| [Background and Objective]Metastasis is responsible for as much as90%of cancer-associated mortality. Cancer metastasis is a very complicated biological process involving many sequential steps. Epithelial-Mesenchymal Transition (EMT) occurs during mammal’s embryogenesis, and its role in the development of cancer metastasis has been a breakthrough in the filed of metastasis research. During EMT, epithelial cells lose their characteristic cell-cell adhesion structures, change their polarity, and become motile and invasive, which cause them assume a mesenchymal phenotype.Plenty of experiments carried out in solid tumors have demonstrated that carcinoma cells can acquire a mesenchymal phenotype through EMT and initiate invasion and metastasis. This discovery provided significant opportunity and challenge for the metastasis studying. Researchers can benefit from the cell model (especially the growth factors induced EMT), which can closely mimic the EMT process in vitro, and appreciate the complexity of EMT regulation network.Nasopharyngeal carcinoma (NPC) is a malignancy that occurs at high incidence in southern China.95%of the NPC is poorly differentiated squamous with high chance to recurrence and metastasis. Through the growth factor (such as EGF and TGF-β1) induced NPC cell EMT model, we had the possibility to analysis the mRNA and microRNA expression profile changes during EMT, and to investigate the regulation mechanism as well as to screen the biomarkers for cancer invasion and metastasis.[Methods]NPC cell HK-1was treated with EGF (final concentration50ng/ml) or TGF-β1(final concentration10ng/ml) for36h to inducing EMT. Cellular RNAs were isolated and then mRNA and microRNA expression were profiled by means of high-throughput chips.Pathways, GO and Gene Set Enrichment Analysis (GSEA) were employed to investigate the mRNA expression profiles. The changes of EGFR, TGF-β and EMT signaling were also detected. The association between "EGF signature" and "TGF-β signature" with NPC clinic specimens was analyzed.microRNA expression profiles were analyzed and the function of differential expressed microRNAs during EMT were probed. Their target genes were also predicted and screened.A "microRNA-target gene-pathway" network has been constructed based on the differential expressed microRNA and their target genes during EMT.[Results]Upon treatment with EGF and TGF-β1for36h, the HK-1cells undergo EMT and their mRNA and microRNA expression profiles had been significantly modulated. There were383genes and8microRNAs up-regulated upon EGF induction, while161genes and4microRNAs were down-regulated.124genes and12microRNAs were up-regulated upon TGF-β1induction, while165genes and28microRNAs were down-regulated.Most of the co-upregulated genes among the EGF and TGF-β1treatment group were related to cell motility, while most of the co-downregulated genes were related to cell cycle and division. The differential expressed genes were involved in the pathways such as: Jak-STAT signaling, cytokine-cytokine receptor interaction, focal adhesion, TGF-β signaling, apoptosis, ECM-receptor interaction and ErbB signaling pathway etc.Significant similarities between EGF-induced stress response and inflammation/infection-induced stress response had been observed by means of GSEA. TGF-β1mostly affected the gene set of Tp53and Tp63targets. EGF has a big role on the gene sets of transcription factor (such as IRF, STAT5, HMGIY, BACH2, HNF4A and E47); while TGF-β1affect the gene sets of transcription factor (such as ATF3, SP1, TCF11, AML and AP1). Further study revealed that EGF-induced EMT was dependent on the ETS family-EGR1axis, while TGF-β1-induced EMT was dependent on the SMAD transcription factors network. We carried a correlations analysis of EMT-inducer profile (i.e. EGF signature) or TGF-β-inducer profile (i.e. TGF-β signature) with the gene expression profiles of patient tumors in a large cohort of NPC patients. Pearson correlation coefficients were calculated and found that EMT-inducer profile displayed a higher correlation to the NPC patient specimens profiles, while TGF-β-inducer profile had a lower correlation comparing to control group. This result suggested that EGF signature contributed to NPC carcinogenesis, while TGF-(3signature did not.By searching for the upstream sequences of the differential expressed microRNAs and their binding transcription factors, we revealed that there was obvious difference between mRNA and microRNA transcription regulation system during growth factor-induced EMT.We constructed a "microRNA-target gene-pathway" network for the regulation mechanism during EMT, by taking advantage of the microRNA and mRNA expression profile data and the cross-talk between them.miR-505and miR-1207-5p were up-regulated during the growth factor-induced EMT in NPC cells. Preliminary study showed their tumor suppressor function, which implies the roles of miR-505and miR-1207-5p in conferring robustness to EMT processes.[Conclusion] 1. Both of EGF and TGF-β1can induce NPC cells undergoing EMT, and have significant impact on mRNA and microRNA expression profiles.2. EGF-induced EMT was dependent on the ETS family-EGR1axis, while TGF-β1-induced EMT was dependent on the SMAD transcription factors network.3. We described a "microRNA-target gene-pathway" network underlies the EMT.4. miR-505and miR-1207-5p conferred robustness to growth factor-induced EMT processes in NPC cells. |
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