CircESRP1 Enhances Metastasis And Epithelial-Mesenchymal Transition In Endometrial Cancer Via The MiR-874-3p/CPEB4 Axis

Objective:To investigate the expression and localization of circESRP1 in endometrial cancer tissues by studying its own characteristics.To investigate the effect of up-or downregulation of circESRP1 on the biological behavior of endometrial cancer cells.To investigate the mechanism of action of circESRP1 in regulating endometrial cancer cell migration,invasion,and Epithelial-to-mesenchymal Transition（EMT）through the sponge adsorption of miR-874-3p.To explore miR-874-3p regulates the biological behavior of endometrial cancer cells through binding to CPEB4.To investigate the regulatory mechanism of circESRP1 on EMT in endometrial cancer cells.Methods:Bioinformatics analysis was performed to screen out circRNAs with increased expression in endometrial cancer.Real-time fluorescence quantitative polymerase chain reaction（qRT-PCR）was used to verify the expression of different circRNAs in endometrial cancer tissues.Sanger sequencing was performed to detect the shear sites of circESRP1.Genomic DNA（gDNA）,actinomycin assay and enzyme elimination assay were applied to explore the self-characterization of circESRP1.Nucleoplasmic separation assay and immunoprecipitation（FISH）were utilized to determine the sublocalization of circESRP1 in endometrial cancer cell lines.CircESRP1 small interfering RNAs（shRNAs）and overexpression lentivirus were constructed and transfected with Ishikawa cells and RL95-2 cells.Cell activity was detected by cell counting kit（CCK8）.Cell proliferation was examined by EdU assay and clone formation assay.Cell invasion and migration were detected by Transwell assay and wound healing assay.E-cadherin and Vimentin protein levels were detected by Western Blot assay.Online databases（miRanda,RNAhybrid,CSCD）were used to predict miRNAs binding to circESRP1,and RNA Pulldown assay was used to validate which miRNA was sponge-adsorbed to circESRP1.The binding relationship of miR-874-3p to circESRP1RNA was reversed by protein immunoprecipitation（RIP）assay.The specific binding site of circESRP1 to miR-874-3p was probed by using a dual-luciferase reporter gene assay.The relative expression of miR-874-3p in endometrial cancer tissues and normal endometrial tissues was verified using qRT-PCR and the correlation between circESRP1 and miR 874-3p correlation was analyzed.The role of miR-874-3p on endometrial cancer cell migration and invasion was validated by Transwell assay.Online databases（miRanda,RNAhybrid,circBank,circInteractome）were used to predict genes interacting with miR-874-3p.qRT-PCR,Western Blot and luciferase reporter gene assay were used to explore the downstream target genes of miR-874-3p.qRT-PCR was applied to detect the difference expression of CPEB4 in endometrial cancer tissues and normal endometrium.Small interfering RNA of CPEB4（sh-CPEB4）was constructed to explore the effect of CPEB4 on the migration and invasion ability of endometrial cancer cells.CircESRP1 lentiviral overexpression virus was constructed.Ishikawa cells and RL95-2 cells were transfected,and the stable transfected cell lines were constructed.miR-874-3p mimic/miR-874-3p mimic control（miR-874-3p mimic NC）or CPEB4 small interfering RNA（sh-CPEB4）/sh-CPEB4 control（sh-CPEB4 NC）were transfected in Ishikawa cells and RL95-2 cells.The migration and invasion ability of Ishikawa cells and RL95-2 cells were examined by Transwell assay,and the protein expressions of EMT-related proteins（Ecadherin and Vimentin）were analyzed using Western Blot.6-week-old female Babl/c nude mice were selected for subcutaneous implantation and Ishikawa cells were injected subcutaneously,and the tumour sizes were measured weekly.28 days later,mice were sacrificed.The subcutaneous tumours were removed,weighed,and the sizes were measured.The cell morphology of the subcutaneous tumour tissues was observed using HE staining.The protein levels of E-cadherin and Vimentin of ubcutaneous tumour tissues were detected by immunohistochemistry.Results:1.Bioinformatics analysis revealed that 692 circRNAs were differentially expressed in endometrial cancer relative to normal endometrial tissues.hsa_circ₀133954,hsa_circ₀084927,hsa_circ₀085173,and hsa_circ₀001681 in endometrial cancer tissues were highly expressed.The expressions of hsa_circ₀133954,hsa_circ₀084927,hsa_circ₀085173,and hsa_circ₀001681 in endometrial cancer were verified using qRTPCR,and the most significant difference was found for hsa_circ₀084927（circESRP1）（p<0.001）.2.The circESRP1 reverse shear site were verified by Sanger sequencing;amplification was performed in cDNA and gDNA using divergent and convergent primers;convergent primers were amplified in both cDNA and gDNA,divergent primers were amplified in cDNA and no amplification in gDNA.CircESRP1 was more stable than ESRP1 mRNA by actinomycin assay and RNase R digestion assay.CircESRP1 was more abundant in the cytoplasm than in the nucleus and was mainly localized in the cytoplasm.3.CircESPRl silenced,the proliferation,migration,and invasion ability of Ishikawa cells and RL95-2 cells were decreased.The protein expression of E-cadherin in Ishikawa cells and RL95-2 cells was increased with circESRP1 knocking out,and the protein expression of Vimentin was decreased.4.CircESRP1 overexpressed,the proliferation,migration,and invasion ability of Ishikawa cells and RL95-2 cells were significantly enhanced.The protein expression level of E-cadherin was decreased in Ishikawa cells and RL95-2 cells and the protein level of Vimentin was increased when circESRP1 was overexpressed.5.MiR-8743p,miR-4694-5p and miR-4732-3p were predicted by online databases.6.In Ishikawa cells and RL95-2 cells,miR-874-3p was pulled down more than the other two miRNAs by immunoprecipitation with a biotin-labelled probe,which targeted the circESRP1 backspliced sequence（p<0.001）.3.Specific enrichment of circESRP1 was detected by qRTPCR in the miR-874-3p probe group compared with the control probe,which was consistent with previous results in Ishikawa cells and RL95-2 cells（p<0.01）.7.In Ishikawa cells and RL95-2 cells,circESRP1 and miR-874-3p were pulled down more abundantly in the AGO2 group than in the IgG group（p<0.001）.Luciferase activity was reduced more in the circESRP1-WT group than in the mutant group by the miR-874-3p mimic.8.MiR-874-3p expression level was lower in endometrial cancer tissues than in normal endometrial tissues and was negatively correlated with circESRP1（R2=0.1433,p=0.0428）.9.The migration and invasion ability of Ishikawa cells and RL95-2 cells were diminished by miR-874-3p mimic;the migration and invasion ability of Ishikawa cells and RL95-2 cells were enhanced by miR-874-3p inhibitor.10.The mRNA and protein levels of CPEB4 but not CRCP,which were predicted to bind to miR-874-3p,were significantly increased with miR-874-3p inhibitor in Ishikawa cells and RL95-2 cells.The mRNA and protein levels of CPEB4 but not CRCP,were significantly reduced with miR-874-3p mimic in Ishikawa cells and RL952 cells.11.The activity of the luciferase reporter containing the wild-type CPEB4 sequence（WT）was significantly decreased by the miR-874-3p mimic;however,this effect was eliminated upon mutation of the miR-874-3p binding site（p<0.01）.12.The mRNA level of CPEB4 was higher in EC tissues than in normal tissues.13.The migration and invasion ability of Ishikawa cells and RL95-2 cells were diminished by silencing CPEB4.E-cadherin protein level was increased and Vimentin protein level was reduced.13.The migration and invasion ability of Ishikawa cells and RL95-2 cells were enhanced after overexpression of circESRP1,and their migration and invasion ability were reversed by miR-874-3p mimic or sh-CPEB4.14.E-cadherin protein level was reduced in cells overexpressing circESRP1 and were reserved by miR-874-3p mimic or sh-CPEB4;Vimentin protein levels were elevated in Ishikawa cells and RL95-2 cells overexpressing circESRP1 and were restored by miR-874-3p mimic or sh-CPEB4.15.Compared to the control group,the volumes and weights of subcutaneous graft tumours were decreased by silencing circESRP1.The protein expressions of Vimentin and CPEB4 were down-regulated and the protein expression of Ecadherin was increased in subcutaneous graft tumour tissues.The volumes and weights of subcutaneous graft tumours were increased by circESRPl overexpressed.Vimentin and CPEB4 protein expressions were upregulated in tumor tissues and E-cadherin protein expression was decreased.The volumes and weights of subcutaneous graft tumours and the protein expressions of Vimentin,CPEB4,and E-cadherin were reserved by sh-CPEB4.Conclusion:1.CircESRP1 is a circular RNA formed by reverse shearing of exons 7,8 and 9 of ESPR1,which is more stable than ESPT1 mRNA.circESRP1 expression in endometrial cancer tissues was significantly higher than that in normal endometrial tissues.2.CircESRP1 enhances the proliferation,migration,and invasion ability of endometrial cancer cells.3.CircESRP1 may regulate the migration and invasion ability of endometrial cancer cells through sponge adsorption of miR-874-3p.4.MiR-874-3p regulates the biological behavior of endometrial cancer cells through CPEB4.5.CircESRP1 regulates the migration and invasion ability of endometrial cancer cells through miR-874-3p/CPEB4 axis.circESRP1 can increase the migration and invasion ability of endometrial cancer cells through CPEB4 in vivo.