RU 13. IRF8 is silenced by CpG methylation in RCC and inhibits RCC cell growth

Purpose: To investigate the promoter methylation status of Interferon regulatory factor 8 (IRF8) and its relation with IRF8 transcriptional silencing or downregulation in renal cell carcinoma (RCC), which further clarifies the effects of IRF8 on RCC tumorigenesis and identifies a new epigenetics biomarker for early diagnosis of RCC.

Materials and Methods: IRF8 expression and methylation was detected by semi-quantitative RT-PCR and methylation specific PCR (MSP) in multiple RCC cell lines and primary tumors. We examined the cell cycle of IRF8-transfected RCC cell lines by flow cytometry method, and assessed the colony formation ability of IRF8 by colony formation assay. We also tested the target genes of IRF8 by quantitative PCR.

Results: IRF8 was highly expressed in normal human adult tissues, but was greatly down-regulated or transcriptional silenced in RCC cell lines. The methylation specific PCR revealed that IRF8 promoter was methylated in 50% RCC cell lines, and in 31.25% renal primary tumors. The silencing of IRF8 in RCC, due to aberrant DNA methylation, could be reversed by pharmacologic demethylation reagent AZA. Ecotopic expression of IRF8 inhibited RCC cell lines (786-O, A498) colony formation, and caused G2/M arrest, suggesting its tumor-suppressive function in RCC cell lines. In addition, Caspase8, a target gene of IRF8, was up-regulated 3 times in IRF8-transfected RCC cell lines.

Conclusions: IRF8 exerts tumor-suppressive function in RCC, and its promoter methylation could lead to its transcriptional silencing or downregulation. Our results indicated that IRF8 may serve as a biomarker for early detection and prognosis prediction of RCC.