Homeobox (HOX) transcript antisense RNA (HOTAIR) is a long intergenic noncoding RNA (lincRNA) that is significantly overexpressed in breast and hepatocellular cancers. It remains unclear, however, whether HOTAIR plays an oncogenic role in human laryngeal squamous cell cancer (LSCC). We therefore investigated the expression and functional role of HOTAIR in LSCC. HOTAIR levels were significantly higher in LSCC than in corresponding adjacent non-neoplastic tissues, and patients with poor histological grade or advanced clinical stage had higher HOTAIR expression. Log-rank test showed a significant association between high levels of HOTAIR and poor prognosis in LSCC patients. Multivariate Cox analysis suggested that HOTAIR is an independent prognostic factor of LSCC. siRNA-mediated knockdown of HOTAIR led to reduced invasion and increased apoptosis of Hep-2 cells in vitro and significantly reduced growth of LSCC xenograft tumors in mice. Moreover, PTEN methylation was significantly reduced in Hep-2 cells depleted of HOTAIR. Taken together, these results suggest that the oncogenic role of HOTAIR in LSCC is related to promotion of PTEN methylation. HOTAIR could serve as a marker for LSCC prognosis and a potential target for therapeutic intervention.

BackgroundLong noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) plays key role in the progression of some human cancers. However, the role of NEAT1 in human laryngeal squamous cell cancer (LSCC) is still unknown. We therefore investigated the expression and function of NEAT1 in LSCC.MethodsNEAT1 level in LSCC and adjacent non-neoplastic tissues were detected by qRT-PCR. NEAT1 was knockdown in LSCC cells and cell proliferation, apoptosis and cell cycle were examined. The growth of xenografts with NEAT1 knockdown LSCC cells was analyzed.ResultsNEAT1 level was significantly higher in LSCC than in corresponding adjacent non-neoplastic tissues, and patients with neck nodal metastasis or advanced clinical stage had higher NEAT1 expression. Moreover, siRNA mediated NEAT1 knockdown significantly inhibited the proliferation and induced apoptosis and cell cycle arrest at G1 phase in LSCC cells. The growth of LSCC xenografts was significantly suppressed by the injection of NEAT1 siRNA lentivirus. Furthermore, NEAT1 regulated CDK6 expression in LSCC cells which was mediated by miR-107.ConclusionNEAT1 plays an oncogenic role in the tumorigenesis of LSCC and may serve as a potential target for therapeutic intervention.

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SummaryMesenchymal stem cells (MSCs) from bone marrow are main cell source for tissue repair and engineering, and vehicles of cell-based gene therapy. Unlike other species, mouse bone marrow derived MSCs (BM-MSCs) are difficult to harvest and grow due to the low MSCs yield. We report here a standardised, reliable, and easy-to-perform protocol for isolation and culture of mouse BM-MSCs. There are five main features of this protocol. (1) After flushing bone marrow out of the marrow cavity, we cultured the cells with fat mass without filtering and washing them. Our method is simply keeping the MSCs in their initial niche with minimal disturbance. (2) Our culture medium is not supplemented with any additional growth factor. (3) Our method does not need to separate cells using flow cytometry or immunomagnetic sorting techniques. (4) Our method has been carefully tested in several mouse strains and the results are reproducible. (5) We have optimised this protocol, and list detailed potential problems and trouble-shooting tricks. Using our protocol, the isolated mouse BM-MSCs were strongly positive for CD44 and CD90, negative CD45 and CD31, and exhibited tri-lineage differentiation potentials. Compared with the commonly used protocol, our protocol had higher success rate of establishing the mouse BM-MSCs in culture. Our protocol may be a simple, reliable, and alternative method for culturing MSCs from mouse bone marrow tissues.

Laryngeal squamous cell carcinoma (LSCC) is a highly aggressive malignant cancer. The regulation of LSCC progression by long non-coding RNA (lncRNA) was not well understood. In this study, we reported that the lncRNA H19 was upregulated in LSCC. The expression levels of H19 were inversely correlated with the survival rate of LSCC patients. Knockdown of H19 expression inhibited LSCC cell migration, invasion and proliferation. We identified microRNA miR-148a-3p as an inhibitory target for H19. Overexpression of miR-148a-3p reduced LSCC migration, invasion and proliferation cell, while inhibition of miR-148a-3p did the opposite. The inhibition of LSCC progression induced by H19 knockdown required the activity of miR-148a-3p. We also identified DNA methyltransferase enzyme DNMT1 as a target of miR-148a-3p. Cellular DNA methylation levels were inhibited by both miR-148a-3p overexpression and H19 knockdown. In summary, our study demonstrated that the lncRNA H19 promoted LSCC progression via miR-148a-3p and DNMT1.

Biopolymer-based supramolecular hydrogels cross-linked by host−guest interactions are usually mechanically weak as shown in "inverted vials" instead of freestanding 3D constructs. Herein, we describe a novel host−guest macromer (HGM) approach for preparation of biopolymerbased freestanding supramolecular hydrogels. Host−guest macromers are formed by molecular self-assembly between adamantane-functionalized hyaluronic acid (AD x HA) guest polymers and monoacrylated β-cyclodextrins (mono-Ac-βCD) host monomers. Supramolecular hydrogels are readily prepared by UV-induced polymerization of the preassembled host−guest macromers. Such hydrogels are soely cross-linked by in situ formed multivalent host−guest nanoclusters and show significantly reinforced mechanical properties yet still retain desirable supramolecular features. They can self-heal and be remolded into freestanding 3D constructs which afford effective protection on the encapsulated stem cells during the compression remolding, making them promising carriers for therapeutic cells that can quickly adapt to and integrate with surrounding tissues of the targeted defects. We demonstrate that such hydrogels not only sustain extended release of encapsulated proteinaceous growth factors (TGF-β1) but also support chondrogenesis of the human mesenchymal stem cells (hMSCs) and promote cartilage regeneration in a rat model.

Tendon injures are common orthopedic conditions, but tendon development and the pathogenesis of tendon injures, such as tendinopathy, remain largely unknown and have limited the development of clinical therapy. Studies on tenogenic differentiation at the molecular level may help in developing novel therapeutic strategies. As novel regulators, long noncoding RNAs (lncRNAs) have been found to have widespread biological functions, and emerging evidence demonstrates that lncRNAs may play important regulatory roles in cell differentiation and tissue regeneration. In this study, we found that lncRNA H19 stimulated tenogenesis of human tendon-derived stem cells. Stable overexpression of H19 significantly accelerated TGF-β1-induced tenogenic differentiation and accelerated tendon healing in a mouse tendon defect model. H19 directly targeted miR-29b-3p, which is considered to be a negative regulator of tenogenesis. Furthermore, miR-29b-3p directly suppressed the expression of TGF-β1 and type I collagen, thereby forming a novel regulatory feedback loop between H19 and TGF-β1 to mediate tenogenic differentiation. Our study demonstrated that H19 promotes tenogenic differentiation both and by targeting miR-29b-3p and activating TGF-β1 signaling. Regulation of the TGF-β1/H19/miR-29b-3p regulatory loop may be a new strategy for treating tendon injury.-Lu, Y.-F., Liu, Y., Fu, W.-M., Xu, J., Wang, B., Sun, Y.-X., Wu, T.-Y., Xu, L.-L, Chan, K.-M., Zhang, J.-F., Li, G. Long noncoding RNA H19 accelerates tenogenic differentiation and promotes tendon healing through targeting miR-29b-3p and activating TGF-β1 signaling.