Effect of estrogen-related receptor silencing on miRNA protein machinery expression, global methylation, and deacetylation in bank vole (Myodes glareolus) and mouse tumor Leydig cells.

Abstract

The function of estrogen-related receptor (ERR) in testicular cells is at the beginning of exploration. Our previous findings showed that expression pattern of estrogen-related receptor (ERR) in mouse Leydig cell depends on physiological status of the cell. Exogenous hormones/hormonally active chemicals affect ERR expression. In Leydig cells in\xa0vitro, ERRα and ERRγ show opposing regulatory properties. The aim of this study was to examine the role of ERR in epigenetic processes in cells with altered level of secreted estrogens; mouse tumor Leydig cells and bank vole Leydig cells, respectively. In Leydig cells, ERRα and ERRγ were silenced via siRNA. mRNA and protein expression and protein localization of molecules required for miRNA biogenesis and function (Exportin 5, Dicer, Drosha and Argonaute 2; Ago2) were studied with the use of qRT-PCR, Western blotting, and immunohistochemistry. Global DNA methylation and histone deacetylation status together with estradiol secretion were determined with fluorometric, and immunoenzymatic assays. Regardless of ERR type knockdown in tumor Leydig cells, downregulation (P\u202f<\u202f0.05; P\u202f<\u202f0.01; P\u202f<\u202f0.001) of Exportin5, Dicer, Drosha but not Ago2 was revealed while at protein level only Drosha was downregulated (P\u202f<\u202f0.01) by both ERRα and ERRγ. Oppositely, Exportin5, Dicer and Ago2 showed ERR type-dependent regulation (downregulation; P\u202f<\u202f0.01 by ERRα and upregulation; P\u202f<\u202f0.01; P\u202f<\u202f0.001 by ERRγ). In ERR-silenced vole Leydig cells, expression of Exportin5, endonucleases and Ago2 was not changed. Immunolocalization of Dicer and Ago2 was independent of the cell origin in contrast to localization of Exportin5 and Drosha which was dependent on the cell origin and ERR type knockdown. Absence of ERR effected on cell methylation status (ERRα increased it; P\u202f<\u202f0.01 while ERRγ decreased it; P\u202f<\u202f0.01, P\u202f<\u202f0.001) but it not changed histone deacetylates activity. ERRα and ERRγ silencing decreased (P\u202f<\u202f0.01, P\u202f<\u202f0.001) estradiol secretion in both tumor and vole Leydig cells. In mouse and bank vole Leydig cell, Exportin5, Dicer, Drosha and Ago2 expression as well as methylation status are regulated by ERR in a manner related to receptor type, molecule type, cell origin and level of secreted estrogen.