Differentially Expressed MicroRNAs in Conservatively Treated Nontraumatic Osteonecrosis Compared with Healthy Controls

Deregulation of microRNAs (miRNAs) contributes to nontraumatic osteonecrosis of the femoral head (ONFH-N), but the differentially expressed circulating miRNAs in patients with ONFH-N receiving nonsurgical therapy are unknown. This study aimed to determine the miRNAs expression profile of patients with ONFH-N receiving conservative treatments. This was a case-control prospective study of 43 patients with ONFH-N and 43 participants without ONFH-N, enrolled from 10/2014 to 10/2016 at the Department of Orthopedics of the Linyi People’s Hospital (China). The two groups were matched for age, gender, and living area. Microarray analysis and quantitative RT-PCR were used to examine the differentially expressed miRNAs. Bioinformatics was used to predict miRNA target genes and signaling pathways. Microarray and quantitative RT-PCR revealed that nine miRNAs were downregulated and five miRNAs were upregulated in ONFH-N (n=3) compared with controls (n=3). Bioinformatics showed that calcium-mediated signaling pathway, regulation of calcium ion transmembrane transporter activity, cytoskeletal protein binding, and caveolae macromolecular signaling complex were probably regulated by the identified differentially expressed miRNAs. In the remaining 80 subjects (n=40/group), miR-335-5p was downregulated (P=0.01) and miR-100-5p was upregulated (P=0.02) in ONFH-N compared with controls. In conclusion, some miRNAs are differentially expressed in conservatively treated ONFH-N compared with controls. Those miRNAs could contribute to the pathogenesis of ONFH-N

Long non-coding RNA HOTAIR inhibits miR-17-5p to regulate osteogenic differentiation and proliferation in non-traumatic osteonecrosis of femoral head.

BACKGROUND AND AIM:The biological functions of non-coding RNAs (ncRNAs) have been widely identified in many human diseases. In the present study, the relationship between long non-coding RNA HOTAIR and microRNA-17-5p (miR-17-5p) and their roles in osteogenic differentiation and proliferation in non-traumatic osteonecrosis of femoral head (ONFH) were investigated. METHODS:The expression levels of HOTAIR and miR-17-5p in the mesenchymal stem cells (MSCs) derived from patients with non-traumatic ONFH and osteoarthritis (OA) were examined by real-time PCR. BMP-2 induced human MSCs from bone marrow (hMSC-BM) were used for osteogenic differentiation. RESULTS:It was observed that the expression level of miR-17-5p was lower and the level of HOTAIR was higher in samples of non-traumatic ONFH compared with OA. HOTAIR downregulation induced by si-HOTAIR led to the increase of miR-17-5p expression and the decrease of miR-17-5p target gene SMAD7 expression. The values of osteogenic differentiation markers, including RUNX2 and COL1A1 mRNA expression and ALP activity, were also elevated by si-HOTAIR. However, the increase of these values was canceled by miR-17-5p inhibitor or SMAD7 upregulation. CONCLUSION:HOTAIR played a role in regulating osteogenic differentiation and proliferation through modulating miR-17-5p and its target gene SMAD7 in non-traumatic ONFH

The expression levels of miR-17-5p and HOTAIR in MSCs of patients with non-traumatic necrosis of femoral head and osteoarthritis.

<p>The MSCs were isolated from patients with non-traumatic necrosis of femoral head (ONFH, n = 15), osteoarthritis (OA, n = 13) and healthy donor (n = 10), the expression levels of miR-17-5p (A) and HOTAIR (B) were detected by real-time PCR. Each sample was repeated three times. ** <i>P</i><0.01.</p

The effects of methylation and histone acetylation alteration on miR-17-5p expression regulation.

<p>The MSCs were isolated from patients with non-traumatic necrosis of femoral head (ONFH, n = 15) and osteoarthritis (OA, n = 13). The expression levels of miR-17-5p in MSCs derived from patients with non-traumatic ONFH was detected by real-time PCR after the treatment of 5 μmol/l 5-Aza-2-deoxycytidine (5-Aza-CdR, A) or 100 nmol/l Trichostatin A (TSA, B) for 48h. The DNA methylation level of miR-17-5p promotor in MSCs from two groups were determined by bisulphite sequencing (C). Each sample was repeated three times. ** P<0.01.</p

HOTAIR regulates osteogenic differentiation markers through mediating SMAD7 expression.

<p>Human mesenchymal stem cells from bone marrow (hMSC-BM) cell line was treated with co-transfection of siRNA-HOTAIR (si-HOTAIR) and Adenovirus-SMAD7 (Ad-SMAD7), with si-control and Ad-GFP acting as controls, respectively (A) or co-transfection of Ad-HOTAIR and si-SMAD7, with Ad-GFP and si-control acting as control, respectively (B) for 48h, then incubated with osteoblastic-inductive BMP-2 for six days. The mRNA and protein levels of RUNX2 and COL1A1 were detected by real-time PCR and western blot, and ALP activity was measured. n = 3, each sample was repeated three times. ** <i>P</i><0.01.</p

The effects of HOTAIR downregulation on the expression levels of miR-17-5p and its target gene.

<p>The MSCs were isolated from patients with non-traumatic necrosis of femoral head (ONFH), and transfected with siRNA-HOTAIR (si-HOTAIR) or siRNA-control (si-control). The HOTAIR expression level was measured by real-time PCR (A). The DNA methylation level of miR-17-5p promoter was determined by bisulphite sequencing assay (B). The miR-17-5p expression level was measured by real-time PCR (C). The SMAD7 expression in mRNA and protein levels was measured by real-time PCR and western blot (D). n = 3, each sample was repeated three times. ** <i>P</i><0.01.</p

HOTAIR regulates osteogenic differentiation markers through mediating miR-17-5p expression.

<p>Human mesenchymal stem cells from bone marrow (hMSC-BM) cell line was treated with co-transfection of siRNA-HOTAIR (si-HOTAIR) and miR-17-5p inhibitor, with si-control and NC acting as controls, respectively (A) or co-transfection of Adenovirus-HOTAIR (Ad-HOTAIR) and miR-17-5p mimic, with Ad-GFP and Pre-NC acting as controls, respectively (B) for 48h, then incubated with osteoblastic-inductive BMP-2 for six days. The mRNA levels of RUNX2 and COL1A1 were detected by real-time PCR and ALP activity was measured. n = 3, each sample was repeated three times. ** <i>P</i><0.01.</p

HOTAIR expression in BMP-2-induced osteoblastic differentiation.

<p>HOTAIR expression level was detected by real-time PCR in human mesenchymal stem cells from bone marrow (hMSC-BM) cell line after the treatment of osteoblastic-inductive BMP-2 for six days. n = 3, each sample was repeated three times. ** <i>P</i><0.01.</p

The effects of HOTAIR on osteogenic differentiation markers.

<p>Human mesenchymal stem cells from bone marrow (hMSC-BM) cell line was transfected with siRNA-HOTAIR (si-HOTAIR, with si-control acting as control, A) or Adenovirus-HOTAIR (Ad-HOTAIR, with Ad-GFP acting as control, B) for 48h, then incubated with osteoblastic-inductive BMP-2 for six days. The mRNA levels of RUNX2 and COL1A1 were detected by real-time PCR and ALP activity was measured by Kits. n = 3, each sample was repeated three times. ** <i>P</i><0.01.</p