Experimental Verification of a Predicted Intronic MicroRNA in Human NGFR Gene with a Potential Pro-Apoptotic Function

Abstract

Neurotrophins (NTs) are a family of secreted growth factor proteins primarily involved in the regulation of survival and appropriate development of neural cells, functioning by binding to their specific (TrkA, TtkB, and TrkC) and/or common NGFR receptor. NGFR is the common receptor of NTs, binding with low-affinity to all members of the family. Among different functions assigned to NGFR, it is also involved in apoptosis induction and tumorigenesis processes. Interestingly, some of the functions of NGFR appear to be ligand-independent, suggesting a probable involvement of non-coding RNA residing within the sequence of the gene. Here, we are reporting the existence of a conserved putative microRNA, named Hsa-mir-6165 [EBI accession#: FR873488]. Transfection of a DNA segment corresponding to the pre-mir-6165 sequence in Hela cell line caused the generation of mature exogenous mir-6165 (a ∼200,000 fold overexpression). Furthermore, using specific primers, we succeeded to detect the endogenous expression of mir-6165 in several glioma cell lines and glioma primary tumors known to express NGFR. Similar to the pro-apoptotic role of NGFR in some cell types, overexpression of pre-mir-6165 in U87 cell line resulted in an elevated rate of apoptosis. Moreover, coordinated with the increased level of mir-6165 in the transfected U87 cell line, two of its predicted target genes (Pkd1 and DAGLA) were significantly down-regulated. The latter findings suggest that some of the previously attributed functions of NGFR could be explained indirectly by co-transcription of mir-6165 in the cells.

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

“…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.…”

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

“…Therefore, HEK293t cells were used for exogenous expression of TrkC-mir1 precursor to test for the production of mature miRNA similar to the approach used by others [18,28] (Fig. 2a).…”

“…According to the protocol [18], cDNA was made from polyadenylated RNAs and each cDNA sample was amplified using specific primers (Table 1) in a 7500 RealTime PCR system (Applied Biosystems), using the following conditions for 45 cycles: stage 1, 95°C for 5 s; stage 2, 60°C for 20 s; stage 3, 72°C for 34 s. RT-qPCR was performed according to MIQE guidelines using SYBRPremix Ex TaqTM II PCR Mastermix (Takara, Japan) in experimental duplicates. Expression data were analyzed using endogenous U48 small nucleolar RNA (SNORD48), B2m and GAPDH as the reference genes and were normalized using the 2 ÀDC t and 2 ÀDDC t method [19].…”

“…Error bars indicate SD of duplicate experiments. U48 RNA and GAPDH were used as internal controls for the amplifications or lack of partner(s) or target(s) of miRNA in each cell line [18,50,51].…”

<p>MicroRNA Profile Identifies miR-6165 Could Suppress Gastric Cancer Migration and Invasion by Targeting STRN4</p>

“…7,8 MiR-6165 was reported to express in brain tumorderived cell lines and tissues, and overexpressing miR-6165 in brain tumor-derived cell line induced the cancer cell apoptosis. 9 Studies have shown that the same cancer-related miRNA could be involved in different cancers. [10][11][12] However, little is known about the role of miR-6165 in GC.…”