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. 2018 Jun 1:11:345-353.
doi: 10.1016/j.omtn.2018.03.004. Epub 2018 Mar 12.

Linc-ROR Promotes Osteogenic Differentiation of Mesenchymal Stem Cells by Functioning as a Competing Endogenous RNA for miR-138 and miR-145

Lu Feng  1 , Liu Shi  1 , Ying-Fei Lu  2 , Bin Wang  1 , Tao Tang  3 , Wei-Ming Fu  4 , Wei He  5 , Gang Li  6 , Jin-Fang Zhang  7
Affiliations

Linc-ROR Promotes Osteogenic Differentiation of Mesenchymal Stem Cells by Functioning as a Competing Endogenous RNA for miR-138 and miR-145

Lu Feng et al. Mol Ther Nucleic Acids. .

Abstract

Long noncoding RNAs (lncRNAs), which serve as important and powerful regulators of various biological activities, have gained widespread attention in recent years. Emerging evidence has shown that some lncRNAs play important regulatory roles in osteoblast differentiation of mesenchymal stem cells (MSCs), suggesting a potential therapeutic strategy for bone fracture. As a recently identified lncRNA, linc-ROR was reported to mediate the reprogramming ability of differentiated cells into induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs) self-renewal. However, other functions of linc-ROR remain elusive. In this study, linc-ROR was found to be upregulated during osteogenesis of human bone-marrow-derived MSCs. Ectopic expression of linc-ROR significantly accelerated, whereas knockdown of linc-ROR suppressed, osteoblast differentiation. Using bioinformatic prediction and luciferase reporter assays, we demonstrated that linc-ROR functioned as a microRNA (miRNA) sponge for miR-138 and miR-145, both of which were negative regulators of osteogenesis. Further investigations revealed that linc-ROR antagonized the functions of these two miRNAs and led to the de-repression of their shared target ZEB2, which eventually activated Wnt/β-catenin pathway and hence potentiated osteogenesis. Taken together, linc-ROR modulated osteoblast differentiation by acting as a competing endogenous RNA (ceRNA), which may shed light on the functional characterization of lncRNAs in coordinating osteogenesis.

Keywords: ceRNA; linc-ROR; mesenchymal stem cells; osteogenesis.

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Figures

Figure 1
Figure 1
Linc-ROR Promoted Osteogenesis of Human BM-MSCs (A) The osteoblast differentiation of human BM-MSCs was initiated by osteogenic inducer and harvested at indicated time points. At days 0, 3, 6, and 9, the expression level of linc-ROR was monitored by qRT-PCR assays. (B and C) The human BM-MSCs were transfected with sh-Linc-ROR or pLinc-ROR overexpression vectors, and osteogenesis was initiated. At day 3, the alkaline phosphatase activity was visualized by NBT/BCIP precipitation (B). At day 14, the calcified nodules of human BM-MSCs were visualized by alizarin red S staining (C). (D) The mRNA expression level of osteogenesis-related marker genes after linc-ROR overexpression or sh-linc-ROR transfection was examined by qRT-PCR assays. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 2
Figure 2
Linc-ROR Directly Targeted miR-138 and miR-145 (A) Venn diagram illustrating the overlap between 43 miRNA targets of linc-ROR interaction predicted by miRanda (dark gray) and 48 miRNA targets predicted by miRcode. Thirty-two common targets were further applied to TargetScan analysis. (B) Schematic diagrams of the mutual interplays between miRNA and linc-ROR. miRNA binding position and calculated ΔG values are shown on the top (kcal/mol). (C) miR-138 or miR-145 mimics were co-transfected with pmirGLO-ROR luciferase reporter into HEK293 cells, and the luciferase activities were measured. (D) Linc-ROR expression level was examined by qRT-PCR assays in human BM-MSCs transfected with miR-138 or miR-145 mimics. n = 3. *p < 0.05; ***p < 0.001.
Figure 3
Figure 3
miR-138 and miR-145 Suppressed Osteoblast Differentiation (A and B) The human BM-MSCs were transfected with miR-138 or miR-145 mimics, and ALP activity was visualized by NBT/BCIP precipitation at day 3 (A). The calcified nodules were visualized by alizarin red S staining at day 14 (B). (C and D) The expression of osteogenesis marker genes was suppressed by miR-138, whereas pLinc-ROR partially rescued the suppressive effect in MSCs (C). miR-145 also suppressed the osteogenic marker genes expression, whereas pLinc-ROR partially reverse the suppressive effects (D). n = 3. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4
Figure 4
miR-138 and miR-145 Suppressed the Wnt/β-Catenin Signaling by Targeting ZEB2 (A) miR-138 or miR-145 mimics were co-transfected with the luciferase reporter harboring 3′ UTR of ZEB2 mRNA into HEK293 cells, and the luciferase activities were measured. (B–E) ZEB2 expression was suppressed by miR-138, whereas it was slightly rescued by the pLinc-ROR vector at mRNA (B) and protein (D) levels. Expression of ZEB2 was also repressed by miR-145, and the suppressive expression was partially reversed by miR-145 pLinc-ROR at mRNA (C) and protein (E) levels. (F) miR-138 or miR-145 were co-transfected with TOPFlash luciferase reporter into HEK293 cells, and the luciferase activities were measured. (G–J) Beta-catenin mRNA (G) and protein (I) expression levels were suppressed by miR-138, and the suppressive effect was partially reversed by pLinc-ROR. Moreover, Beta-catenin mRNA (H) and protein (J) expression levels were also reduced by miR-145, and the suppressive expression was rescued in part by pLinc-ROR. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 5
Figure 5
Linc-ROR Activated the Wnt/β-Catenin Signaling (A) pLinc-ROR vector was co-transfected with ZEB2 3′ UTR luciferase reporter into HEK293 cells, and the luciferase activities were measured. (B and C) ZEB2 mRNA (B) and protein (C) expression levels were evaluated with linc-ROR overexpression. (D) pLinc-ROR vector was co-transfected with TOPFlash luciferase reporter into HEK293 cells, and the luciferase activities were measured. (E and F) Beta-catenin mRNA (E) and protein (F) expression levels were also upregulated by pLinc-ROR vector. (G) Several downstream target genes of Wnt/β-catenin signaling were examined in linc-ROR-overexpressing BM-MSCs by qRT-PCR assays. n = 3. *p < 0.05; **p < 0.01.
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