Lingfei Jia

Expression, regulation and roles of miR-26a and MEG3 in tongue squamous cell carcinoma.

MicroRNA miR‐26a and long noncoding RNA (lncRNA) MEG3 gene have been independently reported to be tumor suppressor genes in various cancers, but neither has been previously associated with tongue squamous cell carcinoma (TSCC). We report here that miR‐26a and lncRNA MEG3 gene expression were both strongly reduced in TSCC compared with levels in matched nonmalignant tissues, and combined low expression levels of both miR‐26a and MEG3 emerged as an independent prognostic factor for poor clinical outcome in TSCC patients. Assays in the human TSCC cell lines SCC‐15 and CAL27 showed that miR‐26a targets the DNA methyltransferase 3B transcript and that its inhibition may result in the upregulation of MEG3, providing a plausible link between the observed reduction of miR‐26a and MEG3 in TSCC tissue. Furthermore, the overexpression of miR‐26a or MEG3 in SCC‐15 and CAL27 cells inhibited cell proliferation and cell cycle progression, and promoted cell apoptosis. Considering the poor prognostic outcomes associated with reduced miR‐26a and MEG3, our findings imply that these factors likely play important antitumor effects in TSCC pathogenesis. Furthermore, they represent potential prognostic biomarkers for stratification of TSCC patients.

Long Noncoding RNA H19 Promotes Osteoblast Differentiation Via TGF‐β1/Smad3/HDAC Signaling Pathway by Deriving miR‐675

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‐β1 (TGF‐β1). The downregulation of TGF‐β1 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‐β1/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

Prognostic Implications of MicoRNA miR-195 Expression in Human Tongue Squamous Cell Carcinoma

Background miR-195 is aberrantly expressed in multiple types of disease. But little is known about the dysregulation of miR-195 in tongue squamous cell carcinoma (TSCC). In this study, we investigated the roles of miR-195 in the development and progression of TSCC. Methods Using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we evaluated miR-195 expression in TSCC samples from 81 patients. Overall survival of these patients was examined using Kaplan–Meier curves with log-rank tests and the Cox proportional hazards model. The expression of two known miR-195 target genes, Cyclin D1 and Bcl-2, was also examined in the TSCC samples by immunohistochemistry. The effects of miR-195 overexpression on cell cycle progression and apoptosis and its effects on the expression of Cyclin D1 and Bcl-2 were examined in transfected TSCC cell lines (SCC-15 and Cal27) using fluorescence-activated cell sorting assays, luciferase reporter assays, and Western blots. Results Reduced miR-195 expression was associated with tumor size and the clinical stage of TSCC tumors. Kaplan–Meier survival analysis indicated that the TSCC patients with reduced expression of miR-195 had poor overall survival and in multivariable analyses low levels of miR-195 emerged as an independent prognostic factor for this clinical outcome. Levels of miR-195 expression were inversely correlated with the expression of Cyclin D1 and Bcl-2. Overexpression of miR-195 inhibited cell cycle progression, promoted apoptosis, and reduced Cyclin D1 and Bcl-2 expression in two TSCC cell lines. Conclusions miR-195 may have potential applications as a prognostic factor for TSCC patients.

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.

Inhibition of lncRNA MIR31HG Promotes Osteogenic Differentiation of Human Adipose‐Derived Stem Cells

Osteogenic differentiation and bone formation is suppressed under condition of inflammation induced by proinflammation cytokines. A number of studies indicate miRNAs play a significant role in tumor necrosis factor‐α‐induced inhibition of bone formation, but whether long non‐coding RNAs are also involved in this process remains unknown. In this study, we evaluated the role of MIR31HG in osteogenesis of human adipose‐derived stem cells (hASCs) in vitro and in vivo. The results suggested that knockdown of MIR31HG not only significantly promoted osteogenic differentiation, but also dramatically overcame the inflammation‐induced inhibition of osteogenesis in hASCs. Mechanistically, we found MIR31HG regulated bone formation and inflammation via interacting with NF‐κB. The p65 subunit bound to the MIR31HG promoter and promoted MIR31HG expression. In turn, MIR31HG directly interacted with IκBα and participated in NF‐κB activation, which builds a regulatory circuitry with NF‐κB. Targeting this MIR31HG–NF‐κB regulatory loop may be helpful to improve the osteogenic capacity of hASCs under inflammatory microenvironment in bone tissue engineering. Stem Cells 2016;34:2707–2720

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

BACKGROUND The present study aims to investigate the distinct expression pattern of circular RNAs (circRNAs) in periodontal ligament stem cells (PDLSCs) during osteogenesis. METHODS PDLSCs 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. RESULTS A 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. CONCLUSION Expression 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.