Downregulation of microRNA-200 in EBV-associated gastric carcinoma.

EBV-associated gastric carcinoma is a distinct gastric carcinoma subtype with characteristic morphologic features similar to those of cells that undergo epithelial-to-mesenchymal transition. The effect of microRNA abnormalities in carcinogenesis was investigated by measuring the expression of the epithelial-to-mesenchymal transition-related microRNAs, miR-200a and miR-200b, in 36 surgically resected gastric carcinomas using quantitative reverse transcription-PCR analysis. MiR-200 family expression was decreased in EBV-associated gastric carcinoma, as compared with that in EBV-negative carcinoma. Downregulation of the miR-200 family was found in gastric carcinoma cell lines infected with recombinant EBV (MKN74-EBV, MKN7-EBV, and NUGC3-EBV), accompanied by the loss of cell adhesion, reduction of E-cadherin expression, and upregulation of ZEB1 and ZEB2. E-cadherin expression was partially restored by transfection of EBV-infected cells with miR-200 family precursors. Reverse transcription-PCR analysis of primary precursors of miR-200 (pri-miR-200) revealed that the transcription of pri-miR-200 was decreased in EBV-infected cells. Transfection of MKN74 cells with BARF0, EBNA1, and LMP2A resulted in a decrease of pri-miR-200, whereas transfection with EBV-encoded small RNA (EBER) did not. These four latent genes contributed to the downregulation of the mature miR-200 family and the subsequent upregulation of ZEB1/ZEB2, resulting in the reduction of E-cadherin expression. These findings indicate that all the latency type I genes have a synergetic effect on the downregulation of the miR-200 family that leads to reduced E-cadherin expression, which is a crucial step in the carcinogenesis of EBV-associated gastric carcinoma.