RNA interference-mediated osteoprotegerin silencing increases the receptor activator of nuclear factor-kappa B ligand/osteoprotegerin ratio and promotes osteoclastogenesis

Abstract

BACKGROUND

In vivo degradation of bone scaffolds is significantly influenced by osteoclast (OC) activity, which is orchestrated by the interplay between receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG). The ratio of RANKL/OPG is a crucial determinant of OC-mediated bone resorption, which plays an integral role in bone remodeling and scaffold degradation. Elevated levels of RANKL relative to OPG enhance osteoclastogenesis, thereby accelerating the degradation process essential for integrating bone scaffolds into the host tissue.

AIM

To elucidate the effects of OPG gene silencing on osteoclastogenesis within rat bone marrow-derived mesenchymal stem cells (BMSCs). By investigating these effects, the study aimed to provide deeper insights into the regulatory mechanisms that influence bone scaffold degradation, potentially leading to improved bone repair and regeneration strategies.

METHODS

We employed recombinant lentiviral plasmids to silence the OPG gene in rat BMSCs to achieve the aims. The efficacy of gene silencing was assessed using quantitative reverse transcription polymerase chain reaction and western blot analysis to measure the expression levels of OPG and RANKL. Tartrate-resistant acid phosphatase staining was utilized to evaluate the formation of OCs. Additionally, co-immunoprecipitation assays were conducted to explore the interactions between RANKL and OPG proteins, further assessing the biochemical pathways involved in osteoclastogenesis.

RESULTS

The silencing of the OPG gene in BMSCs resulted in a significant increase in the RANKL/OPG ratio, evidenced by decreased expression levels of OPG and increased levels of RANKL. Enhanced osteoclastogenesis was observed through tartrate-resistant acid phosphatase staining, which indicated a substantial rise in OC formation in response to the altered RANKL/OPG balance. The co-immunoprecipitation assays provided concrete evidence of the direct interaction between RANKL and OPG proteins, substantiating their pivotal roles in regulating OC activity.

CONCLUSION

The findings from this study underscore the critical role of the RANKL/OPG axis in osteoclastogenesis. Silencing of the OPG gene in BMSCs effectively increases the RANKL/OPG ratio, promoting OC activity and potentially enhancing bone scaffold degradation. This regulatory mechanism offers a promising avenue for modulating bone remodeling processes, which is essential for effective bone repair and the successful integration of bone scaffolds into damaged sites. Future research might focus on optimizing the control of this axis to better facilitate bone tissue engineering and regenerative therapies.

Keywords: Osteoprotegerin; Receptor activator of nuclear factor-kappa B ligand; Bone marrow-derived mesenchymal stem cells; RNA interference; Osteoclast; Bone scaffold

Core Tip: This study reports, for the first time, that enhancing the receptor activator of nuclear factor-kappa B ligand/osteoprotegerin (RANKL/OPG) ratio through RNA interference promotes osteoclastogenesis. Our findings reveal a significant upregulation of RANKL mRNA levels after OPG gene silencing. The study demonstrates a significant downregulation of OPG mRNA and protein levels. The increase in the RANKL/OPG ratio significantly promotes osteoclastogenesis. This study provides a new theoretical basis and molecular targets for degrading bone scaffolds and bone tissue repair.