A novel microRNA located in TrkC gene regulates Wnt signaling pathway and is differentially expressed in colorectal cancer specimens [Cell Biology]

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis, differentiation and tumorigenesis. TrkC diverse functions might be attributed to the hypothetical non-coding RNAs, embedded within the gene. Using bioinformatics approaches, a novel miRNA named TrkC-miR2 was predicted within TrkC gene, capable of regulating Wnt pathway. For experimental verification of this miRNA, predicted TrkC-premir2 sequence was overexpressed in SW480 cells, which led to the detection of two mature TrkC-miR2 isomiRs, and their endogenous forms were detected in human cell lines as well. Later, an independent promoter was deduced for TrkC-miR2 following the treatment of HCT116 cells with 5-Azacytidine, which resulted in differential expression of TrkC-miR2 and TrkC host gene. RT-qPCR and luciferase assay indicated that APC2 gene is targeted by TrkC-miR2 and Wnt signaling is upregulated. Also, Wnt-inhibition by using small molecules, along with TrkC-miR2 overexpression and TOP/FOP flash assay, confirmed the positive effect of TrkC-miR2 on the Wnt pathway. Consistently, TrkC-miR2 overexpression promoted SW480 cell survival, detected by flow cytometry, MTT assay and crystal violate analysis. RT-qPCR analysis revealed that TrkC-miR2 is significantly upregulated (~70 times) in colorectal tumor tissues compared to their normal pairs. Moreover, TrkC-miR2 expression level discriminated grades of tumor malignancies, which was consistent with its endogenous levels in HCT116, HT29 and SW480 colorectal cancer cell lines. Finally, an opposite expression pattern was observed for TrkC-miR2 and APC2 gene in CRC specimens. In conclusion, here we introduce TrkC-miR2 as a novel regulator of Wnt signaling which might be a candidate oncogenic CRC biomarker.

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