No reduction in luciferase activities was observed of 293 T cells transfected with the mutant TrkB 3 UTR. Our finding that TrkB may be targeted by miR 204 5p led us to further delineate the actions of miR 204 5p on TrkB in endometrial cancer cells. Our RT PCR assays revealed that IshikawaTrkB cells transfected with miR 204 5 m had markedly reduced TrkB mRNA transcript protein inhibitors levels compared to IshikawaTrkB cells transfected with scrambled miRNA. Immunoblotting assays also showed that IshikawaTrkB cells transfected with miR 204 m had markedly reduced TrkB levels. Furthermore, HEC 1BshTrkB cells transfected with a miR 204 5p inhibitor had markedly higher TrkB mRNA transcript levels than HEC 1BshTrkB cells transfected with scrambled miRNA.

Our immunoblotting assays additionally showed that HEC 1BshTrkB cells transfected with miR 204i also had noticeably increased levels of TrkB. These findings in dicated Inhibitors,Modulators,Libraries that miR 204 5p negatively regulated the expres sion of TrkB. TrkB represses miR 204 5p expression by activating the JAK2/STAT3 pathway in vitro Our microRNA microarray Inhibitors,Modulators,Libraries showed that miR 204 5p was downregulated in IshikawaTrkB cells. Further examination by qRT PCR assays revealed that miR 204 5p expression was noticeably suppressed in IshikawaTrkB cells, but markedly Inhibitors,Modulators,Libraries enhanced in HEC 1BshTrkB cells. These results further corroborated the microRNA array findings and in dicated that TrkB suppresses miR 204 5p expression. Our qRT PCR assays also showed that the mRNA transcript levels of TRPM3, which shares the same regulatory motif for transcription with miR 204, were significantly decreased in IshikawaTrkB cells, but markedly elevated in HEC 1BshTrkB cells, lending support to an inhibitory role of TrkB at the TRPM3/miR 204 5p locus.

The TrkB signaling pathway overlaps with the ERK/ MAPK or JAK2/STAT3 signaling pathway in myeloma or endometrial cancer cells, which prompted us to examine whether TrkB suppressed miR 204 5p expres sion via these signaling pathways. Our immunoblotting Inhibitors,Modulators,Libraries as says revealed that TrkB overexpression in IshikawaTrkB cells noticeably increased the phosphorylation of JAK2 and STAT3, which, however, was aborted by TrkB knockdown in HEC Inhibitors,Modulators,Libraries 1BshTrkB cells. No obvious activation was found by TrkB overexpression in ERK/MAPK pathway, and effect of its inhibition on miR 204 5p expression.

Moreover, inhibition of STAT3 by small interference RNA elevated the miR 204 5p Wortmannin DNA-PK expression in IshikawaTrkB and HEC 1B cells, indicating that, indeed, TrkB activates the JAK2/STAT3 pathway in endometrial cancer cells. We further examined whether phospho STAT3 could bind to three putative STAT binding sites that are located near the TRPM3 promoter region upstream of miR 204 5p. Our ChIP assays showed that phospho STAT3 could bind to all the three putative STAT binding sites in Ishika waTrkB and HEC 1B cells, with the greatest binding to DW2.