MicroRNA-495 downregulates AQP1 and facilitates proliferation and differentiation of osteoblasts in mice with tibial fracture through activation of p38 MAPK signaling pathway.

Osteoblasts are implicated in the building of the vertebrate skeleton. The current study aimed to investigate the role of microRNA-495 (miR-495) in the osteoblasts of mice with tibial fractures and the underlying mechanism involving in aquaporin-1 (AQP1) and the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. Initially, a microarray-based analysis was performed to screen the differentially expressed genes and miRNAs associated with tibial fracture. Following the establishment of a tibial fracture mouse model, the positive rate of the AQP1 protein in the fracture tissue was detected by immunohistochemistry (IHC). Next, to verify the binding site between miR-495 on AQP1, bioinformatics data were employed in addition to the application of a dual-luciferase reporter gene assay. The osteoblast cell line MC3T3-E1 was treated with miR-495 mimic, miR-495 inhibitor and Anisomycin to explore the potent effects of miR-495 on proliferation and differentiation of osteoblasts in mice with tibial fracture. The expression of miR-495, AQP1, p38 MAPK, PCNA, Cyclin D1, OCN, and OPN was subsequently evaluated by RT-qPCR and Western blot analysis. Cell viability, the number of calcium nodules and alkaline phosphatase (ALP) activity were detected by MTT assay, alizarin red staining, and ALP activity assay, respectively. Our results revealed that miR-495 was down-regulated while AQP1 was up-regulated in the mice with tibial fractures. AQP1 was verified as a target gene of miR-495. When the cells were treated with overexpressed miR-495 or activated p38 MAPK signaling pathway, elevated expression of PCNA, Cyclin, D1, OCN, and OPN along with an increased amount of calcium nodules, higher cell viability, and enhanced ALP activity was detected, while the expression of AQP1 was reduced. Collectively, the key findings of the present study support the notion that overexpressed miR-495 may activate the p38 MAPK signaling pathway to inhibit AQP1 and to promote the proliferation and differentiation of osteoblasts in mice with tibial fracture.

Retracted: Inhibition of microRNA-940 suppresses the migration and invasion of human osteosarcoma cells through the secreted frizzled-related protein 1-mediated Wnt/ß-catenin signaling pathway.

Osteosarcoma (OS) is the most common malignant tumor of bone with a high potential for metastasis. This study intends to explore whether microRNA-940 (miR-940) affects the development of OS cells and the underlying mechanism. OS and adjacent normal tissues were collected from OS patients; the OS cell line with the highest expression of miR-940 was selected, which was then subjected to transfection of miR-940 mimic, miR-940 inhibitor, siRNA-secreted frizzled-related protein 1 (SFRP1) or LiCl (agonists of Wnt/ß-catenin pathway) to identify regulation of miR-940 to OS cells through SFRP1. The targeting relationship between miR-940 and SFRP1 was verified using dual-luciferase reporter gene assay. Reverse-transcription quantitative polymerase chain reaction and Western blot assay were performed to determine miR-940, SFRP1, ß-catenin, and cyclinD1 and apoptosis-related genes Fas, Bax, and Bcl-2. MTT (3-(4, 5-dimethylthiazol-2-Yl)-2, 5-diphenyltetrazolium bromide) assay, scratch test, transwell assay, and flow cytometry were carried out to detect proliferation, migration, invasion, and apoptosis, respectively. Nude mice models were established to observe the tumor formation. Higher expression of miR-940, ß-catenin, and cyclinD1 and lower SFRP1 expression were identified in OS tissues. miR-940 targeted and negatively regulated SFRP1 expression. Furthermore, upregulated miR-940 expression activated the Wnt/ß-catenin signaling pathway in OS. With the treatment of miR-940 mimic, LiCL, or siRNA-SFRP1, OS cells showed promoted proliferation, migration, invasion, tumor formation, and impeded apoptosis (further reflected by elevated Bcl-2 expression and reduced Fas and Bax expression). The study demonstrates that miR-940 can promote the proliferation, migration, and invasion but suppress the apoptosis of human OS cells by downregulating SFRP1 through activating Wnt/ß-catenin signaling pathway.

[Differential expression profile of microRNA between hyperplastic scar and normal skin].

OBJECTIVE: To explore the miRNA differential expression profiles of hyperplastic scar and normal skin so as to further elucidate the pathogenesis of hyperplastic scar and search for new therapeutic targets. METHODS: The total RNA was extracted from 5 human hyperplastic scar and normal skin tissues by Trizol. The specimens were collected from the First Affiliated Hospital of Nanchang University from November 2010 to May 2011, and purified by mirVana(TM) miRNA Isolation Kit and then labeled and hybridized by miRNA Complete Labeling and Hyb Kit. The images of hybridization were analyzed by the Feature Extraction (v10.7) software and the microarray results confirmed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In hyperplastic scar, 92 miRNA genes were up-regulated and 13 down-regulated. The most significantly up-regulated miRNAs were hsa-miR-564 and hsa-miR-936, etc. while hsa-miR-451, hsa-miR-223, hsa-miR-363 and hsa-miR-29b-1* became significantly down-regulated. The findings of RT-PCR on hsa-miR-21 and hsa-miR-451 of regulation were in a high concordance with the microarray results. CONCLUSION: Distinct differences of miRNA expression between human hyperplastic scar and normal skin, it may be closely correlated with the formation, development and evolution of hyperplastic scar.