Gene set enrichment analysis revealed NF-κB signaling as a potential downstream regulator of Efp.

(A and B) RNA from siEfp #A- or siControl-treated Ishikawa cells were subjected to microarray analysis and, subsequently, gene set enrichment analysis (GSEA) was performed using the GSEA software (https://software.broadinstitute.org/gsea/index.jsp). GSEA revealed that the gene set ‘TNFA SIGNALING VIA NFKB’ was enriched among the genes that had low expressions in siEfp #A-treated Ishikawa cells in the microarrays (A). Top 10 downregulated genes among “TNFA SIGNALING VIA NFKB” gene set members in siEfp #A-treated Ishikawa cells are shown (B). (C) Silencing of Efp decreases the expression of IL6ST and IL18 genes. Ishikawa and HEC-1A cells were transfected with siEfp #A, #B, or siControl, and the expression levels of IL6ST and IL18 mRNAs were quantified by qRT-PCR. Data are presented as means ± s.d. (n = 3). **, P D and E) Decrease of IL6ST and IL18 mRNA levels in siEfp #A-treated tumors in vivo. RNAs were isolated from the siEfp #A- or siControl-treated tumors generated in the subcutaneous inoculation experiment using Ishikawa cells shown in Fig 5(D) and orthotopic inoculation experiment using HEC-1A cells shown in Fig 6(E). The expression levels of IL6ST and IL18 mRNAs were quantified by qRT-PCR. Data are presented as means ± s.d. (n = 3). *, P P F) Model for the role of Efp in endometrial cancer. Efp could act as an E3 ubiquitin ligase to promote degradation of 14-3-3σ protein. Moreover, Efp is suggested to promote endometrial cancer by enhancing the NF-κB-mediated transcription and its donwnstream signals including IL6ST and IL18.