Circulating Long Non-Coding RNA GAS5 Is Overexpressed in Serum from Osteoporotic Patients and Is Associated with Increased Risk of Bone Fragility

Abstract

Osteoporosis (OP) is a multifactorial disorder in which environmental factors along with genetic variants and epigenetic mechanisms have been implicated. Long non-coding RNAs (lncRNAs) have recently emerged as important regulators of bone metabolism and OP aetiology. In this study, we analyzed the expression level and the genetic association of lncRNA GAS5 in OP patients compared to controls. Quantitative RT-PCR analysis of GAS5 was performed on the serum of 56 OP patients and 28 healthy individuals. OP subjects were divided into three groups of analysis: 29 with fragility fractures of lumbar spine (OP_VF), 14 with fragility fractures of femoral neck (OP_FF) and 13 without fractures (OP_WF). Genotyping of the rs145204276 insertion/deletion polymorphism has also been performed by Restriction fragment length polymorphism (RFLP) and direct sequencing analyses. Expression of circulating GAS5 is significantly increased in OP patients compared to controls (p < 0.01), with a statistically higher significance in fractured OP individuals vs. healthy subjects (p < 0.001). No statistically significant change was found in female OP patients; conversely, GAS5 is upregulated in the subgroup of fractured OP women sera (p < 0.01) and in all OP males (p < 0.05). Furthermore, a direct correlation between GAS5 expression level and parathyroid hormone (PTH) concentration was found in OP patients (r = 0.2930; p = 0.0389). Genetic analysis of rs145204276 revealed that the deletion allele was correlated with a higher expression of GAS5 in OP patients (0.22 ± 0.02 vs. 0.15 ± 0.01, ** p < 0.01). Our results suggest circulating GAS5 as a putative biomarker for the diagnosis and prognosis of OP and OP-related fractures.

Keywords: biomarker; bone fragility; epigenetics; lncRNA GAS5; osteoporosis.

Figure 1

Schematic representation of the rs145204276 located in the promoter region of GAS5 gene.

Figure 2

Serum expression level of GAS5 in OP patients. GAS5 expression level was analyzed in serum from 56 OP patients and 28 healthy controls. GAPDH mRNA level was used to normalize the relative amount of GAS5 and relative expression values are expressed as 2^−ΔCt. Individual data points represent the mean of duplicate assays for each sample. Statistical differences between OP and CTR groups were analyzed by non-parametric Mann–Whitney U-test. Error bars indicate mean ± standard error mean (s.e.m.) ** p < 0.01.

Figure 3

Serum expression level of GAS5 in OP patients with and without fragility fractures. The GAS5 expression level in the serum from 43 OP patients with fragility fractures (OP_F) was compared to 13 OP patients without fractures (OP_WF) and 28 healthy samples (CTR). The GAPDH mRNA level was used to normalize the relative amount of GAS5 and relative expression values are expressed as 2^−ΔCt. Statistical differences between groups were analyzed using ANOVA with Tukey’s multiple comparison post-test analysis. Error bars indicate mean ± s.e.m. *** Represents a significant difference between OP_VF and CTR (p < 0.001); ## represents a significant difference between OP_VF and OP_FF (p < 0.01).

Figure 4

Serum expression level of GAS5 in OP male patients. (A) GAS5 expression level was analyzed in the serum of 14 OP total male, with and without fragility fractures, and 8 healthy male controls. * p < 0.05; (B) GAS5 expression level was analyzed in the serum of 10 OP fractured male and 8 healthy male controls. GAPDH mRNA level was used to normalize the relative amount of GAS5 and relative expression values are expressed as 2^−ΔCt. Individual data points represent the mean of duplicate assays for each sample. Statistical differences between groups were analyzed for significance using unpaired Student’s t-test. Error bars indicate mean ± s.e.m. * p < 0.05.

Figure 5

Serum expression level of GAS5 in OP female patients. (A) GAS5 expression level was analyzed from serum of 42 OP females, with and without fragility fractures, and 20 healthy females. NS; (B) GAS5 expression level was analyzed in the serum from 33 OP fractured females and 20 healthy females. GAPDH mRNA level was used to normalize the relative amount of GAS5 and relative expression values are expressed as 2^−ΔCt. Individual data points represent the mean of duplicate assays for each sample. Statistical differences between groups were analyzed for significance using unpaired Student’s t-test. Error bars indicate mean ± s.e.m. ** p < 0.01.

Figure 6

Expression level of GAS5 in OP and CTR individuals with different rs145204276 genotypes (A) GAS5 expression in OP patients with Ins/Del (n = 17) and Ins/Ins (n = 39) genotypes (0.22 ± 0.02 vs. 0.15 ± 0.01, ** p < 0.01). (B) GAS5 expression CTR subjects with genotype Ins/Del (n = 7) and Ins/Ins (n = 21), respectively (0.12 ± 0.05 vs. 0.11 ± 0.03 p = 0.5). Statistical differences between groups were analyzed for significance using unpaired Student’s t-test. Error bars indicate mean ± s.e.m.