The results showed that treat ment of A549 cells with the COX 2 inhibitor

The Dicer1e transcript consisted of 2,822 nucleotides that were predicted AP24534 価格 to encode an 820 amino acid truncated protein form of Dicer1 comprising both RNase III domains, a nuclear localization signal and the dsRNA binding domain. the nuclear fraction in all cell types examined. No differ ences in cellular localization were detected between the oral cancer cell lines and HOKs. However, in the OSCC cell lines exhibiting high expression levels of Dicer1e, Dicer1e was also detected in the cytoplasm. Interestingly, the Doyle et al. study found that a C terminal fragment of Dicer1, a construct structurally similar to Dicer1e, could localize to the cytoplasm, but failed to localize to the nucleus of HeLa cells.<br><br> In an effort to resolve this discre pancy and to confirm our observed nuclear localization of Dicer1e, the localization of a recombinant FLAG tagged Dicer1e AT7519 溶解度 protein was subsequently tested in transiently trans fected HeLa cells. Indirect immunofluorescent analyses of these transfected cells confirmed nuclear localization of the recombinant Dicer1e protein, with nuclei verified by DAPI staining. Additionally, the recombinant Dicer1e protein was also found to be either equally distributed between both nuclear and cytoplasmic compartments or exclusively localized within the cytoplasm of trans fected cells. Together, these data demonstrated that Dicer1e could primarily localize to the nucleus in cells, especially in low Dicer1e expressing cells with the ability to also accumulate in the cytoplasm, particularly in high Dicer1e expressing cells or in transfected HeLa cells overexpressing a recom binant form of the Dicer1e protein.<br><br> Moreover, these data suggested that the 210 amino acid sequence N terminal of the first RNase III domain within Dicer1e appeared to be important for enabling the nuclear accumulation of Dicer1e. Depletion of Dicer1e expression inhibits the cell proliferation and clonogenic potential of oral cancer cell lines To determine whether the higher levels of Dicer1e contri bute to buy Alisertib the proliferation and clonogenic potential of oral cancer cells, we employed siRNA knockdown of Dicer1e expression. Using an siRNA designed to specifically target a unique sequence in Dicer1e mRNA, we found the Dicer1e protein levels to be considerably reduced compared to cells transiently transfected with a control non targeting siRNA 48 hours post treatment.<br><br> Of note, the Dicer1e silencing effect was also assessed 7 and 9 days post transfection and ob served to persist up to 9 days, with maximum silencing occurring 7 days post transfection for several of the cell lines. To ensure the tar geting specificity of siDicer1e, we also analyzed the protein levels of Dicer1 and found Dicer1 expression to be un affected upon treatment with siDicer1e compared to siNT. Having de monstrated that siDicer1e was capable of suppressing Dicer1e protein levels, but not Dicer1, we next examined the effects of Dicer1e depletion on cancer cell proliferation. The cell proliferation experiment was carried out where CAL 27, SCC 4, and SCC 25 cells were either transfected with siNT or siDicer1e, after which cell numbers were assayed 2, 4, and 7 days post transfection. The growth curves showed that silencing of Dicer1e sig nificantly inhibited cell proliferation over a period of 7 days in all treated oral cancer cell lines compared to control siNT transfected cells.