Circular RNA CDR1as regulates osteoblastic differentiation of periodontal ligament stem cells via the miR-7/GDF5/SMAD and p38 MAPK signaling pathway.

BACKGROUND: Periodontal 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. METHODS: The 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. RESULTS: We 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. CONCLUSIONS: Our 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.

LncRNA NEAT1 promotes dexamethasone resistance in multiple myeloma by targeting miR-193a/MCL1 pathway.

Although dexamethasone (DEX) remains a first-line agent for multiple myeloma (MM) therapy, the development of DEX resistance has become an indicator of poor prognosis in MM patients. It is thus urgent to develop strategies to restore the vulnerability of MM to DEX. This study demonstrated long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) was highly expressed in DEX-resistant myeloma cell lines, and upregulation of NEAT1 was tightly linked to poor prognosis. The in-depth study revealed that during the development of DEX resistance in these cells, the miR-193a levels were decreased, which resulted in the increased expression of the target gene myeloid cell leukemia-1 (MCL1). We also found knockdown of NEAT1, the DEX-induced sensitivity was enhanced in the resistant cells. Meanwhile, overexpression of NEAT1 increased the DEX-induced resistance in the sensitive cells. In conclusion, the NEAT1/miR-193a/MCL1 pathway is closely associated with the development of DEX resistance in myeloma cells, and knockdown of NEAT1 can significantly improve DEX sensitivity in MM.

Overexpression of CircRNA BCRC4 regulates cell apoptosis and MicroRNA-101/EZH2 signaling in bladder cancer.

Emerging evidence has indicated that circular RNAs (circRNAs) play pivotal roles in the regulation of cellular processes and are found to be aberrantly expressed in a variety of tumors. However, the clinical role of circRNAs in bladder cancer (BC) and the molecular mechanisms have yet to be fully understood. In this study, the clinical specimens were obtained and the expression level of a circRNA BCRC4 was detected by real-time PCR in both BC tissues and cell line. The circular RNA over-expression plasmid was constructed and transfected into BC cells and related cell line. The cell cycles and apoptosis were observed using inverted microscope and flow cytometry. Western blotting was used to compare the relative protein expression of groups with different treatments. It was found that circRNA BCRC4 expression was lower in BC tissues than in adjacent normal tissues. Furthermore, consequences of forced-expression of BCRC4 promoted apoptosis and inhibited viability of T24T and UMUC3 cells, and up-regulated BCRC4-increased miR-101 level, which suppressed EZH2 expression in both RNA and protein levels. In addition, gambogic acid (GA) is a promising natural anticancer compound for BC therapy, and GA treatment increased the BCRC4 expression in T24T and UMUC3 cells in a dose-dependent manner. Altogether, our findings suggest that BCRC4 functions as a tumor suppressor in BC, and mediates anticancer function, at least in part, by up-regulating the expression of miR-101. Targeting this newly identified circRNA may help us develop a novel strategy for treating human BC.