The Study Of Activation Between Transcriptional Factors EGFR & STAT3 And CyclinD1 Gene Regulated By LMP

The tumor is the results of the disorder of the cell signaling pathway and its network adjustment. Abnormal activation of various oncogenic factors intracellular signal transduction pathway, and cause adjustment out of the control, furthermore induce the cycle checkpoint of the important cell disorder, which results in the growth advantage of malignant tumor cells. D-type cyclin, for short cyclinD1, shows over-expression in a variety of tumors, and overexpression cyclinD1, whose gene transcription and expression is affected by many factors regulation, is one of the essential positive regulatory protein for G1. Eukaryotic gene expression regulation is mainly on transcriptional level, which regulated by the cis-acting elements,trans-acting factors and the interaction of them who must be combined together to achieve the regulation of transcription. Eukaryotic gene cis-acting element includes the promoter, enhancer, silencer, which are the specific DNA sequences regulating gene transcription.Transcription of target genes involved in the regulation of trans-acting factors, namely transcription factors (transcriptional factor, TF) are a class of proteins that directly or indirectly identify or in combination with cis-acting elements in the corresponding transcription factor binding sites sequences involved in transcription inhibition or promotion of transcription initiation and regulation of the efficiency of downstream gene activity, EGFR and STAT3 transcription factor including which are cyclinD1 gene transcription inducer.Epidermal growth factor receptor (epidermal growth factor receptor, EGFRs, ErbB1), a type of membrane receptor tyrosine kinases is the transmembrane tyrosine kinase growth factor receptor. EGFR was found over-expression and a nuclear translocation in many tumors, EGFR ligand stimulation in all of its nuclear translocation and increases the activity of downstream genes, nuclear translocation of EGFR can bind to the promoter region and up-regulate the transcriptional activity of cyclinD1.Signal transducer and activator of transcription factor STAT3 (signal transducer and activation of transcription 3, STAT3) plays a key role for the action of variety of cytokines and growth factors signaling molecule, which not only transduce extracellular signal into the nucleus, but also be directly involved in gene regulation; phosphorylation and activation of STAT3 nuclear translocation from the cytoplasm activating a variety of related downstream of target gene expression. Many studies show that the activation of STAT3 and cyclinDl gene expression was positively correlated. EB virus (Epstein-Barr Virus, EBV) is a external pathogenic factors which is closely related to the development of a variety of tumors, with nasopharyngeal carcinoma, Burkitt's lymphoma, lung cancer and gastric cancer and other tumors of the occurrence, the encoding latent membrane proteins (latent membrane protein 1, LMP1) is an important oncogenic protein, studies have found that LMP1 can activate the expression of cyclinD1 and can control EGFR nuclear translocation and phosphorylation of STAT3 activation.Tumor development is a complex regulatory network. We will explore whether transcription factors EGFR and STAT3 mediates the regulatory role of LMP1 on cyclinD1. The significance of this study is to testify that cyclinD1 is the final target gene regulated via LMP1. Our study will provides a more extensive theory for Molecular targeted therapy of tumors.We adopted the method of liposome transfection using the reporter gene technology to detect changes of cyclinD1 promoter reporter gene activity in LMP1 positive cells,we found that the activity is significantly higher than LMP1-negative cells, and transfection with EGFR, STAT3 expressional plasmid can up-regulate cyclinDl promoter activity, whereas transfection with their respective dominant negative mutant significantly inhibited the promoter activity cyclinD1, which confirmed that regulate transcription factor EGFR, STAT3 can Transactivate cyclinDl activity regulated by LMP1. Further more,we applied siRNA technology specifically silencing transcription and expression of transcription factor EGFR, STAT3, and then used the reporter gene to detecte promoter activity of cyclinD1,it was found that EGFR, STAT3-silenced respectively and the Joint Group of cyclinD1 promoter activity decreased significantly compared with the control group, which confirmed that EGFR, STAT3 on the positive regulation of cyclinD1 from the negative aspect.Similarly, specifically silenced by siRNA technology transcription factor EGFR, STAT3,then use RT-PCR to detect cyclinD1 mRNA level, results suggest that EGFR, STAT3, respectively, and the Joint silent group cyclinD1 mRNA levels were significantly higher than the control group decreased, while detected with Western-Blot cyclinD1 protein expression changes also occurred in a consistent, proven LMP1 can regulate transcription factor EGFR, STAT3 positive regulator of mRNA cyclinD1 gene transcription and protein expression. Based on the above findings, we believe that CyclinD1 was the target gene that is regulated by LMP1 through transcription factors EGFR and STAT3; LMP1 can regulate transcription factor EGFR and STAT3 to trans-activate cyclinD1 activity, therefore up-regulate cyclinD1 gene transcription and expression.