Further Study Of The Tumor Suppressing Mechnisms Of Cytochrome B5 Reductase 2 In Nasopharyngeal Carcinoma

Cytochrome b5 reductase 2 (CYB5R2) Contains one ferredoxin reductase-type flavin adenine dinucleotide-binding domain and belongs to the flavoprotein pyridine nucleotide cytochrome reductase family. It is involved in multiple biological processes, such as electron transportation, oxidization-reduction, lipid metabolism, extension and desaturation of fatty acid, biosynthesis of cholesterol, P450-mediated internal drug metabolism and methemoglobin reduction. Our previous study found that CYB5R2 is transcriptional inactivated due to promoter hypermethylation in nasopharyngeal carcinoma(NPC). Re-expression of CYB5R2 could inhibit the proliferation, migration, clonogenicity ability of NPC cells. Moreover, in vivo tests indicated that ectopic expression of CYB5R2 reduces the tumorigenicity of NPC cells. However, the detail tumor suppressing mechanisms of CYB5R2 is far from clear. In the present study, we aim to further explore the molecular mechanism of CYB5R2 in the tumorigenesis and development of NPC.We first established transient CYB5R2-expressing NPC cell lines, then Human Cancer PathwayFinder PCR Array was applied to analyze the the effect of CYB5R2 on the expression of 84 genes involved in the six tumor-related biological pathways. We find that the transcriptional level of 10 genes were significantly regulated by CYB5R2. Among them, the expression of CDKN2A, ITGB3, MTSS and IFNB1 were increased while ITGB5, VEGF, IGF1, TEK and TGFBR1 decreased. The mRNA expression level of pro-apoptotic gene, FAS, increases by 1.81 times. The mRNA expression of CDKN2A, which is involved in cell cycle control and related to DNA damage repairing, increases by 1.57 times. The mRNA expression of ITGBS and MTSS1 that are related to cellular adhesion and invasion, increases by 1.68 times and 1.59 times respectively, while the mRNA expression of ITGB5 decreases by 8.72 times. The mRNA expression of IFNB1, an anti-angiogenesis gene, increases by 1.84 times, while the mRNA expression of VEGF, IGF1, TEK, TGFBR1, angiogenesis genes, decreases by 1.69 times,1.57 times,10.28 times and 1.71 times respectively. Real-time quantitative RT-PCR was used to verity the expression of the 10 genes. The results tended to be the same as the result of PCR array.To further analyze the biological functions of CYB5R2, we conducted flow cytometric analysis, caspase activity assay, transwell assay and chicken chorioallantoic membrane (CAM) experiment. Our findings revealed that when compare to control group, ectopic expression of CYB5R2 in NPC cells could induce cell apoptotic, increase the activity of caspase 8 and caspase 9, lead to cell cycle arrest and inhibit cell invasion and metastasis. What’s more, ectopic expression of CYB5R2 in NPC cells in CAM model significantly inhibited angiogenesis. Down-regulation of VEGF was found in CYB5R2-overexpressing NPC cells by HE straining and immunohistochemical staining.In conclusion, CYB5R2 could up-regulate the genes participating angiogenesis and down-regulate VEGF, thereby suppressing the tumorigenesis...