LncRNA LOC100506178 promotes osteogenic differentiation via regulating miR-214-5p-BMP2 axis in human bone marrow mesenchymal stem cells

Abstract

Osteogenic differentiation is an important role in dental implantation. Long no coding RNAs (lncRNAs) are a novel class of noncoding RNAs that have significant effects in a variety of diseases. However, the function and mechanisms of LOC100506178 in osteogenic differentiation and migration of bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation of human bone marrow mesenchymalstem cells (hBMSCs) remain largely unclear. BMP2 was used to induce osteogenic differentiation of hBMSCs. Quantitative real time PCR (qRT-PCR) was used to examine the expression of LOC100506178, miR-214-5p, Runt-related transcription factor 2 (RUNX2), Osterix (Osx), and Alkaline Phosphatase (ALP) in BMP2-induced osteogenic differentiation of hBMSCs. The function of LOC100506178 and miR-214-5p was explored in vitro using Alizarin Red S Staining, ALP activity, as well as in vivo ectopic bone formation. Luciferase reporter assay was performed to assess the association between LOC100506178 and miR-214-5p, as well as miR-214-5p and BMP2. The miR-214-5p sponging potential of LOC100506178 was evaluated by RNA immunoprecipitation. In the present study, the expression of LOC100506178 was found to be increased in BMP2-induced osteogenic differentiation of hBMSCs, accompanied with decreased miR-214-5p expression and increased RUNX2, Osx and ALP expression. LOC100506178 significantly induced, while miR-214-5p suppressed the BMP2-induced osteogenic differentiation of hBMSCs. Mechanistically, LOC100506178 was directly bound to miR-214-5p and miR-214-5p targeted the 3'-untranslated region of BMP2 to negatively regulate its expression. In conclusion, our data indicate a novel molecular pathway LOC100506178/miR-214-5p/BMP2 in relation to hBMSCs differentiation into osteoblasts, which may facilitate bone anabolism.

Keywords: BMP2; LOC100506178; hBMSCs; miR-214-5p; osteogenic differentiation.

Figure 1. LOC100506178 is increased in BMP2-induced osteogenic differentiation of hBMSCs.

(A, B) Alizarin Red S Staining was performing on day 28 of osteoblast differentiation. (C) To quantify the amount of alizarin red staining in different groups, ^∗∗∗p < 0.001. (D) Quantitative evaluation of the osteogenic differentiation capacity using alkaline phosphatase (ALP) activity assay, ^∗∗∗p < 0.001. (E) The expression levels of RUNX2, Osx and ALP mRNA were examined by qPCR, ^∗∗∗p < 0.001. (F) qPCR results also showed that the expression of LOC100506178 was significantly up-regulated in BMP2 stimulated hBMSCs, ^∗∗∗p < 0.001.

Figure 2. LOC100506178 promotes BMP2-induced osteogenic differentiation of hBMSCs.

(A) qPCR analyzed the expression of LOC100506178 in hBMSCs after transfection of LOC100506178 overexpression plasmid and LOC100506178 knockdown plasmid, different letters mean significantly difference in different groups. (B) Alizarin Red S Staining was performing in hBMSCs on day 28 after induction. (C) To quantify the amount of alizarin red staining in different groups, different letters mean significantly difference in different groups. (D) Quantitative evaluation of the osteogenic differentiation capacity using alkaline phosphatase (ALP) activity assay during osteogenesis differentiation, different letters mean significantly difference in different groups. (E) The mRNA expression of RUNX2, Osx and ALP was measured in BMP2-induced hBMSCs transfected with LOC100506178. Different letters mean significantly difference in different groups.

Figure 3. LOC100506178 functions as an endogenous sponge of miR-214-5p.

(A) Putative binding sites of miR-214-5p in LOC100506178 were shown. (B) miR-214-5p was increased in shLOC100506178 transfected hBMSCs and decreased in LOC100506178 overexpression plasmids transfected hBMSCs, ^∗∗p < 0.01. (C) Correlation analysis between LOC100506178 and miR-214-5p levels in hBMSCs at 0, 1, 3, 5, 7, 14 and 28 d after osteogenic differentiation. (D) Luciferase reporter assay showed the directly reaction between LOC100506178 and miR-214-5p. (E) RIP assay demonstrated that LOC100506178 and miR-214-5p expression levels were significantly higher in the anti-AGO1 group compared with the anti-normal IgG group.

Figure 4. miR-214-5p inhibits BMP2-induced osteogenic differentiation of hBMSCs.

(A) qPCR analyzed the expression of miR-214-5p in hBMSCs, ^∗∗∗p < 0.001. (B) Alizarin Red S Staining was performing in hBMSCs on day 28 after induction. (C) To quantify the amount of alizarin red staining in different groups, different letters mean significantly difference in different groups. (D) Quantitative evaluation of the osteogenic differentiation capacity using alkaline phosphatase (ALP) activity assay during osteogenesis differentiation, different letters mean significantly difference in different groups. (E) Overexpression of miR-214-5p suppressed the expression of RUNX2, Osx and ALP and knockdown of miR-214-5p promoted the expression of RUNX2, Osx and ALP in BMP2-induced hBMSCs, different letters mean significantly difference in different groups.

Figure 5. LOC100506178 positively regulates BMP2 expression through sponging miR-214-5p.

(A) Schematic diagram of miR-214-5p target site in the 3′UTR of BMP2. (B) The BMP2-WT 3′UTR luciferase reporter activity was significantly decreased by miR-214-5p mimics, which was abolished in the mutation of BMP2 3′UTR, ^∗∗p < 0.01 compared with BMP-2-3′UTR-WT+NC. (C) Western blot showed that endogenous BMP2 protein level was obviously suppressed by miR-214-5p mimics transfection. (D) The luciferase activity of BMP2, which was inhibited by miR-214-5p mimics, was significantly rescued after co-transfection of LOC100506178 WT, compared with vector or LOC100506178 MUT. ^∗p < 0.05, ^∗∗p < 0.01, compared with NC + vector, NC + LOC100506178 WT or NC + LOC100506178 MUT; #p < 0.05, compared with miR-214-5p mimics + LOC100506178 MUT or miR-214-5p mimics + vector.

Figure 6. LOC100506178 increases in vivo ectopic bone formation in hBMSCs by suppression miR-214-5p.

(A–D) Representative image of transplanted specimens in different treated groups by H&E staining; NB, new bone; CT, connective tissue. (B) New bone volume was quantified as per total specimen area. The data represent the mean ± SD, ^∗p < 0.05, ^∗∗p < 0.01, compared with control; ^#p < 0.05, compared with LOC100506178.