Abstract:

Objective To discuss the regulatory role of ETS translocation variant 4 （ETV4） in the biological behavior of esophageal cancer cells， and to clarify its possible molecular mechanism. Methods The expression levels of ETV4 mRNA in esophageal cancer tissue and normal esophageal tissue were analyzed using the Gene Expression Profiling Interactive Analysis 2 （GEPIA2） database. The esophageal cancer transcriptome dataset GSE199967 was obtained from the Gene Expression Omnibus （GEO） database， and the ETV4 mRNA expression level was analyzed using R software. Based on the TCGA-ESCA dataset， the expression distribution characteristics of ETV4 were systematically evaluated according to clinical stage， pathological grade， and histological subtype using the UALCAN online platform. The human esophageal cancer KYSE-150 cells were divided into control group and ETV4 interference group （shETV4 group）； the cells were transfected with control lentivirus or ETV4-targeted short hairpin RNA （shRNA） lentivirus， and after puromycin selection， the control cells and cells with stable ETV4 interference were established. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the ETV4 mRNA expression level in the cells in two groups； Western blotting method was used to detect the ETV4 protein expression levels in the cells in two groups； cell counting kit-8 （CCK-8） method was used to detect the proliferation activities of the cells in the two groups； Transwell chamber assay was used to count the numbers of migration cells in two groups； wound healing assay was used to detect the wound healing rates of the cells in two groups. The target genes of ETV4 were predicted using the TF-Target Finder platform； potential binding sites of ETV4 in the promoter region of MDM2 binding protein （MTBP） were screened using the JASPAR database； dual-luciferase reporter assay was used to verify the regulatory effect of ETV4 on the MTBP promoter. The KYSE-150-shMTBP cells with stable MTBP interference was established； RT-qPCR method was used to detect the expression levels of MTBP， cyclin-dependent kinase inhibitor 1A （CDKN1A）， and cyclin-dependent kinase inhibitor 1B （CDKN1B） mRNA in the cells in two groups； flow cytometry was used to detect the percentages of the cells at different cell cycles. Results The bioinformatics analysis results showed that compared with normal esophageal tissue， the expression level of ETV4 mRNA in esophageal cancer tissues was increased （P<0.05）. In the TCGA-ESCA cohort， the expression levels of ETV4 mRNA in esophageal cancer tissues at clinical stages Ⅰ-Ⅳ， in tumor tissues with pathological grades G₁-G₃， and in adenocarcinoma and squamous cell carcinoma subtypes were all higher than those in normal tissue （P<0.001）； however， there was no statistically significant difference in ETV4 mRNA expression levels among the subgroups stratified by clinical stage， pathological grade， and histological subtype （P>0.05）. The KYSE-150-shETV4 cells with stable ETV4 interference was successfully constructed. Compared with control group， the ETV4 mRNA and protein expression levels in the cells in shETV4 group were decreased （P<0.001）. The CCK-8 assay results showed that compared with control group， the proliferation activity of the cells in shETV4 group was decreased （P<0.001）. The Transwell chamber assay results showed that compared with control group， the number of migration cells in shETV4 group was decreased （P<0.01）. The wound healing assay results showed that compared with control group， the wound healing rate of the cells in shETV4 group was decreased （P<0.001）. The KYSE-150-shMTBP cells with stable MTBP interference was successfully constructed. Compared with control group， the MTBP mRNA expression level in the cells in shMTBP group was decreased （P<0.001）. The bioinformatics analysis and dual-luciferase reporter assay results confirmed that MTBP was a downstream target gene of ETV4， and TFBS2 in its promoter region was the key binding site for ETV4. The RT-qPCR results showed that compared with control group， the expression levels of CDKN1A and CDKN1B mRNA in the cells in shMTBP group were increased （P<0.001）. The flow cytometry results showed that compared with control group， the percentage of cells in shMTBP group at G₀/G₁ phase was increased （P<0.001）， and the percentage of the cells at S phase was decreased （P<0.001）. Conclusion The transcription factor ETV4 activates the expression of MTBP by binding to its promoter， regulates the cell cycle， and promotes the proliferation and migration of esophageal cancer cells； its mechanism is related to the ETV4/MTBP axis.

Key words: Esophageal neoplasm, Transcription factor, ETS translocation variant 4, Cyclin-dependent kinase inhibitor 1A, Cell cycle