MiR‐485‐3p modulates neural stem cell differentiation and proliferation via regulating TRIP6 expression

Abstract Recent references have showed crucial roles of several miRNAs in neural stem cell differentiation and proliferation. However, the expression and role of miR‐485‐3p remains unknown. In our reference, we indicated that miR‐485‐3p expression was down‐regulated during NSCs differentiation to neural and astrocytes cell. In addition, the TRIP6 expression was up‐regulated during NSCs differentiation to neural and astrocytes cell. We carried out the dual‐luciferase reporter and found that overexpression of miR‐485‐3p decreased the luciferase activity of pmirGLO‐TRIP6‐wt but not the pmirGLO‐TRIP6‐mut. Ectopic expression of miR‐485‐3p decreased the expression of TRIP6 in NSC. Ectopic miR‐485‐3p expression suppressed the cell growth of NSCs and inhibited nestin expression of NSCs. Moreover, elevated expression of miR‐485‐3p decreased the ki‐67 and cyclin D1 expression in NSCs. Furthermore, we indicated that miR‐485‐3p reduced proliferation and induced differentiation of NSCs via targeting TRIP6 expression. These data suggested that a crucial role of miR‐485‐3p in self‐proliferation and differentiation of NSCs. Thus, altering miR‐485‐3p and TRIP6 modulation may be one promising therapy for treating with neurodegenerative and neurogenesis diseases.

including cell differentiation, growth, migration, metastasis and invasion. 26,27 More recently, growing evidence suggested that miR-NAs play important roles in the differentiation and proliferation of NSCs.
In this study, we indicated that miR-485-3p expression was down-regulated during NSCs differentiation to neural and astrocytes cell. Ectopic miR-485-3p expression suppressed the cell growth of NSCs and inhibited nestin expression of NSCs.

| Cell culture and transfection
NSCs were isolated and cultured using previous standard way. 28,29 These cells were isolated from embryos of rat and kept in growth medium supplement with bFGF, EGF and N2. This reference was agreed with our hospital's ethical board and complied with Helsinki Declaration. miR-485-3p, miR-485-3p control (scramble), pcDNAcontrol and pcDNA-TRIP6 were bought from GenePharma and then transfected to NSCs by Lipofectamine with the final concentration of 10 nmol/L.

| Dual-luciferase assay
Full-length 3′UTR of TRIP6 gene and one fragment consisting of putative miR-485-3p binding site was amplified from genomic DNA and then specific cloned into pGl3-promoter plasmid (Promega). A mutant plasmid in seed area of miR-485-3p binding site was also established. Cell was treated with one mixture of Renilla, miR-485-3p mimic, miR-NC, pLuc-3′-UTR and mut or WT pGl3-TRIP6 plasmid using Lipofectamine. Luciferase activity was detected with Promega Dual-Luciferase kit.

| Western blot analysis
Western blot assay was done using the standard way. Protein was isolated with SDS-PAGE (12%) and diverted to PVDF membrane (Millipore, USA). After blocking with milk (5%) for 2 hours, membrane was stained in primary antibodies (anti-TRIP6 and anti-GAPDH, 1:1,000, Abcam) at 4°C overnight. After washing in TBST, membrane was incubated in second antibody. Blot was observed with ECL detection reagent.

| Immunohistochemistry
Cell was fixed by paraformaldehyde (4%) in PBS for about 10 min and blocked with TritonX-100 (0.1%), FBS (1%) and serum. Then, cell was stained in primary antibodies (anti-nestin and anti-Tuj1, 1:2,000, Abcam) at 4°C overnight. After washed three times in PBS, cell was incubated with second antibody for 1 hour at 37°C. Cell was observed by Leica camera (Leica Germany).

| Statistical analysis
Result was present as the mean ± standard deviation and was calculated via SPSS 17.0 software. Student's t test was utilized to determine the difference between these two groups. A P value < .05 was regarded to be significant.

| NSCs have self-proliferation and differentiation capacity
NSCs were isolated from mouse forebrain and they can form neurosphere ( Figure 1A). After withdraw of bFGF, these cells differentiated to astrocytes and neurons ( Figure 1B). Moreover, NSCs were expressed the nestin, which is the NSC maker ( Figure 1C).

| miR-485-3p is down-regulated a during cell differentiation of NSC
miR-485-3p expression level was measured by qRT-PCR assay during differentiation of NSCs. It was shown that miR-485-3p expression was down-regulated during NSCs differentiation to neural cell ( Figure 2A). We also found that expression of miR-485-3p was reduced during NSCs differentiation to astrocytes cell ( Figure 2B).

| TRIP6 is overexpressed during NSC differentiation
TRIP6 expression level was determined by qRT-PCR assay during differentiation of NSCs. It was shown that TRIP6 expression was upregulated during NSCs differentiation to neural cell ( Figure 3A). We also found that expression of TRIP6 was overexpressed during NSCs differentiation to astrocytes cell ( Figure 3B).
F I G U R E 1 NSCs have self-proliferation and differentiation capacity. A, NSCs were isolated from mouse forebrain and they can form neurosphere. B, These cells differentiated to astrocytes and neurons. C, NSCs were expressed the nestin, which is the NSC maker

| miR-485-3p reduced proliferation and induced differentiation of NSCs
Ectopic expression of miR-485-3p suppressed the cell growth of NSCs by using MTT assay ( Figure 5A). Elevated expression of miR-485-3p decreased the expression of nestin, which is one maker of NSCs ( Figure 5B). As determined by qRT-PCR, ectopic expression of miR-485-3p suppressed the expression of ki-67 ( Figure 5C).

| miR-485-3p reduced proliferation and induced differentiation of NSCs via targeting TRIP6 expression
To further consider contribution of TRIP6 to cell biological effect of miR-485-3p on differentiation and proliferation of NSCs, we induced TRIP6 expression in the NSCs and co-transfected with miR-485-3p mimic. Ectopic expression of TRIP6 increased miR-485-3p-overexpressing NSCs proliferation with using MTT assay ( Figure 6A). Elevated expression of TRIP6 promoted the expression of nestin in miR-485-3p-overexpressing NSCs ( Figure 6B).

Elevated expression of TRIP6 enhanced the expression of ki-67
( Figure 6C) and cyclin D1 ( Figure 6D) in the miR-485-3p-overexpressing NSCs. Restoration expression of TRIP6 over-turned the function effect of miR-485-3p on NSCs differentiation ( Figure 6E). induced the Tuj1 expression, which is a maker of neuronal. F, As measured by Tuj1 immunofluorescence analysis, data showed that elevated expression of miR-485-3p increased the Tuj1 expression. *P < .05 and **P < .01 As measured by Tuj1 immunofluorescence analysis, results indicated that restoration expression of TRIP6 decreased the Tuj1 expression in the miR-485-3p-overexpressing NSCs ( Figure 6F).

| D ISCUSS I ON
In the present research, we found that miR-485-3p expression was down-regulated during NSCs differentiation to neural and astrocytes cell. In addition, the TRIP6 expression was up-regulated during NSCs differentiation to neural and astrocytes cell.
We carried out the dual-luciferase reporter and found that over- overexpression decreased breast tumour development and promoted chemosensitivity partly via modulating survivin expression. 35 Previous study indicated that miR-485-5p expression was down-regulated in the serum of NSCLC cells and patients.
TRIP6 is one member of zyxin family of the LIM proteins and is one focal adhesion element with the capacity to the shuttle between cell nucleus and surface. 38,39 TRIP6 was played roles in modulation of signal transduction and actin dynamics during cell migration and adhesion. 40,41 Increasing studies showed that TRIP6 was expressed in neurons of hippocampal and regulated biological function of neurological. 42 Previous reference indicated that TRIP6 was sufficient and essential for proliferation and self-renewal of NSCs, but suppressed NSCs differentiation. 43 Another reference suggested that miR-138-5p modulated differentiation and proliferation of NSCs via inhibiting TRIP6 expression. 44 In our reference, we exploited TargetScan software to find potential target gene of miR-485-3p and found that miR-485-3p has target sites in 3′-UTR of TRIP6. Ectopic expression of miR-485-3p decreased the expression of TRIP6 in NSC. Dual-luciferase reporter analysis was carried out to confirm that overexpression of miR-485-3p decreased the luciferase activity of pmirGLO-TRIP6-wt but not the pmirGLO-TRIP6-mut. Furthermore, we found that miR-485-3p reduced proliferation and induced differentiation of NSCs via targeting TRIP6 expression.
In summary, this reference revealed that miR-485-3p expression was down-regulated during NSCs differentiation and miR-485-3p reduced proliferation and induced differentiation of NSCs via targeting TRIP6 expression. These data suggested that a crucial role of miR-485-3p in self-proliferation and differentiation of NSCs. Thus, altering miR-485-3p and TRIP6 modulation may be one promising therapy for treating with neurodegenerative and neurogenesis diseases.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.