C/EBPβ Is Implicated In Esophageal Squamous Cell Carcinoma By Regulating MicroRNAs

Background and ObjectiveEsophageal cancer is one of the most common human malignancies, characterized byhigh incidence and mortality rate. Esophageal cancer includes esophageal squamous cell(ESCC) and esophageal adenocarcinoma (EA). ESCC is the predominant histologicalsubtype of esophageal cancer in China. At present, the prognosis and outcome ofesophageal cancer is very poor, the overall5-year survival rate is only15%~25%. So it isvery important to investigate the mechanism of ESCC and find novel target for earlydiagnosis and therapy.ESCC has been generally considered as one of the epithelial malignant tumor. Both anaggressive potential and distant metastasis are the major causes of treatment failure, most ofthe patients die of metastasis and recurrence after tumor resection. It has been reported thatepithelial mesenchymal transition (EMT) is a primary molecular event during tumormetastasis, which is correlated with aggressiveness and poor prognosis. In the past severaldecades, a series of transcription factors (TFs), including β-catenin, snail, slug and twistwere shown to be involved in EMT of several cancers, and might be promising therapeutictargets. The critical EMT modulators largely vary in different types of cancer, and therefore,the central context-dependent regulatory molecules participating in EMT and metastasis inESCC should be clearly defined.Apart from EMT, there are some other biological events such as abnormal cellularproliferation and apoptosis also contribute to tumor malignant behavior. A broken balancebetween proliferation and apoptosis is a common phenomenon during the tumordevelopment. The excessive proliferation and reduced apoptosis make tumor growth out ofcontrol. It is of great clinical importance to study the dysregulatory mechanism ofproliferation and apoptosis in esophageal cancer cells.Also known as nuclear factor for IL-6, the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) is generally regarded as a regulator of cell proliferation anddifferentiation and is implicated in many biological activities including adipogenesis,inflammatory responses and tumor progression. C/EBPβ exists in three isoforms: LAP1(Liver-enriched transcriptional activator protein1), LAP2(Liver-enriched transcriptionalactivator protein2) and LIP (Liver-enriched transcriptional inhibitory protein). Emergingevidence has shown that LAP1and LAP2are generally transcriptional activators, whereasLIP frequently acts as a dominant-negative inhibitor of transcription, and that dysregulationof C/EBPβ is markedly correlated with malignancy of several tumors including glioma,Wilms tumors, renal cell carcinoma and ovarian tumor. It has been reported that C/EBPβ isimplicated in many biological activities during tumor progression including EMT, invasion,proliferation and apoptosis. As far as we know, however, the roles of C/EBPβ gene inesophageal cancer have been rarely reported.Apart from transcription factors, increasing evidence indicates that miRNAs regulatekey genetic programs in cancers as well. In particular, miR-203is defined as a tumorsuppressor for controlling EMT and metastasis. Additionally, miRNA profiling has shownthat miR-146b is overexpressed in many cancer types, such as thyroid tumor, breast cancer,colorectal cancer and melanoma. In ESCC, higher miR-146b expression level is anindependent risk factor for prognosis. Although both miR-203and miR-146b have greatdiagnostic value in predicting outcome of ESCC for miR-203down-regulation andmiR-146b up-regulation are associated with poor overall survival rate, the biological rolefor miR-203and miR-146b in esophageal cancer cells and the underlying mechanism ofmiR-203and miR-146b dysregulation are still obscure.In our prior study, we examined the protein levels of C/EBPβ in clinical tissuesand probed the significance of the C/EBPβ dysregulation in ESCC. Next, we furtherexplored the functional roles of C/EBPβ on EMT, invasion, migration, proliferation andapoptosis in esophageal cancer cells and the related mechanisms.Methods and contents:1. Western blot analysis and densitometric analysis were applied to detect C/EBPβprotein levels in esophageal tumor tissues and the corresponding adjacent non-tumor tissuesfrom36surgical ESCC patients.2. Immunofluorescence stainings were applied to detect C/EBPβ and vimentin proteins in normal tissue sections and esophageal squamous cell carcinoma tissue sections.3. Microscope was applied to observe the morphological changes of esophageal cancerEC109and EC9706cells after exposure to EGF condition; Western blot analysis anddensitometric analysis were applied to detect expression changes of EMT markers andC/EBPβ after exposure to EGF condition. RT-qPCR analysis was applied to measureC/EBPβ mRNA levels after exposure to EGF condition.4. EC109cells were transfected with C/EBPβ siRNA in EGF condition; EC109cellswere transfected with C/EBPβ expressing vectors. Western blot analysis and densitometricanalysis were applied to detect EMT markers.5. EC109cells were transfected with C/EBPβ expressing vectors. Migration andinvasion ability were measured by transwell assay; the proliferation ability was detected byEdU assay; cellular apoptosis was measured by flow cytometric analysis.6. EC109cells were transfected with miR-203mimic in EGF condition. Western blotanalysis and densitometric analysis were applied to detect EMT markers.7. EC109cells were transfected with miR-203inhibitor. Migration and invasion abilitywere measured by transwell assay.8. EC109cells were transfected with C/EBPβ siRNA before miR-203and miR-203target gene expressions were detected by RT-qPCR. EC109cells were transfected withC/EBPβ LIP expressing vector before RT-qPCR analysis for miR-203expression detection.9. The putative C/EBPβ binding motifs located within the5-KB regions upstream ofTSSs of miR-203loci were predicted with the MAPPER database. ChIP and EMSA assayswere performed to determine the C/EBPβ enrichment.10. To investigate the functional consequences of C/EBPβ binding sites on miR-203transcriptional activaty, wild type and mutant reporters were constructed and dual-luciferasereporter assays were performed.11. EC109cells were transfected with miR-146b mimic. Cellular proliferation andapoptosis were measured by EdU assay and flow cytometric analysis, respectively.12. EC109cells were transfected with C/EBPβ expressing vectors before RT-qPCRanalysis for miR-146b expression detection.13. The putative C/EBPβ binding motifs located within the5-KB regions upstream ofTSSs of miR-146b loci were predicted with the MAPPER database. ChIP assay was performed to determine the C/EBPβ enrichment.14. To investigate the functional consequences of C/EBPβ binding sites on miR-146btranscriptional activaty, wild type and mutant reporters were constructed and dual-luciferasereporter assays were performed.Results:1. The dysregulation of C/EBPβ LIP and LIP: LAP ratio in esophageal squamous cellcarcinoma (ESCC) is associated with lymph node metastasis.2. C/EBPβ was co-expressed with vimentin in ESSC samples.3. Both EC109and EC9706cells exhibited typical polygonal cobblestone epithelialmorphology when grown in normal culture, whereas exposure to EGF induced obviousmorphological changes, including a more scattered pattern of growth, and some cellsexhibited an elongated spindle shape. EGF treatment increased the expression ofmesenchymal markers (β-catenin and vimentin), and decreased the expression of epithelialmarkers (ZO-1and pan-cytokeratin). EGF treatment not only upregulated C/EBPβ LIP andLIP: LAP ratio but also increased total transcripts of C/EBPβ in parallel with protein levels.4. C/EBPβ interference significantly increased the expressions of epithelial markersZO-1and pan-cytokeratin, and decreased the expressions of mesenchymal markersβ-catenin and vimentin in EGF conditions. On the contrary, ectopic expression of C/EBPβLIP altered EMT-related marker levels in a dose-dependent manner. Meanwhile, forcedoverexpression of C/EBPβ LAP1and C/EBPβ LAP2could not change EMT-related markerlevels.5. Compared with the empty vector, overexpression of C/EBPβ LIP significantlyenhanced cellular migration and invasion potential; overexpression of C/EBPβ LAP2increased EdU positive cells and decreased cell apoptosis rate.6. Similar to the effects of C/EBPβ disruption, the miR-203mimic blockedEMT-related marker gene alterations in the EGF condition.7. miR-203inhibitor significantly enhanced the migration and invasion potential ofEC109cells.8. EGF significantly reduced miR-203expression; Disruption of C/EBPβ attenuatedthe suppression of EGF on miR-203and decreased the expressions of miR-203downstreamgenes E2F1, BIRC5, snail and slug. Ectopic overexpression of C/EBPβ LIP was sufficient to decrease miR-203in a dose-dependent manner.9. Using in silico analysis, we found the presence of putative C/EBPβ binding motifslocated within the5-KB regions upstream of TSSs of miR-203loci. The miR-203locuscontains two putative C/EBPβ binding sites, located at-3628/-3615(203R1) and-3266/-3253(203R2).The C/EBPβ enrichment increased in both C/EBPβ motifs upstreamof miR-203after EGF treatment. In EC109cells, LAP/LIP heterodimers were the mainform that binds to miR-203regulatory regions in control group, and EGF treatment causeda dramatic increase in the binding of LIP/LIP homodimers.10. Transfection of C/EBPβ LIP vector significantly decreased the activity ofwild-type203CNS-Luc without affecting mutant m203CNS-Luc activity.11. Compared with mimic control，miR-146b mimic increased EdU positive cells anddecreased cell apoptosis rate.12. Ectopic overexpression of C/EBPβ LAP2increased miR-146b level in adose-dependent manner, while C/EBPβ LAP1and C/EBPβ LIP overexpression could notchange miR-146b expression.13. Using in silico analysis, we found the presence of putative C/EBPβ binding motifslocated within the5-KB regions upstream of TSSs of miR-146b loci. The miR-146b locuscontains two putative C/EBPβ binding sites, located at-1736/-1722(146R1)and-1492/-1478(146R2). Compared with negative group (IgG), the C/EBPβ enrichmentwas higher in both C/EBPβ motifs upstream of miR-146b locus. There was no C/EBPβenrichment in two other negative regions Neg.ctrl1and Neg.ctrl2.14. Transfection of C/EBPβ LAP2vector significantly increased the activity ofwild-type146CNS-Luc without affecting mutant m146CNS-Luc activity.Conclusion:1. The dysregulation of C/EBPβ expression in ESCC is associated with lymph nodemetastasis.2. C/EBPβ is involved in EGF-induced EMT, cellular invasion/migration, proliferationand apoptosis.3. C/EBPβ LIP mediates EGF-induced EMT and promotes cellular invasion/migrationby positively regulating miR-203.4. C/EBPβ LAP2promotes cell proliferation and inhibits cell apoptosis by negatively regulating miR-146b.In summary, the aberrantly expressed C/EBPβ may play an important role in thedevelopment of ESCC. By regulating miRNAs, C/EBPβ is implicated in many biologicalactivities, including EMT, cell invasion, migration, proliferation and apoptosis, therebypromoting tumor malignancy. Our research is helpful for further understanding the complexmechanisms in the development of esophageal squamous cell cancer and provides newevidence for ESCC diagnosis and therapy. C/EBPβ may represent an attractive target fortherapeutic intervention and prognosis.