LncRNA Riken‐201 and Riken‐203 modulates neural development by regulating the Sox6 through sequestering miRNAs

Abstract Objectives Long non‐coding RNAs (LncRNAs) play important roles in epigenetic regulatory function during the development processes. In this study, we found that through alternative splicing, LncRNA C130071C03Riken variants Riken‐201 (Riken‐201) and Riken‐203 (Riken‐203) are both expressed highly in brain, and increase gradually during neural differentiation. However, the function of Rik‐201 and Rik‐203 is unknown. Materials and methods Embryonic stem cells (ESCs); RNA sequencing; gene expression of mRNAs, LncRNAs and miRNAs; over‐expression and RNA interference of genes; flow cytometry; real‐time quantity PCR; and Western blot were used in the studies. RNA pull‐down assay and PCR were employed to detect any miRNA that attached to Rik‐201 and Rik‐203. The binding of miRNA with mRNA of Sox6 was presented by the luciferase assay. Results Repression of Rik‐201 and Rik‐203 inhibited neural differentiation from mouse embryonic stem cells. Moreover, Rik‐201 and Rik‐203 functioned as the competing endogenous RNA (ceRNA) to repress the function of miR‐96 and miR‐467a‐3p, respectively, and modulate the expression of Sox6 to further regulate neural differentiation. Knockout of the Rik‐203 and Rik‐201 induced high ratio of brain developmental retardation. Further we found that C/EBPβ might potentially activated the transcription of Rik‐201 and Rik‐203. Conclusions These findings identify the functional role of Rik‐201 and Rik‐203 in facilitating neural differentiation and further brain development, and elucidate the underlying miRNAs‐Sox6‐associated molecular mechanisms.


| INTRODUC TI ON
Neural differentiation is an important developmental event during gastrulation of the embryonic development. 1 The abnormal neural differentiation results in severe neural tube defects, 2 which may further induce the risk of post-natal lethality or lifelong disability. 3 However, there are still many regulatory mechanisms should be investigated.
Embryonic stem cells (ESCs) have the capacity to generate many differentiated types of cells in the body and are excellent sources for regenerative medicine and development research. Neural stem cells (NSCs) differentiated from the ESCs could be used for treatment of Alzheimer's and Parkinson's disease. 4,5 Previous study indicated that Sirt1 regulated neural differentiation of mouse embryonic stem cells (mESCs). 3 Sox2 mediates proliferation and neurogenesis of neural precursors derived from the ESCs by regulating Lin28. 6 In addition to the protein-coding genes, long non-coding RNAs (LncRNAs) function as critical modulators in many biological processes over the course of human development by regulating complicated and multiple signalling pathways. [7][8][9][10] Analyses of the genome-wide transcriptome indicate that there are thousands of LncRNAs. 11 Many of the LncRNAs are expressed spatially and temporally, contributing to neural differentiation, further brain development or nerve-related diseases. 8,[12][13][14][15][16] However, very little is known about the specific function of LncRNAs, particularly the role of related regulating pathway in neural differentiation.
MicroRNAs (miRNAs) are 20-25 nucleotide (nt) non-coding RNAs that partially bind to the mRNA 3′ untranslated regions (3′UTRs) to induce the translational repression. 17,18 miRNAs are involved in many biological and physiological processes, such as the regulation of disease formation, and embryonic development. [19][20][21][22] miRNAs are abundant in the CNS and are critically involved in all stages of neural differentiation during brain development. 23,24 miR-96 has been reported to repress neural induction from human embryonic stem cells (hESCs) by targeting Pax6, the critical regulator of neural differentiation. 25 Whether miR-96 regulated the neural differentiation and the regulatory mechanism remains unclear.
LncRNAs can be the competing endogenous RNA (ceRNA) to function as miRNA sponge by sequestrating the miRNAs and releasing them away from mRNA 3′UTR binding sites to further repress the function of miRNAs. 26,27 LncRNA ND regulates expression of Notch genes by sequestering miR-143-3p. 8 LncRNAN2 was found to harbour neurogenesis-associated miRNAs, miR-125b and let-7, in its intronic regions. 28 A recent study identified the novel Rik-201 and its homologous gene ECONEXIN, 29 and demonstrated its ceRNA functional role in gliomagenesis. There is, however, still much that needs to be understood about the numerous mechanisms of LncRNAs in regulating neural development by functioning as the ceRNA, and inducing further influence of the downstream signalling pathways.
SOX6 is a critical neural differentiation-related gene.It was found to regulate the specification of dopamine neurons 30 and also controls dorsal progenitor identity and inter-neuron diversity during neocortical development. 31 Disruption of Sox6 is associated with dopa-responsive movement disorder. 32 Whether Sox6 could be regulated by miRNAs during the neural differentiation remains unknown.

| Neural differentiation form mESCs
We performed the neural differentiation of mESCs by using the methods described in our previous study. 33

| Sevoflurane anaesthesia for mice
We used C57BL/J6 mice at post-natal day 6 (Shanghai SLAC

| Nuclear and cytoplasm extraction
We performed the nuclear and cytoplasm extraction studies using the methods described in our previous study. 36  Primer of Stem-loop reverse transcription: 5′-GTCGTAT 5′-AGTGCGTGTCGTGGAGTCG-3′.

| Over-expression of Sox6 by pcDNA3.1-Sox6 vector
The whole RNA was isolated, and inverse transcription to cDNA was then performed using cDNA Synthesis Kit (TaKaRa). Sox6

| Over-expression of miR-96 and miR-467a-3p
We transfected the miR-96 and miR-467a-3p mimics overexpressed vector (Biogot Technology, Nanjing, China) into the embryonic bodies derived from 46c mESCs during the neural differentiate on every 48 hours to maintain the certain concentration using Lipofectamine 2000 (Thermo Fischer Scientific), following the instructions given to overexpress the miR-96 and miR-467a-3p. The expression level of over-expression is detected by qRT-PCR.

| Statistics
The data were presented as mean + standard deviation (SD) with more than three independent experiments. The significance of statistics was determined by a student's t test, one-way ANOVA and two-way ANOVA. * and #P < 0.05, **and ##P < 0.01, *** and ###P < 0.001. The studies employed two-tailed hypothesis, and statistically significant P values were <0.05. We used GraphPad

| LncRNA Rik-201 and Rik-203 is critically involved in mouse neural differentiation
We performed neural differentiation of the mESCs to neural progenitor cells (NPCs), and found that alternative splicing of LnRNA-Rik (C130071C03Riken) variants, Rik-201 and Rik-203, was up-regulated during neural differentiation from day 3 ( Figure 1A).
Additionally, these two variants were also more enriched in the embryonic brain than in other tissues at day 14.5 ( Figure 1B). We constructed the Rik-201 and Rik-203 knockdown mESCs lines, respectively, and found that there was no significant change in the stemness marker expression of the mESCs compared with the control mESC with only empty pLKO-tet-on vector ( Figure 1C,D). markers Sox1 and Nestin detected at day 7 were also downregulated ( Figure 1H).

| miR-96 and miR-467a-3p combine with Rik-201 and Rik-203 respectively and both repress neural differentiation
A recent study identified the novel Rik-201 and its homologous gene ECONEXIN and demonstrated its ceRNA functional role in gliomagenesis. 29 We also found that most of Rik-201, and part of Rik-203, are located in the cytoplasm (Figure 2A  target in previous study 42 ( Figure S2B). Inhibition of miR-467a-3p restored the neural differentiation that was repressed by Rik-203 knockdown ( Figure 3D). FACS studies further indicated that knockdown of Rik-203 reduced the rate of Sox1-positive cells, and that miRNA-467a-3p inhibitor rescued such reductions ( Figure 3E) and also the marker expression of the NPCs ( Figure 3F).

| miR-96 and miR-467a-3p target the Sox6 to control over the neural differentiation
We detected the potential downstream targets of the miR-96 and miR-467a-3p by using online miRNA target predication tools (mi-Randa, targetscan, miRBD) and found that miR-96 and miR-467a-3p could both target Sox6 3′UTR ( Figure 4A). During the neural differentiation, Sox6 was up-regulated ( Figure 4B). Luciferase reporter assay also showed that miR-96 or miR-467a-3p binds to the wild-type 3′UTR of Sox6 to downregulate the luciferase expression, but has no influence on the mutant one ( Figure 4C). We further confirmed that over-expression of miR-96 and miR-467a-3p downregulated the Sox6 protein expression in the NPCs ( Figure 4D). We then performed the rescue experiment to confirm that over-expression of Sox6 restored miR-96 inducing downregulation level of protein ( Figure S3A) and also blocked the neural differentiation inhibition caused by miR-96, which was detected at day 7 ( Figure 4E,F). The decrease of the related NPCs markers influenced by miR-96 was also rescued ( Figure 4G). Additionally, over-expression of Sox6 blocked miR-467a-3p inducing downregulation level of protein ( Figure S3B) to restore the neural differentiation inhibited by miR-467a-3p ( Figure 4H,I) and also the regulation of NPCs markers ( Figure 4J).
F I G U R E 4 miR-96 and miR-467a-3p target the Sox6 to regulate the neural differentiation. A, The expression of Sox6 during the neural differentiation from ESCs to NPCs. B, Target validation of the binding of Sox6 3′UTR by miR-96 or miR-467a-3p. C, Luciferase report assay indicated that miR-96 or miR-467a-3p targeted wild-type Sox6 3′UTR but not mutant UTR. D, Over-expression of miR-96 or miR-467a-3p decreased the protein level of Sox6. E, The Sox6 restored the neural differentiation repression and the Sox1-positive cells decrease caused by over-expression of miR-96. F, The quantification of Sox1-positive cells using FACS indicated that over-expression of miR-96 reduced Sox1-positive cells, and that Sox6 mitigated such reductions. G, RT-PCR showed that the mRNA levels of Sox1 and Nestin repressed by over-expression of miR-96 were also rescued by Sox6. H, The Sox6 restored the neural differentiation repression and the Sox1-positive cells decrease caused by over-expression of miR-467a-3p. I, The quantification of Sox1-positive cells using FACS indicated that over-expression of miR-467a-3p reduced Sox1-positive cells, and that Sox6 mitigated such reductions. J, RT-PCR showed that the mRNA levels of Sox1 and Nestin repressed by over-expression of miR-467a-3p were also rescued by Sox6. The scale bar represents 100 μm. Ctrl, control; Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; by one-way ANOVA (A, C, F, G, I and J). n = 3 independent experiments

| Sox6 restored the neural differentiation repressed by downregulating Rik-201 or Rik-203
A knockdown of Rik-201 or Rik-203 inhibited the Sox6 expression during neural differentiation, which is detected at day 7 ( Figure 5A,B). Over-expression of Sox6 also restored the inhibition of process of neural differentiation ( Figure 5C), reduction of   Figure 6B).

| Rik-201 and Rik-203 may be regulated by C/EBPβ and associated with anaesthesia-induced neurotoxicity
C/EBPβ is a transcription factor that is involved in neurogenesis. 43 By performing a double luciferase assay, the transcription factor

| D ISCUSS I ON
We found that Rik-201 and Rik-203 were significantly higher expressed in the mouse brain than in other tissues of E14.5 embryo.
This miR-96 families have been reported to be the most differentially expressed miRNA between epidermis and neuroectoderm, and could repress neural induction from human embryonic stem cells. 25 We also found that over-expression of miR-96 repressed mouse neural differentiation.
miR-467a-3p has been reported to be related to breast cancer growth, 47 and to target some skin-related genes. 48 However, there is currently no available data on the neural regulation of the miR-467a-3p. We found that over-expression of miR-467a-3p also reduced neural differentiation.
Rescue experiments further showed the miR-96 and miR-467a-3p are the downstream targets of Rik-201 and Rik-203 during neural differentiation.
These results reveal the novel LncRNAs/miRNAs regulatory pathways regulating neural differentiation of NPCs induction from mESCs.
Sox6 was reported to be a critical mediator of maintaining the stemness of the mouse neural stem cells. 49 Sox6 also forms a positive feedback loop with Sox2, a key transcription factor for NPCs self-renewal which inhibits neuronal differentiation in the developing central nervous system. 50 Here, we found that over-expression of The abnormal neural differentiation leads to further disability of brain. 3 In mouse embryonic retinas, miR-96 expression level is minimal and up-regulates following birth, peaking in adult retinas. 53  Sevoflurane, the most commonly used general anaesthetic in children, induces neurotoxicity and cognitive impairment in young mice 35 and inhibits neurogenesis in vitro. 60,61 The mechanisms by which these effects are created have yet to be identified, impeding further research in anaesthesia neurotoxicity in the developing brain.
In December 2016, the FDA issued a warning about the use of general anaesthetics in young children (0-3 years of age) and pregnant women, and required warnings to be added to the labels of general anaesthetic agents. The widespread and growing use of anaesthesia in children makes its safety a major health issue of interest. 62 It has become a matter of even greater concern as evidence shows that multiple exposures to anaesthesia and surgery may induce cognitive impairment in children. [63][64][65][66][67][68] In this study, the administration of sevoflurane decreased the levels of Rik-201 and Rik-203. This not only suggests that Rik-201 and Rik-203 could be a potential novel target of the anaesthesia neurotoxicity, but also indicated the critical relationship between lncRNA Rik and the neural function.

CO N FLI C T S O F I NTE R E S T
The authors declare no competing financial interests.