Society of University SurgeonsAn MAPK-dependent pathway induces epithelial-mesenchymal transition via Twist activation in human breast cancer cell lines
Section snippets
RNA aptamer
The osteopontin-R3 aptamer, mutant, and all test variants were synthesized by Dharmaco (Lafayette, CO). Synthesized osteopontin-R3 and its mutant osteopontin-R3 with 2′-O-methylated purines, 5′ cholesterol, and 3′-IDT modification were used for in vitro studies. This aptamer has the usual stem-loop structure of other RNA aptamers. Osteopontin-R3 had been tested previously for affinity and specificity for osteopontin by using RNA electrophoretic mobility shift assays and its in vitro half-life
MEK1/2 inhibition decreased Twist serine phosphorylation in MB-231
The potential kinases targeting Twist were identified by sequence analysis using NetPhosK. In MB-231 control, the relative band density of Twist serine phosphorylation standardized to beta actin was 1.30. Among the kinase inhibitions, MEK1/2 inhibition caused a significant decrease in Twist serine phosphorylation (Fig 1, A) with a relative band density of 0.61 (P < .05). The remaining kinase inhibitions decreased Twist serine phosphorylation but not to the extent of MEK1/2 inhibition. The
Discussion
Osteopontin is upregulated in a variety of metastatic tumors; it plays a substantial role in tumor invasion, migration, and differentiation. In this study we built on previous findings that osteopontin mediates the serine phosphorylation of Twist, an EMT-TF. We found that inhibition of MEK1/2 kinase, a component of the MAPK cascade, causes Twist serine phosphorylation to decrease compared to MB-231 control and other kinase inhibitors. When inhibiting osteopontin with aptamer, which decreases
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