Eb Virus Latent Membrane Protein Lmp1 By Transcription Factors Egfr And Total Factor Tif2 Regulation Mechanism Of Cyclind1 Gene Research

Epidermal growth factor receptor (EGFR), a ubiquitously expressed receptor tyrosine kinases (RTKs), is an important factor in carcinogenesis. In previous studies, nuclear EGFR could directly bind to the cyclinD1promoter under the regulation of the oncoprotein latent membrane protein1(LMP1), but it also indicated that other factors were involved in the activation of target genes. Transcriptional intermediary factor2(TIF2) interacts with nuclear receptors and serves as transcriptional co-activator of various transcription factors regulating a variety of gene expression. It is reported that TIF2is involved in the transcriptional regulation of cyclinD1, suggesting that it may have some new relationship between EGFR and TIF2. To explore the molecular mechanisms of EBV important oncoprotein LMP1, we used the NPC (nasopharyngeal carcinoma cells) cell lines as model to investigate how EBV-LMP1is involved in the progression of cell cycle via EGFR and TIF2.To explore the interaction and subcelluar localization of EGFR and TIF2triggered by EBV LMP1, RT-PCR analysis, western blotting, Co-IP, mammalian two-hybrid assays and confocal laser analysis were performed on LMP1-negative and LMP1-positive NPC cells. The results suggested that LMP1could not regulate TIF2at mRNA level but induced protein level in a dose dependent manner. We also found that TIF2 interacted with EGFR and LMP1induced an increase of the immunocomplex level. Besides, The date indicated EGFR and TIF2co-localization was discovered in the nucleus in the presence of LMP1.To further address whether the intact complex of TIF2and EGFR was involved with gene activatin, ChIP/Re-ChIP, luciferase reporter assays, site-directed mutagenesis and RNA interference were performed. We first demonstrated that EGFR and TIF2could bind directly to the cyclinD1promoter region in vivo. Then we detected the effect of the EGFR/TIF2complex on cyclin D1at the protein, transcription level and mRNA level, the dates showed that nuclear EGFR could cooperate with TIF2target the promoter region of cyclinD1and increase its expression in the presence of LMP1. Moreover, the stimulatory effect of the EGFR/TIF2complex on cyclinD1promoter activity depends on the ATRS of the cyclin D1promoter.To understand the physiological function of the intact complex in nasopharyngeal carcinoma cells, we performed a cell growth analysis in both CNE1and CNE1-LMP1cells after transient transfection or depletion with either EGFR or TIF2or both. The data indicate that LMP1enhanced cell proliferation by stimulating cyclinDl transcription via a complex that contains nuclear EGFR and TIF2. Then we performed FACS analysis on both CNE1and CNE1-LMP1cells, The data indicated that the stimulation of LMP1could promote cell cycle progression including S and G2/M phase. Besides the intact complex of EGFR and TIF2affects cell cycle progression through the cyclinDl signaling pathway.This project for the first time observed that LMP1could induce EGFR and TIF2co-localization in the nuclei of NPC cells, and also found TIF2is a novel binding partner that interacts with the EGFR in the presence of LMP1, which, in turn, upregulates cyclinDl gene expression in nasopharyngeal carcinoma cells. Furthermore, the intact complex contributes to cell proliferation and cell cycle progression. These findings demonstrate a novel link between the activated EGFR in the nucleus and coactivators in cell cycle progression.