Modulation effects of MicroRNAs on sclerostin expression in osteoblast cell lines

Sclerostin is a protein encoded by the SOST gene that acts as a negative regulator of osteoblastic bone formation by blocking the Wnt/β-catenin signaling pathway. The right amount of sclerostin production is key for healthy bone formation. Agents that inhibit sclerostin are currently being studied for their bone anabolic potential. The main objective of this study is to identify microRNAs (miRNAs), a small non-coding RNA molecule that serves as post-transcriptional regulator of sclerostin production. In this study, human osteoblast-like cell lines were cultured and used for analysis. The bioinformatics prediction algorithms were employed to identify potential miRNAs that may target SOST/sclerostin. In-silico approaches were also applied to identify the features of the selected miRNA/mRNA complex and to perform functional enrichment analyses. Mimics and inhibitors of the selected miRNAs, along with their corresponding negative controls, were transfected into Saos-2 cells using Lipofectamine™ 3000 reagent to validate their regulatory functions. The post-transcriptional regulatory effects of the selected miRNAs on SOST mRNA expression were measured using RT-qPCR, while the effects on extracellular sclerostin protein expression were validated using ELISA. Additionally, in-situ trypan blue exclusion assays, CFSE/PI assays, and MTT assays were conducted to examine post-transfection cell viability and cytotoxic effects. The bioinformatics results identified hsa-miR-378c and hsa-let-7b-5p as potential miRNAs that may modulate SOST/sclerostin expression, thereby being examined for further in-vitro validation analyses. Moreover, functional annotation analyses demonstrated that both selected miRNAs have crosstalk with sclerostin and other genes involved in various pathways, including the Wnt pathway. RT-qPCR results from this study showed a reduction of SOST mRNA expression when the Saos-2 cells were transfected with 5 nM (non-significant), 50 nM (p<0.01), and 100 nM (p<0.0001) of hsa-miR-378c mimic, compared to the mimic negative controls. ELISA results showed a significant decrease in extracellular sclerostin protein expression when Saos-2 cells were transfected with 100 nM (p<0.05) of hsa-miR-378c mimic, relative to the mimic negative control. Despite that, transfection with hsa-let- 7b-5p mimic and hsa-miR-378c inhibitor did not yield significant hypothesized results, as analyzed with RT-qPCR and ELISA. Furthermore, all the transfected Saos-2 cells retained high cell viability and showed no notable cell cytotoxic effects following transfection. In conclusion, up-regulation of hsa-miR-378c may promote new bone regeneration by modulating osteoblast functions through targeting its putative target, SOST/sclerostin. Therefore, future studies are required to validate the predicted miRNAs that may potentially modulate healthy bone development, as such information can provide a platform for the development of a novel therapy treating various bone diseases.

Text 125083 - Full Text.pdf Download (17MB) Official URL or Download Paper: http://ethesis.upm.edu.my/id/eprint/18802

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