Long non-Coding RNA, RP11-399K21.11, Suppresses Osteogenesis by Diminishing Promoter Activity

Objectives: Non-coding RNAs (ncRNAs) play significant roles in the control of epigenetics. Long ncRNAs (LncRNAs), which exceed 500 bps and are synthesized predominantly by RNA polymerase II, exhibit expression patterns particular to certain tissues. We established induced pluripotent stem cells (iPSCs) from patients with clavicular craniocranial dysplasia (CCD) syndrome which caused by alterations in the Runx2 gene and examined the role of non-coding RNAs (ncRNAs) in bone development. In this study, we demonstrated the regulatory mechanism underlying the LncRNA RP11-399K21.11.
Methods: SAOS2 cells, human osteoblast-like osteosarcoma cells, were grown in osteoblast differentiation medium (OBM) for 7 days. The overexpression and downregulation vectors were transfected in SAOS2 cell and cultured with 1000ul/ml G418 for 10 days to establish stable cell lines.
Results: The overexpression of RP11-399K21.11 in SAOS2 cells resulted in a reduction in the staining of both ALP and alizarin compared to the control group. Significant reductions in the expression of ALP and IBSP mRNA were detected. Conversely, in SAOS2 cells where RP11-399K21.11 was suppressed, there was an increase in ALP staining and a corresponding increase in ALP mRNA expression in OBM. Long non-coding RNAs (lncRNAs) are involved in regulating gene activity through epigenetic mechanisms. We investigated DNA methylation patterns using PCR analysis. However, there is no DNA methylation on the ALP and SP7 GpC island. In contract, H3K4me3 and H3K27ac were depleted at ALP upstream/promoter region in RP11-399K21.11 overexpressed SAOS2 compared to that in control SAOS2. Lastly, we observed that the expression of the lncRNA RP11-399K21.11 decreased when SAOS2 cells were treated with CHIR99021 during their development into osteoblasts.
Conclusions: The present findings indicate that the non-coding RNA RP11-399K21.11 hinders osteoblast differentiation in a repressive manner. However, the activation of the WNT pathway counteracts its inhibitory effect on bone formation.