Yunfei Zheng

Long Non-coding RNA H19 Inhibits Adipocyte Differentiation of Bone Marrow Mesenchymal Stem Cells through Epigenetic Modulation of Histone Deacetylases.

Bone marrow mesenchymal stem cells (BMSCs) exhibit an increased propensity toward adipocyte differentiation accompanied by a reduction in osteogenesis in osteoporotic bone marrow. However, limited knowledge is available concerning the role of long non-coding RNAs (lncRNAs) in the differentiation of BMSCs into adipocytes. In this study, we demonstrated that lncRNA H19 and microRNA-675 (miR-675) derived from H19 were significantly downregulated in BMSCs that were differentiating into adipocytes. Overexpression of H19 and miR-675 inhibited adipogenesis, while knockdown of their endogenous expression accelerated adipogenic differentiation. Mechanistically, we found that miR-675 targeted the 3′ untranslated regions of the histone deacetylase (HDAC) 4–6 transcripts and resulted in deregulation of HDACs 4–6, essential molecules in adipogenesis. In turn, trichostatin A, an HDAC inhibitor, significantly reduced CCCTC-binding factor (CTCF) occupancy in the imprinting control region upstream of the H19 gene locus and subsequently downregulated the expression of H19. These results show that the CTCF/H19/miR-675/HDAC regulatory pathway plays an important role in the commitment of BMSCs into adipocytes.

miR-34a Inhibits Migration and Invasion of Tongue Squamous Cell Carcinoma via Targeting MMP9 and MMP14

Background miR-34a is an important tumor suppressor gene in various cancer types. But little is known about the dysregulation of miR-34a in tongue squamous cell carcinoma (TSCC). In this study, we investigate the expression and potential role of miR-34a in TSCC. Methods We evaluated miR-34a expression and its relationship with clinicopathological characters in 75 pairs of TSCC samples, and confirmed the role of miR-34a for predicting lymph node metastases from a further 15 pairs of paraffin-embedded TSCC specimens with stringent clinicopathological recruitment criteria using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The effects of miR-34a on cell proliferation, migration and invasion were examined in TSCC cell lines using Cell Counting Kit-8 assay, wound healing assay and transwell assay, respectively. The effects of miR-34a on the expression of matrix metalloproteinase (MMP) 9 and 14 were detected by luciferase reporter assays and Western blot analysis. The expression of miR-34a, MMP9 and MMP14 were also confirmed in TSCC samples by in situ hybridization and immunohistochemistry. Results miR-34a expression in tumor tissues from TSCC patients with positive lymph node metastases was significantly lower than that with negative lymph node metastases. Overexpression of miR-34a significantly suppressed migration and invasion in TSCC cells and simultaneously inhibited the expression of MMP9 and MMP14 through targeting the coding region and the 3′untranslated region, respectively. Moreover, miR-34a expression in TSCC was inversely correlated with protein expression of MMP9 and MMP14 in the TSCC samples. Conclusions miR-34a plays an important role in lymph node metastases of TSCC through targeting MMP9 and MMP14 and may have potential applications in prognosis prediction and gene therapy for lymph node metastases of TSCC patients.

Long non-coding RNA MEG3 inhibits adipogenesis and promotes osteogenesis of human adipose-derived mesenchymal stem cells via miR-140-5p

AbstractlncRNAs are an emerging class of regulators involved in multiple biological processes. MEG3, an lncRNA, acts as a tumor suppressor, has been reported to be linked with osteogenic differentiation of MSCs. However, limited knowledge is available concerning the roles of MEG3 in the multilineage differentiation of hASCs. The current study demonstrated that MEG3 was downregulated during adipogenesis and upregulated during osteogenesis of hASCs. Further functional analysis showed that knockdown of MEG3 promoted adipogenic differentiation, whereas inhibited osteogenic differentiation of hASCs. Mechanically, MEG3 may execute its role via regulating miR-140-5p. Moreover, miR-140-5p was upregulated during adipogenesis and downregulated during osteogenesis in hASCs, which was negatively correlated with MEG3. In conclusion, MEG3 participated in the balance of adipogenic and osteogenic differentiation of hASCs, and the mechanism may be through regulating miR-140-5p.

The pro-apoptotic and pro-inflammatory effects of calprotectin on human periodontal ligament cells.

Calprotectin, a heterodimer of S100A8 and S100A9 subunits, is associated with inflammatory disorders such as rheumatoid arthritis and cystic fibrosis. Although calprotectin levels are increased significantly in the gingival crevicular fluid (GCF) of periodontitis patients, its effects on periodontal ligament cells (PDLCs) remain largely unknown. The aim of this study was to evaluate calprotectin levels in the GCF of generalized aggressive periodontitis (AgP) patients and to investigate the effects of recombinant human calprotectin (rhS100A8/A9) and its subunits (rhS100A8 and rhS100A9) in PDLCs. Both the concentration and amount of crevicular calprotectin were significantly higher in the AgP group compared with healthy controls. In addition, the GCF calprotectin levels were correlated positively with clinical periodontal parameters including bleeding index, probing depth, and clinical attachment loss. rhS100A8/A9 promoted cell apoptosis, whereas rhS100A8 and rhS100A9 individually exerted little effect on apoptosis in PDLCs. rhS100A9 and rhS100A8/A9 increased the activation of nuclear factor-κB (NF-κB) by promoting the nuclear translocation of p65 in PDLCs, subsequently inducing expression of the pro-inflammatory cytokines IL-6, IL-8, TNFα, and COX2. Treatment with an NF-κB inhibitor partially reversed the rhS100A9- and rhS100A8/A9-induced upregulation of the pro-inflammatory cytokines. rhS100A9, and not rhS100A8, was mainly responsible for the pro-inflammatory role of calprotectin. Collectively, our results suggest that calprotectin promotes apoptosis and the inflammatory response in PDLCs via rhS100A9. These findings might help identify novel treatments for periodontitis.

Long non-coding RNA MIAT knockdown promotes osteogenic differentiation of human adipose derived stem cells†

Recently, long non‐coding RNAs (lncRNAs) have emerged as critical players in gene regulation for multiple biological processes. However, their roles and functions in human adipose‐derived stem cells (hASCs) differentiation remain unclear. In the present study, we investigated the role of lncRNA myocardial infarction‐associated transcript (MIAT) in the osteogenic differentiation of hASCs. We found that the expression of MIAT was downregulated in a time‐dependent manner during hASCs osteoinduction. MIAT knockdown promoted osteogenic differentiation of hASCs both in vitro and in vivo. Moreover, MIAT expression was increased upon tumor necrosis factor‐α treatment and MIAT knockdown reversed the negative effects of inflammation on osteoblastic differentiation. This study improves our knowledge of lncRNAs in governing the osteogenic differentiation of hASCs and may provide novel therapeutic strategies for treating bone diseases.

The Circular RNA Landscape of Periodontal Ligament Stem Cells During Osteogenesis

BACKGROUNDnThe present study aims to investigate the distinct expression pattern of circular RNAs (circRNAs) in periodontal ligament stem cells (PDLSCs) during osteogenesis.nnnMETHODSnPDLSCs were isolated and cultured in osteogenic medium. Total RNA was extracted from cells at day 0 (D0), day 3 (D3), day 7 (D7), and day 14 (D14) and submitted to RNA-sequencing to detect expression profiles of circRNAs, messenger RNAs (mRNAs), and microRNAs (miRNAs). Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to validate expression of circRNAs and miRNAs. Differential expression analysis and gene ontology analysis were performed. A circRNA-miRNA-mRNA network was constructed to reveal the potential regulatory role of circRNAs.nnnRESULTSnA total of 12,693 circRNA transcripts were detected, and circRNAs displayed stage-specific expression. Expression of four well-known circRNAs was validated by qRT-PCR. In total, 118 circRNAs were differentially expressed at D3, 128 circRNAs were differentially expressed at D7, and 139 circRNAs were differentially expressed at D14 compared with D0. Host genes of differentially expressed circRNAs were enriched in cytoplasmic or membrane-bound vesicles and extracellular matrix, indicating their potential roles in modulating biogenesis of extracellular vesicles. Moreover, mRNAs that were potentially regulated by circRNAs were enriched in bone-formation-associated processes, including extracellular matrix organization, cell differentiation, and bone morphogenetic protein signaling pathway.nnnCONCLUSIONnExpression profiles of circRNAs were significantly altered during osteogenic differentiation of PDLSCs, providing a clue for future studies on the role of circRNAs in osteoblast differentiation.

miR-375 inhibits cell growth and correlates with clinical outcomes in tongue squamous cell carcinoma

miR-375 has been implicated in various types of cancers. However, its role in tongue squamous cell carcinoma (TSCC) remains unclear. This study aimed to investigate the effects of miR-375 on cell growth and the prognosis of TSCC patients. Using quantitative reverse transcription-polymerase chain reaction, we evaluated miR-375 expression in TSCC samples and TSCC cell lines. The results showed that miR-375 expression was significantly reduced in the TSCC tissues and cell lines. A low level expression of miR-375 in TSCC patients was related to poor of prognosis. Moreover, the effects of miR-375 overexpression on cell proliferation, the cell cycle and the expression of Sp1 and cyclinxa0D1 were examined in TSCC cells. We demonstrated that overexpression of miR-375 significantly inhibited the cell proliferation and cell cycle progression in TSCC cell lines. Overexpression of miR-375 inhibited Sp1 expression by targeting the 3 untranslated region of the Sp1 transcript. The knockdown of Sp1 expression resulted in the subsequent downregulation of cyclinxa0D1. Taken together, our study suggests that miR-375 inhibits the cell growth, and its expression is correlated with clinical outcomes in TSCC.

S100A9-induced release of interleukin (IL)-6 and IL-8 through toll-like receptor 4 (TLR4) in human periodontal ligament cells

S100A8, S100A9, and calprotectin (the S100A8/S100A9 complex) are calcium-binding proteins that promote extracellular pro-inflammatory functions and may play an important role in periodontal disease. Both toll-like receptor 4 (TLR4) and the receptor for advanced glycation end-products (RAGE) are thought to be important receptors for S100A8, S100A9, and calprotectin, but the specific pathways in periodontal ligament (PDL) cells are not yet clear. Our study was designed to identify the specific receptors for S100A9 in human PDL cells. Additionally, we investigated the specific pathways that activate the secretion of pro-inflammatory cytokines interleukins (IL)-6 and IL-8 in PDL cells. The role of nuclear factor (NF)-κB, mitogen-activated protein kinase (MAPK) and reactive oxygen species (ROS) in S100A9-induced pro-inflammatory cytokines were investigated through western blot analysis, dichlorodihydrofluorescein diacetate (H2DCFDA) probe and the application of specific pathway inhibitors. Our results suggest that the S100A9-induced release of IL-6 and IL-8 from human PDL cells is dependent on TLR4, but not RAGE. We provide evidence that S100A9 promotes the secretion of IL-6 and IL-8 through different pathways. Specifically, S100A9 up-regulates the secretion of IL-6 from human PDL cells through NF-κB and p38 pathways and up-regulates the release of IL-8 from human PDL cells through the NF-κB, extracellular-regulated kinase (ERK) 1/2, c-Jun amino-terminal kinase (JNK) 1/2, and p38 signaling pathways. In addition, the release of both cytokines depends on ROS production. The release of both cytokines depends on ROS production. These results suggest that S100A9 promotes pro-inflammatory responses in PDL cells through the TLR4-mediated NF-κB and MAPK signaling pathways.

Promotion Effects of miR-375 on the Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells

Summary MicroRNA plays an important role in bone tissue engineering; however, its role and function in osteogenic differentiation warrant further investigation. In this study, we demonstrated that miR-375 was upregulated during the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hASCs). Overexpression of miR-375 significantly enhanced hASCs osteogenesis both in vitro and in vivo, while knockdown of miR-375 inhibited the osteogenic differentiation of hASCs. Mechanistically, microarray analysis revealed DEPTOR as a target of miR-375 in hASCs. Knockdown of DEPTOR accelerated the osteogenic differentiation of hASCs by inhibiting AKT signaling, which mimics miR-375 overexpression. Furthermore, we confirmed that miR-375 regulated osteogenesis by targeting YAP1, and that YAP1 reversely bound to miR-375 promoter to inhibit miR-375 expression. Taken together, our results suggested that miR-375 promoted the osteogenic differentiation of hASCs via the YAP1/DEPTOR/AKT regulatory network, indicating that miR-375-targeted therapy might be a valuable approach to promote bone regeneration.

Proinflammatory effects and mechanisms of calprotectin on human gingival fibroblasts

BACKGROUND AND OBJECTIVEnCalprotectin (S100A8/A9) is a heterodimer of S100A8 and S100A9 and is associated with multiple inflammatory diseases, including Crohns disease, rheumatoid arthritis and periodontitis. Levels of calprotectin are elevated in the gingival crevicular fluid of patients with periodontitis; however, the effects of calprotectin on human gingival fibroblasts (HGFs) remain unknown. This study investigated the proinflammatory activity of calprotectin on HGFs and the functional receptors and signaling pathways engaged by calprotectin.nnnMATERIAL AND METHODSnHGFs were stimulated by equimolar concentrations of S100A8 and/or S100A9, and the expression levels of interleukin (IL)-6 and IL-8 were detected using real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. The calprotectin receptors were identified by pre-incubating HGFs with the toll-like receptor (TLR) 4 inhibitor or the antibody targeting the advanced glycation end product receptor (RAGE). The involvement of reactive oxygen species (ROS) and signaling pathways were also investigated by treating HGFs with ROS inhibitor or specific pathway inhibitors, respectively.nnnRESULTSnS100A9 and S100A8/A9 significantly upregulated IL-6 and IL-8 expression, which was inhibited upon treatment with the TLR4 inhibitor TAK242. Pretreatment with RAGE-blocking antibodies did not affect cytokine expression. Additionally, S100A9 promoted the production of IL-6 and IL-8 from HGFs via different signaling pathways. IL-6 expression was upregulated via the NF-κB, c-Jun amino-terminal kinase (JNK) 1/2 and p38 mitogen-activated protein kinase (MAPK) pathways, and IL-8 expression was upregulated via NF-κB, p38, JNK1/2 and extracellular-regulated kinase 1/2 MAPK pathways. The release of both cytokines was dependent upon the production of ROS.nnnCONCLUSIONnOur findings suggest that calprotectin exerts proinflammatory effects on HGFs via the S100A9 subunit and TLR4-mediated NF-κB and MAPK signaling pathways.