To reveal the molecular mechanism by which nuclear epidermal growth factor receptor (nEGFR) synergistically regulates the expression of cell migration-inducing protein (CEMIP) by forming a complex with the transcription factor Yin Yang 1 (YY1), and to investigate the biological functions of the nEGFR-YY1-CEMIP signaling axis in invasion of hepatocellular carcinoma (HCC).

After HCC cells were serum-starved for 24 h, the cells were treated with 100 ng/mL EGF. Thus, the cells were divided into a control group and EGF-treated groups at different time points. Nuclear expression and localization changes of EGFR were detected by Western blotting and immunofluorescence (IF). To investigate the interaction between nEGFR and YY1, their nuclear colocalization and interaction were examined by IF and co-immunoprecipitation (Co-IP), respectively. Transcriptional profiling was performed using RNA sequencing (RNA-seq) to identify differentially expressed genes at the genome-wide level. Combined with Gene Ontology (GO) functional enrichment analysis and transcription factor binding profiles via using the JASPAR database, CEMIP was identified as a candidate target gene. To validate the regulatory mechanism, the following experimental groups were established, Control, EGF, siYY1, and siYY1+EGF. The expression of CEMIP at protein and mRNA levels was detected by Western blotting and RT-qPCR. To elucidate the molecular mechanism of nEGFR/YY1 binding to the CEMIP promoter, the control and EGF-treated groups were established. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was performed to assess the enrichment of nEGFR/YY1 at the CEMIP promoter region. Luciferase reporter assay was conducted following transfection with either wild-type EGFR (EGFR-WT), nuclear localization-deficient mutant (EGFR-dNLS), YY1 overexpression plasmid (YY1-OE), or dominant-negative YY1 mutant (YY1-DN) to evaluate changes in promoter activity. Subsequently, cell migration and invasion capabilities were evaluated using scratch wound healing assay and Transwell assay, while hyaluronic acid (HA) level was quantified by ELISA. The expression of matrix metalloproteinases (MMP2/9) was analyzed via Western blotting to assess the regulatory role of the nEGFR/YY1-CEMIP axis in the migration and invasion of HCC cells. By analyzing the CEMIP expression profiles in HCC patients from National Center for Biotechnology Information (NCBI) public databases, its potential association with tumor metastasis risk was validated.

Western blotting and IF demonstrated that EGF treatment significantly induced nuclear translocation of EGFR, peaking at 30 min (P<0.001). Co-IP and IF assays indicated both physical interaction and nuclear co-localization between nEGFR and YY1. RNA-seq analysis identified CEMIP as a significantly differentially expressed gene. GO enrichment analysis revealed that CEMIP was significantly enriched in biological processes related to cell invasion promotion. JASPAR prediction identified conserved YY1 potential binding region within the CEMIP promoter region. Western blot and RT-qPCR analyses confirmed that EGF treatment up-regulated CEMIP at both protein and mRNA levels (P<0.05). Notably, YY1 knockdown significantly suppressed CEMIP expression, while exogenous EGF supplementation restored CEMIP level in YY1-deficient cells (P<0.05). ChIP-qPCR analysis demonstrated specific enrichment of the nEGFR/YY1 complex at the CEMIP promoter region, with EGF stimulation significantly enhancing its binding affinity (P<0.001). Luciferase reporter assay confirmed that nEGFR/YY1 robustly enhanced CEMIP promoter activity (P<0.01), while either the EGFR-dNLS or the YY1-DN substantially attenuated this transcriptional activation. Functional phenotyping showed that the nEGFR/YY1-CEMIP axis significantly enhanced the migration and invasion of HCC cells by promoting HA catabolism and up-regulating MMP2/9 expression (P<0.05). Analysis of NCBI datasets revealed that CEMIP expression was significantly up-regulated in HCC tumor tissues than adjacent normal tissues (P<0.001). Moreover, HCC patients with elevated CEMIP expression exhibited higher risk of metastasis (P<0.001).

nEGFR promotes HCC invasion by forming a transcriptional complex with YY1 to cooperatively activate CEMIP expression.