Mechanism Research On Tumor Suppressor DLC-1Gene In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is an infrequent cancer with specific race and geographical distribution and high incidence in Southeast Asia and Southern China. Etiological studies have demonstrated that NPC may be caused by a combination of Epstein-Barr virus infection, genetic variation and environmental factors. Because there is no obvious symptom at the early stage of NPC, most patients are diagnosed at advanced tumor stage. The pathogenesis of NPC remains unclear, which leads to difficult early diagnosis and clinical treatment failure, even if great progress has been obtained in this field. Therefore, identification of NPC diagnosis-associated biomarkers and further clarification of the mechanism of NPC carcinogenesis are of great importance for its clinical diagnosis and therapy.Deleted in liver cancer-1(DLC1) gene was first discovered in liver by representational difference analysis (RDA). In our previous work, we found that the abnormal methylation of DLC1promoter and loss of heterozygosity (LOH) of microsatellite locus are related to high-frequency down-regulation or absent expression of DLC1gene in NPC. For further research, we established5-8F cell line stably expressing DLC1(5-8F-DLC1) and control cell line transfected with empty vector (5-8F-vector). Compared with5-8F-vector cells,5-8F-DLC1cells showed decreased proliferation, invasion, metastatic abilities and the cell cycle was arrested at G0/G1phase. In this study, we further investigated the underlying mechanism of DLC1in NPC.Objectives:In order to further study DLC1mechanism in NPC, differentially expressed genes caused by up-regulation of DLC1were screened and their internal correlation was analyzed by using microarray technology.Methods:1. Gene chip was used to analyze the differences of gene expression between5-8F-DLC1and5-8F-vector cells. Then KEGG and GO softwares in MAS3.0were used to analyze the functional classification of differentially expressed genes and predict potential pathways which may be regulated by DLC1.2. RT-PCR and real-time quantitative RT-PCR (Real-time PCR) were used to identify differentially expressed genes.3. Flow cytometry was used to detect the apoptosis ratio of5-8F-DLC1cells and5-8F-vector cells.4. Mitochondrial membrane potentials of5-8F-DLC1cells and5-8F-vector cells were monitored by detection kit.5. Apoptosis-associated factors were detected by western blot. 6. Metastasis-associated factors were detected by western blot.Results:1. Chip results showed that454genes (e.g. IGFBP7, TNS1, TP53and TP63) were up-regulated and386genes were down-regulated (e.g. EGFR, KRAS and TGFβ2) in5-8F-DLC1, compared to5-8F-vector cells. The GO results showed that DLC1were related to21kinds of biological function. The KEGG results showed that DLC1were related to30pathways.2. The expression of up-regulated genes, such as WNT5A, TNS1, FHL1, S100A2, RECK, DUSP, CASP9, IGFBP7, and down-regulated genes, such as EGFR, CDCP1, KRAS, TGFβ2, Akt3, MMP7, NUC4, BCL10, PTK6, were detected by RT-PCR and Real-time PCR. The results were consistent with chip analysis.3. The apoptosis ratio of5-8F-DLC1cells was higher than that of5-8F-vector cells.4. The mitochondrial membrane potential of5-8F-DLC1cells was lower than that of5-8F-vector cells.5. The apoptosis-associated molecules, such as caspase9, caspase-3, Cytochrome c, Bax, Bcl-2, and metastasis-associated molecules, such as MMP2, MMP7, MMP9, Snail, E-cadherin, TRAF5, NF-κB, were notably altered in5-8F-DLC1cells, compared with5-8F-vector cells.Conclusions 1. DLC1participates and regulates some signal pathways. Up-regulation of DLC1influences some oncogenes and tumor suppressor genes in NPC cells.2. Up-regulation of DLC1can induce apoptosis through affecting apoptosis pathway.3. Up-regulation of DLC1can suppress metastasis of NPC cells through down-regulating PI3K/Akt/NF-κB/Snail or TRAF5/NF-KB/Snail.