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    • Further analysis sought to understand the mechanism associat

    2024-04-13

    Further analysis sought to understand the mechanism associated with the autotaxin-mediated increase of miR-489-3p in circulation. Based upon sequence complementarity, DIANA TarBase predicted miR-489-3p targets MAPK2K1, or MEK1, an oncogene widely dysregulated in cancer (Fig. 4A). To test this relationship, exogenous addition of a miR-489-3p mimic to parental OVCAR-3 resulted in a marked reduction in total MEK1, ERK1/2, pMEK1 and pERK1/2 (Fig. 4B and C). Although OVCAR-3-ATX-wt and OVCAR-3-ATX-mut cell lines displayed decreases in pMEK1, a notable increase in total MEK1 was observed when OVCAR-3-ATX-wt WAY-262611 sale were treated with miR-489-3p with limited changes to ERK1/2 or pERK1/2. We further confirmed that miR-489-3p is secreted from cells in vitro (Fig. 4D and E). Taken together, these data confirm the prediction of TarBase in that miR-489-3p targets MEK1 (Fig. 4F) and also that upregulated autotaxin overrides the function of miR-489-3p and increases MEK1. To verify that MEK1 was increased in tumor tissue as a result of miR-489-3p export, we assessed the protein levels in specimens. Comparing tumor tissue to liver tissue of non-tumor AT-ATX animals, there was a significant increase in MEK1 (*p < 0.05) in the tumors (Fig. 5A). Further, immunohistochemistry data confirms MEK1 expression in tumor tissues compared to corresponding unstained sections (Fig. 5B). Localization of MEK1 staining to certain regions of the tissue may be attributable to the exportation of miR-489-3p from the tumors into circulation, resulting in the lack of MEK1 inhibition in those areas. We next determined the effects of miR-489-3p on the activity of an emerging class of inhibitors in ovarian cancer. OVCAR-3 cells transiently transfected with 50 nM miR-489-3p followed by 48 h of treatment with the PARP inhibitors Olaparib and Niraparib at 25 μM and 50 μM resulted in a significant decrease in cell viability, particularly in the presence of miR-489-3p, either alone or in combination with either PARP inhibitor (Fig. 6A). This indicates that miR-489-3p may have an additive affect with Olaparib or Niraparib to further reduce cell viability. SDS-PAGE analysis displays a similar effect of miR-489-3p and PARP inhibitors at the protein level on the inhibition of MEK1 in OVCAR-3 (Fig. 6B). Since Olaparib is FDA-approved for use in triple-negative breast cancer, MDA-MB-231 cells were also subjected to miR-489-3p and PARP inhibitor co-treatment, resulting in a marked decrease in MEK1 expression (Fig. 6B). However, in HER2-positive, ER/PR-negative BT474 breast cancer cells, miR-489-3p had negligible effects on MEK1 alone, but synergized together with Olaparib to inhibit MEK1 (Fig. 6C). Niraparib treatment on BT474 cells was highly toxic and therefore excluded from analysis (data not shown).
    Discussion MiR-489-3p has been shown to target several oncogenes and largely appears to have a tumor suppressive role across various cancer types. For example, studies in pancreatic cancer show a repression of miR-489-3p expression driven by KRAS and inflammatory NF-kB signaling [32]. Similarly, miR-489-3p has been found to be significantly decreased in hepatocellular carcinoma patients with late recurrence compared to early recurrence, suggesting its role in tumor suppression and possibly, progression free survival [30]. Our data shows that exogenous miR-489-3p addition inhibits MEK1, which is a target for miR-489-3p predicted by DIANA TarBase miRPath v.3. Interestingly, a substantial increase in MEK1 among OVCAR-3-ATX-wt cells treated with miR-489-3p (Fig. 4B and C) indicates the likely presence of an override mechanism or feedback loop triggered by autotaxin. Indeed, autotaxin reverses the effects of miR-489-3p from inhibiting MEK levels to increasing MEK levels. Other studies have demonstrated that a feedback loop encompassing HER2, SHP2 and MAPK is inhibited by miR-489-3p to control breast cancer cell proliferation, further lending significance to the role of miR-489-3p in our results [31].