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Long Non-coding RNA Maternally Expressed 3 Increases the Expression of Neuron-Specific Genes by Targeting miR-128-3p in All-Trans Retinoic Acid-Induced Neurogenic Differentiation From Amniotic Epithelial Cells

Gao, Yuhua, Zhang, Ranxi, Wei, Guanghe, Dai, Shanshan, Zhang, Xue, Yang, Wancai, Li, Xiangchen, Bai, Chunyu·2019·Frontiers in Cell and Developmental Biology
DOI 10.3389/fcell.2019.00342PMC PMC6936004
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FIGURE 1 Neurogenic differentiation derived from amniotic epithelial cells (AECs). AECs were induced with all-trans retinoic acid (ATRA) for 18 days. (A,B) Neurogenic markers were detected by immunofluorescence staining and flow cytometry. Neuron-specific genes (β III-tubulin) and astrocyte-specific genes (glial fibrillary acidic protein: GFAP) were detected. Scale bar = 100 μm. (C–E) RT-qPCR was used to determine the relative expression of β III-tubulin, GFAP, and miR-128-3p in non-induced AECs and induced AECs. (F,G) Comparative analysis of miR-128-3p and β III-tubulin or GFAP in neurogenic differentiation. (H) The expression of Jagged 1 (JAG1), a putative target for miR-128-3p in neurogenic differentiation, was significantly altered in induced AECs. ∗P < 0.05.
FIGURE 1 Neurogenic differentiation derived from amniotic epithelial cells (AECs). AECs were induced with all-trans retinoic acid (ATRA) for 18 days. (A,B) Neurogenic markers were detected by immunofluorescence staining and flow cytometry. Neuron-specific genes (β III-tubulin) and astrocyte-specific genes (glial fibrillary acidic protein: GFAP) were detected. Scale bar = 100 μm. (C–E) RT-qPCR was used to determine the relative expression of β III-tubulin, GFAP, and miR-128-3p in non-induced AECs and induced AECs. (F,G) Comparative analysis of miR-128-3p and β III-tubulin or GFAP in neurogenic differentiation. (H) The expression of Jagged 1 (JAG1), a putative target for miR-128-3p in neurogenic differentiation, was significantly altered in induced AECs. ∗P < 0.05.CC BY · PubMed Central
FIGURE 2 Role of miR-128-3p in neurogenic differentiation from AECs. (A,B) Neurocyte markers were tested using Western blotting after AECs were exposed to miR-128-3p, miR-128-3p inhibitor, and Jagged 1, respectively. Protein abundance was analyzed using ImageJ tools. The data revealed that Jagged1 could rescue the β III-tubulin expression after overexpressed miR-128 in AECs. ∗P < 0.05; ∗∗P < 0.01; ns, non-significant. (C) Immunoprecipitation of Myc-tagged Argonaute 2 (AGO2) from AECs co-transfected with Myc-AGO2 and either miR-128-3p or Let-7a (negative control). The empty vector (EV) served as the Myc-AGO2-related negative control. Jag 1 and β-actin mRNA levels were quantified using qPCR, and the relative immunoprecipitate (IP)/input (cell total RNA) values were plotted. ∗P < 0.05. (D) The effect of miR-128-3p on JAG1 expression was evaluated using luciferase reporter assays. ∗∗P < 0.01. (E) Western blot analysis of JAG1 expression was performed following siRNA targeting. Protein abundance was analyzed using ImageJ tools. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control and Scramble was used as a control. Western blot images are representative of at least three independent replicates. ∗P < 0.05. (F,G) Neurocyte markers were tested using flow cytometry after induced AECs were exposed to various small RNAs or Jagged 1. iAECs, induced AECs; miR-128 i, miR-128 inhibitor; miR Con, miR-128 mimics control.
FIGURE 2 Role of miR-128-3p in neurogenic differentiation from AECs. (A,B) Neurocyte markers were tested using Western blotting after AECs were exposed to miR-128-3p, miR-128-3p inhibitor, and Jagged 1, respectively. Protein abundance was analyzed using ImageJ tools. The data revealed that Jagged1 could rescue the β III-tubulin expression after overexpressed miR-128 in AECs. ∗P < 0.05; ∗∗P < 0.01; ns, non-significant. (C) Immunoprecipitation of Myc-tagged Argonaute 2 (AGO2) from AECs co-transfected with Myc-AGO2 and either miR-128-3p or Let-7a (negative control). The empty vector (EV) served as the Myc-AGO2-related negative control. Jag 1 and β-actin mRNA levels were quantified using qPCR, and the relative immunoprecipitate (IP)/input (cell total RNA) values were plotted. ∗P < 0.05. (D) The effect of miR-128-3p on JAG1 expression was evaluated using luciferase reporter assays. ∗∗P < 0.01. (E) Western blot analysis of JAG1 expression was performed following siRNA targeting. Protein abundance was analyzed using ImageJ tools. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control and Scramble was used as a control. Western blot images are representative of at least three independent replicates. ∗P < 0.05. (F,G) Neurocyte markers were tested using flow cytometry after induced AECs were exposed to various small RNAs or Jagged 1. iAECs, induced AECs; miR-128 i, miR-128 inhibitor; miR Con, miR-128 mimics control.CC BY · PubMed Central
FIGURE 3 Notch pathway inhibition by miR-128-3p decreases the expression of neurocyte markers in neurogenic differentiation. (A) Immunofluorescence staining illustrates activation of the Notch pathway based on the nuclear expression of intercellular activated notch 1 (Notch intracellular domain: NICD) and Hes 5 after ATRA induction and miR-128 addition. (B) Fluorescence intensity was analyzed using ImageJ tools. (C,D) Western blot data of NICD and HES5 expression after JAG1, BMS-708163, or miR-128 overexpression treatment. Protein abundance was analyzed using ImageJ tools. ∗P < 0.05; ∗∗P < 0.01; ns, non-significant. (E) Neurocyte markers were tested using flow cytometry in induced AECs exposed to 708163 (an inhibitor of the Notch pathway) or Jagged 1 (activator for the Notch pathway). iAECs, induced AECs.
FIGURE 3 Notch pathway inhibition by miR-128-3p decreases the expression of neurocyte markers in neurogenic differentiation. (A) Immunofluorescence staining illustrates activation of the Notch pathway based on the nuclear expression of intercellular activated notch 1 (Notch intracellular domain: NICD) and Hes 5 after ATRA induction and miR-128 addition. (B) Fluorescence intensity was analyzed using ImageJ tools. (C,D) Western blot data of NICD and HES5 expression after JAG1, BMS-708163, or miR-128 overexpression treatment. Protein abundance was analyzed using ImageJ tools. ∗P < 0.05; ∗∗P < 0.01; ns, non-significant. (E) Neurocyte markers were tested using flow cytometry in induced AECs exposed to 708163 (an inhibitor of the Notch pathway) or Jagged 1 (activator for the Notch pathway). iAECs, induced AECs.CC BY · PubMed Central
FIGURE 4 HES5 promotes transcription of the neurocyte marker genes β III-tubulin, NSE, and PSA-NCAM via physical interactions with their promoters. (A) HES5 promoter-binding sites were predicted using bioinformatics algorithms in the promoter region of β III-tubulin, NSE, and PSA-NCAM. Predicted sequences were calculated according to the relative frequency of promotor sequences, which have the potential to bind Hes 5 but do not completely conform to the sequence logo of Hes 5. Consensus sequences are shown in red. (B–D) The amount of HES5 enriched within neurocyte marker gene promoter regions in normal and induced AECs was quantified by ChIP-coupled real-time PCR. The percentage of input was calculated according to threshold cycle values. The negative control for Hes 5 binding sites is shown in the Supplementary Material. Data are presented as the mean ± SD (three independent experiments).
FIGURE 4 HES5 promotes transcription of the neurocyte marker genes β III-tubulin, NSE, and PSA-NCAM via physical interactions with their promoters. (A) HES5 promoter-binding sites were predicted using bioinformatics algorithms in the promoter region of β III-tubulin, NSE, and PSA-NCAM. Predicted sequences were calculated according to the relative frequency of promotor sequences, which have the potential to bind Hes 5 but do not completely conform to the sequence logo of Hes 5. Consensus sequences are shown in red. (B–D) The amount of HES5 enriched within neurocyte marker gene promoter regions in normal and induced AECs was quantified by ChIP-coupled real-time PCR. The percentage of input was calculated according to threshold cycle values. The negative control for Hes 5 binding sites is shown in the Supplementary Material. Data are presented as the mean ± SD (three independent experiments).CC BY · PubMed Central
FIGURE 5 lncRNA MEG3 targets miR-128-3p in neurogenic differentiation from AECs. (A) The expression of MEG3 was analyzed in normal and induced AECs using RT-qPCR. (B) The binding site between MEG3 and miR-128-3p was predicted using bioinformatics algorithms and subsequently mutated to verify interactions. (C) Full-length MEG3 (WT) and sequences containing different mutated variants of the miR-128-3p binding site (MUT) were tested to determine the effect on miR-15/16 expression. The pRL-40 vector alone was used as an internal control. Results are expressed as relative luciferase activity and represent the mean ± SD of at least three replicates. (D) Immunoprecipitation of Myc-tagged Argonaute 2 (AGO2) from AECs co-transfected with Myc-AGO2 and either miR-128-3p or Let-7a (negative control). Empty vector (EV) served as the Myc-AGO2-related negative control. MEG3 and β-actin mRNA levels were quantified using qPCR, and relative immunoprecipitate (IP)/input (cell total RNA) values were plotted. ∗P < 0.05; ∗∗P < 0.01.
FIGURE 5 lncRNA MEG3 targets miR-128-3p in neurogenic differentiation from AECs. (A) The expression of MEG3 was analyzed in normal and induced AECs using RT-qPCR. (B) The binding site between MEG3 and miR-128-3p was predicted using bioinformatics algorithms and subsequently mutated to verify interactions. (C) Full-length MEG3 (WT) and sequences containing different mutated variants of the miR-128-3p binding site (MUT) were tested to determine the effect on miR-15/16 expression. The pRL-40 vector alone was used as an internal control. Results are expressed as relative luciferase activity and represent the mean ± SD of at least three replicates. (D) Immunoprecipitation of Myc-tagged Argonaute 2 (AGO2) from AECs co-transfected with Myc-AGO2 and either miR-128-3p or Let-7a (negative control). Empty vector (EV) served as the Myc-AGO2-related negative control. MEG3 and β-actin mRNA levels were quantified using qPCR, and relative immunoprecipitate (IP)/input (cell total RNA) values were plotted. ∗P < 0.05; ∗∗P < 0.01.CC BY · PubMed Central
FIGURE 6 ATRA increases the expression of MEG3 through the cAMP/CREB pathway to modulate neurogenic differentiation. (A) A siRNA for MEG3 (si-MEG3) effectively depressed the expression of MEG3 in induced AECs (P < 0.05). (B) The concentration of cAMP was detected between normal significantly increased in induced compared with normal AECs (P < 0.05). (C) Expression of cAMP response element-binding protein (CREB) was detected under ATRA or 666-15 (a CREB inhibitor) treatment using immunofluorescence staining. (D) MEG3 expression was analyzed under 666-15 or Jagged 1 treatment by RT-qPCR. (E,F) Expression of β III-tubulin and GFAP were as assessed using Western blotting. (G) β III-tubulin and GFAP expression was analyzed following single or assorted overexpression of MEG3 (o-MEG3), si-MEG3 or 666-15 treatment in induced AECs. iAECs, induced AECs; siMEG3-NC, siMEG3 negative control. ∗P < 0.05; ∗∗P < 0.01; ns, non-significant.
FIGURE 6 ATRA increases the expression of MEG3 through the cAMP/CREB pathway to modulate neurogenic differentiation. (A) A siRNA for MEG3 (si-MEG3) effectively depressed the expression of MEG3 in induced AECs (P < 0.05). (B) The concentration of cAMP was detected between normal significantly increased in induced compared with normal AECs (P < 0.05). (C) Expression of cAMP response element-binding protein (CREB) was detected under ATRA or 666-15 (a CREB inhibitor) treatment using immunofluorescence staining. (D) MEG3 expression was analyzed under 666-15 or Jagged 1 treatment by RT-qPCR. (E,F) Expression of β III-tubulin and GFAP were as assessed using Western blotting. (G) β III-tubulin and GFAP expression was analyzed following single or assorted overexpression of MEG3 (o-MEG3), si-MEG3 or 666-15 treatment in induced AECs. iAECs, induced AECs; siMEG3-NC, siMEG3 negative control. ∗P < 0.05; ∗∗P < 0.01; ns, non-significant.CC BY · PubMed Central
FIGURE 7 Statistical analysis of FCM data in miR-128-3p and its upstream or downstream genes affecting neurogenic differentiation. miR-128-3p reduces the amount of neurons and increases the amount of gliocytes by targeting JAG1 to prevent Notch pathway activation, which may enhance the transcription of neuron-specific markers. MEG3 increases the amount of neurons and reduces the amount of gliocytes via targeting miR-128-3p to activate the Notch pathway (∗P < 0.05; ∗∗P < 0.01; blue asterisk: neurons; pink asterisk: gliocyte).
FIGURE 7 Statistical analysis of FCM data in miR-128-3p and its upstream or downstream genes affecting neurogenic differentiation. miR-128-3p reduces the amount of neurons and increases the amount of gliocytes by targeting JAG1 to prevent Notch pathway activation, which may enhance the transcription of neuron-specific markers. MEG3 increases the amount of neurons and reduces the amount of gliocytes via targeting miR-128-3p to activate the Notch pathway (∗P < 0.05; ∗∗P < 0.01; blue asterisk: neurons; pink asterisk: gliocyte).CC BY · PubMed Central
FIGURE 8 Schematic of promoting neurogenic differentiation from human amniotic epithelial cells.
FIGURE 8 Schematic of promoting neurogenic differentiation from human amniotic epithelial cells.CC BY · PubMed Central

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Authors
Gao, Y., Zhang, R., Wei, G., Dai, S., Zhang, X., Yang, W., Li, X., & Bai, C.
Venue
Frontiers in Cell and Developmental Biology
Publisher
Frontiers Media SA
Published
2019-12-23
Volume
7
ISSN
2296-634X
DOI
10.3389/fcell.2019.00342
PMC
PMC6936004
Cited by
25
References
0
Licence
creativecommons.org/licenses/by/4.0/ (vor)

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