BackgroundTGF-β promotes tumor invasion and metastasis through inducing epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Circular RNAs (circRNAs) are recognized as functional non-coding RNAs involved in human cancers. However, whether and how circRNAs contribute to TGF-β-induced EMT and metastasis in NSCLC remain vague. Here, we investigated the regulation and function of Circular RNA hsa_circ_0008305 (circPTK2) in TGF-β-induced EMT and tumor metastasis, as well as a link between circPTK2 and transcriptional intermediary factor 1 γ (TIF1γ) in NSCLC.MethodsCircular RNAs were determined by human circRNA Array analysis, real-time quantitative reverse transcriptase PCR and northern blot. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), RNA pull-down and fluorescence in situ hybridization (FISH) assays were employed to test the interaction between circPTK2 and miR-429/miR-200b-3p. Ectopic overexpression and siRNA-mediated knockdown of circPTK2, TGF-β-induced EMT, Transwell migration and invasion in vitro, and in vivo experiment of metastasis were used to evaluate the function of circPTK2. Transcription and prognosis analyses were done in public databases.ResultsCircPTK2 and TIF1γ were significantly down-regulated in NSCLC cells undergoing EMT induced by TGF-β. CircPTK2 overexpression augmented TIF1γ expression, inhibited TGF-β-induced EMT and NSCLC cell invasion, whereas circPTK2 knockdown had the opposite effects. CircPTK2 functions as a sponge of miR-429/miR-200b-3p, and miR-429/miR-200b-3p promote TGF-β-induced EMT and NSCLC cell invasion by targeting TIF1γ. CircPTK2 overexpression inhibited the invasion-promoting phenotype of endogenous miR-429/miR-200b-3p in NSCLC cells in response to TGF-β. CircPTK2 overexpression significantly decreased the expression of Snail, an important downstream transcriptional activator of TGF-β/Smad signaling. In an in vivo experiment of metastasis, circPTK2 overexpression suppressed NSCLC cell metastasis. Moreover, circPTK2 expression was dramatically down-regulated and positively correlated with TIF1γ expression in human NSCLC tissues. Especially, circPTK2 was significantly lower in metastatic NSCLC tissues than non-metastatic counterparts.ConclusionOur findings show that circPTK2 (hsa_circ_0008305) inhibits TGF-β-induced EMT and metastasis by controlling TIF1γ in NSCLC, revealing a novel mechanism by which circRNA regulates TGF-β-induced EMT and tumor metastasis, and suggesting that circPTK2 overexpression could provide a therapeutic strategy for advanced NSCLC.Electronic supplementary materialThe online version of this article (10.1186/s12943-018-0889-7) contains supplementary material, which is available to authorized users.
Epithelial-mesenchymal transition (EMT), a key step in the early stages of cancer metastasis, is orchestrated by several signaling pathways, including IL-6/JAK/STAT3 and TGF-β/Smad signaling. However, an association between the two signaling pathways during the EMT process is largely unknown. Here, we focused on lung cancer and demonstrated that TGF-β1 induced the phosphorylation of Smad3 (p-Smad3), upregulation of Snail, a fibroblast-like morphology, and downregulation of E-cadherin as well as upregulation of vimentin in lung cancer cell lines. SIS3 (an inhibitor of Smad3) suppressed TGF-β1-induced activation of Smad3, upregulation of Snail and the EMT process. Importantly, the JAK2/STAT3-specific inhibitor AG490 blocked Stat3 phosphorylation, resulting in attenuated levels of TGF-β1-induced p-Smad3, Snail, MMP2, and Smad-mediated PAI-1 promoter reporter gene activity in A549 and H1650 cells. Subsequently, AG490 inhibited TGF-β-induced cell migration and invasion. Moreover, exogenous IL-6 treatment stimulated Stat3 activation, enhanced TGF-β-induced expression of p-Smad3 and Snail, aggravated the EMT process, and increased lung cancer cell migration and invasion induced by TGF-β1. Our findings show that the JAK/STAT3 pathway is required for TGF-β-induced EMT and cancer cell migration and invasion via upregulation of the expression of p-Smad3 and Snail, and the IL-6/JAK/STAT3 and TGF-β/Smad signaling synergistically enhance EMT in lung carcinomas. The present study suggests a novel rationale for inhibiting cancer metastasis using anti-IL-6/JAK/STAT3 and anti-TGF-β/Smad therapeutic strategies.
Background: Platelet-rich fibrin (PRF) by Choukroun's technique is derived from an autogenous preparation of concentrated platelets. Little is known about the effects of PRF on periodontal ligament fibroblasts (PDLFs) and the application of PRF for periodontal regeneration. Methods: PDLFs were derived from healthy individuals undergoing extraction for orthodontic reasons. Blood collection was carried out from healthy volunteers. PRF was obtained from a table centrifuge centrifuged at 3000 rpm for 12 minutes. The effects of PRF on PDLFs were determined by measuring the expression of phosphorylated extracellular signal-regulated protein kinase (p-ERK), osteoprotegerin (OPG) and alkaline phosphatase (ALP) activity. Moreover, we retrospectively examined the feasibility and safety of reconstructing the periodontal infrabony defects with PRF in six patients. Results: PRF was found to increase ERK phosphorylation and OPG in PDLFs in a time-dependent manner (p < 0.05). ALP activity was also significantly upregulated by PRF (p < 0.05). Application of PRF in infrabony defects exhibited pocket reduction and clinical attachment gain after six months. Periapical radiography revealed radiographic defect filled in grafted teeth. Conclusions: The enhancement of p-ERK, OPG and ALP expression by PRF may provide benefits for periodontal regeneration. Clinical and radiologic analysis showed that the use of PRF is an effective modality for periodontal infrabony defects.
Background: Platelet-rich fibrin (PRF) prepared by Choukroun's technique is derived from an autogenous preparation of concentrated platelets without any manipulation. PRF was found to increase osteoblast growth and proliferation. However, the underlying mechanisms are not yet completely understood. This study aimed to determine the effects of PRF on cell attachment, proliferation, phosphorylated Akt, heat shock protein 47 (HSP47) and lysyl oxidase (LOX) expression on human osteoblasts. Methods: Blood collection was carried out from 10 healthy volunteers. Cell attachment and proliferation were measured by colorimetric assay with WST-1 and alamar blue in human osteoblast cell line U2OS cells, respectively. Western blot was employed to evaluate the expression of p-Akt, HSP47 and LOX. Results: PRF alone was found to stimulate U2OS cell attachment compared with untreated controls (p < 0.05). PRF was found to increase osteoblast proliferation during a 5-day incubation period (p < 0.05). PRF was found to increase Akt phosphorylation in a time-dependent manner (p < 0.05). Collagen-related proteins HSP47 and LOX were significantly elevated by stimulation with PRF compared with untreated controls (p < 0.05). Conclusions: It is suggested that PRF is capable of increasing osteoblast attachment, proliferation and simultaneously upregulating collagen-related protein production. These actions in combination would effectively promote bone regeneration.
TIF1γ is a novel regulator of transforming growth factor (TGF)-β/Smad signaling. Our previous studies show that dysregulated expression of transcriptional intermediary factor 1 γ (TIF1γ) and abnormal TGF-β/Smad signaling are implicated in non-small-cell lung cancer (NSCLC) separately. However, how TIF1γ contributes to NSCLC by controlling TGF-β/Smad signaling is poorly understood. Here, we investigated the mechanistic role of TIF1γ in TGF-β-induced epithelial-mesenchymal transition (EMT), as well as a link between TIF1γ and SOX2 in NSCLC. We show that TIF1γ is a downstream target of SOX2 in NSCLC cells. SOX2 overexpression negatively regulated TIF1γ promoter activity and thereby attenuated TIF1γ mRNA and protein expression levels; SOX2 knockdown significantly enhanced TIF1γ promoter activity and augmented TIF1γ expression. Moreover, TIF1γ mRNA expression was downregulated in human NSCLC tissues and negatively correlated with SOX2 protein, which was upregulated in NSCLC tissues. Importantly, knockdown of TIF1γ or SOX2 overexpression augmented SMAD4 (human Mad (mothers against decapentaplegic)-related homologous protein 4)-dependent transcriptional responses, and enhanced TGF-β-induced EMT and human NSCLC cell invasion; knockdown of SOX2 impaired TGF-β-induced EMT and NSCLC cell invasion. In an in vivo model of metastasis, knockdown of TIF1γ promotes NSCLC cell metastasis. In addition, our data suggested that TIF1γ inhibited TGF-β-induced EMT through competing with SMAD4 in NSCLC cells. Taken together, our findings reveal a new mechanism by which SOX2-mediated transcription repression of TIF1γ promotes TGF-β-induced EMT in NSCLC.
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