Identification of Aberrantly-Expressed Long Non-Coding RNAs in Osteoblastic Cells from Osteoporotic Patients

Abstract

Osteoporosis (OP) is a multifactorial disease influenced by genetic, epigenetic, and environmental factors. One of the main causes of the bone homeostasis alteration is inflammation resulting in excessive bone resorption. Long non-coding RNAs (lncRNAs), have a crucial role in regulating many important biological processes in bone, including inflammation. We designed our study to identify lncRNAs misregulated in osteoblast primary cultures derived from OP patients (n = 4), and controls (CTRs, n = 4) with the aim of predicting possible RNA and/or protein targets implicated in this multifactorial disease. We focused on 84 lncRNAs regulating the expression of pro-inflammatory and anti-inflammatory genes and miRNAs. In silico analysis was utilized to predict the interaction of lncRNAs with miRNAs, mRNAs, and proteins targets. Six lncRNAs were significantly down-regulated in OP patients compared to controls: CEP83-AS1, RP11-84C13.1, CTC-487M23.5, GAS5, NCBP2-AS2, and SDCBP2-AS1. Bioinformatic analyses identified HDCA2, PTX3, and FGF2 proteins as downstream targets of CTC-487M23.5, GAS5, and RP11-84C13.1 lncRNAs mediated by the interaction with miRNAs implicated in OP pathogenesis, including miR-21-5p. Altogether, these data open a new regulatory mechanism of gene expression in bone homeostasis and could direct the development of future therapeutic approaches.

Keywords: bone homeostasis; epigenetics; inflammation; long non-coding RNAs; osteoblast; osteoporosis.

Figure 1

Primary human osteoblast cultures were observed under phase-contrast microscope at 10× field. Scale bar: 500 μm.

Figure 2

Volcano plot of LncRNAs levels in OP vs. CTRs. The Volcano plot shows the significant lncRNAs that we found down-regulated in osteoblast cell lines on the left (down-regulated) and above the solid vertical line (statistically significant), according to significant p-value and fold-regulation change. The solid vertical line represents no change in gene expression (log2 (1) = 0). The dotted lines represent a selected threshold or boundary for fold change. The default setting is 2. The solid vertical line represents a selected threshold for the p-value and statistical significance. The default setting is 0.05. Statistical significance versus fold-change is shown on the y- and x-axes, respectively [24].

Figure 3

Hypothetical pathogenetic mechanisms emerging from this study. (A) Growth arrest-specific 5 (GAS5) lncRNA masks miR-21-5p binding sites, (B) CTC-487M23.5 lncRNA inhibits the translation of HDAC2 mRNA, (C) NCPB2-AS2 lncRNA masks miRNA binding sites implicated in osteoblast differentiation, and (D) RP11-84C13.1 lncRNA masks miR-576-3p binding sites. ↑ and ↑: increase; ↓: decrease; : no repression; : repression.