Abstract
The dysregulation of microRNAs (miRNAs) is a crucial molecular signature of disease development. The potential implication of miRNAs in neurofibromatosis type 1 (NF1) remains poorly investigated. The expression levels of miR-27a-3p, miR-27b-3p, and neurofibromin 1 (NF1) were detected by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. The functional roles of miR-27a-3p and miR-27b-3p in NF1 were explored by CCK8 (Cell Counting Kit-8), 5-ethynyl-2′-deoxyuridine (EdU), terminal deoxynucleoitidyl transferase dUTP nick-end labeling (TUNEL), and transwell assays. Luciferase reporter, RNA pull-down, and RNA binding protein immunoprecipitation (RIP) assays were employed to study the probable target relationship between miRNA and messenger RNA (mRNA). MiR-27b-3p and miR-27a-3p were upregulated in dermal and plexiform human Schwann cells (HSC) from NF1 neurofibromas as well as cell lines of malignant peripheral nerve sheath tumors (MPNSTs). MiR-27a-3p/miR-27b-3p mimics promoted the proliferative, migratory, and invasive ability of dermal HSC and MPNST cell ST88-14, while inhibiting the apoptotic capacity. MiR-27a-3p/miR-27b-3p inhibitors elicited the opposite impacts on the above cellular behaviors in dermal HSC and ST88-14. Intriguingly, NF1 was revealed to be the target of both miR-27a-3p and miR-27b-3p, and was negatively modulated by them. MiR-27a-3p/miR-27b-3p upregulation suppressed the expression of NF1 in dermal HSC and ST88-14. Furthermore, NF1 depletion counterbalanced the functional alteration induced by miR-27a-3p/miR-27b-3p inhibition. Our study suggests that both miR-27b-3p and miR-27a-3p are involved in upstream molecular activity responsible for the depletion of NF1, representing promising targets for therapeutic application in NF1.
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Abbreviations
- NF1:
-
Neurofibromatosis type 1
- NF1:
-
Neurofibromin 1
- MPNSTs:
-
Malignant peripheral nerve sheath tumors
- HSC:
-
Human Schwann cells
- UTR:
-
Un-translated region
- mRNA:
-
Messenger RNA
- miRNAs:
-
MicroRNAs
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- FBS:
-
Fetal bovine serum
- RT-qPCR:
-
Real-time quantitative polymerase chain reaction
- CCK8:
-
Cell Counting Kit-8
- PFA:
-
Paraformaldehyde
- EdU:
-
5-Ethynyl-2′-deoxyuridine
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- TUNEL:
-
Terminal deoxynucleotidyl transferase dUTP nick-end labeling
- RIP:
-
RNA binding protein immunoprecipitation
- WT:
-
Wild-type
- Mut:
-
Mutant
- SD:
-
Standard deviation
- ANOVA:
-
Analysis of variance
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12031_2020_1779_MOESM1_ESM.tif
Supplementary file1 (TIF 1316 KB) Figure S1. A. The expression of miR-27a-3p/miR-27b-3p in normal HSC was analyzed after transfection with miR-27a-3p/miR-27b-3p mimics. B. The cell vitality was studied by CCK8 after upregulation of miR-27b-3p and miR-27a-3p. C. Transwell assay was carried out to assess the migratory ability of the transfected normal HSC. **P < 0.01
12031_2020_1779_MOESM2_ESM.tif
Supplementary file2 (TIF 2089 KB) Figure S2. A. The expression of miR-27a-3p/miR-27b-3p in ST88-14 was analyzed after transfection with miR-27a-3p/miR-27b-3p mimics. B–C. The cell vitality and proliferation were studied by CCK8 and EdU assays, respectively, after upregulation of miR-27b-3p and miR-27a-3p. D. Transwell assay was carried out to assess the migratory ability of the transfected ST88-14. **P < 0.01
12031_2020_1779_MOESM3_ESM.tif
Supplementary file3 (TIF 1677 KB) Figure S3. A. The interference effectiveness of miR-27a-3p/miR-27b-3p inhibitor in ST88-14 was detected by RT-qPCR analysis. B–F. A series of functional experiments were performed to elucidate the effects of miR-27a-3p/miR-27b-3p inhibitor on proliferation, apoptosis, and migration by CCK8 (B), TUNEL (C), flow cytometry analysis (D), caspase-3 detection assay (E), and transwell assay (F). **P < 0.01.
12031_2020_1779_MOESM4_ESM.tif
Supplementary file4 (TIF 2903 KB) Figure S4. A. RT-qPCR assay was performed to study the expression of NF1 mRNA in the transfected ST88-14 with miR-27a-3p/miR-27b-3p mimics. B. RNA pull-down assay was completed to elucidate the molecular relationship between miR-27a-3p/miR-27b-3p and NF1. C. RIP assay was utilized to verify the interaction between target miRNAs and NF1. D-I. The rescue impact of NF1 depletion on miR-27b-3p/miR-27a-3p inhibitor-induced cell viability, proliferation, apoptosis, and migration phenotype was studied via CCK8 (D), EdU assay (E), TUNEL (F), flow cytometry analysis (G), caspase-3 detection assay (H), and transwell assay (I). **P < 0.01.
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Lu, H., Liu, P. & Pang, Q. MiR-27a-3p/miR-27b-3p Promotes Neurofibromatosis Type 1 via Targeting of NF1 . J Mol Neurosci 71, 2353–2363 (2021). https://doi.org/10.1007/s12031-020-01779-2
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DOI: https://doi.org/10.1007/s12031-020-01779-2