Experimental verification of a conserved intronic microRNA located in the human TrkC gene with a cell type-dependent apoptotic function

Abstract

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis induction and tumorigenesis. We hypothesized that, similar to p75(NTR) receptor, some of the diverse functions of TrkC could be mediated by a microRNA (miRNA) embedded within the gene. Here, we experimentally verified the expression and processing of two bioinformatically predicted miRNAs named TrkC-miR1-5p and TrkC-miR1-3p. Transfecting a DNA fragment corresponding to the TrkC-premir1 sequence in HEK293t cells caused ~300-fold elevation in the level of mature TrkC-miR1 and also a significant downregulation of its predicted target genes. Furthermore, endogenous TrkC-miR1 was detected in several cell lines and brain tumors confirming its endogenous generation. Furthermore, its orthologous miRNA was detected in developing rat brain. Accordingly, TrkC-miR1 expression was increased during the course of neural differentiation of NT2 cell, whereas its suppression attenuated NT2 differentiation. Consistent with opposite functions of TrkC, TrkC-miR1 overexpression promoted survival and apoptosis in U87 and HEK293t cell lines, respectively. In conclusion, our data report the discovery of a new miRNA with overlapping function to TrkC.

A novel microRNA located in the TrkC gene regulates the Wnt signaling pathway and is differentially expressed in colorectal cancer specimens

“…Similar to the approaches used by others (19,27,28), exogenous production of mature TrkC-miR2 was detected when a DNA fragment corresponding to TrkC-premir2 sequence was overexpressed in SW480 cells ( Fig. 2A).…”

“…Hence, discovery of common regulatory factors for the Wnt signaling pathway may provide the possibility of cell fate manipulation in the cases of diseases like CRC and tissue regeneration. Our previous attempts resulted in discovery of hsa-miR-6165 (27) and hsa-miR-11181 (19), which are located in NGFR and TrkC genes, respectively. Herein, we gathered bioinformatics and supportive experimental evidences showing the presence of a second novel miRNA located in the TrkC gene that has the potential of being considered as a regulator of Wnt signaling pathway and also has the potential to be used as a valuable biomarker for diagnosis of CRC.…”

“…After our successful bioinformatics prediction and experimental verification of hsa-miR-11181 (19) as the first miRNA located in TrkC gene, we predicted and verified TrkC-miR2, which is located in the vicinity of hsa-miR-11181. Functional analysis of the novel TrkC-miR2 confirmed its regulatory effect on Wnt signaling pathway and suggested its potential as a CRC biomarker.…”

A novel miRNA located in the GATA4 gene regulates the expression of IGF‐1R and AKT1/2 genes and controls cell proliferation

“…Despite the great advances made in the field of RNA sequencing and related bioinformatics tools, novel functional RNA transcripts are still being added to the bank of RNA families . Relying on our previous successful discovery of novel miRNAs within important genes, we intended to search the GATA4 gene sequence for novel miRNA(s). Bioinformatics analysis suggested 90 stem‐loop structures within this genomic region, one of which, we named GATA4‐miR1 precursor, is located in the second intron of the gene and was chosen for further validations (Figure A).…”

Alternative splicing of the OCC-1 gene generates three splice variants and a novel exonic microRNA, which regulate the Wnt signaling pathway

“…1D,E). Therefore, production of an OCC-1-originated miRNA was investigated following a protocol we have used for other miRNA discoveries (Parsi et al 2012;Dokanehiifard et al 2015;Saleh et al 2016). Briefly, the predicted miRNA precursor and its mature form (miR-ex1) were PCR amplified from a cDNA library of U87 cell lines (Supplemental File S4-3).…”