ESCC cells.In vivo experiments confirmed that high GTF2E2 level increased the growth and metastasis of ESCC cells.Conclusion:GTF2E2 is highly expressed in ESCC and early postoperative recurrence tissues,and high GTF2E2 expression level in ESCC patients is a predictor of poor clinical outcome.Moreover,the in vitro and in vivo results collectively suggest that GTF2E2 promotes the proliferation,migration and invasion of ESCC cells and is required for tumorigenesis and metastasis.PART Ⅱ The effect and mechanism of miR-139-5p regulating GTF2E2 on the progression of ESCC cellsObjective:To explore the expression of miR-139-5p,an upstream regulatory target of GTF2E2,and its relationship with GTF2E2 in ESCC,and to explore the effects of interfering with the expression of miR-139-5p and GTF2E2 on the progression and metastasis of ESCC cells.Methods:The miRNAs regulating GTF2E2 were predicted by TargetScan,miRWALK,and StarBase websites.It was verified by RT-PCR to obtain miR-139-5p and predict its binding site sequence on GTF2E2 3’ UTR by TargetScan.Dual-luciferase assay and anti-Ago2 RNA immunoprecipitation(RIP)assay were used to verify whether miR-139-5p was a potential upstream target of GTF2E2.The expression of miR-139-5p and the correlation between miR-139-5p and GTF2E2 in ESCC were explored by TCGA database.miR-139-5p overexpression or knockdown cell lines were constructed by transfecting mimics or inhibitor into ESCC cells.The effects of miR-139-5p on GTF2E2 mRNA and protein expression were detected by RT-PCR and Western blot.Western blot was subsequently used to determine the protein levels of GTF2E2 after transfecting miR-139-5p mimics in Eca-109 and KYSE-150 cells with high endogenous GTF2E2 level and miR-139-5p inhibitor in TE-1 cells with low endogenous GTF2E2 level.The effects of co-transfection of miR-139-5p and GTF2E2 on the proliferation of ESCC cells were detected by CCK8,colony formation and EdU assays.Transwell assay was employed to verify the effects of co-transfection on the migration and invasion abilities of ESCC cells.Results:Eight miRNAs were co-predicted in the websites,and we overexpressed them in Eca-109 and KYSE-150 cells by miRNA mimics,respectively.RT-PCR revealed the level of miR-139-5p was negatively correlated with GTF2E2 in Eca-109 and KYSE-150 cells.TargetScan predicted that the position 387-394 of GTF2E2 3’ UTR was a potential binding site of miR-139-5p.The dual luciferase reporter assay suggested that miR-139-5p mimics inhibited and miR-139-5p inhibitor enhanced reporter luciferase activity,and the changes were diminished after mutating the binding position.The RIP assay suggested that Ago2 enriched miR-139-5p and 3’UTR of GTF2E2.Based on the TCGA database,the expression of miR-139-5p was shown to be significantly higher in normal tissues relative to ESCC tumor samples.The mRNA level of miR-139-5p was found in negative correlation with GTF2E2 mRNA in TCGA ESCC samples.RT-PCR and western blot assays demonstrated that overexpression of miR-139-5p downregulated and inhibition of miR-139-5p upregulated the GTF2E2 mRNA and protein level.Western blot assay suggested that transfection with miR-139-5p mimics into the GTF2E2 overexpressed ESCC cells could reverse the inhibitory effect of miR-139-5p on the protein level of GTF2E2,and transfection with miR-139-5p inhibitor into the GTF2E2 downregulated ESCC cells showed the opposite results.CCK8,EdU,and colony formation assays revealed that overexpression of GTF2E2 reversed the inhibited cell proliferation in miR-139-5p upregulated cells.Transwell assay showed that the impaired migration and invasion abilities in miR-139-5p-upregulated cells were recovered by overexpression of GTF2E2.In contrast,the enhanced proliferation,migration,and invasion abilities in miR-139-5p downregulated cells were impeded by knockdown of GTF2E2.Conclusion:miR-139-5p negatively regulates GTF2E2 expression by binding to a specific site on the 3’UTR of GTF2E2,thereby inhibiting the malignant biological behavior of ESCC cells.PART Ⅲ Identification of whole-genome DNA-binding sites and transcription targets for GTF2E2 and exploration of the mechanism of FUS mediating the GTF2E2-induced progression in ESCC cellsObjective:To clarify the molecular mechanism of GTF2E2 promoting the malignant biological behavior of ESCC,this study validated the downstream target gene FUS transcriptionally regulated by GTF2E2 and identified the affected signaling pathways.Methods:ChIP-sequencing and transcriptome sequencing were used to find the target gene FUS transcriptionally regulated by GTF2E2.According to the ChIP-seq results,the primers of FUS promoter were designed and ChIP followed by RT-PCR was performed to detect whether GTF2E2 could bind directly to the FUS promoter region.With the help of bioinformatics tools,all predicted motifs were compared with the promoter-binding region of FUS to obtain the potential sequences of binding site and mutation site.Dual-luciferase reporter assay was applied to detect the effect of GTF2E2 on the reporter luciferase activity of the FUS promoter region.The expression of FUS and the correlation between GTF2E2 and FUS in ESCC were verified by TCGA database.RT-PCR and Western blot were used to examine the effect of interfering with GTF2E2 expression on the FUS level,and to detect the FUS transfection efficiency.Western blot was used to detect the expression of FUS after upregulating FUS in GTF2E2-downregulated cells and transfecting a FUS downregulating lentivirus in GTF2E2-upregulated cells.CCK8 and EdU assays were used to detect the effect of co-transfection of GTF2E2 and FUS on the proliferation of ESCC cells.Trans well was employed to verify the effect of co-transfection on the metastatic ability of ESCC cells.IHC was performed to detect FUS expression in subcutaneous tumors,lung and liver metastases of nude mice constructed in the first part.Western blot was used to examine the effect of co-transfection of GTF2E2 and FUS on the expression of molecular markers related to EMT.GO and KEGG enrichment analysis was performed to predict the signaling pathways regulated by GTF2E2,and the pathways were validated by Western blot.Results:The overlapping gene sets were obtained between the differentially expressed genes after GTF2E2 knockdown and ChIP-seq.ChIP followed by RT-PCR confirmed that GTF2E2 bound directly to the promoter region of FUS in Eca-109 and KYSE-150 cells.Dual-luciferase reporter assay suggested that GTF2E2 downregulation inhibited and GTF2E2 upregulation enhanced reporter luciferase activity,and the changes were diminished after mutating the FUS promoter-binding position.Based on the TCGA database,ESCC tissues expressed FUS at a significantly high level and the mRNA level of FUS was found in positive correlation with GTF2E2.RT-PCR and western blot showed that GTF2E2 downregulation inhibited and GTF2E2 upregulation enhanced FUS level,and we successfully overexpressed FUS and stably knocked down FUS in Eca-109 and KYSE-150 cells.Western blot suggested that upregulated FUS in GTF2E2-downregulated cells could reverse the inhibitory effect of GTF2E2 on the level of FUS,and transfection with FUS downregulating lentivirus in GTF2E2-upregulated ESCC cells showed the opposite results.FUS overexpression restored the changes in CCK8,EdU,and transwell assay induced by GTF2E2 downregulation.In contrast,the enhanced proliferation,migration,and invasion abilities in GTF2E2-upregulated cells were impaired by FUS knockdown.The reduction of FUS expression was observed in the tumor xenografts,lung and liver metastatic nodules derived from Eca-109 and KYSE-150 cells with GTF2E2 downregulation,and the opposite trend was found in the above tissues derived from TE-1 cells with GTF2E2 upregulation by IHC.In western blot analysis,FUS downregulation or upregulation restored the changes in EMT markers induced by GTF2E2 overexpression or knockdown.Besides,the results of GO and KEGG analysis found that GTF2E2 could activate AKT/ERK/mTOR pathway.Western blot showed that FUS downregulation or upregulation restored the changes in the phosphorylation level of AKT,ERK,and mTOR induced by GTF2E2 overexpression or knockdown.Conclusion:In ESCC tissues,FUS was highly expressed and positively correlated with the GTF2E2 level.GTF2E2 positively regulated FUS expression by binding to a specific site on the promoter region of FUS,so as to promote the progression and metastasis of ESCC cells and enhance the activity of the AKT/ERK/mTOR pathway.