Implication of TrkC‐miR2 in neurotrophin signalling pathway regulation through NGFR transcript targeting

Abstract TrkC and NGFR neurotrophin receptors are associated with cell death, cancer and differentiation. TrkC‐miR2, which is located in TrkC gene, is known to regulate Wnt signalling pathway, and its influence on other signalling pathways is under investigation. Here, through RT‐qPCR, dual‐luciferase assay and Western blotting we reveal that TrkC‐miR2 targets NGFR. Overexpression of TrkC‐miR2 also affected TrkA, TrkC, NFKB, BCL2 and Akt2 expressions involved in neurotrophin signalling pathway, and elevated survival rate of HEK293t and U87 cells was distinguished by flow cytometry and MTT assay. Consistently, an opposite expression correlation was obtained between TrkC‐miR2 and NGFR or TrkC for the duration of NT2 differentiation. Meanwhile, TrkC‐miR2 down‐regulation attenuated NT2 differentiation into neural‐like cells. Overall, here we present in silico and experimental evidence showing TrkC‐miR2 as a new controller in regulation of neurotrophin signalling pathway.

differentiation. 9 We have also introduced TrkC-miR2 at the vicinity of hsa-miR-11181 regulating Wnt pathway. 10,11 Here, we hypothesized interplay between neurotrophin receptors through the TrkC-miR2 and confirmed the effect of TrkC-miR2 on NGFR gene transcript.

| In silico analysis for prediction of TrkC-miR2 candidate target genes
In order to predict TrkC-miR2 potential target genes, we used DIANA-microT and RNAHybrid online tools. DAVID, Geneset2 and Diana-miRPath online software packages were applied to find the pathways are affected by TrkC-miR2. 12

| Cell lines and tissue samples
RPMI 1640 was used as the media for culturing HeLa, HepG2, U87MG, 1321N1, Daoy, A172 and SK-N-MC cell lines. SW480, HEK293 t and NT2 cells were maintained in DMEM-HG. These media were supplemented with 10% foetal bovine serum (FBS). 10,12 Tissue samples were freshly obtained from Imam Hospital, in Tehran, Iran, and then stored in −80 until used.

| DNA constructs
Human genomic DNA extraction, 13 TrkC-miR2 precursor cloning using the primers named Int-F and Int-R both in sense and in antisense directions, cloning of the scrambled sequence as a control construct, 14 and the strategy used for NGFR 3'UTR cloning [10][11][12]15 have been previously reported. In order to clone the region corresponding to TrkC-premir2 sense and antisense sequences, about 802bp of human TrkC-intron-14 was PCR-amplified using Int-F and Int-R primers (Table 1) and cloned into pEGFP-C1 expression vector (Clontech) downstream of GFP sequence, both in sense and in antisense directions. 11

| RNA extraction
TRIzol kit (Invitrogen) was used for total RNA extraction according to the protocol reported by its manufacturer and then was treated with DNase I purchased from Fermentas.

| RNA polyA adenylation, cDNA synthesis and RT-qPCR
In order to examine TrkC-miR2 expression level, polyA tail was initially added to the extracted total RNA by using polyA polymerase enzyme (NEB), and cDNA was then synthesized by using two anchored oligodT (Table 1) primers against both TrkC-miR2 isomiRs according to the previously reported protocol. 8,10,11 TrkC-miR2 has two isomiRs, which are different in 2 last nucleotides at their 3'ends. These two isomiRs were identified in our previous study. 11 The sequences of TrkC-miR2-5p-GC and TrkC-miR2-5p-CT are GGCTGGGGATTCTGAGCTGC and GGCTGGGGATTCTGAGCT, respectively. 1 μL of cDNA products was then applied for RT-qPCR. 8,11 U48 and GAPDH were used as the control genes.

| NT2 cell differentiation
In order to differentiate NT2 cells into neural-like cells, retinoic acid (RA) treatment was applied, according to the previously reported procedure. 17 Also, NT2 cells were transfected with the vector overexpressing anti-TrkC-premir2, 10 days after beginning differentiation. Expression alteration of Oct4, PAX6, hsa-miR-302 and hsa-miR-145 differentiation markers along with morphological changes was used for following up the successful differentiation process.

| Overexpression of TrkC-premir2
A pEGFP-C1 vector containing and expressing TrkC-miR2 precursor was surrounded in Lipofectamine 2000 purchased from Invitrogen, and utilized for transfection of the studied cell lines.
Successful transfection was then ensured via GFP microscopy one day post-transfection.

| Western blotting
After loading of 30μg of each protein on to 12% SDS-PAGE, protein transferring was performed into PVDF membrane. 5% skim milk was used for membrane blocking for 1h at room temperature. The primary antibody incubation was done for 12h at 4°C and then followed by secondary antibody incubation for 1h at RT. Amersham ECL Prime Western Blotting Detection Reagent Kit was used for visualization of blot. Western blotting data were quantitated using the TotalLab Quant software.

| Cell Cycle Analysis
The protocol performed for cell cycle analysis has been described in our previous paper. 11

| Statistical Analysis
Statistical study was completed by GraphPad Prism 5.04. In order to analyse the apoptosis experiment statistically, the Bonferroni test was employed following the repeated-measures ANOVA test.
Following the RT-qPCR assessment of NGFR endogenous expression level within SW480 cells (data not shown), TrkC-premiR2 was overexpressed in these cells, which resulted in 50% down-regulation of NGFR expression ( Figure 1B). Furthermore, Western blotting verified about 8% decrease in NGFR protein level following the TrkC-premir2 overexpression in comparison with the cells transfected by scrambled construct. Consistently, overexpression of a vector containing an antisense sequence against TrkC-premir2 resulted in NGFR protein level elevation ( Figure 1C). When NGFR 3'UTR was cloned in a vector at downstream of luciferase ORF, and coexpressed with TrkC-premir2, dual-luciferase assay showed about 50% reduction in luciferase counts supporting a direct interaction between TrkC-miR2 and 3'UTR of NGFR ( Figure 1D).

| TrkC-premir2 overexpression effect on the expression of the genes implicated in neurotrophin signalling pathway
The global consequence of TrkC-premir2 overexpression effect on downstream genes of neurotrophin signalling in U87 cell line was additionally examined using RT-qPCR. Results indicated that the expression levels of TrkA, Akt2, NF-κB and BCL2 genes have been highly elevated following the TrkC-premir2 overexpression, compared with the mock control. Nevertheless, TrkC gene expression level has been reduced within the same cells ( Figure 2).

| Detection of endogenous TrkC-miR2 in human cell lines and brain tumour specimens
Status of endogenous expression level of TrkC-miR2 was identified through RT-qPCR in astrocytoma (1321N1), glioblastoma (A172 and U87MG), medulloblastoma (Daoy) and neuroblastoma (SK-N-MC) human brain tumour cell lines ( Figure 3A). The highest expression level of TrkC-miR2 was identified in A172.
The endogenous TrkC-miR2-5p-GC isomiR was also detected in primary brain tumour specimens ( Figure 3B,C). Although, TrkC-miR2-5p-GC was relatively expressed at low level in most of the examined brain cancer biopsies, the highest expression level of it was detected in glioma biopsies ( Figure 3B,C) compared with meningioma transition type 1 tissue samples as the control. On the other hand, both TrkC (significant) and NGFR (non-significant) genes were down-regulated in the examined tumour samples ( Figure 3B). Interestingly, it seemed that TrkC-miR2-5p-GC is expressed independent of TrkC (as the TrkC-miR2 host gene) in all of the tested tumour samples ( Figure 3C).

| Anti-apoptotic effect of TrkC-premir2 in cell lines
In order to look at the outcome of TrkC-premir2 overexpression on the cell cycle status, U87 and HEK293 t cell lines were transfected overexpression in U87 was also confirmed by using annexin V test ( Figure 4C). Further, MTT assay results confirmed survival effect of TrkC-premir2 overexpression in U87 and HEK293 t cells ( Figure 4D).

| TrkC-miR2 expression alteration for the duration of NT2 cell differentiation
As TrkC is primarily expressed in neural cells, the expression status of TrkC-miR2 was explored for the period of NT2 cell differentiation towards the neural-like cells ( Figure 5). This process was successfully accomplished in three weeks, and then Sox2, Oct4A,  Figure 5A). Unlike TrkC-miR2-5p-GC, a major TrkC-miR2-5p-CT expression elevation was noticed at the second week of NT2 differentiation course, which was coincident with notable TrkC expression decline ( Figure 5B). Consistently, the expression of NGFR was reduced at the time that the expression of TrkC-miR2-5p-CT was increased ( Figure 5C).
TrkC-miR2 down-regulation effect against the neural cell-like differentiation was also investigated. To this aim, NT2 cells were first treated with RA (retinoic acid) in order to convince the cell differentiation and then were transfected with the vector containing anti-TrkC-premir2, 10 days after beginning of differentiation induction.

Real-time PCR results revealed a significant reduction (about 35% )
in TrkC-miR2 expression in these cells compared with the NT2 cells only treated with RA, as a control ( Figure 5D). Following TrkC-miR2 suppression via anti-TrkC-premir2, PAX6 and hsa-miR-145 differentiation markers were significantly down-regulated, whereas OCT4A and hsa-miR-302 pluripotent markers were up-regulated, 21 days after starting differentiation ( Figure 5E).

| D ISCUSS I ON
MiRNAs are small non-coding RNAs regulating many important cell processes such as differentiation via translation inhibition or mRNA degradation. 18 It has been reported that TrkC receptor is TrkC-miR2, which is located in TrkC gene and implicated in Wnt signalling pathway regulation. 11 Also, in our previous research, following the overexpression of TrkC-miR2 precursor, we tested the production of both predicted TrkC-miR2-5p and TrkC-miR2-3p levels using specific RT-qPCR. However, only TrkC-miR2-5P was amplifiable, probably due to the more stability of it. Consistently, the number of reads for TrkC-miR2-3p sequence in the RNAseq data F I G U R E 2 Consequence of TrkC-premir2 overexpression on the expression level of the downstream genes in neurotrophin signalling pathway. Elevated expression of the genes implicated in subpathways of neurotrophin signalling pathway following TrkC-premir2 overexpression in comparison with the cells transfected with empty vector as a negative control. Except TrkC, most of tested genes were up-regulated following overexpression of TrkC-premir2. SD of duplicate experiments is shown by the error bars. GAPDH was applied as an internal control. Expression data were normalized using 2 -ΔΔCt method was much lower than TrkC-miR2-5p. 11 Here, we presented in silico study and supportive experimental evidence revealing TrkC-miR2 has the potential to be under consideration as a main regulator implicated in neurotrophin signalling pathway.

| Association between neurotrophin signalling pathway and TrkC-miR2
MiRNAs, as the key regulatory factors in the cells, apply their effects via target mRNAs degradation or their translation inhibition. 21 RT-qPCR data revealed that NGFR (a key gene in neurotrophin signalling pathway) is down-regulated following the overexpression of TrkC-premir2 ( Figure 1B), which was then verified by performing Western blotting against NGFR ( Figure 1C). Furthermore, direct interaction of NGFR 3'UTR with TrkC-miR2 was supported by dual-luciferase assay ( Figure 1D). following TrkC-premir2 overexpression, whereas NGFR was downregulated ( Figure 1). It suggests that TrkC-miR2 might be involved in neurotrophin signalling pathway in an NGFR-independent pathway. 26

| Uncovering of TrkC-miR2 expression in brain cell lines and tumour specimens
Both isomiRs of TrkC-miR2 were identified in several cancer tissues and cell lines ( Figure 3A,

| Induction of cell survival through ectopic expression of TrkC-premir2
Flow cytometry, annexin V test and also MTT assay in U87 and HEK293 t cells transfected with a construct overexpressing TrkC-premir2 showed significant increase in the rate of cell survival ( Figure 4). These results were consistent with the survival effect of TrkC gene, which has been previously described 25

| TrkC-miR2 expression is altered in the course of NT2 differentiation into neural-like cells
As Trk genes are identified to be implicated in neural cell differ-

| CON CLUS ION
In conclusion, here we introduced accumulative evidence showing the function of TrkC-miR2 against the components of neurotrophin signalling pathway. Altogether, the presented evidence identifies this miRNA candidate as a controller of neurotrophin pathway and its implication in differentiation of neural cells.

ACK N OWLED G EM ENTS
The authors thank Dr Saman Hosseinkhani for his kind advice.

CO N FLI C T O F I NTE R E S T S
The authors declare that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

E TH I C A L A PPROVA L
Tissue samples were obtained from Imam Hospitals, Tehran/Iran. All these samples have been used with getting satisfying with all donors.
The Tarbiat Modares University Ethics Committee approved the study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.