Results TrkB overexpression is associated with global changes in miRNA expression in endometrial cancer cells We selleck chemicals llc examined TrkB protein and mRNA expression in the normal endometrium and endometrial cancer tissues using laser capture microdissection /quantitative reverse transcription polymerase chain reaction and immunohistochemistry. Our RT PCR assays of normal and endometrial cancer cells captured by LCM revealed that mRNA transcript levels of TrkB appeared to be higher in the tumors than in the normal Inhibitors,Modulators,Libraries specimens, but overall the difference in TrkB mRNA expression between endometrial cancer tissues and the normal endometrium was not Inhibitors,Modulators,Libraries statistically significant. Immunohistochemistry, on the other hand, demonstrated Inhibitors,Modulators,Libraries a markedly higher expression of TrkB in endometrial cancer tissues compared with the normal endometrium.

The data suggest that TrkB is upregulated mainly at the posttranscriptional level in human endometrial cancer tissues. We were interested in whether changes in TrkB expres sion impacted on the Inhibitors,Modulators,Libraries global profile of miRNA expression in endometrial cancer cells. Our microRNA array consisting of 1347 capture probes for mature human miRNAs showed marked changes in the expression of 98 miRNAs in HEC 1BshTrkB cells whose TrkB expression was suppressed by short hairpin RNA against TrkB compared to HEC 1B cells. TrkB overexpression also caused marked changes in 126 miRNAs in IshikawaTrkB cells ectopi cally expressing TrkB compared to Ishikawa cells. Consistently, 76 miRNAs were found among the differen tially expressed miRNAs in both HEC 1BshTrkB cells and IshikawaTrkB cells.

MiR 204 5p is a negative modulator of TrkB expression in endometrial cancer cells Separately, we surveyed the 3 UTR of the TrkB promoter using three target prediction algorithms, TargetScan, Pictar and MiRanda, to identify candidate miRNAs which may act as posttranscriptional Inhibitors,Modulators,Libraries modulators of TrkB expression. TargetScan, Pictar and MiRanda revealed 4, 3 and 37 candidate miRNAs, respectively. Examination of the 76 differentially expressed miRNAs as identified by microarray analysis showed that miR 211 5p and miR 204 5p were the only two candidate miRNAs that were also identified by all three target prediction algorithms to potentially bind to the 3 UTR of the TrkB. Furthermore, the miR 204 5p targeting site within the 3 UTR of TrkB was highly conserved across five mammalian species.

These intriguing findings suggest that miR 211 5p and miR 204 5p and TrkB are likely mutual modula tors of their expression. MiR 204 5p is reportedly dysregulated in endometrial car cinoma. references To examine whether miR 204 5p modulated TrkB expression, we constructed vectors containing a wildtype or mutant TrkB 3 UTR directly fused down stream of the Firefly luciferase reporter gene. The wildtype or mutant vector was co transfected into 293 T cells with miR 204 5p mimic or its scrambled control.