Long noncoding RNAs (lncRNAs) are emerging as important regulatory molecules at the transcriptional and post-transcriptional levels and may play essential roles in the differentiation of human bone marrow mesenchymal stem cell (hMSC). However, their roles and functions remain unclear. Here, we showed that lncRNA H19 was significantly upregulated after the induction of osteoblast differentiation. Overexpression of H19 promoted osteogenic differentiation of hMSCs in vitro and enhanced heterotopic bone formation in vivo, whereas knockdown of H19 inhibited these effects. Subsequently, we found that miR-675, encoded by exon1 of H19, promoted osteoblast differentiation of hMSCs and was partially responsible for the pro-osteogenic effect of H19. Investigating the underlying mechanism, we demonstrated that H19/miR-675 inhibited mRNA and protein expression of transforming growth factor-b1 (TGF-b1). The downregulation of TGF-b1 subsequently inhibited phosphorylation of Smad3. Meanwhile, H19/miR-675 downregulated the mRNA and protein levels of histone deacetylase (HDAC) 4/5, and thus increased osteoblast marker gene expression. Taken together, our results demonstrated that the novel pathway H19/miR-675/TGF-b1/Smad3/HDAC regulates osteogenic differentiation of hMSCs and may serve as a potential target for enhancing bone formation in vivo. STEM CELLS 2015;33:3481-3492 SIGNIFICANCE STATEMENTLong noncoding RNAs (lncRNAs) are emerging as important regulatory molecules in biology control and pathology. However, their roles and functions in osteogenic differentiation of mesenchymal stem cell (MSC) remain unclear. Our studies provide important insights into the prodifferentiation effect of lncRNA H19 in osteogenesis both in vitro and in vivo. miR-675 is partially responsible for this pro-osteogenic effect of H19 via the transforming growth factor-b1 (TGF-b1)/Smad3/histone deacetylase (HDAC) pathway. These findings provide important clues for understanding the role of lncRNA-miRNA-mRNA functional network in osteogenic differentiation, and would be helpful in developing stem cell-based therapy for bone defect.

BackgroundPeriodontal ligament stem cells (PDLSCs) are considered as candidate cells for the regeneration of periodontal and alveolar bone tissues. Antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), which is a newly discovered circular RNA (circRNA), has been reported to act as an miR-7 sponge and to be involved in many biological processes. Here, we investigated the potential roles of CDR1as and miR-7 in the osteogenic differentiation of PDLSCs.MethodsThe expression pattern of CDR1as and miR-7 in PDLSCs during osteogenesis was detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Then we overexpressed or knocked down CDR1as or miR-7 to confirm whether they were involved in the regulation of osteoblast differentiation in PDLSCs. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were used to detect the activity of osteoblasts and mineral deposition. Furthermore, a dual luciferase reporter assay was conducted to analyze the binding of miR-7 to growth differentiation factor (GDF)5. To further verify the role of CDR1as in osteoblast differentiation, we conducted animal experiments in vivo. New bone formation in specimens was analyzed by microcomputed tomography (micro-CT), hematoxylin and eosin staining, and immunofluorescence staining.ResultsWe observed that CDR1as was significantly upregulated during the osteogenic differentiation, whereas miR-7 was significantly downregulated. Moreover, knockdown of CDR1as and overexpression of miR-7 inhibited the ALP activity, ARS staining, and expression of osteogenic genes. Overexpression of miR-7 significantly reduced the activity of luciferase reporter vectors containing the wild-type, but not the mutant, 3’ untranslated region (UTR) sequence of GDF5. Furthermore, knockdown of GDF5 partially reversed the effects of miR-7 inhibitor on osteoblast differentiation. Downregulation of CDR1as or GDF5 subsequently inhibited phosphorylation of Smad1/5/8 and p38 mitogen-activated protein kinases (MAPK), while upregulation of miR-7 decreased the level of phosphorylated Smad1/5/8 and p38 MAPK. In vivo, CDR1as knockdown lead to less bone formation compared with the control group as revealed by micro-CT and the histological analysis.ConclusionsOur results demonstrated that CDR1as acts as a miR-7 inhibitor, triggering the upregulation of GDF5 and subsequent Smad1/5/8 and p38 MAPK phosphorylation to promote osteogenic differentiation of PDLSCs. This study provides a novel understanding of the mechanisms of osteogenic differentiation, and suggests a potential method for promoting bone formation.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0976-0) contains supplementary material, which is available to authorized users.

Head and neck squamous cell carcinoma (HNSCC) cells exposed to cisplatin (CIS) displayed a dramatic ATM-dependent phosphorylation of DNp63a that leads to the transcriptional regulation of downstream mRNAs. Here, we report that phospho (p)-DNp63a transcriptionally deregulates miRNA expression after CIS treatment. Several p-DNp63a-dependent microRNA species (miRNAs) were deregulated in HNSCC cells upon CIS exposure, including miR-181a, miR-519a, and miR-374a (downregulated) and miR-630 (upregulated). Deregulation of miRNA expression led to subsequent modulation of mRNA expression of several targets (TP53-S46, HIPK2, ATM, CDKN1A and 1B, CASP3, PARP1 and 2, DDIT1 and 4, BCL2 and BCL2L2, TP73, YES1, and YAP1) that are involved in the apoptotic process. Our data support the notion that miRNAs are critical downstream targets of p-DNp63a and mediate key pathways implicated in the response of cancer cells to chemotherapeutic drugs.

Cisplatin was shown to induce the ataxia telangiectasia mutated (ATM)-dependent phosphorylation of tumor protein p63 isoform, (ΔNp63α), leading to a transcriptional regulation of specific genes implicated in the control of cell death of squamous cell carcinoma (SCC) cells. We previously observed that the cisplatin-induced phosphorylated (p)-ΔNp63α transcriptionally regulates the expression of specific microRNAs (miRNAs) in SCC cells. We found here that cisplatin exposure of SCC cells led to modulation of the members of the autophagic pathway, such as Atg1/Ulk1, Atg3, Atg4A, Atg5, Atg6/Becn1, Atg7, Atg9A and Atg10, by a direct p-ΔNp63α-dependent transcriptional regulation. We further found that specific miRNAs (miR-181a, miR-519a, miR-374a and miR-630), which are critical downstream targets of the p-ΔNp63α, modulated the protein levels of ATG5, ATG6/BECN1, ATG10, ATG12, ATG16L1 and UVRAG, adding another level of expression control for autophagic pathways in SCC cells upon cisplatin exposure. Our data support the notion that the cisplatin-induced p-ΔNp63α could regulate key pathways implicated in response of cancer cells to chemotherapeutics.